Cellular Implications of Redox Signaling

Cellular Implications of Redox Signaling

Editors

Carlos Gitler Avihai Danon

Imperial College Press

Cellular Implications of Redox Signaling

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Cellular Implications of Redox Signaling

Editors

Carlos Gitler Avihai Danon Weizmann Institute of Science, Israel

Imperial College Press

Published by Imperial College Press 57 Shelton Street Covent Garden London WC2H 9HE Distributed by World Scientific Publishing Co. Pte. Ltd. 5 Toh Tuck Link, Singapore 596224 USA office: Suite 202, 1060 Main Street, River Edge, NJ 07661 UK office: 57 Shelton Street, Covent Garden, London WC2H 9HE

British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library.

CELLULAR IMPLICATIONS OF REDOX SIGNALING Copyright © 2003 by Imperial College Press All rights reserved. This book, or parts thereof, may not be reproduced in any form or by any means, electronic or mechanical, including photocopying, recording or any information storage and retrieval system now known or to be invented, without written permission from the Publisher.

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ISBN

1-86094-331-4

Typeset by Stallion Press.

This book is printed on acid-free paper. Printed in Singapore by Mainland Press

Preface

Electron transfer between proteins is an essential cellular process. It underlies primary metabolic reactions such as in photosynthesis and respiratory oxidative phosphorylation. Recently, electron transfer reactions between specific proteins have also been found to play a key regulatory role in many fundamental cellular phenomena, including cell proliferation, regu­ lation of specific gene expression, programmed cell death, and cell responses to oxygen levels, free radicals, and oxidants. Increasing number of genetic, molecular and biochemical studies in bacteria, plants, and animals point to the importance and the ubiquity of redox signaling. These redox-regulated phenomena are most likely controlled by specific factors that use intrinsic redox changes to regulate their biological activity. The intrinsic redox changes of the regulatory proteins, typically involve, similarly to protein phosphorylation, covalent modifications that allosterically modulate the protein catalytic activity or its capacity to bind other proteins in a selective manner. The covalent modifications that the regulatory proteins undergo derive from electron transfer between the regulatory protein and specific electron donors or acceptors. In the majority of cases, the regulatory redox reactions involve transfer of two electrons. The most common regulatory chemical groups involved are two proximal cysteinyl moieties (vicinal thiols) that alter­ nate between the oxidized intraprotein disulfide and the reduced dithiol configurations. Another type of two electron-redox reaction involves a protein monothiol that reacts with glutathione to form a proteinglutathione mixed disulfide and an acceptor intraprotein disulfide (Chapter Thomas and col.). Historically, the electron donor proteins were discovered in reactions that involved the catalytic turnover of the enzyme ribonucleotide reductase. The active site disulfhydryl form was found to be oxidized to a disulfide during one round of conversion of a ribonucleotide to a deoxyribonucleotide. Reichart and Holmgren discovered that subsequent reduc­ tion of the active site disulfide, necessary for a new round of catalysis, was mediated by the reactive dithiol of reduced thioredoxin acting as an elec­ tron donor. The thioredoxin disulfide was reduced in turn by thioredoxin reductase utilizing NADPH as the electron source. Holmgren furthermore

V

vi Preface

showed that another protein, glutaredoxin, can use glutathione to function as an alternative reductase of the active site intraprotein disulfide of ribonucleotide reductase (Chapter Holmgren). Subsequent studies have shown that cells contain many proteins with surface localized vicinal thiols. In many cases, the dithiols oxidized to disulfides are not involved directly in the catalytic activity of the proteins. Rather, oxidation of these dithiols to the disulfides functions as an allosteric site regulating the activity of enzymes (Chapter Schurmann). The intraprotein disulfides formed on oxidation are typically reduced to the dithiols by the thioredoxin-NADPH (or ferredoxin) — thioredoxin reductase systems (Chapter Holmgren, Buchanan, Gitler and col.). Selenium has been found to be present as selenocysteine in thioredoxin reductase (Chapter Holmgren, Arner). The glutaredoxin-glutathione pair can also participate in selected reactions as an electron donor or acceptor (Chapter Carmel-Harel and Storz). Thioredoxin is the main cellular intraprotein disulfide reductase. Coupled with NADPH- thioredoxin reductase it maintains the majority of the cell dithiol proteins in the reduced state. It also functions as a regula­ tory subunit in a growing number of protein complexes (Chapter Yodoi and col.). In addition, thioredoxin migrates to the nucleus and has a cen­ tral function in DNA repair and in regulation of transcription factors activity. As mentioned above, reduced thioredoxin is essential for the synthesis of deoxyribonucleotides, which because of their toxicity do not accumulate in cells. Thus, every round of DNA synthesis or repair requires the nuclear presence of reduced thioredoxin. Nuclear-localized reduced thioredoxin may thus become available for its known 2-electron transfer to Ref-1. The Ref-1 protein, in addition to its apurine nuclease activity, may act as a key nuclear protein disulfide reductase regulating activity of many transcription factors that must be in the reduced state to bind to DNA. Thioredoxin is also secreted from cells by an unknown mechanism. Thioredoxin has been shown to play a role as an autocrine growth factor, as a cytokine and as a cytokine modulator. (Chapter Yodoi and col.) Redox-dependent regulatory conformational changes in proteins can also result from alteration in the stability or in the valence of prosthetic iron-sulfur centers. Protein thiols participate in the formation of a diverse set of iron-sulfur centers that are sensitive to electron transfer (Chapter Beinert). In most cases, a change in the oxidation of the iron results in a loss of the stability of the iron-sulfur center leading to an altered activity of the regulatory protein (Chapters Beinert, Kaplan and col., and Kuhn).

Preface vii

Thus, the catalytic activity or the specific binding of a protein may be modified in cells by this type of mechanism. Noteworthy, reversible changes in redox state of iron in the regulatory iron-sulfur center of SoxR, and not iron-sulfur center stability, were found to modulate SoxR transcriptional activity. In addition, recent data suggest that valence changes in metalophosphatases could also modulate activity of these enzymes (Chapter Gitler and col.). Because the above redox reactions involve electron transfer between two proteins as a redox donor/acceptor pair, the reacting moieties have to be in or close to the protein surface (Chapter Gitler and col.). Furthermore, selective site recognition is possible by specific proteinprotein interaction. This is best exemplified by the selective interaction of plant thioredoxin isoforms with different enzymes (Chapters Buchanan, Schurmann). However, because the redox reactions can also occur with small oxidants, the target proteins are highly sensitive to oxidants such as diamide, alkylhydroperoxides and hydrogen peroxide. For this reason, redox changes in selected transcription factors function as sensitive cellu­ lar sensors of peroxide (Chapter Carmel-Harel and Storz), superoxide (Chapter Beinert and col.) or oxygen (Chapter Kaplan and col.). Changes in cellular iron levels are detected by alterations in the stability of the iron-sulfur center of cytoplasmic aconitase. The loss of the iron sulfur cluster activates the RNA-binding activity of the protein which then acts simultaneously as regulator of translation of ferritin mRNA and stability of transferrin receptor mRNA (Chapter Kuhn). Selective protein dithiol oxidation plays a key role in the maturation of nascent polypeptides to form the native disulfide-linked proteins. Recent work has illustrated the electron pathway in the Dsb system that forms disulfides in nascent polypeptides in the bacterial periplasmic space (Chapter Beckwith). In protein disulfide-bond formation in the endoplasmic reticulum (ER) of eukaryotes, oxidizing equivalents are transferred from a conserved ER-membrane protein, Erolp, to substrate proteins via protein disulfide isomerase (Chapter Gilbert and col.). Recently, the ERV1/ALR family of proteins has been found to function as protein dithiol oxidases in the cellular cytoplasm and to interact with glutaredoxins. In growth initiation, and probably in other cellular reactions involving activation of receptor phosphotyrosine kinases, a dual initial calciummediated burst in hydrogen peroxide formation and inhibition of thioredoxin reductase occurs (Chapter Gitler and col.). Thus, early in these ligand-receptor mediated reactions, an oxidizing cellular milieu is required for activation of the phosphorylation cascades. A central role is

viii Preface

suggested for thioredoxin-dependent peroxidases or peroxiredoxins (Chapters Gitler and col., Yodoi and col.). Furthermore, the ensuing oxida­ tion of cellular thioredoxin probably leads to the activation of ASK1. This protein is negatively regulated by its selective binding to reduced thiore­ doxin. ASK1 activation by its dissociation from the oxidized thioredoxin may be critical for its role in the normal activation of the so-called stress pathway of MAP kinases and for cellular commitment to apoptosis (Chapters Gitler and col., and Yodoi and col.). The required inhibition of excess phosphotyrosine phosphatases to allow regulation by kinases could also require the redox oxidizing environment that ensues in cells on ligand-binding to receptor phosphotyrosine kinases. The regulation of gene expression requires the transduction of specific redox signals via unique signaling pathways. These regulatory redox reactions must occur in an otherwise highly reductive intracellular milieu, suggesting that specific oxidation of regulatory factors must occur. Dissection of the reactions that regulate light-mediated translation of chloroplast mRNAs shows that illumination directs the unique oxidation of a protein disulfide isomerase-like protein, acting as a translational activator. Both the reduction and oxidation reactions that govern the redox state of the regulatory PDI-like protein were found to be specific, resulting in coupling of redox regulated translation and light. The mani­ fested selective redox regulation of this process is not transient and occurs throughout the day, demonstrating that the redox state of regulators of gene expression could be uniquely controlled according to their biological function (Chapter Danon and col.). Mitochondria are key organelles in the cell redox status. They have a basic role in defining the cellular levels of reduced pyridine nucleotides by means of electron-transfer and of transhydrogenases. In addition, mitochondria are a constant cellular source of significant levels of superoxide and hydrogen peroxide. The redox regulation of the permeability transition pore, a cyclosporin A-sensitive mitochondrial channel, is a good example of the complex redox interactions that occur in this organelle (Chapter Bernardi). The basic bioenergetic aspects of pore modulation are discussed, with some emphasis on the links between oxidative stress and pore regulation as a potential cause of mitochondrial dysfunction that may be relevant to a variety of forms of cell death. One electron transfer to protein vicinal thiols can occur making redox regulatory proteins highly sensitive to cellular radical reactions. Mounting evidence suggests that ascorbic acid may play a key role in reducing tocopheryl radicals and thus may be essential for radical termination in cell membranes. Dismutation of the ascorbyl radical may be linked to

Preface ix

thioredoxin reductase while dehydroascorbate reduction may be carried out by the cell glutaredoxins. Furthermore, a general mechanism of radicalchain termination may use vicinal thiol proteins to convert radicals to superoxide. Thus, vicinal thiol proteins, superoxide and superoxide dismutase could function as a general cell radical chain-termination system (Chapter Winterbourn). In plant cells, the interplay between radicals and H 2 O z on the one hand and the antioxidants ascorbic acid and glutathione varies in different cellular compartments. The ensuing changes in the lev­ els of ascorbic acid and/or glutathione (and their oxidized forms) can function as a signal for cells to activate different systems that guard against cell damage and disease (Chapter Foyer). Cells contain two different systems involved in reduction of dioxygen and disulfides. One is thioredoxin-based and the other is glutathionebased. The thioredoxin systems regulate protein disulfide reduction by direct reaction of reduced thioredoxin with intraprotein disulfides. Thioredoxin peroxidases or peroxiredoxins are a large family of proteins involved in the reduction of cellular peroxides (dioxygen). The selenoprotein thioredoxin reductase links this system to NADPH as an electron source. On the other hand, glutaredoxins use reduced glutathione to reduce intraprotein disulfides while the selenoprotein glutathione peroxidase functions in the reduction of cellular peroxides. Thus, cells have evolved two different systems — both selenium-dependent — to regulate the redox state of cellular proteins. Genetic links can be found in, for example, hydroperoxide reductases that utilize a thioredoxin-like domain in their function. Processing of peroxides and disulfides evolved presum­ ably in parallel to transfer electrons to reduce the products of radicalchain termination and to reduce disulfides and iron-sulfur centers for redox regulation. The purpose of this book is to present an overall picture of the current state of redox regulation. Not all facets could be covered because of the extensive nature of the subject matter. Rather, we hope that this joint effort will serve as a timely and integrated presentation describing the underlying principles of this key regulatory mechanism. The Editors would like to acknowledge the support of the Weizmann Institute of Science through the Aharon Katzir-Katchalski Center, The Goldshlager Fund, The Dr. Josef Cohn Minerva Center for Biomembrane Research and the Dean of the Faculty of Biology. CG would like to thank Ana and Pablo Brener for their continued support of his research. Our personal thanks also to Beky Gitler and Tami Danon for their active part and total commitment over the long and trying times that were needed to bring this book to fruition.

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Contents

Preface 1

v

The Role of Thioredoxin and Glutaredoxin Systems in Disulfide Reduction and Thiol Redox Control

1

2 Selenocysteine Insertion and Reactivity: Mammalian Thioredoxin Reductases in Relation to Cellular Redox Signaling

27

3

Iron-Sulfur Proteins: Properties and Functions

47

4

The Ferredoxin Ferredoxin/Thioredoxin Thioredoxin System. A light-Dependent Redox Regulatory System in Oxygenic Photosynthetic Cells

73

Thioredoxin and Redox Regulation: Beginnings in Photosynthesis Lead to a Role in Germination and Improvement of Cereals

99

5

6

The Role of Thioredoxin in Regulatory Cellular Functions

115

7

Protein S-Thiolation, S-Nitrosylation, and Irreversible Sulfhydryl Oxidation: Roles in Redox Regulation

141

Radical Scavenging by Thiols: Biological Significance and Implications for Redox Signaling and Antioxidant Defense

175

Ascorbate and Glutathione Metabolism in Plants: H 2 0 2 -Processing and Signalling

191

8

9

10 Disulfide Bond Formation in the Periplasm and Cytoplasm of Escherichia Coli 11 The Thiol Redox Paradox in the Requirement for Disulfide Isomerization in the Eukaryotic Endoplasmic Reticulum

xi

213

233

xii

Cellular Implications of Redox Signalling

12 Mechanisms Controlling Redox Balance in Cells. Inhibition of Thioredoxin and of Thioredoxin Reductase

257

13 Regulatory Disulfides Controlling Transcription Factor Activity in the Bacterial and Yeast Responses to Oxidative Stress

287

14 Redox Signaling During Light-Regulated Translation in Chloroplasts

311

15 Regulation of mRNA Translation and Stability in Iron Metabolism: Is there a Redox Switch?

327

16 Redox Flow as an Instrument for Gene Regulation

361

17 The Permeability Transition Pore as Source and Target of Oxidative Stress Author Index

393 421

Subject Index

423

Chapter 1 The Role of Thioredoxin and Glutaredoxin Systems in Disulfide Reduction and Thiol Redox Control Arne Holmgren Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77, Stockholm, Sweden [email protected]

Keywords: Selenium, thioredoxin reductase, thioredoxin, glutaredoxin

1. Summary The intracellular redox environment in Escherichia coli and mammalian cells is reducing with a high level (1-10 mM) of the tripeptide thiol glutathione (GSH) and the proteins contain free sulfhydryl groups and disulfides are very rare. This is in contrast to the outer cell surface or the extracellular environment where oxidizing conditions prevail due to the presence of oxygen and proteins have stabilizing disulfides and no or few free sulfhydryl groups. The thioredoxin (thioredoxin reductase and thioredoxin) and the glutaredoxin (glutathione reductase, GSH and glutaredoxin) systems are responsible for maintaining the low intracellu­ lar redox potential using electrons from NADPH. Thioredoxin and glutaredoxin are required also in essential metabolic reactions like the synthesis of deoxyribonucleotides for DNA synthesis by ribonucleotide reductase, one of several enzymes which requires disulfide reduction for each catalytic turnover. Other such enzymes are the family of thioredoxin peroxidases or peroxiredoxins, which use cysteine sulfur residues to reduce hydrogen peroxide with a mechanism-derived disulfide inter­ mediate. Thioredoxin, via its classical active site Cys-Gly-Pro-Cys dithiol, is used to maintain protein SH-groups reduced, but can also make disulfides

l

2

Cellular Implications ofRedox Signalling

via its disulfide form. Thioredoxins are regulating the activity of enzymes, transcription factors and receptors by reversible disulfide bond formation (thiol redox control) oxidized thioredoxin is made following rapid and temporal generation of superpoxide and hydrogen peroxide. Analogous reactions for the glutaredoxins are to also catalyze reversible S-glutathionylation of protein SH-groups, from glutathione disulfide (GSSG) another mechanism of thiol redox control of protein activity. Recent studies of the FAD-containing mammalian thioredoxin reductase has resulted in the determination of the structure and mechanism. This has shown surprisingly large differences to the conserved family of thioredoxin reductases from bacteria, fungi and plants. The larger mam­ malian thioredoxin reductases are structurally built from a glutathione reductase scaffold with a 16-residue elongation containing the conserved active site sequence: -Gly-Cys-SeCys-Gly, where the penultimate SeCys is selenocysteine. In its oxidized form, each active site in the dimeric enzyme contains a selenenylsulfide which is reduced to a selenolthiol in the reduced enzyme with electrons from the active site disulfide in the second subunit. The reductive half-reaction is identical to that of glu­ tathione reductase leading to reduction of the identical disulfide in thiore­ doxin reductase. A 3 A resolution X-ray structure of the rat enzyme demonstrates the close similarity to glutathione reductase including conserved residues involved in GSSG binding. However the C-terminal 16-residue swinging arm blocks GSSG binding, but enables electron trans­ port to the SeCys-Cys selenenylsulfide and the enzyme surface. The open active site enables docking of oxidized thioredoxin without any large conformational changes in sharp contrast to the bacterial thioredoxin reduc­ tases. The selenium is essential for enzyme activity, since without selenium, the truncated polypeptide, lacking the terminal SeCys-Gly dipeptide arising from the UGA in the mRNA acting as a stop codon is folded, but lacks all enzymatic activity. Replacement of selenium by sulfur yields an active enzyme but with a 100-fold major loss in Kcat but with a lower Km-value for thioredoxin. The selenium is also essential for the inher­ ent NADPH-dependent lipid hydroperoxide and hydrogen peroxide reductase activities of mammalian thioredoxin reductase. The selenazol drug ebselen, known to act as a glutathione peroxidase mimick, is a direct efficient substrate for mammalian thioredoxin reductase strongly enhanc­ ing its hydrogen peroxide reducing activity particularly with thioredoxin present. Future studies of the use of drugs that affect the thioredoxin sys­ tem will be useful for developing treatments of diseases either involving damage caused by overproduction of reactive oxygen species.

The Role of Thioredoxin and Glutaredoxin Systems

3

Glutaredoxins operate as disulfide reductases and have a CysPro-Tyr-Cys active site and a GSH-binding site which is used in binding GSH for reduction of the active site disulfide to a dithiol via a glutathione mixed disulfide intermediate. Glutaredoxins are species specific electron donors for ribonucleotide reductase. Recently, the structure of the large unusual £. coli glutaredoxin-2 (23 kDa), which is a powerful GSH-disulfide oxidoreductase and an electron donor for arsenate reductase was deter­ mined in solution and shown to be similar to glutathione-S-transferases. This structure also defines a novel family of mammalian large monothiol glutaredoxins, which have only the N-terminal nucleophilic Cys-residue and catalyze GSH-disulfide oxidoreductions. Future research should define how thiol redox control via thioredoxin and glutaredoxin systems is integrated with phosphorylation. Also the control of thioredoxin activity and expresssion in cells by specific binding proteins remains to be classified. Almost nothing is yet known about the mechanism by which thioredoxin is secreted without a leader sequence, how it is located on the cell surface and how it can move between different compartments within the cell like the cytoplasm and the nucleus. Future goals will also be to utilize specific drugs targeted to induce thioredoxin or thioredoxin reductase and also to develop gene therapy vectors with appli­ cation to prevent degenerative diseases in e.g. the brain. Thus, both thiore­ doxin and glutaredoxin have in preliminary experiments been shown to protect nerve cells from apoptosis. The redoxin electron donor identity for ribonucleotide reductase in tumor cells of different tissues is another area where more knowledge is required, also for rational use of current chemotherapy.

2. Introduction and Historical Perspective Thioredoxin was discovered 1964 by Peter Reichard and coworkers in E. coli1 as a small heatstable protein cofactor containing a dithiol required to enable the synthesis of dCDP from CDP by a partially purified enzyme today known as ribonucleotide reductase. This essential enzyme catalyzes the reduction of all four ribonucleotides to deoxyribonucleotides by replacing the 2'-OH-group in the ribose of the nucleotide by a hydrogen using a free radical mechanism. 2 The enzymatic reduction of CDP to dCDP required a hydrogen donor and the dithiol of dihydrolipoic acid gave activity, whereas monothiols like glutathione (GSH) or mercaptoethanol were inactive. NADPH was active as a hydrogen donor when coupled with

4

Cellular Implications ofRedox Signalling

an enzyme activity, thioredoxin reductase, required to regenerate a dithiol from the disulfide in the oxidized thioredoxin. E. coli thioredoxin (12 kDa) contained a single cystine disulfide group and after improve­ ment of the purification procedure to get homogeneous protein cleavage with cyanogen bromide at the single methionine residue yielded two peptide fragments, where the N-terminal 37-residues contained the disulfide group. 3 The complete amino acid sequence of thioredoxin with its now classical active site sequence: -Cys-Gly-Pro-Cys- was published in 1968. After 2 years of fruitless attempts to crystallize thioredoxin, useful single crystals were obtained in 1970 from the oxidized protein by addition of cupric ions 5 and in, 1975, the three-dimensional structure of thioredoxinS2 was solved. 6 The active site was located in a protrusion of the thiore­ doxin molecule, which was described as a first example of a male protein. 6 Thioredoxin consists of a central core of 5 B-strands surrounded by 4 a-helices with more than 75% of the residues in well defined secondary structures explaining the high stability of the structure (the thioredoxin fold). The structure of reduced thioredoxin remain elucive for many years although a localized conformational change was early observed from the three-fold increase in tryptophan fluorescence following reduction of oxi­ dized thioredoxin. 7 This unusual large increase in tryptophan fluorescence unique to the bacterial thioredoxin with its Trp-28 apart from the con­ served Trp-31 has proven to be of great importance to enable direct mea­ surements of the kinetics of thiol-disulfide exchange for thioredoxin.8,9 For a long time up to the mid-1970s, thioredoxin was almost exclu­ sively connected to ribonucleotide reductase and DNA synthesis as well as sulfate reduction or methionine sulfoxide reduction. The isolation of viable E. coli cells, which lacked thioredoxin, called into question its role in ribonucleotide reduction and lead to the discovery of glutaredoxin as a glutathione-dependent hydrogen donor for ribonucleotide reductase. 1011 Studies of thioredoxin and thioredoxin reductase in mammalian cells by purification and characterization of the proteins was initiated around 1970 based on use of ribonucleotide reductase as an assay system. This complicated assay and the fact that mammalian thioredoxins now known to contain additional sulfhydryl groups, which upon air oxidation lead to aggregation and inactivation made progress slow. A major break-through was the real­ ization that only the reduced form obtained after incubation with a thiol like dithiothreithol could be purified as a single peak component from liver or thymus extracts.12 Oxidized form show multiple artefactial peaks involving aggregation by different mechanisms. Since E. coli thioredoxin reductase shows no cross-reactivity with the human, rat or bovine thioredoxin it was of

The Role of Thioredoxin and Glutaredoxin Systems

5

no use for coupling to NADPH using reduction of 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB). Reduction of the latter is an easily used assay for thiore­ doxin from E. coli or yeast.3 In contrast, the mammalian thioredoxin reductase of calf thymus showed completely different properties with a higher molecular weight and a wide substrate specificity, which involved direct reduction of DTNB13 and rather complete inhibition in reduction of DTNB by the addition of bovine thioredoxin. In order to avoid ribonucleotide reductase as an assay system, the use of insulin in disulfide reduc­ tion was developed. 1213 Furthermore, the general role of thioredoxin and thioredoxin reductase as main disulfide reductase system of cells was shown e.g. by selective reduction of 5 out of 28 disulfide bonds in human fibrinogen by the thioredoxin system.14 This was later followed by selective disulfide reduction in a number of different proteins and has been used as a selective tool to probe disulfides and get wild selective reduction. The speci­ ficity is impressive since 3 of 5 disulfides in trypsin react, but none of 17 disulfides in albumin. A review of the method is found in Ref. 15 The wide distribution of thioredoxin in mammalian cells and its pres­ ence irrespective of DNA synthesis and ribonucleotide reductase activity was proven by studies of the distribution of calf thioredoxin. 16 This demonstrated thioredoxin in the nucleus, the microsomal fraction and mitochondrial fraction using radioimmunoassays. The first evidence of human thioredoxin was found in extracts from human platelets 14 and later in extracts from cultured fibroblasts.16 The localization of thioredoxin and thioredoxin reductase in adult rats 17 demonstrated a general cytoplasmic staining with prominent expression in epithelial cells including large amounts of thioredoxin in the nervous system, 18 axoplasmic transport of both thioredoxin and thioredoxin reductase in nerves and functional related changes in pancreatic B-cells and the gastric mucosa.19,20 A new area in thiredoxin research was initiated when Yodoi and coworkers identified adult T-cell leukemia derived factor (ADF) as a thiore­ doxin present in conditioned medium from lymphocytes and involved as a growth factor in upregulation of IL-2 recptor.21 This started a novel field in thioredoxin research regarding redox regulation of extracellular phenomena and growth control.22 Furthermore, thioredoxin released from B-cells infected with the EBV-virus is shown to be involved in lymphocyte immortalization. 23 Also from CD-4 T-cells a secreted factor, growthpromoting for normal and leukemic B-cells was identified as thioredoxin.24 Thus, in the last decade there has been an intense search for new functions of thioredoxin in redox control of cell growth, transcription factors and apoptosis.

6

Cellular Implications ofRedox Signalling

The concept of thiol redox control of redox regulation of cellular phenomena by changes in the structure of SH-groups on proteins has a long history and was suggested early to involve thioredoxin. 8 Only in the last decade it has been realized that there is an oxidizing mechanism con­ trolling the disulfide status in cells, via the generation of reactive oxygen species, which are converted into a disulfide signal by glutathione peroxidases generating GSSG or thioredoxin peroxidases (peroxiredoxins) generating disulfide forms of thioredoxins. The latter can then be trans­ formed to generation of disulfides in proteins as part of thiol redox con­ trol. Thus, the discovery of the peroxiredoxins 25 is a major concept in thioredoxin-dependent regulation of cellular activation. A human thiore­ doxin was first purified to homogeneity from placenta by A. Ernberg in Stockholm (1979) (cited in Ref. 26) and the antibodies against this protein was used by Sitia and coworkers to demonstrate a leaderless secretory pathway for thioredoxin 27 . Escherichia coli glutaredoxin was purified to homogeneity and its activity with ribonucleotide reductase, showed a higher turnover number as seen by the ten-fold lower Kra-value (0.13 uM) compared with thiore­ doxin. It was also discovered that pure glutaredoxin had inherent glutathione-disulfide oxidoreductase activity or was a member of glutathione disulfide transhydrogenase. This rapid spectrophotometric assay and showed that bacteria were indeed a very rich source of such activity. In fact, the activity in E. coli crude extracts was a 100-fold higher than what was specifically measured as glutaredoxin assayed with ribonucleotide reductase.28,29 The additional activities were found to be due to two new glutaredoxins called glutaredoxin-2 and -3, 30 which have no or some activity with ribonucleotide reductase, but represent major proteins in E. coli. Homogeneous preparations of calf thymus glutaredoxin, 31 which acts as a species-specific electron donor for calf thymus ribonucleotide reductase 32 contained the same conserved active site sequence Cys-Pro-TyrCys33 as E. coli glutaredoxin. 34 However, it was not clear at that time, as discussed in Ref. 31, if glutaredoxin and a GSH-homocystine transhydro­ genase from rat liver renamed thioltransferase 35 were identical proteins. The latter was reported to contain 8.6% carbohydrate. 35,36 However, the carbohydrate content was not confirmed and sequencing of proteins including a revised sequence of calf thymus glutaredoxin 37 showed the identity of the two proteins. Earlier work on thioredoxin and glutaredoxin have been summarized in review articles such as Refs. 26 and, 38—40. The proceedings of a Nobel

The Role of Thioredoxin and Glutaredoxin Systems 7

conference in 1985 on thioredoxin and glutaredoxin systems has been published. 41 Recent reviews with particular foccus on mammalian thiore­ doxin and thioredoxin reductase may be consulted42,43 for details. In this article some novel data on the structure and function of mammalian thioredoxin reductase will be described. General aspects of thiol redox control will be discussed.

3. The Thioredoxin System Thioredoxin reductase (TrxR) will reduce oxidized thioredoxin (Trx-S2) at the expense of NADPH [Reaction (1)] and reduced thioredoxin (Trx-(SH)2) is reoxidized by disulfides in proteins generating sulfhydryl groups [Reaction (2)]:

Trx-S2

+

Trx-(SH)2

NADPH +

Protein-S2

+

H+

TrxR ► Trx-(SH)2 ► Trx-S2

+ +

NADP+

(1)

Protein-(SH)2.

(2)

The Km-value for Trx-S2 is typically from 1 to 3 uM. Thioredoxin is an effi­ cient reductant with a low redox potential of - 270 mV.44 Today we know that there are some major differences between the thioredoxin systems of prokaryotes like E. coli and that of mammalian organisms. Thus, E. coli and mammalian cytosolic thioredoxins are very similar proteins in term of substrate specificity and reactivity with a con­ served -Cys-Gly-Pro-Cys-active site. However, mammalian thioredoxin must be purified and stored in the fully reduced form since they contain structural SH-groups which form additional disulfides upon oxida­ tion.13,45 This may have autoregulatory function of thioredoxin activity in resting cells or upon oxidative stress yet incompletely known in vivo. Thioredoxin reductases from mammalian cells have very different prop­ erties when compared with the enzymes from E. coli, yeast or plants; review in Ref. 46. The mammalian cytosolic enzyme has subunits with 55 kDa or larger instead of the 35 kDa in the E. coli enzyme with known three-dimensional structure. 46 As will be described below, the mam­ malian enzyme has an unusually broad substrate specificity entirely different from the species-specific enzymes only reducing Trx-S2 present in prokaryotes, yeast and plant cytosol.

8 Cellular Implications ofRedox Signalling

3.1 Thioredoxin Reductase and Selenium The fact that administration of selenium compounds like selenite (Se032~) cause inhibition of tumor cell proliferation in vivo and the knowledge that thioredoxin reductase appeared to be more highly expressed in malignant cells prompted us to start investigations on the reactivity of selenium compounds with pure mammalian thioredoxin reductase and thiore­ doxin. Contrary to expections, we discovered that selenite is a direct sub­ strate for thioredoxin reductase as well as an efficient oxidant of Trx-(SH)2.47'48 With 200 uM NADPH and 50 nM calf thymus thioredoxin reductase, addition of 10 uM selenite caused oxidation of 40 uM NADPH in 12 min and 100 uM NADPH after 30 min demonstrating a direct reduc­ tion of selenite with redox cycling by oxygen.47'48 This was demonstrated by incubation under anaerobic conditions where only 3 mol of NADPH was oxidized per mol of selenite according to Reaction (3):

Se032" + 3 NADPH

+

3 H+

TrxR ►

Se2- +

3NADP+

+ 3H 2 0.

(3)

Addition of thioredoxin stimulated the reaction further since selenite rapidly reacts with Trx-(SH)2 to oxidize it to Trx-S2.47-49 Since glutathione reductase will not react with selenite, Reaction (3) should provide cells with selenide, a required precursor for selenophosphate and selenocysteine synthesis. 50 Selenite and glutathione react to form selenodiglutathione (GS-Se-SG) which has been suggested to be a major metabolite of inor­ ganic selenium salts in mammalian tissues. 51 Reaction of selenodiglu­ tathione by NADPH and glutathione reductase was demonstrated by Ganther 51 and it has been proposed to be a source of selenide in cells as well as an inhibitor of neoplastic growth. We synthesized GS-Se-SG48 and discovered that is a direct efficient substrate for mammalian thioredoxin reductase and a highly efficient oxidant of reduced thioredoxin. Since GSSG is not a substrate for mammalian thioredoxin reductase,13,52 the insertion of the selenium atom in the GSSG molecule to form GS-Se-SG makes this molecule highly reactive with the enzyme. Reduction of GS-Se-SG to yield selenide by glutathione reductase requires 2 mol of NADPH. We found only the first stoichiometric reduction to be fast with GS-Se- as a product. 48 The second reaction was slow and inefficient. These results strongly suggest that the major selenide generation in cells is via thioredoxin reductase and thioredoxin. Thus, in mammalian cells the selenoenzyme thioredoxin reductase is also responsible for the synthesis

The Role of Thioredoxin and Glutaredoxin Systems 9

of selenide required for its own synthesis. An oxygen dependent non-stoichiometric consumption of NADPH is given by the thioredoxin system in the presence of selenite, selenodiglutathione and selenocystine.47"49 The latter is an efficient substrate for mammalian thioredoxin reductase with a Km of 6 uM. 49 The mechanism may be that the XSe" reacts with a dithiol (or selenolthiol) to catalyze oxidation according to Reaction (4): XSe"

+

R-(SH)2

+

02

►

XSe"

R-S2

+

H 2 0.

(4)

The effect will be 0 2 -dependent consumption of NADPH and provides an explanation for the lack of an autooxidizable free pool of selenocysteine as well as the acute toxic effects of selenium compounds on cells, for example, leading to apoptosis. Mammalian thioredoxin reductases dis­ play a surprisingly very wide substrate specificity as first observed dur­ ing purification.13,52 This is in contrast to the smaller prokaryotic enzymes, which do not react with mammalian thioredoxins despite the identical active sites and closely related three-dimensional structures of the thiore­ doxins. As summarized in Table 2, a truly wide range of direct reductions are catalyzed by the mammalian cytosolic thioredoxin reductases. Thiore­ doxin from E. coli is a substrate with a similar Kcat, but with a 15-fold higher Km value (35 uM) compared with the rat liver protein. 52 The mam­ malian cytosolic thioredoxins generally show full crossreactivity with the enzymes from different sources and vice versa.

3.2 Structure of Mammalian Thioredoxin Reductase Recent biochemical studies, sequencing and cloning of mammalian thioredoxin reductases has revealed that the enzymes are selenoproteins and entirely different from the corresponding enzymes in bacteria, yeast and plants (review in Ref. 46). Stadtman and coworkers serendipitously discovered that a human tumor cell thioredoxin reductase is a selenoprotein using labeling of selenoproteins with radioactive selenite.63 This also explained 64 why a putative clone of the human enzyme, 65 where the TGA codon for selenocysteine (SeCys) was interpreted as the stop codon (Fig. 1) gave no enzyme activity. The TGA acts as a stop codon in E. coli due to the fact that the species-specific machinery for synthesis of seleno­ proteins is different in bacteria and mammalian cells.66 By sequencing large parts of the cytosolic bovine enzyme, we directly identified the C-terminal peptide as containing selenocysteine. The bovine peptides were used to identify a rat cDNA clone which was

10

Cellular Implications ofRedox Signalling CVNVGC

GCUG

,\ / H,N-|

V FAD

NADPH

Inierrace

]-COOH

-Gln-Ala-Gly-Cys-Sec-Gly-Ter (human TrxR) CAG GCT GGC TGC TGA GGT TAA GCC CCA . . . CAG TCT GGC TGC TQA GGT TAA GCC CCA . . . -Gln-Ser-Gly-Cys-Sec-Gly-Ter (rat TrxR)

Fig. 1. Structure of the subunit of human and rat cytosolic thioredoxin reductase. The N-terminal glutathione reductase-like active site disulfide (CVNVGC) is shown in the upper portion of the figure as well as the FAD, NADPH and interface domains. The active site is shown in the C-terminus with GCUG denoting Gly-Cys-SeCys-Gly. Below, the region of that the part of the human and rat cytosolic genes with the TGA codon encoding selenocysteine (Sec) is shown. sequenced 67 and showed a polypeptide chain with a high homology to glutathione reductase including an identical active site disulfide (CVNVGC) (Fig. 1), but with a 16-residue elongation containing the con­ served C-terminal sequence -Gly-Cys-SeCys-Gly. A selenocysteine inser­ tion sequence (SECIS) was identified in the 3'untranslated region. 67 Furthermore, digestion of thioredoxin reductase by carboxypeptidase after reduction by NADPH released selenocysteine with loss of activity; the oxidized form of the enzyme was resistant to carboxypeptidase digestion.67 Redox titrations with dithionite and NADPH demonstrated that the mechanism of the human placenta enzyme is similar to that of lipoamide dehydrogenase and glutathione reductase and distinct from the mechanism of thioredoxin reductase from E. coli.68 The results also demon­ strated that the SeCys residue of human thioredoxin reductase is redox active and communicates with the redox active disulfide, since more than 4 electrons per subunit are required to completely reduce the FAD of the oxidized enzyme. Furthermore, the SeCys residue is alkylated with loss of activity only after reduction by NADPH. 67 The SeCys residue is also the target of the irreversible inhibitor l-chloro-2,4-dinitrobenzene only after reduction by NADPH 69 as shown by peptide analysis.70 The essential role of selenium in the catalytic activities of mammalian thioredoxin reductase was revealed by characterization of recombinant enzymes with selenocysteine mutations. 56 This was done by removing the selenocysteine insertion sequence in the rat gene and changing the SeCys49s encoded by TGA to Cys or Ser codons by mutagenesis. The trun­ cated protein having the C-terminal dipeptide deleted, which is expected

The Role of Thioredoxin and Glutaredoxin Systems

NADPH domain

Interface domain

11

16 aa elongation with Cys-SeCys

FAD domain

FAD domain 16 aa elongation with Cys-SeCys

Thioredoxin

Interface domain

NADPH domain

Reductase

Fig. 2. Structure model of mammalian TrxR (71). The 16-residue C-terminal extension with the active site is displayed as well as the head to tail arrangement of the subunits in the dimer as in glutathione reductase. The FAD, NADPH and interface domains are shown (see also Fig. 1). to mimic selenium deficiency, was also engineered. All three mutants were successfully overexpressed in E. coli and purified to homogeneity with 1 mol of FAD per monomeric subunit. All three mutant proteins rapidly gen­ erated the AJJO absorbance resulting from the thiolate-flavin charge transfer complex characteristic of mammalian TrxR.56 Only the SeCys498 Cys enzyme showed catalytic activity with thioredoxin, with a 100-fold lower Kcat, but also a 10-fold lower Km compared to the wild type rat enzyme. The pH-optimum of the SeCys-containing wild type enzyme was 7 whereas the SeCys498 Cys enzyme showed a pH optimum of 9. This strongly suggested the involvement of the low pKa SeCys selenol in the enzyme mechanism. Also selenium was required for hydrogen peroxide reductase activity.56 Thus, selenium is required for the catalytic activities of thioredoxin reduc­ tase explaining the essential role of this trace element in cell growth. Based on the homology to glutathione reductase, we proposed a schematic structure of mammalian thioredoxin reductase (Fig. 2). The active enzyme is a head to tail dimer with the 16-residue elonga­ tion in principle taking the place of GSSG in glutathione reductase. The catalytic site of the enzyme is a selenolthiol in its reduced form and a

12

Cellular Implications ofRedox Signalling

selenenylsulfide formed from the conserved cysteine-selenocysteine sequence in the oxidized form.71 The selenenylsulfide was isolated by peptide sequencing and also confirmed by mass spectrometry. 71 The reductive half-reaction is similar to that of glutathione reductase leading to reduction of the active site disulfide (Figs. 1 and 2). Electrons are thereafter transferred from the redox-active dithiols to the selenenylsulfide of the other subunit generating the selenolthiol (see below). Characteriza­ tion of the Cys mutant enzyme revealed that the selenium atom with its larger radius is critical for the formation of the unique selenenylsulfide,71 since the C-terminal dithiol remains reduced in the Cys mutant. 71 The presence of selenocysteine in the mammalian enzyme precludes direct recombinant expression of the enzyme in E. coli, although, engi­ neered constructs have overcome this and given promising results.72 Attempts to crystallize the native enzyme to get X-ray quality crystals from regular preparations have not been successful probably due to microheterogeneity in the selenium content also reflected in varying specific activities. The active SeCys498Cys mutant enzyme in contrast can be pre­ pared in large quantities and has been crystallized 73 in three different forms. Recently, the X-ray crystal structure of the rat SeCys498Cys mutant enzyme in complex with NADP + was solved to 3 A resolution respresenting the first structure of this unique class of selenoenzymes. 74 The most impressive result (Fig. 3) is the close similarity overall to the structure of glutathione reductase, including conserved amino acid residues binding the cofactors FAD and NADPH. Surprisingly, all residues interacting directly with the substrate GSSG in glutathione reductase are conserved despite the fact that GSSG is not acting as a substrate for thioredoxin reductase. The 16-residue C-terminal tail, which is unique to mammalian thioredoxin reductase and carries the SeCys residue, folds in such a way that it can approach the active site disulfide of the other subunit in the dimer. A model of the complex of rat thioredoxin reductase with human thioredoxin-S 2 (Fig. 4) suggests that electron transfer from NADPH to the disulfide of the substrate is possible without large conformational changes. Thus, the C-terminal extension to glutathione reductase scaffold typical of mammalian thioredoxin reductase has two main func­ tions. First, it extends the electron transport chain from the catalytic disul­ fide to the enzyme surface, where it can react with thioredoxin and a range of other substrates. Second, the C-terminal extension prevents the enzyme from acting as a glutathione reductase by blocking acess of GSSG to the redox active disulfide. The structure of the enzyme is also compatible with

The Role of Thioredoxin and Glutaredoxin Systems 13

Fig. 3. Ribbon representation of the dimer of rat TrxR. The two subunits are shown in light or dark colors respectively. Red, FAD-binding domain; yellows, NADP-binding domain, blue interface domain. Bound FAD and NADP are shown as ball and sticks models. Also the positions of SeCys498 and His 472 are shown as ball and sticks model. Taken from Ref. 74. The figure was kindly made by Dr Tarjana Sandalova.

evolution of mammalian thioredoxin reductase from a glutathione reductase scaffold rather than from the prokaryotic counterpart. Such an evolu­ tionary switch also rendered cell growth dependent upon selenium.

14 Cellular Implications ofRedox Signalling

Fig. 4. Model of complex of mutant rat TrxR with human thioredoxin. The

structure of rat TrxR is shown as a ribbon model with red, FAD-binding domain and FAD shown as a ball and sticks model; yellow NADPH-binding domain and the blue interface domain from the second subunit with His 472, Cys 479 and Cys 498. Thioredoxin is shown in green with active site disulfide C32-C35 and other residues indicated.

3.3 Mechanism of Mammalian Thioredoxin Reductase A mechanism of reduction of thioredoxin is shown in Fig. 5. The low pKa value and the high nucleophilicity of the selenium atom in the selenol makes this an excellent reducing agent, but also the likely target of drugs

The Role of Thioredoxin and Glutaredoxin Systems 15 (A) -FAD +

64 - S 59

J V

2NADPH+H1'-

497' 498' 2NADP^

(D) -FAD

(E) -FAD

-S

NADPH+H

(B) l-FAD

^ ^

« - * ^

^ i

59 -SH

HS- 497' "Se- 498'

Fig. 5. Mechanism of mammalian TrxR in reduction of thioredoxin-S2. A, oxidized enzyme; B, reduced enzyme; B with charge transfer complex and the selenenylsulfide being reduced to the selenolate anion. This attacks the disulfide in Trx-S2 and forms a Trx-TrxR mixed selenenyl sulfide as shown in C. As Trx(SH)2 is released the selenenyl sulfide is formed (D), which will be reduced by the active site thiolate from the other subunit to give E. like goldthioglucose 75 known to inhibit mammalian thioredoxin reductase. For reduction of hydrogen peroxide and lipid hydroperoxides the selenol which is available at the enzyme surface will take up the oxygen from hydrogen peroxide. 71 To the large group of substrates for mammalian thioredoxin reductase can also be added the selenazol drug ebselen (2-phenyl-l,2-benzoisoselenazol-3(2H)-one) an antioxidant and anti-inflammatory agent, which is a known glutathione peroxidase mimic.76 We have recently shown 77 that ebselen is an excellent substrate for mammalian thioredoxin reductase and stimulates its hydrogen peroxide reductase activity quite dramatically. Ebselen is also an efficient oxidant of reduced thioredoxin. 77

4. The Glutaredoxin System The glutaredoxin system is comprised of GSH, NADPH, glutathione reductase and glutaredoxin.40,78 The level of GSH in cells are generally

16

Cellular Implications ofRedox Signalling

/ GSH YmonothlolV I mechanism I Sll * \

/^~\

/

y^tJ] I mductes* j

V GSH *

«Nol mechanism

* NADP* / '

Fig. 6. Mechanism of glutaredoxin catalyzed monothiol or dithiol reactions. The monothiol mechanism involves formation and cleavage of GSH-mixed disulfides. The dithiol mechanism as in ribonucleotide reductase involves reduction of a disulfide. h i g h (1-10 m M ) a n d it is kept r e d u c e d b y N A D P H a n d glutathione r e d u c t a s e (GR) a s s h o w n in Reaction (5): GSSG +

NADPH

H+

GS

2GSH

NADP+.

(5)

Via g l u t a r e d o x i n (Grx) electrons from G S H are u s e d to r e d u c e disulfides [Reactions (6) a n d (7)] Grx-S2 Grx-S,

2 GSH Protein-S,

Grx-(SH)2 Grx-S,

GSSG Protein-(SH)2.

(6) (7)

Grx also catalyzes formation a n d cleavage of glutathionylated p r o t e i n s [(Reaction (8)]: Protein-SH

GSSG

Protein-S-SG

GSH

(8)

A s s h o w n in Fig. 6, glutaredoxins can either catalyze m o n o t h i o l or dithiol reactions. T o d a y glutaredoxins a r e a multifunctional family of G S H disulfide oxidoreductases w h i c h b e l o n g t o t h e thioredoxin fold s u p e r family. 79 ' 80 T h e y h a v e a GSH-binding site a n d a redoxactive disulfide w i t h the concensus sequence -Cys-Pro-Tyr-Cys-. O n l y t h e N-terminal nucleophilic Cys-residue 8 1 is required for catalyzing reversible glutathiorylation

The Role of Thioredoxin and Glutaredoxin Systems 17

Fig. 7. Left: NMR solution structure of E. coli Grx 1 in mixed disulfide with GSH (Glygs) via Cys 11. Right: Molecular surface with residues interacting with the GSH molecule; GSH shown as a sticks model.

reaction (monothiol mechanism) whereas both Cys residue are required for disulfide reduction (dithiol mechanism) (Fig. 6). Structural studies by NMR have solved the solution structure of glutaredoxin (Fig. 7).82 As molecular machines catalyzing thiol-disulfide oxidoreductions by cysteine thiols glutaredoxins are particularly interesting. A recent superb study of the structure, dynamics and electrostatics of the active site in glutaredoxin-3 from £. coli has recently been published. 83 This gives a unifying theme for the chemistry of the active site cysteine residues in the whole thioredoxin superfamily explaining why the pKa value of the N-terminal CXXC Cys-residue varies. This is primarily due to direct hydrogen bonding with the thiol proton of the other C-terminal Cys residue 84 and amide protons of the other residues inside the CXXC loop. 83 Glutaredoxins in mammalian cells have a growing list of functions such as reduction of dehydroascorbic acid,85 cellular differentiation or regulation of transcription factor activity.86 A new class of monothiol glutaredoxins in yeast and many other organisms appear to be particu­ larly important in defense against oxidative stress.87 Glutaredoxin also pro­ tects cerebellar granulae neurons from dopamine induced oxidative stress by activating NF-kappaB via Ref l.88 Recently, a novel human glutaredoxin

18

Cellular Implications ofRedox Signalling

with both mitochondrial and nuclear isoforms has been cloned.89 The structure of E. colt Grx2 in solution demonstrates a similarity in structure to glutathione-S-transferases and defines a novel family of large monothiol glutaredoxins. 90

5. Redox Regulation of Cellular Function Control of the activity of proteins by the reversible oxidation of SH-groups or thiol redox control26,40 is now recognized as a major mecha­ nism for signal transduction. Oxidants generated upon cell activation or exposure to oxidative stress are converted to a disulfide signal via GSHperoxidases or thioredoxin peroxidases and are balanced by antioxidants from the thioredoxin and glutaredoxin systems. Transcription factor binding to DNA is particularly sensitive to the redox state of critical SHgroups. 91 The outside of cells is an oxidizing environment dominated by disulfides whereas the cytosol is rich in SH-groups. Changes in the levels of GSH and GSSG will be an important global parameter in determining the intracellular redox potential since glutathione is the major redox buffer of mammalian cells.92

6. Future Perspectives Today we have a relatively good idea about the catalytic activity of various forms of thioredoxins and glutaredoxins in the cytosol. Obvi­ ously, it will take a long time to understand the flux through these cat­ alytic proteins and the dynamics in the regulation via thiol redox control. Of particular interest in the future will be to understand phenomena at the cell surface and mechanisms of global regulation of secretion and movement of thioredoxin and glutaredoxin isoforms within cells. The use of specific inhibitors and pharmacological agents inducing or supressing the activities of thioredoxins and glutaredoxins will be future goals. Such potential use of the antiapoptotic effects of thioredoxins may be to rescue cells from tissues undergoing degenerative cell death. In other situations, like in cancer therapy, directed inhibitors or use of gene therapy would be possible to selectively block the growth promoting and growth advantage of cells expressing high thioredoxin or glutaredoxin. Clearly, the complexity of SH-groups and their interplay in a cellular environment will keep us busy for the forseen future.

The Role of Thioredoxin and Glutaredoxin Systems

19

Acknowledgments Research support by the Swedish Medical Research Council, the Swedish Cancer Society and the K.A. Wallenberg Foundation is gratefully acknowledged.

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20

Cellular Implications ofRedox Signalling

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The Role ofThioredoxin and Glutaredoxin Systems 21

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22

Cellular Implications ofRedox Signalling

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The Role of Thioredoxin and Glutaredoxin Systems

23

51. Ganther HE. 1971. Reduction of the selenotrisulfide derivative of glutathione to a persulfide analog by glutathione reductase. Bio­ chemistry 10: 4089-4098 52. Luthman M, Holmgren A. 1982. Rat liver thioredoxin thiore­ doxin reductase: Purification characterization. Biochemistry 21: 6628-6633 53. Lundstrom J, Holmgren A. 1990. Protein disulfide-isomerase is a sub­ strate for thioredoxin reductase has thioredoxin-like activity. /. Biol. Chem. 265: 9114-9120 54. Nikitovic D, Holmgren A. 1996. S-nitrosoglutathione is cleaved by the thioredoxin system with liberation of glutathione redox regulat­ ing nitric oxide. /. Biol. Chem. 271:19180-19185 55. Bjornstedt M, Xue J, Huang W, Akesson B, Holmgren A. 1994. The thioredoxin glutaredoxin systems are efficient electron donors to human plasma glutathione peroxidase. /. Biol. Chem. 269: 29382-29384 56. Zhong L, Holmgren A. 2000. Essential role of selenium in the cat­ alytic activities of mammalian thioredoxin reductase revealed by characterization of recombinant enzymes with selenocysteine muta­ tions. /. Biol. Chem. 275:18121-18128 57. Bjornstedt M, Hamberg M, Kumar S, Xue J, Holmgren A. 1995. Human thioredoxin reductase directly reduces lipid hydroperoxides by NADPH selenocystine strongly stimulates the reaction via catalytically generated selenols. /. Biol. Chem. 270:11761-11764 58. Holmgren A, Lyckeborg C. 1980. Enzymatic reduction of alloxan by thioredoxin NADPH-thioredoxin reductase. Proc. Natl. Acad. Sci. USA 77: 5149-5152 59. Andersson M, Holmgren A, Spyrou G. 1996. NK-lysin a disulfide containing effector peptide of T-lymphocytes is reduced inactivated by thioredoxin reductase. Implication for a protective mechanism against NK-ysin cytotoxicity. /. Biol. Chem. 271: 10116-10120 60. Arner ESJ, Nordberg J. Holmgren A. 1996. Efficient reduction of lipoamide lipoic acid by mammalian thioredoxin reductase. Biochem. Biophys. Res. Commun. 225y: 268-274 61. May JM, Mendiratta S, Hill KE, Burk RF. 1997. Reduction of dehydroascorbate to ascorbate by the selenoenzyme thioredoxin reduc­ tase. /. Biol. Chem. 272: 22607-22610 62. May JM, Cobb CE, Mendiratta S, Hill KE, Burk RF. 1998. Reduction of the ascorbyl free radical to ascorbate by thioredoxin reductase. /. Biol. Chem. 27: 23039-23045

24

Cellular Implications ofRedox Signalling

63. Tamura T, Stadtman TC. 1996. A new selenoprotein from human lung adenocarcinoma cells: Purification properties thioredoxin reductase activity. Proc. Natl. Acad. Sci USA 9: 1006-1011 64. Gladyshev VN, Jeang K-T, Stadtman TC. 1996. Selenocysteine identi­ fied as the penultimate C-terminal residue in human T-cell thiore­ doxin reductase corresponds to TGA in the human placental gene. Proc. Natl. Acad. Sci. USA 9: 6146-6151 65. Gasdaska PY, Gasdaska JR, Cochran S, Powis G. 1995. Cloning sequencing of a human thioredoxin reductase FEBS Lett. 37: 5-9 66. Bock A, Forchhammer K, Heider L, Leinfelder W, Sawers G, Veprek B, Zinoni F. 1991. Selenocysteine: The 21st amino acid. Mol. Microbiol. 5: 515-520 67. Zhong L, Arner ESJ, Ljung J, Aslund F, Holmgren A. 1998. Rat calf thioredoxin reductase are homologous to glutathione reductase with a carboxyterminal elongation containing a conserved catalytically active penultimate selenocysteine residue. /. Biol. Chem. 273: 8581-8591 68. Arscott LD, Gromer S, Schirmer RH, Becker Williams CH. 1997. The mechanism of thioredoxin reductase from human placenta is similar to the mechanism of lipoamide dehydrogenase glutathione reductase is distinct from the mechanism of thioredoxin reductase from Escherichia coll Proc. Natl Acad. Sci. USA 94: 3621-3626 69. Arner ESJ, Bjornstedt M, Holmgren A. 1995. l-chloro-24-dinitrobenzene DNCB. is an irreversible inhibitor of human thioredoxin reductase: Loss of thioredoxin disulfide reductase activity is accom­ panied by a large increase in NADPH oxidase activity. /. Biol. Chem. 270: 3479-3482 70. Nordberg J, Zhong L, Holmgren A, Arner ESJ 1998. Mammalian thioredoxin reductase is irreversibly inhibited by dinitrohalobenzenes by alkylation of both the redox active selenocysteine its neigh­ bouring cysteine residue. /. Biol. Chem. 27:10835-10842 71. Zhong L, Arner ESJ, Holmgren A. 2000. Structure mechanism of mammalian thioredoxin reductase: The active site is a redoxactive selenolthiol/selenenylsulfide formed from the conserved cysteineselenocysteine sequence. Proc. Natl. Acad. Sci. USA 9: 5854-5859 72. Arner ESJ, Sarioglu H, Lottspeich F, Holmgren A, Bock A. 1999. High level expression in Escherichia coli of selenocysteine-containing rat thioredoxin reductase utilizing gene fusions with engineered bacterialtype SECIS elements co-expression with the selA, selB and selC genes. /. Mol. Biol. 29: 1003-1016

The Role of Thioredoxin and Glutaredoxin Systems

25

73. Zhong L, Persson K, Sandalova T, Schneider G, Holmgren A. 2000. Purification crystallization preliminary crystallographic data for rat cytosolic selenocysteine-498 to cysteine mutant thioredoxin reductase. Acta Cryst. D5:1191-1193 74. Sandalova T, Zhong L, Lindqvist Y, Holmgren A, Schneider G. 2001. Three-dimensional structure of a mammalian thioredoxin reductase: Implications for mechanism evolution of a selenocysteine-dependent enzyme. Proc. Natl. Acad. Sci. USA, 98: 9533-9538 75. Gromer S, Arscott LD, Williams CH Jr, Schirmer RH, Becker K. 1998. Human placenta thioredoxin reductase. Isolation of the selenoenzyme steady state kinetics inhibition by therapeutic gold com­ pounds. /. Biol. Chem. 273: 20096-20101 76. Schewe T. 1995. Molecular actions of ebselen — An antiinflammatory antioxidant, Gen. Pharmacol. 26:1153-1169 77. Zhao R, Masayasu H, Holmgren A. 2002. Ebselen: a substrate for human thioredoxin reductase strongly stimulating its hydroperoxide reductase activity and a superfast thioredoxin oxidaut Proc. Natl. Acad. Sci. USA 99: 8579-8584 78. Holmgren A, Aslund F. 1995. Glutaredoxin. Meth. Enzymol. 252: 283-292 79. Martin JL. 1995. Thioredoxin — A fold for all reason. Structure 3: 245-250 80. Holmgren A. 1995. Thioredoxin structure mechanism: Conformational changes on oxidation of the active site sulfhydryls to a disulfide. Structure 3: 239-243 81. Bushweller JH, Aslund F, Wuthrich K, Holmgren A. 1992. Structural functional characterization of the mutant Escherichia coli glutaredoxin C14-»S. Its mixed disulfide with glutathione. Biochemistry 31:9288-9293 82. Bushweller JH, Billeter M, Holmgren A, Wuthrich K. 1994. The nuclear magnetic resonance solution structure of the mixed disulfide between Escherichia coli glutaredoxin C14S. Glutathione. /. Mol. Biol. 235:1585-1597 83. Foloppe N, Sagemark J, Nordstrand K, Berndt KD, Nilsson L, 2001. Structure dynamics electrostatics of the active site of glutaredoxin-3 from Escherichia coli: Comparison with functionally related proteins. /. Mol. Biol. 310: 449-470 84. Jeng M-F, Holmgren A, Dyson HJ. 1995. Proton sharing between cysteine thiols in Escherichia coli thioredoxin: Implications for the mechanism of protein disulfide reduction. Biochemistry 34: 10101-10105

26

Cellular Implications ofRedox Signalling

85. Wells WW, Xu DP, Washburn MP. 1995. Glutathione: Dehydroascorbate oxidoreductases. Meth. Enzymol. 252: 30-38 86. Nakamura T, Ohno T, Hirota K, Nishiyama A, Nakamura H, Wada H, Yodoi J. 1999. Mouse glutaredoxin — cDNA cloning high level expression in £ coli. Its possible implication in redox regulation of the DNA binding activity in transcription factor PEBP2. Free Radio. Res. 4: 357-365 87. Rodriguez-Mazaneque MT, Ros J, Cabiscol E, Sorribas A, Herrero E. 1999. Grx5 glutaredoxin plays a central role in protection against protein oxidative damage in Saccharomyces cerevisiae. Mol. Cell. Biol. 12: 8180-8190 88. Daily D, Vlamis-Gardikas A, Offen D, Mittelman L, Melamed E, Holmgren A, Barzilai A. 2001. Glutaredoxin protects cerebellar gran­ ule neurons from dopamine induced apoptosis by activating N F - K B via Ref-1. /. Biol. Chem. 276:1335-1344 89. Lundberg M, Johansson C, Chandra J, Enoksson M, Jacobsson G, Ljung J, Johansson M, Holmgren A. 2001. Cloning expression of a novel h u m a n glutaredoxin Grx2 with mitochondrial nuclear isoforms. /. Biol. Chem. 276: 26269-26275 90. Xia B, Vlamis-Gardikas A, Holmgren A, Wright PE, Dyson HJ. 2001. Solution structure of Escherichia coli glutaredoxin-2 shows similarity to mammalian glutathione-S-transferases. /. Mol. Biol, 310: 907-918 91. Zheng M, Aslund F, Storz G. 1998. Activation of the OxyR transcrip­ tion factor by reversible disulfide bond formation. Science 279: 1718-1721 92. Gilbert HF. 1990. Molecular cellular aspects of thiol-disulfide exchange. Adv. Enzymol. Relat. Areas Mol. Biol. 63: 69-172

Chapter 2

Selenocysteine Insertion and Reactivity: Mammalian Thioredoxin Reductases in Relation to Cellular Redox Signaling Elias S.J. Arner Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77, Stockholm, Sweden [email protected]

Keywords: Selenocysteine, selenoprotein, thiol redox control, reactive oxygen species, intracellular signaling

1. Summary Selenocysteine (Sec) — the 21st amino acid — is incorporated into selenoproteins at the position of specific TGA codons (UGA in the mRNA), normally conferring termination of translation. Stretching the genetic code by insertion of Sec at the Sec-specific UGA involves a highly intricate trans­ lation machinery, which differs significantly between species and which is dependent upon a secondary structure in the selenoprotein mRNA; a SECIS (Selenocysteine Insertion Sequence) element. In man, at least 20 selenoproteins are known, including the glutathione peroxidases and thyroid hormone deiodinases. Since the mid-1990s, the mammalian thio­ redoxin reductase (TrxR) isoenzymes are also known to be selenoproteins, in contrast to the smaller non-selenoprotein thioredoxin reductases from bacteria, plants or yeast. In E. coli, the three formate dehydrogenase H, O and N isoenzymes are the only natural selenoproteins. In most instances, selenoproteins are oxidoreductases dependent upon the high reactivity of the selenocysteine residue. Mammalian thioredoxin reductases (the cytosolic, mitochondrial and testis specific TrxR iso­ enzymes) all carry a Sec residue within a rather unique carboxyterminal motif being -Gly-Cys-Sec-Gly-COOH. A high reactivity of cytosolic 27

28 Cellular Implications ofRedox Signalling

TrxR with diverse electrophilic agents, including dinitrohalobenzenes (e.g. DNCB), iodoacetic acid, 4-vinylpyridine or platinum drugs, has been demonstrated. All these compounds irreversibly inactivates the enzyme, but only when in a reduced form. This is explained by derivatization of the reactive Sec residue being exposed when the enzyme is reduced by NADPH. An additional unique effect in inhibition of TrxR with dinitro­ halobenzenes is a pronounced induction of an NADPH oxidase activity in the dinitrophenyl-derivatized enzyme. This can be mechanistically explained by a functional half-reaction with subsequent interaction of the enzyme-bound FAD and/or disulfide/dithiol motif in the N-terminal domain, with the nitro groups of the dinitrophenyl moieties at the derivatized C-terminus. Moreover, this reaction may be proposed to mediate some of the strong inflammatory components of the immunostimulatory effects seen upon topical treatment with dinitrohalobenzenes. This model for inflammation is based upon the induced intracellular oxidative stress due to the inactivated TrxR with a superoxide-producing NADPH oxidase activity, in combination with an increased synthesis of thioredoxin with secretion to the extracellular space where it is known to have cytokine-like activity. Recent studies of the human promoter for cytosolic TrxR reveal that the gene seems to be the first known to have a housekeeping-type promoter with regulation of mRNA levels through AUUUA motifs in the 3'-untranslated region. Such AU-rich elements are otherwise known to be present in cytokines or proto-oncogenes regulated in response to intracel­ lular redox signaling. The reactivity, function and regulation of cytosolic TrxR indicates that this selenoprotein plays a central role in cellular redox signaling, which shall be discussed in this chapter.

2. The Mammalian Thioredoxin System The mammalian thioredoxin system consists of thioredoxin (Trx), thio­ redoxin reductase (TrxR) and NADPH. Thioredoxin is reduced by TrxR and participates in many different types of reactions, including synthesis of deoxyribonucleotides, redox control of transcription factors, reduction of peroxides and redox regulation of apoptosis. Extracellularly thiore­ doxin has immunoregulatory activities as co-cytokine or chemokine. These functions are reviewed elsewhere 1 " 3 and will not be discussed at length in this chapter. It is of importance, however, to note that the redox status of thioredoxin is essential for most, if not all of its many vital cellular functions. Consequently, perturbations of the TrxR activity

Selenocysteine Insertion and Reactivity

29

are implicated in a number of cell proliferative or immunological diseases and the enzyme is increasingly being recognized as an important pharmacological target in a number of medical conditions, as reviewed in Ref. 4. In addition to reduction of the active site disulfide in thioredoxin, mammalian TrxR also reduces disulfides in other proteins like protein disulfide isomerase or NK-lysin, low molecular weight disulfides like 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman's reagent) or lipoic acid, low molecular weight non-disulfide substrates like selenite or alloxan, or even lipid hydroperoxides (see Ref. 5). The mammalian TrxR enzymes are implied to play central roles in cell proliferation, redox regulation and protection against oxidative damage, but conclusive experimental insights regarding the cellular functions of these seleno­ proteins are yet, however, quite scarce. Here we shall discuss the selenoprotein nature and characteristics of mammalian TrxR with a focus on the possible functional roles this enzyme may indeed play in cellular redox control.

3. Selenocysteine in Thioredoxin Reductase 3.1 Co-Translational Insertion of Selenocysteine In 1996, Theresa Stadtman and coworkers reported that human cytosolic TrxR contains the rare amino acid selenocysteine at its penultimate carboxyterminal position. 6,7 It was subsequently shown that bovine, rat and human cytosolic TrxR all contain a conserved carboxy-terminal tetrapeptide motif-Gly-Cys-Sec-Gly-COOH (where Sec is selenocysteine) and that the selenocysteine residue is essential for the enzymatic activity.8 More­ over, the overall amino acid sequence of the enzyme is not homologous to that of TrxR from lower organisms but instead closely similar to the sequence of glutathione reductase, with the addition of a 16-residue carboxy-terminal elongation carrying the selenocysteine-containing amino acid motif.8 Selenoproteins containing selenocysteine residues are found in most bacteria, archaea as well as eukarya. Selenocysteine is the selenium analogue of cysteine and is due to the electrophilicity of selenium nor­ mally ionized at physiological p H to a selenolate, in contrast to cysteine which is most often present in the protonated sulfhydryl form (Fig. 1). This difference between selenocysteine and cysteine usually lead to a higher reactivity of selenoproteins in comparison to their cysteine mutants and known selenoproteins are most often oxidoreductases with

30

Cellular Implications ofRedox Signalling H

H

I

I

'HgH-O-COO"

*H3N-C-COO"

Se~

SH

Selenocysteine

Cysteine

(U, Stc)

(C. Cy>)

Fig. 1. Selenocysteine versus cysteine. The figure depicts the difference between selenocysteine (U, Sec) and cysteine (C, Cys) with a selenium atom taking the place of the sulfur in cysteine, and with selenocysteine usually being present in the ionized selenolate form at physiological pH (free Sec pKa = 5.2) in comparison to the usually protonated thiol of cysteine (pKa = 8.3). a catalytic selenocysteine residue in their active site. In bacteria and archaea these selenoproteins include formate dehydrogenases, hydrogenases or glycine reductase, whereas in mammals other known seleno­ proteins apart from thioredoxin reductases constitute the glutathione peroxidase family and the thyroid hormone deiodinases. In addition, mammalian selenoproteins have been described with yet unknown func­ tion such as selenoprotein P or W.9"11 Recent database searches indicate that the list of mammalian selenoproteins will continue to grow. 1213 Selenocysteine is in all organisms cotranslationally inserted at the position of an opal (UGA) codon, normally conferring termination of translation. The UGA codon is encoded as selenocysteine by a highly complex translation machinery, characterized in detail for E. coli by August Bock and coworkers, using formate dehydrogenase H as a model system — for reviews, see Refs. 10, 11 and 14. In short, mRNA for E. coli selenoproteins contain an about 40 nucleotides long selenocysteine inser­ tion sequence (SECIS) positioned immediately 3 ' of the UGA codon. These nucleotides have dual functions; they provide codons for the trans­ lation of amino acids following the selenocysteine residue, and they fold into a stem-loop type secondary structure — a SECIS element. The SECIS element binds the SELB protein, the selB gene product. SELB is homolo­ gous to elongation factor EF-Tu but, in addition, carries a carboxy-terminal domain binding the loop region of the SECIS element. SELB also binds to a selenocysteine specific tRNA (tRNA 3 "), the selC gene product, in its selenocysteinylated form. Thereafter, in analogy with EF-Tu, SELB is at the ribosome catalyzing selenocysteine insertion at the specific position of the selenocysteine UGA codon. The tRNA560 is originally charged with a seryl-residue which by utilization of selenophosphate is converted to

Selenocysteine Insertion and Reactizrity 31

Fig. 2. The selenocysteine insertion machinery in E. coli. In translation of a non-selenoprotein mRNA, the elongation factor Ef-Tu catalyzes insertion of amino acids into the elongating polypeptide chain utilizing tRNA's for any of the common 20 amino acids and if a UGA codon is encountered, the release factor RF2 terminates the elongation. If a bacterial-type SECIS element structure is present in the mRNA next to the UGA, however, this will be recognized by the selenocysteine-specific elongation factor SELB. The SELB only utilizes the selenocysteine-specific tRNA8*0 (the SelC gene product) in its selenocysteinylated form, thereby elongating the polypeptide chain with selenocysteine insertion at the correct UGA codon. The tRNA 5 " is originally charged with a seryl moiety, which is converted to selenocysteinyl while bound to the tRNA. See text for further details and references to reviews on this subject. selenocysteinyl b y selenocysteine synthase, a n oligomer of the selA g e n e p r o d u c t . The s e l e n o p h o s p h a t e , in t u r n , is p r o v i d e d b y s e l e n o p h o s p h a t e synthetase, the selD gene p r o d u c t . T a k e n together, selenocysteine inser­ tion in E. coli involves: an E. co/z'-type SECIS e l e m e n t at the right position after the U G A c o d o n in the selenoprotein m R N A , a n d the selA, selB, selC a n d selD g e n e p r o d u c t s . This E. coli selenoprotein translation m a c h i n e r y is schematically s u m m a r i z e d in Fig. 2. A SECIS e l e m e n t is found also in the m R N A of m a m m a l i a n selenop r o t e i n s b u t this h a s other s e c o n d a r y structures a n d conserved features

32

Cellular Implications ofRedox Signalling

than found in E. coli and, moreover, is situated in the 3'-untranslated region several hundred nucleotides downstream of the UGA codon. 915 Thereby mammalian selenoprotein genes are generally incompatible with direct recombinant expression in E. coli. A technique, however, to by-pass the barriers to heterologous expression of selenoproteins in E. coli16 enabling bacterial production of recombinant mammalian TrxR was developed utilizing engineered variants of the bacterial SECIS element, encoding the C-terminal motif of TrxR.17 Use of this recombinant method­ ology is likely to facilitate further studies of mammalian TrxRs as well as other selenoproteins.

3.2 Selenocysteine in TrxR as a Drug Target The catalytic mechanism of mammalian TrxR shall not be described in detail here but can be concluded to be similar to that of glutathione reductase 18 but in addition involving a reversible selenolthiol/selenenylsulfide formed by the penultimate selenocysteine and its neighboring cysteine, constituting a second non-flavin redox active center.19 The oxidized selenenylsulfide-containing form of the enzyme is highly resistant to modi­ fication with electrophilic agents or to digestion with carboxypeptidase. 8 However, when the enzyme is reduced by NADPH the Cys-Sec site becomes susceptible to modification by the above treatments, which thereby easily inactivate the enzyme.8,20-22 This molecular mechanism hence generally suggests how the many inhibitors of TrxR act. These elec­ trophilic inhibitors include antitumor quinones, 23 doxorubicin, 24 antitumor nitrosourea drugs, 25 retinoic acid,26 anti-rheumatic gold compounds such as gold thioglucose.27,28 Molecular modeling of the C-terminal tetrapeptide of TrxR in both the oxidized and the reduced state may illus­ trate how the selenenylsulfide must induce a beta-turn like bend at the C-terminus protecting this redox active center, and, alternatively, when reduced, how the selenol(ate) of the selenocysteine residue becomes exposed and hence highly susceptible to reactions with either substrates or inhibitors of the enzyme (Fig. 3). It is probable that the inhibition of thioredoxin reductase by electro­ philic drugs in clinical use should contribute to their therapeutic effects, or side effects, which is a notion that has also recently been reviewed elsewhere. 4 Dinitrohalobenzenes are unique in their inactivation of TrxR by derivatizing the enzyme concomitant with an induction of an NADPH oxidase activity in the derivatized enzyme.20'21 The reactivity with

Selenocysteine Insertion and Reactivity

33

Fig. 3. Molecular modelling of the C-terminal tetrapeptide of TrxR in reduced and oxidized form. In (A), a stereo view of a modelled reduced C-terminal Gly-Cys-Sec-Gly tetrapeptide is given, illustrating the highly exposed Sec and Cys residues on opposite sides of the polypeptide backbone. In (B), the same tetra­ peptide has been modelled with a selenenylsulfide bridge between the Cys and Sec residues, as has been experimentally demonstrated to be present in oxidized TrxR, using Edman degradation and mass spectrometry. 19 In order to make possi­ ble this selenenylsulfide, a beta-turn like bend must be imposed on the structure to place the two side chains of Cys and Sec on the same side of the peptide back­ bone. This unique structure may explain why the oxidized enzyme is resistant to carboxypeptidase treatment 8 or derivatization with electrophilic agents (see text). It is also possible that the larger atom radius of the selenium atom may help to form the bridge in the oxidized motif, as the cysteine mutant seem not to be able to easily form a corresponding disulfide but leaves a dithiol motif in the oxidized mutant holoenzyme. 70 Modelling was performed using the CORINA algorithm (see http://www2.ccc.uni-erlangen.de/software/corina/corina.html). The sulfur of cysteine is shown in yellow and the selenium of selenocysteine is purple.

dinitrohalobenzenes will b e discussed b e l o w in m o r e detail, also being the basis for a discussion o n the relation b e t w e e n the activities of cytosolic TrxR a n d diverse intra- as well as extra-cellular signaling systems. T w o additional TrxR i s o e n z y m e s h a v e b e e n identified, one m i t o chondrial 29 " 32 a n d one testis specific, 33 w i t h b o t h h a v i n g the s a m e overall d o m a i n structure as T r x R l . Interestingly, the testis specific isoenzyme, h o w e v e r , also contains a n N - t e r m i n a l m o n o t h i o l g l u t a r e d o x i n d o m a i n w h i c h seems to give this e n z y m e a n a d d i t i o n a l g l u t a t h i o n e r e d u c t a s e

34

Cellular Implications ofRedox Signalling

activity which the other TrxR isoenzymes lack, and was therefore recently named TGR for its thioredoxin and glutathione reductase. 34 The possible role of the cytosolic TrxRl isoenzyme in relation to intracellular redox signaling shall now be discussed in some further detail.

4. Regulation of Cytosolic Thioredoxin Reductase in Relation to Cellular Redox Signaling The 3 ' untranslated region of the mRNA for cytosolic TrxRl contains in addition to the SECIS element also AU-rich elements, AREs, which in untreated cells lead to a rapid TrxRl mRNA turnover.35,36 In fact, TrxRl was independently cloned as KDRF in a study specifically set out to identify genes being regulated through AREs.35 The presence of func­ tional AREs are otherwise typically found in mRNAs of cytokines, protooncogenes, transcription factors and other transiently expressed genes. 37 Post-transcriptional regulation via AREs enables quick expression responses to various stimuli, by a block in the rapid mRNA degradation through specific ARE-interacting proteins responding to intracellular signaling. 37 It is interesting that TrxRl contains functional AREs35,36 since this enzyme is not transiently expressed only under specific growth conditions, but is widely expressed in many diverse tissues and cells.30,38-40 TrxRl is nonetheless known to display significant and fast (within hours) increase of protein as well as mRNA upon treatment of cells with a number of different exogenous agents. Examples of this include human epidermoid carcinoma A431 cells treated with epidermal growth factor, H 2 0 2 or l-chloro-2,4-dinitrobenzene 33 or thyrocytes given calcium ionophore (A23187) and PMA.41 The latter was also seen in human umbili­ cal vein endothelial cells, although less pronounced much due to more than 10-fold higher basal TrxR levels in these cells compared to thyrocytes.42 In human bone marrow-derived stromal cells (KM102) both PMA in com­ bination with A23187 or, alternatively, treatment with interleukin-lp or lipopolysaccharide significantly increased the TrxRl mRNA levels within 4 hrs, being the KDRF study referred to above. 35 In peripheral blood monocytes and myeloid leukaemia cells43 as well as osteoblasts 44 TrxRl mRNA levels were shown to be increased above basal levels in a fast but transient manner by vitamin D3 treatment. How is the increase of TrxRl levels upon diverse exogenous stimuli transmitted and what may this regulation tell us about the cellular func­ tion of TrxRl? We recently found that human TrxRl has an Octl- and

Selenoq/steine Insertion and Reactivity

35

Spl-driven TATA- and CCAAT-less typical housekeeping-type core promoter, with expression in many different cell types. 45 Considering this functional organization with a housekeeping-type promoter in combina­ tion with ARE-mediated post-transcriptional regulation, being quite unique, we propose a novel type of regulation of the enzyme in relation to intracellular redox signaling. The presence of 3' untranslated region AREs may generally enable a quick stabilization of mRNA and can thereby upregulate protein levels in fast response to various signals.37,46 Upon many different exogenous stimuli, reactive oxygen species (ROS) such as superoxide or hydrogen peroxide are also produced as common mediators for intracellular signaling.47 One regulatory protein which is rapidly upregulated upon formation of ROS is the p38 mitogen-activated protein (MAP) kinase, as reviewed in Ref. 48. The stress-activated p38 MAP kinase in turn upregulates the MAP kinase-activated protein kinase-2 (MK2) and, interestingly, MK2 was shown to induce stabilization of ARE-containing mRNAs, thereby exe­ cuting their stabilization under intracellular formation of ROS.49 Since TrxRl contains functional AREs and is also known to be upregulated by many exogenous agents (see above) which in turn are known to mediate intracellular ROS formation as a common denominator, 50 it becomes possible that the AREs participate in mediating a fast response in increased expression of TrxR upon intracellular ROS formation. In addi­ tion, in cells, the TrxRl enzyme has been reported to be rapidly inacti­ vated by ROS, targeting the selenocysteine residue. 28 This chain of events makes it possible to propose the following model for TrxRl regulation and function. With a strong constitutive transcription, suggested from the initial characterizations of the promoter, 45 combined with ARE-regulated mRNA turnover and generally a short mRNA half-life in non-stimulated cells,36 TrxRl thereby has the inherent capacity for a fast response to an increase of intracellular ROS in their role of stabilizing the mRNA via MK2 and the ARE motifs; this would occur concomitant with a momen­ tary inactivation of the enzyme.28 Once more TrxRl rapidly has been syn­ thesized as a result of the stabilized mRNA, the antioxidant properties of the newly produced enzyme would then possibly be able to carry the cells back to a correct basal balance of the intracellular redox status, yet having allowed the transient burst of ROS being a necessary component for the many diverse systems of intracellular signaling. This proposed model for TrxRl activity and regulation in relation to intracellular redox signaling has yet, however, to be experimentally scrutinized. Still the model can indicate how the thioredoxin system may interrelate to intracellular signaling

36

Cellular Implications of Redox Signalling

systems via a fast regulated activity of TrxR. The reactivity of TrxR with dinitrohalobenzenes is another example which may illustrate this interrelationship.

5. Effects of Dinitrohalobenzenes by Interactions with Thioredoxin Reductase l-chloro-2,4-dinitrobenzene (DNCB, CDNB) is an electrophilic compound used as a substrate in assays to determine glutathione S-transferases, being involved in elimination of DNCB in vivo.52 DNCB is therefore also used in cell culture experiments as a GSH-depleting agent. 53 Furthermore, DNCB has an established use as an immunomodulatory agent to provoke delayed-type hypersensitivity reactions.54 Although proposed to function as a hapten, the mechanism of DNCB immunomodulation is however not clear, especially regarding the pro-inflammatory properties of the compound. 55 In 1995, we found that DNCB irreversibly inhibited NADPH-reduced mammalian thioredoxin reductase, with a concomitant induction of an NADPH oxidase activity20 and we later demonstrated that the selenocysteine residue in thioredoxin reductase indeed was the target for derivatization. 21 In addition, the neighboring cysteine residue in the carboxyterminal tetrapeptide of the enzyme (-Gly-Cys-Sec-Gly-COOH), was also derivatized. Also incubation with other electrophilic compounds, like iodoacetic acid or 4-vinylpyridine, inhibited TrxRl irreversibly but in this case no induction of an NADPH oxidase activity was seen.21 This indi­ cated that an inherent property of dinitrohalobenzenes was necessary for the NADPH oxidase activity to be induced and this property was most likely carried by the nitro groups. The induced NADPH oxidase activity was found to produce superoxide anions 21 and since superoxide-producing NAD(P)H-dependent redox cycling of aromatic nitro compounds with flavoenzymes is a known phenomenon,56-59 a model for the interaction between mammalian thioredoxin reductase and dinitrohalobenzenes became possible to propose. 60 In this model, NADPH is proposed to reduce the enzyme-bound oxidized FAD even if the C-teminal Sec-containing redox active motif has been derivatized with dinitrophenyl groups. Of importance for the reac­ tivity of TrxR with dinitrohalobenzenes is that only upon reduction with NADPH is the carboxyterminal motif known to be accessible for alkylation20,21 or digestion with carboxypeptidase Y,8 most likely being

Selenocysteine Insertion and Reactivity

37

PADH2 S — ^ X

Fig. 4. Derivatization of TrxR with DNCB and model for the NADPH oxdiase activity. The selenenylsulfide at the C-terminus of one subunit of the oxidized holoenzyme TrxR must first be reduced to a selenolthiol for derivatization to occur. The reduction to a selenolthiol is NADPH dependent (A) and occurs via the FAD (B) and redox active dithiol (C) of the other subunit, as described in Ref. 19. When the selenolthiol has been exposed (see Fig. 3), two DNCB molecules easily derivatize both the cysteine and the selenocysteine at the C-terminus (E). Possibly the selenolate is first derivatized, which may induce a thiolate at the cysteine by resonance effect, hence leading to alkylation also of the neighboring cysteine residue. The experimentally observed superoxide-producing NADPH oxidase activity by the dinitrophenyl-derivatized enzyme 21 may be explained by a functional half reaction and NADPH-dependent reduction of the FAD in the derivatized enzyme (F) and consecutive one-electron transfers to the nitro groups of the dinitrophenyl moieties. First, a flavin semiquinone and a nitro anion radical is formed (G). The nitro anion radical readily reacts with molecu­ lar oxygen to form one molecule of superoxide (H). The semiquinone subse­ quently forms a second nitro anion radical (I) which also reacts with oxygen to produce another molecule of superoxide (J), returning the enzyme to a fully oxi­ dized dinitrophenyl-derivatized form, which again may go through a cycle of NADPH oxidase acitivity (F-J). This model has been published in a somewhat simplified form in Ref. 60.

38

Cellular Implications of Redox Signalling

explained by the selenenylsulfide of the oxidized enzyme 19 being highly inert. Once reduced, however, the free selenolthiol motif should be highly susceptible to derivatization with dinitrohalobenzenes, explaining the experimentally found 10,000-fold higher reactivity of reduced TrxRl with DNCB compared to derivatization of reduced GSH under the same conditions at pH 7.5.20 See also Fig. 3 illustrating the difference between the reduced (susceptible) and oxidized (protected) C-terminal motif of TrxR. To explain the induced NADPH oxidase activity in the dinitrophenylderivatized TrxR, it is hence proposed that the FAD of the alkylated enzyme still can be reduced by NADPH but that one (or two) of the nitro groups in the dinitrophenyl moieties of the alkylated enzyme in two con­ secutive one-electron transfers are converted to nitro anion radicals that in turn react with oxygen to form superoxide. This would regain dinitrophenyl-derivatized TrxR having oxidized FAD, that again can be reduced with NADPH to give the observed superoxide producing NADPH oxidase activity (Fig. 4). Does the specific and high reactivity with mammalian TrxR of dinitrohalobenzenes like DNCB play a role in the molecular mechanism behind the immunomodulatory properties of these compounds? In dis­ cussing this question, it is of importance to note that a number of DNCB analogs which failed to inhibit TrxR or to induce any NADPH oxidase activity21 previously had been tested in vivo for induction of hypersensitivity reactions in mice and shown to provoke no reaction.55 In that same study, 0 2 utilization, H 2 0 2 production and NADPH consumption in skin or liver microsomes was also measured upon addition of dinitrohaloben­ zenes or the DNCB analogs. All of these properties correlated well to mouse ear swelling upon application of the compounds, whereas changes in levels of GSH or GSSG did not.55 The enzyme(s) responsible for the NADPH consumption and superoxide (or H 2 0 2 ) production were not identified, but it should be safe to conclude that TrxR is a strong candi­ date. How would the interaction with TrxR by dinitrohalobenzenes take part in the mechanism of immunostimulation by these compounds? Two mechanisms are possible. First, thioredoxin is known to play a central role in redox regulation of cell function1'3'61 and an irreversible inhibition of TrxR with concomitant superoxide production would therefore with cer­ tainty affect thioredoxin-related functions in the immune system, possibly mimicking the natural intracellular signaling conveyed by increased lev­ els of intracellular ROS.3'47,48 Second, it may be proposed that secretion of thioredoxin a n d / o r its shorter truncated variant Trx-80 is stimulated upon oxidative stress, resulting in immunostimulation through co-cytokine or

Selenocysteine Insertion and Reactimty

39

chemokine activities of full-length thioredoxin62"65 or Trx80,66 the latter also involving stimulation of interleukin-12 production from monocytes thereby favouring a Thl response. 67 It should also be noted that if excessive, increased oxidative stress is a known initiatiator of cell death by either apoptosis or necrosis. This may also be exaggerated through inhibition of the thioredoxin system; e.g. by the facts that reduced thioredoxin binds to and thereby inhibits the proapoptotic apoptosis signaling kinase-1 (ASK-1)68 or that peroxiredoxins, being dependent upon thioredoxin activity, seem to counteract apoptosis at an early stage upstream of bcl-2.69 The actual difference between mimicking intracellular signaling through induction of ROS formation and thereby stimulating cells, or inducing cell death by excessive ROS forma­ tion through the inactivation of TrxR, may therefore possibly be dose dependent with regard to the TrxR inhibitor being utilized. The interrelationships between the thioredoxin system and cellular signaling are certainly intimate but also complex. It has been the aim of this chapter to focus on the reactivity of the selenocysteine residue in TrxR and the relation of this enzyme to the intracellular redox signaling pathways as well as to the inflammatory response seen upon use of dinitrohalobenzenes. The idea of TrxR as a cellular redox sensor33 is intriguing and certainly deserves further functional studies on the cellular or organism level.

Acknowledgements The research of the author is supported by the Karolinska Institute and the Swedish Cancer Society (projects 3775-B00-05XAC and 4056-B99-02PBD).

References 1. Arner ESJ, Holmgren A. 2000. Physiological functions of thioredoxin and thioredoxin reductase. Eur. J. Biochem. 267: 6102-6109 2. Holmgren A. 1985. Thioredoxin. Ann. Rev. Biochem. 54: 237-271 3. Nakamura H, Nakamura K, Yodoi J. 1997. Redox regulation of cellular activation. Ann. Rev. Immunol. 15: 351-369 4. Becker K, Gromer S, Schirmer RH, Miller S. 2000. Thioredoxin reduc­ tase as a pathophysiological factor and drug target. Eur. ]. Biochem. 267: 6118-6125

40

Cellular Implications ofRedox Signalling

5. Arner ESJ, Zhong L, Holmgren A. 1999. Preparation and assay of mammalian thioredoxin and thioredoxin reductase. Meth. Enzymol. 300: 226-239 6. Gladyshev VN, Jeang K-T, Stadtman TC. 1996. Selenocysteine, iden­ tified as the penultimate C-terminal residue in human T-cell thio­ redoxin reductase, corresponds to TGA in the human placental gene. Proc. Natl. Acad. Sci. USA 93: 6146-6151 7. Tamura T, Stadtman TC. 1996. A new selenoprotein from human lung adenocarcinoma cells: Purification, properties, and thioredoxin reductase activity. Proc. Natl. Acad. Sci. USA 93: 1006-1011 8. Zhong L, Arner ESJ, Ljung J, Aslund F, Holmgren A. 1998. Rat and calf thioredoxin reductase are homologous to glutathione reductase with a carboxyl-terminal elongation containing a conserved catalytically active penultimate selenocysteine residue. /. Biol. Chetn. 273: 8581-8591 9. Low SC, Berry MJ. 1996. Knowing when not to stop: Selenocysteine incorporation in eukaryotes. TIBS 21: 203-208 10. Bock A, Forchhammer K, Heider J, Leinfelder W, Sawers G, Veprek B, Zinoni F. 1991. Selenocysteine: The 21st amino acid. Mol. Microbiol. 5: 515-520 11. Stadtman TC. 1996. Selenocysteine. Ann. Rev. Biochem. 65: 83-100 12. Lescure A, Gautheret D, Carbon P, Krol A. 1999. Novel selenoproteins identified in silico and in vivo by using a conserved RNA structural motif. /. Biol. Chem. 274: 38147-38154 13. Kryukov GV, Kryukov VM, Gladyshev VN. 1999. New mammalian selenocysteine-containing proteins identified with an algorithm that searches for selenocysteine insertion sequence elements. /. Biol. Chem. 274: 33888-33897 14. Hiittenhofer A, Bock A. 1998. RNA structures involved in selenoprotein synthesis. In RNA Structure and Function, eds. Simons RW. Grunberg-Manago M, Cold Spring Harbor Laboratory Press, New York 15. Walczak R, Westhof E, Carbon P, Krol A. 1996. A novel RNA struc­ tural motif in the selenocysteine insertion element of eukaryotic selenoprotein mRNAs. RNA 2: 367-379 16. Tormay P, Bock A. 1997. Barriers to heterologous expression of a selenoprotein gene in bacteria. /. Bacteriol. 179: 576-582 17. Arner ESJ, Sarioglu H, Lottspeich F, Holmgren A, Bock A. 1999. High-level expression in Escherichia coli of selenocysteine-containing rat thioredoxin reductase utilizing gene fusions with engineered

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selenoenzyme, steady state kinetics, and inhibition by therapeutic gold compounds. /. Biol. Chetn. 273: 20096-20101 Hill KE, McCollum GW, Burk RF. 1997. Determination of thioredoxin reductase activity in rat liver supernatant. Anal. Biochem. 253: 123-125 Lee SR, Kim JR, Kwon KS, Yoon HW, Levine RL, Ginsburg A, Rhee SG. 1999. Molecular cloning and characterization of a mitochondrial selenocysteine-containing thioredoxin reductase from rat liver /. Biol. Chem. 274: 4722-4734 Miranda-Vizuete A, Damdimopoulos AE, Pedrajas JR, Gustafsson JA, Spyrou G. 1999. Human mitochondrial thioredoxin reductase cDNA cloning, expression and genomic organization. Eur. } . Biochem. 261: 405-112 Rigobello MP, Callegaro MT, Barzon E, Benetti M, Bindoli A. 1998. Puri­ fication of mitochondrial thioredoxin reductase and its involvement in the redox regulation of membrane permeability. Free Radic. Biol. Med. 24: 370-376 Watabe S, Makino Y, Ogawa K, Hiroi T, Yamamoto Y, Takahashi SY. 1999. Mitochondrial thioredoxin reductase in bovine adrenal cortex its purification, properties, nucleotide/amino acid sequences, and identification of selenocysteine. Eur. }. Biochem. 264: 74-84 Sun QA, Wu Y, Zappacosta F, Jeang KT, Lee BJ, Hatfield DL, Gladyshev VN. 1999. Redox regulation of cell signaling by selenocysteine in mammalian thioredoxin reductases. /. Biol. Chem. 274: 24522-24530 Sun QA, Kirnarsky L, Sherman S, Gladyshev VN. 2001. Selenoprotein oxidoreductase with specificity for thioredoxin and glutathione systems. Proc. Natl. Acad. Sci. USA 98: 3673-3678 Koishi R, Kawashima I, Yoshimura C, Sugawara M, Serizawa N. 1997. Cloning and characterization of a novel oxidoreductase KDRF from a human bone marrow-derived stromal cell line KM-102. /. Biol. Chem. 272: 2570-2577 Gasdaska JR, Harney JW, Gasdaska PY, Powis G, Berry MJ. 1999. Regulation of human thioredoxin reductase expression and activity by 3'-untranslated region selenocysteine insertion sequence and mRNA instability elements. /. Biol. Chem. 274: 25379-25385 Xu N, Chen CY, Shyu AB. 1997. Modulation of the fate of cytoplasmic mRNA by AU-rich elements: Key sequence features controlling mRNA deadenylation and decay. Mol. Cell. Biol. 17: 4611^621

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38. Rozell B, Hansson HA, Luthman M, Holmgren A. 1985. Immunohistochemical localization of thioredoxin and thioredoxin reductase in adult rats. Eur. }. Cell. Biol. 38: 79-86 39. Gasdaska JR, Gasdaska PY, Gallegos A, Powis G. 1996. Human thioredoxin reductase gene localization to chromosomal position 12q23-q24.1 and mRNA distribution in human tissue. Genomics 37: 257-259 40. Rundlof A-K, Carlsten M, Giacobini MMJ, Arner ESJ. 2000. Prominent expression of the selenoprotein thioredoxin reductase in the medullary rays of the rat kidney and thioredoxin reductase mRNA variants dif­ fering at the 5' untranslated region. Biochem. ]. 347: 661-668 41. Howie AF, Arthur JR, Nicol F, Walker SW, Beech SG, Beckett GJ. 1998. Identification of a 57 kDA selenoprotein in human thyrocytes as thioredoxin reductase and evidence that its expression is regulated through the calcium-phosphoinositol signaling pathway. /. Clin. Endocrinol. Metab. 83: 2052-2058 42. Anema SM, Walker SW, Howie AF, Arthur JR, Nicol F, Beckett GJ. 1999. Thioredoxin reductase is the major selenoprotein expressed in human umbilical-vein endothelial cells and is regulated by protein kinase C. Biochem. }. 342:111-117 43. Schutze N, Fritsche J, Ebert-Dumig R, Schneider D, Kohrle J, Andreesen R, Kreutz M, Jakob F. 1999. The selenoprotein thio­ redoxin reductase is expressed in peripheral blood monocytes and THP1 human myeloid leukemia cells-regulation by 1,25-dihydroxyvitamin D3 and selenite. Biofactors 10: 329-338 44. Schutze N, Bachthaler M, Lechner A, Kohrle J, Jakob F. 1998. Identi­ fication by differential display PCR of the selenoprotein thioredoxin reductase as a lalpha,25(OH)2-vitamin D3-responsive gene in human osteoblasts—regulation by selenite. Biofactors 7: 299-310 45. Rundlof A-K, Carlsten M, Arner ESJ. 2001. The core promoter of human thioredoxin reductase 1: Cloning, transcriptional activity and Octl, Spl and Sp3 binding reveal a housekeeping-type promoter for the ARE-regulated gene. /. Biol. Chem., epub ahead of print 46. Chen CY, Shyu AB. 1995. AU-rich elements: Characterization and importance in mRNA degradation. Trends. Biochem. Sci. 20: 465-470 47. Dalton TP, Shertzer HG, Puga A. 1999. Regulation of gene expression by reactive oxygen. Ann. Rev. Pharmacol. Toxicol. 39: 67-101 48. Allen RG, Tresini M. 2000. Oxidative stress and gene regulation. Free Radic. Biol. Med. 28: 463-499

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49. Winzen R, Kracht M, Ritter B, Wilhelm A, Chen Y, Shyu AB, Muller M, Gaestel M. Resch K, Holtmann H. 1999. The p38 MAP kinase pathway signals for cytokine-induced mRNA stabilization via MAP kinase-activated protein kinase-2 and an AU-rich region-targeted mechanism. EMBO J. 18: 4969-4980 50. Finkel T. 2000. Redox-dependent signal transduction. FEBS Lett. 476: 52-54 51. Hori K, Katayama M, Sato N, Ishii K, Waga S, Yodoi J. 1994. Neuroprotection by glial cells, through adult T cell leukemia-derived factor/human thioredoxin (ADF/Trx). Brain Res. 652: 304-310 52. Habig WH, Pabst MJ, Jakoby WB. 1974. Glutathione S-transferases. The first enzymatic step in mercaptopuric acid formation. /. Biol. Chem. 249: 7130-7139 53. Meister A, Anderson ME. 1983. Glutathione. Ann. Rev. Biochem. 52: 711-760 54. Ahmed AR, Blose DA. 1983. Delayed-type hypersensitivity skin testing. A review. Arch. Dermatol. 119: 934-945 55. Schmidt RJ, Chung LY. 1992. Biochemical responses of skin to allergenic and non-allergenic nitrohalobenzenes. Evidence that an NADPH-dependent reductase in skin may act as a prohaptenactivating enzyme. Arch. Dermatol. Res. 284: 400^408 56. Sreider CM, Grinblat L, Stoppani AOM. 1990. Catalysis of nitrofuran redox-cycling and superoxide anion production by heart lipoamide dehydrogenase. Biochem. Pharmacol. 40:1849-1857 57. Sreider CM, Grinblat L, Stoppani AOM. 1992. Reduction of nitrofuran compounds by heart lipoamide dehydrogenase: Role of flavin and the reactive disulfide groups. Biochem. Int. 28: 323-334 58. Mason RP, Josephy PD. 1985. Free radical mechanism of nitroreductase. In Toxicity of Nitroaromatic Compounds, ed. Rickert, DE, Hemisphere, New York, pp. 121-140 59. Mason RP, Holtzman JL. 1975. The role of catalytic superoxide formation in the 0 2 inhibition of nitroreductase. Biochem. Biophys. Res. Commun. 67: 1267-1274 60. Arner ESJ. 1999. Superoxide production by dinitrophenyl-derivatized thioredoxin reductase—A model for the mechanism and correlation to immunostimulation by dinitrohalobenzenes. Biofactors 10:219-226 61. Holmgren A, Arner E, Aslund F, Bjornstedt M, Liangwei Z, Ljung J, Nakamura H, Nikitovic D. 1998. Redox regulation by the thioredoxin and glutaredoxin systems. In Oxidative Stress, Cancer, AIDS and

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Neurodegenerative Diseases, eds. Montagnier L, Olivier R, Pasquier C, Marcel Dekker, Inc., New York, pp. 229-246 Wakasugi N, Tagaya Y, Wakasugi H, Mitsui A, Maeda M, Yodoi J, Tursz T. 1990. Adult T-cell leukemia-derived factor/thioredoxin pro­ duced by both human T-lymphotropic virus type I- and Epstein-Barr virus-transformed lymphocytes, acts as an autocrine growth factor and synergizes with interleukin-1 and interleukin-2. Proc. Natl. Acad. Sci. USA 87: 8282-8286 Blum H, Rollirighoff M, Gessner A. 1996. Expression and co-cytokine function of murine thioredoxin/adult T cell leukaemia-derived factor (ADF). Cytokine 8: 6-13 Bertini R, Howard OM, Dong HF, Oppenheim JL Bizzarri C, Sergi R, Caselli G, Pagliei S, Romines B, Wilshire JA, Mengozzi M, Nakamura H, Yodoi J, Pekkari K, Gurunath R, Holmgren A, Herzenberg LA, Herzenberg LA, Ghezzi P. 1999. Thioredoxin, a redox enzyme released in infection and inflammation, is a unique chemoattractant for neutrophils, monocytes, and T cells. /. Exp. Med. 189: 1783-1789 Schenk H, Vogt M, Droge W, Schulze-Osthof K. 1996. Thioredoxin as a potent costimulus of cytokine expression. /. Immunol. 156: 765-771 Pekkari K, Gurunath R, Arner ESJ, Holmgren A. 2000. Truncated thioredoxin is a mitogenic cytokine for resting human peripheral blood mononuclear cells and is present in human plasma. /. Biol. Chem. 275: 37474-37480 Pekkari K, Avila-Carino J, Bengtsson A, Gurunath R, Scheynius A, Holmgren A. 2001. Truncated thioredoxin (Trx80) induces production of interleukin-12 and enhances CD-14 expression in human mono­ cytes. Blood 97: 3184-3190 Saitoh M, Nishitoh H, Fujii M, Takeda K, Tobiume K, Sawada Y, Kawabata M, Miyazono K, Ichijo H. 1998. Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase-1 (ASK-2). EMBO }. 17: 2596-2606 Zhang P, Liu B, Kang SW, Seo MS, Rhee SG, Obeid LM. 1997. Thio­ redoxin peroxidase is a novel inhibitor of apoptosis with a mechanism distinct from that of bcl-2. /. Biol. Chem. 272: 30615-30618 Zhong L, Holmgren A. 2000. Essential role of selenium in the catalytic activities of mammalian thioredoxin reductase revealed by characterization of recombinant enzymes with selenocysteine muta­ tions. /. Biol. Chem. 275:18121-18128

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Chapter 3 Iron-Sulfur Proteins: Properties and Functions Helmut Beinert Institute for Enzyme Research, University of Wisconsin, 1710 Univ Ave. Madison WI53726-4087 USA, hbeinert@facstaff. wise, edu

Keywords: Iron-sulfur (Fe-S) clusters, cluster stability, cluster interconversions, redox potentials, electron transfer

1. Summary Many biologically active proteins contain clusters of high-spin iron ions in complexation with sulfide (S2~) and thiolate (RS~), called Fe-S clusters. The basic chemical structures and their electronic structure and stability are discussed. There are three basic cluster types: [2Fe-2S], [3Fe-4S], and [4Fe-4S], which can undergo a number of interconversions or even destruction depending on external conditions, such as pH, presence of oxidants or reductants, or of N O and other compounds. Their principal function is in electron transfer, e.g. in biological oxidations. Depending on their environment, Fe-S clusters may have a wide range of redoxpotentials (~1V). Their structural flexibility and sensitivity to external influences has also been exploited by nature in signaling and regulatory pathways. Examples of their use in such functions and their structural flexibility are discussed.

2. Historical Comments Fe-S clusters are most likely among the very first cofactors that were available as life started, because, under suitable conditions, they may be

47

48

Cellular Implications ofRedox Signalling

formed spontaneously. If we consider this, it may seem surprising that we do not know them for more than about 40 years. One of the reasons is that most cofactors, vitamins and prosthetic groups such as heme, flavin, pyridoxal, carotenes, and others were discovered because of their strong color; and the pale yellow, with a reddish or greenish tinge, of Fe-S proteins is not very conspicuous. Another reason is that the rather robust methods that could be used for the purification of known cofac­ tors did not work for Fe-S proteins, but only led to their decomposition. They were eventually found by their catalytic action, as brown bands on columns during protein purification and as EPR signals, which are unique markers of most of them. 10 However, there was no EPR until 1943 and it was not introduced into biology for at least another 10 years.

3. Properties 3.1 Structure Redox regulation, the main theme of this volume, is actually not what applies for Fe-S proteins in most instances of which we know; it is rather regulation by oxidative destruction and reconstitution. This makes the process more complicated than simple one- or two-electron oxidation, followed by reduction, or vice-versa; namely, in addition to a reductant and the simple building blocks, other components such as enzymes, cofactors, chaperones or transport proteins are required for reconstitu­ tion, as far as we know today. In order to understand what we are faced with, we have to take a closer look at Fe-S clusters, Fe-S proteins and their properties. Figure 1 shows the most common structures that we have to deal with.33 The corresponding compounds are formed in mixtures of ferric chloride, thiol and sulfide combined in the indicated proportions in an anaerobic solution of an organic solvent such as ethanol or acetonitrile: a mono-nuclear complex, and the di- and tetra-nuclear clusters and the cage complex (upper right). The 4Fe cluster is the "sink" toward which every­ thing goes, if all ingredients are present in sufficient quantity. The cluster charge is given here as 2-. There is a difference between coordination chemists' and biochemists' nomenclature, which can be confusing to the outsider. In this presentation the charge of the iron-sulfur core only is

Iron-Sulfur Proteins: Properties and Functions

49

FeCI< 2>5RS-

3.5 RS2-

SR

S-^ F %/ R

2RS

SR

n ?FeN RS—Fe-f—S^ SR

R-HT

Fe /

RS

\

/

SR

R-

\

R'S 4S

SR RS

2 RS\

...s-..

/SR

RS^ ^ S ^

^SR

MeOH

S p /?—£* Fe-j—S

RS--b Fe -T7 5 S—Fe \

SR

Fig. 1. Depiction of the course of reactions resulting in assembly of [Fe4S4(SR)4]2" clusters via the intermediates [Fe(SR)4]2", [Fe2S2(SR)4]2~, and [Fe4(SR)10]2~. Note that the symbols shown here are those that are used when the whole complex, including the ligands, is considered (adapted with permission from Hagen et al.,33 copyright 1981, American Chemical Society).

m e a n t , as it is s h o w n in the formulae, i.e. [xFe-yS], w i t h o u t the four negatively charged Cys Kgands; the charge is then 2+ instead of 2 - for the clusters s h o w n in Fig. 1. With the basic m o d u l a r units s h o w n , m o r e complex structures can t h e n b e built u p , as seen in Fig. 2: on the left a n Fe-S-heme c o m b i n a t i o n is s h o w n , o n the right, t w o 4Fe m o d u l e s c o m b i n e d , as in the so-called P- (for protein) clusters of nitrogenase.

50

Cellular Implications ofRedox Signalling

Fig. 2. Schematic structures of native assemblies, in which a cubane-type or cuboidal Fe-S cluster is bridged to the other component of the active site; left: Escherichia coli sulfite reductase; right: the P-cluster of nitrogenase. Irons and carbons are in black, sulfurs white (reprinted with permission from Beinert et al.,n copyright 1997, American Association for the Advancement of Science). 3.2 Electronic Structure Sulfur is considered as a weak ligand; and iron with all-sulfur ligation assumes a state with the maximal number of unpaired electrons allowed by the Pauli exclusion principle; as one says, it is "high-spin", and highspin complexes are less stable than low-spin ones with strong ligands such as, e.g. cyanide. The electronic structure is schematically illustrated in Fig. 3. 1112 The formal charges on the iron atoms (large circles) for the different oxidation states are indicated by shading: Fe34" black, Fe2+ white, and "mixed valence", Fe25+ gray; and the system spins and net charges are given under the structures. The cluster irons are magnetically coupled in pairs: in 2Fe clusters the electron spins are coupled antiparallel (antiferromagnetically) to a spin of zero in the 2+ (the "oxidized") state, and to a spin of 1/2 in the 1+ ("the "reduced") state, in 4Fe clusters, two pairs are formed by coupling of two Fe, in this case each pair with spins parallel (ferromagnetically); the spins of these primary pairs are then coupled antiparallel to the system spin. It has been ascertained by NMR that there may be shifts of electrons between Fe atoms of the pairs within 4Fe clusters as schematically shown in Fig. 46 Usually the formal charges are attributed to and written down for the Fe atoms. However, calculations of the distribution of valence electron

Iron-Sulfur Proteins: Properties and Functions 51

Fig. 3. Localization and delocalization patterns in Fe-S clusters, showing localized Fe3+ (black) and localized Fe2+ (white) sites, delocalized Fe25+Fe25+ (gray), sulfur (white). Indicated are also the spin S of the cluster and its core oxidation state (reprinted with permission from Beinert et al.,n copyright 1997, American Society for the Advancement of Science). density, carried out by density functional methods 61 clearly show that, on oxidation-reduction, by far the greater changes in electron density occur on all sulfur atoms, bridging sulfides and Cys-sulfurs, not on the iron atoms. For instance, for every electron's worth of charge density on a 4Fe cluster, the sulfurs account for about 0.1 electron per sulfur and the irons for slightly more than one half of this. Figure 5 shows the electron density change on reduction of the [4Fe-4S]2+ to the [4Fe-4S]+ state. The gray shaded

52

Cellular Implications ofRedox Signalling

CysS

^rf

SCys

SCys CysS

CysS

Fig. 4. Shift of mixed valence pair of irons (filled squares) in high-potential Fe-S protein between cluster faces, here from irons 3 and 4 to irons 3 and 1. As a result, iron 3 is Fe 25+ and iron 2 is Fe3+, whereas irons 1 and 4 have oxidation numbers between 2.5+ and 3+ (reprinted with permission from Banci et al.,6 copyright 1993, American Chemical Society).

Fig. 5. Total valence electron density difference between 1+ and 2+ states of a [4Fe-4S] cluster. Dark shading shows increased electron density on reduction and white decreased density (reprinted with permission from Noodleman and Case,61 copyright 1992, Academic Press).

Iron-Sulfur Proteins: Properties and Functions

53

areas are those that experience increased electron density, while the white areas show depletion of electron density. Similar changes in the opposite direction occur on oxidation of the cluster from the 2+ to the 3+ state.

3.3 Stability All these properties, such as electron delocalization and spin coupling, are, of course, not unrelated to the stability of Fe-S clusters. Can they be considered as cofactors, such as flavins, hemes or pyridine nucleotides, which can be isolated and put into a bottle? According to the electronic structure, schematically indicated in Fig. 3, and the magnetic coupling between the iron atoms, the 2- and 4-Fe clusters with their thiol ligands are quite stable and self-contained; however they are vulnerable through outside influences: water protons, oxygen, reduction products of oxygen, or other oxidants, N O and also high concentrations of thiols and sulfide and, under some conditions, also chelators. However, reaction with chelators cannot a priori be expected, i.e. failure to observe an effect of iron chelators is no proof of the absence of Fe-S clusters. Fe-S clusters are most stable when embedded into proteins, or better yet membrane proteins, which may prevent access of deleterious compounds, such as solvent which carries oxidants or chelators. 3.4 Complex Fe-S Proteins In addition to these intrinsic properties, nature often introduces modula­ tions, as we have seen in Fig. 2, by welding together various cluster modules or fragments, or by juxtaposing two or more clusters in a protein or other cofactors such as heme, flavin, or by forming heterometal clus­ ters, such as Fe/Ni clusters as in hydrogenases or CO dehydrogenase, 29 or by use of unusual ligands other than cysteines.66 Thus, given the different cluster types to start with, the possibilities of subtle modulation are almost unlimited, and so is also the variety of uses that FeS clusters can be put to.

3.5 Cluster Ligands The availability and location of the ligating Cys residues determine much of the behavior of Fe-S clusters. There are some characteristic patterns of

54 Cellular Implications ofRedox Signalling

16

Leu

26

/

_

Ser 24 Cys

21

Gln ^ — ^

F e lll

14

Cys

^>*—o -S^ JJCys_^

^ S ^ 5*" ^

( 6 0

Felll,

\ Cys-^

II

X

^S o — ^ v,

56Cys-—^

Fig. 6. Tentative scheme of the Fe-S active site environment of the C. ■pasteurianum 2 Fe ferredoxin. Cys56 and Cys60 are indicated as ligands of the reducible Fe. Those residues that may become ligands of the cluster upon muta­ tion into cysteines are shown (reprinted with permission from Golinelli et a/.,31 copyright 1998, American Chemical Society).

Cys distribution in proteins which allow one to make predictions whether an Fe-S cluster is most likely present and also what type it may be;40,57 and from amino acid sequence similarities, conclusions can often be drawn as to what the biological function of the respective proteins is likely to be. In the following we will see a few instructive examples of choice of ligands and cluster interconversions. In a 2Fe Fd from Clostridium pasteurianum it seemed impossible to find out by systematic mutation of residues, which Cys residues of the five present in the molecule were the iron ligands. 31 It turned out that only two of the four were definitely required, and that the other two could be replaced either by the fifth cysteine present or when other neighboring residues in a certain region of the structure were changed to cysteines. This particular region was a flexible loop located on the outside of the protein, whereas the two clearly required cysteines were located in a more structured region toward the core of the protein (Fig. 6, residues 11 and 56). Another example documenting the plasticity of Fe-S clusters in proteins became apparent when the enzyme aconitase, in its inactive 3Fe form, was exposed to elevated p H (>9 ).45 The cubane type 3Fe cluster was stretched out to a linear cluster as shown in Fig. 7, by detaching one of the Cys residues and recruiting two new ones from a helix lying 15-17 A

Iron-Sulfur Proteins: Properties and Functions 55

OH

s

Cys-S 358

/

+Fe +e-

^r •Fe -e

424Cys-S

424Cys-S

£ - * »

S-Cys421

S-Cys421

S ^Fe

S-Cys Fe

cys-s^ \ / W

Fe

\ S-Cys

Fig. 7. Schematic description of the interconversion between cubane-type and linear Fe-S clusters (reprinted with permission from Beinert and Kennedy,13 copyright 1989, Blackwell Science).

away in the crystal structure (Fig. 8). The linear cluster is more stable than the cubane type 3Fe cluster; however, on lowering the p H under reduc­ ing conditions, the 4Fe cluster of the active enzyme was formed again in good yield, indicating that the protein bearing the cluster has regained its original structure. Another oddity is a unique "loosened-up" cluster (Fig. 9) discovered in Desulfovibrio species. 3 This so-called hybrid cluster has sulfide- and oxo-bridges simultaneously and a persulfide group, and may appear as a Fe-S cluster which is either in a precursor form or on the way to destruc­ tion. This seems, however, barely compatible with the fact that it can be crystallized. 3 The function of the respective protein is yet unknown.

3.6 Heterometal Clusters The 3Fe cluster was mentioned and shown repeatedly above (Fig. 3). Obviously this is the ideal starting material for heterometal 4Fe clusters,28,37

56

Cellular Implications ofRedox Signalling

Fig 8. Close-up view of part of the structure of mitochondrial aconitase, showing the active site with substrate bound. When the linear cluster is formed, the two Cys ligands on the right side are maintained and two cysteines (indicated by small arrows at the lower right) from a distant helix are used to complete the ligation. As shown in [11] from unpublished work of SJ Loyd, GS Prasad and CD Stout and reprinted with permission, copyright 2000, Society for Biological Inorganic Chemistry. a n d this h a s b e e n exploited in in vitro work. Yet, such clusters are not found in n a t u r e ; the only exceptions are the Mo-Fe or V-Fe proteins of nitrogenase 6 9 a n d t h e Ni-Fe clusters of h y d r o g e n a s e s a n d C O d e h y d r o g e n a s e s . 2 9

Iron-Sulfur Proteins: Properties and Functions 57

►(His 244) (Cys 459}S

(Gfu 494)

(Glu268)

%e

g

\

1

(Cys406)S \ ^ S^

1

Fe6« k

(Cys 434)8

S(Cys312)

^

Fig. 9. A schematic view of cluster 2, the "hybrid" cluster from Desulfovibrio vulgaris which contains both S and O bridges between the iron atoms. X represents a putative substrate-binding site, which may be partially occupied in the present structure (reprinted with permission from [3], copyright 1998, Society for Biological Inorganic Chemistry).

However, these are unusual, specialized structures. Artificially, though, a whole series of metals have been incorporated into man-made or naturally occurring 3Fe clusters.37,41 These turned out to be useful materials for explor­ ing the electronic structure of Fe-S clusters, because it was possible to incor­ porate non-magnetic metals. Why are such clusters not found in nature? An answer was proposed by Armstrong and Williams:5 They argued that heavy metals are known not to float around freely in tissue; they are carefully guided and chaperoned by special proteins. Thus, metals like Zn, for instance, or Cd, which are known to have, in vitro, a higher affinity for the 3Fe cluster than Fe,22 are bound so tightly to their chaperones — that is, in this example, metallothionein for Zn and Cd — that they cannot compete with Fe for the 3Fe cluster. For instance, it was determined that the 3Fe cluster of aconitase competes for Fe2+ with some success even with EDTA: the formation constant for the iron complex is 10 for 3Fe aconitase and 14 for EDTA.27

58

Cellular Implications ofRedox Signalling

Active

Inactive S-1/2

PRJ-4S]1*

^ *k

(^Fe^f*)

S-1/2

V

-e*

+6"

Fe2*

+©"

-©"

>

S =9

faPo.MIRIO

*_£* >

[4Fe-4S]2+ +0

S-0

.0

I4F©-4SJ1+

S-1/2

Fig. 10. A model showing the relationship between the various cluster forms of aconitase. For each cluster type, its oxidation state (i.e. the charge balance of the core) is presented as a superscript and its spin state at the outer sides. In the model, aconitase is inactive when it contains a 3Fe cluster and active when it contains a 4Fe cluster. Fe2+ plays the role of converting the 3Fe clusters to 4Fe clus­ ters. The existence of a [4Fe-4S]3+ cluster is uncertain and, therefore, the corresponding symbol is placed within parentheses (from [27] with the permis­ sion of the American Society for Biochemistry and Molecular Biology).

3.7 Self-activation of 3Fe-enzymes The high affinity of the 3Fe cluster for iron is the basis for the pheno­ menon of self-activation under reducing conditions that has been observed repeatedly for enzymes that require a 4Fe cluster for activity, but are obtained in the 3Fe form on purification. What happens is that, under reducing conditions, some 3Fe clusters are spontaneously disassembled and the iron that becomes available is used to build up 4Fe clusters, which are, under reducing conditions, more stable. This has repeatedly misled investigators, when they ascribed activity to enzymes containing 3Fe clus­ ters, while in fact 4Fe clusters were being made by self-activation during the assay or during preparations for it.2046 Figure 10 describes the situation encountered with aconitase, which is obtained in its 3Fe form on

Iron-Sulfur Proteins: Properties and Functions

59

routine purification. All these observations show again the enormous flexibility and plasticity of Fe-S clusters. In this context we should also consider the reuse of sulfide, not only of the iron of a cluster. What happens to the sulfide when clusters are dis­ mantled? When oxygen is excluded — oxygen would either lead to for­ mation of disulfides or sulfenate (SO") — it has been found that the sulfide sulfur can also be reused for formation of more stable clusters, such as the 4Fe type. There are indications that iron sulfides may be attached to pro­ tein in some fashion and may be reused as such for rebuilding clusters. 63 All this has a bearing on the transport of Fe-S clusters or their precursors. There is evidence that Fe-S clusters have to cross membranes during Fe-S cluster biosynthesis in eukaryotes.48,52 Such iron-sulfides on proteins, pos­ sibly on specific chaperones, or even whole 2Fe- or, maybe, linear 3Fe clusters might be the forms that can be transported more easily than the cubic 4Fe-clusters. We may also recall here the hybrid cluster shown above (Fig. 9), which could be looked at as a model for such a loosenedu p cluster form.

3.8 Degradation and Biosynthesis of Fe-S Clusters While it is likely that originally Fe-S clusters may have been formed spon­ taneously, their building blocks, iron and sulfide, are much too dangerous materials to be allowed in living tissues unguarded. Thus, a whole set of proteins has evolved that has the task of bringing about the uptake and transport of iron, and the generation and transport of sulfide to the place of synthesis. Iron, of course, has to be imported from the outside or taken from internal stores previously formed; and sulfide is produced from cysteine by pyridoxal-containing enzymes51,75 and safeguarded and trans­ ported in the form of a hydrodisulfide (RSS*H, also called persulfide) attached to protein 75 or a cysteine.51 Under reducing conditions, it can then be released from its carrier as sulfide. The iron, on the other hand, is assembled into a cluster precursor on three cysteines of a different protein, 74 IscU, in E. coli.1 NifU (or IscU) is able to form a complex with the sulfane carrrier NifS (or IscS), in which the transfer of sulfide takes place with the formation of a 2Fe cluster, from which eventually a [4Fe-4S] cluster may be formed. 1 In in vitro work with proteins purified from the cyanobacterium Synechocystis it has been possible to observe transfer of a 2Fe cluster from the IscU homolog of this organism to the apoprotein of a

60

Cellular Implications ofRedox Signalling

Fd isolated from Synechocystis; in this way the specific electron transfer activity of the holo-Fd was restored to the inactive apoFd.60 The last few years have witnessed enormous progress in the area of Fe-S cluster biosynthesis, both in pro- and eukaryotes. As one would expect, the mechanism of synthesis in multicellular organisms is considerably more complex than in unicellular ones and requires a number of additional fac­ tors or proteins. The respective original or review literature26,39'52'59 will have to be consulted about details of the status in this field, which is rapidly moving at present. On the contrary, little detail is available on the degradation of Fe-S clusters. Thorough studies of the decomposition of Fe-S clusters by acid or base are available,56 but this has not as much relevance to biological con­ ditions than degradation by oxygen, its reduction products or by nitric oxide. We know that Fe-S clusters are destroyed by these agents, but little detail is known about the mechanisms involved. The sulfides and cysteine ligands are assumed to be the primary targets and as products proteinbound di- or tri-sulfides have been identified.43'65 As it is not possible to quantitatively account for the originally present sulfide as disulfide or sulfane sulfur (also called sulfur zero, S°), it can be assumed that some sul­ fur oxides are formed such as sulfenate (SO "), sulfinate (SO2-), or yet more oxidized forms. On oxidation of 4Fe clusters the first iron, at least, is released as Fe2"1".7'44 This is clearly the case on oxidation of the 4Fe to the 3Fe cluster; it has also been observed, when the end product is the 2Fe cluster,7 which may suggest, but does not prove, that the 3Fe cluster is an interme­ diate in this process. In the reaction of N O with Fe-S clusters protein-bound DiNitrosyl-Iron Complexes (DNIC), Fe(Cys)2(NO)2, are formed with destruction of the original Fe-S clusters .42'50,71 All these destructive reactions, except for the simple 4Fe to 3Fe conversion, are not readily reversed in vitro by adding reductants with or without iron and sulfide, but can, apparently be repaired in cellular preparations.1819,62'64 The factors, probably proteins, required for such repair, have not been identified. They may or may not68 be identical to the enzymes used in the original synthesis.

4. Functions of Fe-S Proteins 4.1 Electron Transfer Foremost is their use in electron transfer, for which the "respiratory chain" of mitochondria is the prime example (Fig. 11). There are as many

Iron-Sulfur Proteins: Properties and Functions 61 Succinate

1 FAD [2Fe-2S]S-1 [4Fe-4S]S-2 [3Fe-4S]S-3 Cytb Qs Complex I NADH-

I

Complex

a

Complex m

Cytt%66 FMN I - l QM ™ N 6[4Fe-4S] ^ -♦•Q-pool—*• Cytb562 f2Fe-2S]R,este Cytc, 2 2Fe 2S

t

\

[4Fe-4S] FAD

Cytc

t

Complex IV

JoA —♦■ AcylCoA —*• Electron dehydrogenase transferring flavoprotein

1

Cyta Cu-Cu

Cyta3 Cue

\ 02

Fig. 11. Mitochondrial respiratory chain. Qs and QN are protein-associated pools of ubiquinone that can be distinguished from the bulk ubiquinone pool (adapted from Johnson40 with permission from Wiley).

Fe-S clusters in NADH dehydrogenase alone as there are hemes and flavins in the components of the whole system: there are a total of 13-14 Fe-S clusters altogether! Note also that, in order to reach ubiquinone in any pathway, electrons have to pass through Fe-S proteins, more pre­ cisely, Fe-S flavoproteins. There are many variants of such electron transfer systems in different organisms or even within individual macromolecules, which, almost without exception, make use of Fe-S proteins, if the range of low redox potentials is involved.

4.2 Oxidation-Reduction Potentials The function of Fe-S clusters in electron transfer leads to a consideration of redox potentials. As we can see in Fig. 12,23 the redox potentials that are possible with Fe-S proteins cover a wide range, even exceeding 1 volt. On the low side we have the most negative potentials with 7Fe Fds ([4Fe-4S2+/+], [3Fe-4S]+/0) of -650mV, and at the high end the HiPIP clusters

62 Cellular Implications ofRedox Signalling

4Fe cluster in [8Fe-8S] Ferredoxins 4Fe cluster in HiPIP

4Fe cluster in [7Fe-8S] Ferredoxins 4Fe cluster in [4Fe-4S] Ferredoxins 3Fe cluster in (7Fe-8S] Ferredoxins ■ 3Fe cluster in [3Fe-4S] Ferredoxins 2Fe cluster in r2Fe-2S] Ferredoxins

Rubredoxins —I

[

!

1

1

1

j

-700 -600-500-400-300-200-100 0 E°'{mV)

1

1

1

1

100 200 300 400 500

Fig. 12. Experimental ranges of reduction potentials (versus NHE) of various subclasses of Fe-S proteins (from Capozzi et alP with permission from Springer Verlag).

([4Fe-4S3+/2+]) with up to +450 mV; with 4Fe, 3Fe, 2Fe clusters, rubre­ doxins and Rieske clusters (not shown) in between. Rieske clusters, are [2Fe-2S] clusters that have two Cys and two His ligands, asymmetrically disposed. The potentials of these clusters fall into the range of 100 to 320 mV for the bc1 type, and those of the related dioxygenase type Rieske clusters fall between -100 and -150 mV.53 It is easy to understand that the net charge of the clusters is one of the primary determining features of these potentials, but what are the deter­ minants of the enormous spread of values? One might think that with hundreds of Fe-S proteins, and often derivatives of them available that were generated by mutations, one should have been able to come to some answers on this point. However, there is still a lively debate about this subject and there are many opinions represented. 14 Clearly, it must be the environment in which the cluster finds itself that modulates the intrinsic potential of the cluster, and this environment is one of the most com­ plex ones that nature can supply, namely protein in water. Most success in predicting or rationalizing differences in redox potentials of Fe-S proteins has been achieved, when a series of homologous proteins of relatively simple structure were available, such as the rubredoxins, plant

Iron-Sulfur Proteins: Properties and Functions

63

2Fe Fds, HiPIPs, or Rieske proteins; however, it remains very difficult to predict potentials by comparing more complex, less or unrelated Fe-S proteins. A number of factors that influence, or are most likely to influ­ ence the potentials of redox proteins, have been identified, but the extent to which each one of these can influence the values of the potentials in any one case is subject to great variations in an aqueous protein environment. The following factors must be considered: first, the identity of the cluster ligands, hydrogen bonds from protein constituents to cluster sulfides or its Cys ligands, charges or dipoles on adjacent peptide chains, water molecules in the protein as shields or as dipoles; further, of course, other clusters or redox groups such as hemes or flavins; second, the distance of any of the interacting species from the Fe-S cluster, which, when increas­ ing, will attenuate any of these effects; and, for dipoles also their orienta­ tion: if a dipole points toward the cluster, it will increase the redox potential, if it points away it decreases it. Then, it has to be considered that this environment is not static, but is subject to continuous dynamic fluctuations. As the value of the dielectric of the medium, in which all these interactions occur, is of importance, it is a questionable simplification, if a uniform continuum is assumed as the dielectric, instead of a microscopic, heterogeneous one.72 It has also been pointed out that, in addition to the expected random fluctuations, the very process of oxidation-reduction may bring about distinct, reversible conformational changes in the protein; these could influence the redox potential transiently during a reaction, but may not be expressed in the statically measured potential. 4 Thus, it could be misleading to assume that the spatial sequence of redox components in a protein can be simply deduced from their statically measured potentials or vice-versa. Surface charges on the protein may have some influence; and it has been possible, within a series of similar HiPIPs, to relate the electrostatic contributions of such charges to the observed potentials. 15 On the other hand, it has been observed that net charges fully exposed to solvent have little effect on the potential, whereas buried charges have to be considered as impor­ tant factors. In the Rieske (bCj type) protein series it has been possible to relate a sizeable increase in redox potential (+150mV) to the presence of a serine residue, which forms a hydrogen bond to one of the bridging sulfide groups of the cluster in those proteins that use ubiquinone as elec­ tron donor versus those that use menaquinone as donor. 53 An instructive example as to how subtle structural details can influence redox potentials can be found in a recent paper on the comparison of the structures of a Rieske and a Rieske-type Fe-S protein. 24 A peculiar property of Rieske

64

Cellular Implications of Redox Signalling

proteins is also that their redox potentials are pH dependent, which presumably has to do with the ligation of His residues to the reducible iron. This p H dependence of the redox potentials of bcx proteins is an important feature in their ability to generate a proton gradient. It has also been learned that in 2Fe Fds that have all-Cys ligation, the presence of solvent plays a major role in determining the iron of the cluster that becomes ferrous on reduction of the protein. 16 Most recently a thorough study of the thermodynamics of reduction of a variety of Fe-S clusters by variable temperature electrochemistry has been published, 9 which draws particular attention to entropic contributions and should be consulted in connection with the matter discussed above. After these considerations that are more germane to classical one- or two-electron oxidation-reductions, we should also consider other effects of the electronic makeup of Fe-S clusters, which do not necessarily lead to net electron transfer, such as polarization of adjacent structures or elec­ tron storage; in other words, we can look at Fe-S clusters as reservoirs of electrons that can be drawn on in reactions carried out by neighboring cofactors, such as, e.g. flavins, or adenosyl-methionine. 11 ' 25 For instance in the shuffle that leads to oxidative phosphorylation in NADH dehydrogenase, Fe-S clusters are bound to play a crucial role of this kind. By the use of the reducing power of a [4Fe-4S]+ cluster in conjunction with ATP hydrolysis, very low redox potentials can be achieved as required, e.g. for reduction of N 2 to NH 3 by the Fe-protein — Mo-Fe-protein complex of nitrogenase,38 in the anaerobic microbial reduction of aromatic compounds, 17 and in dehydration reactions, when a hydrogen is to be removed from an unactivated carbon.21

4.3 Non-Redox Functions of Fe-S Proteins These functions have to do with the integrity of Fe-S proteins. Thus, Fe-S proteins have the ability to serve as what has been called "circuit breaker"; 30 i.e. there are oxygen sensitive Fe-S proteins that catalyze vital reactions, which require an intact 4Fe cluster for their function. If oxygen, superoxide or hydrogen peroxide are present, these clusters are con­ verted to the 3Fe form, which is more stable toward oxygen, but can be easily reconverted to the 4Fe form, when anaerobicity is restored. Thus the enzymes can be preserved in a quasi-intact state, only temporarily shut off, but readily reconverted to the active form. This, for example, occurs with aconitase, an enzyme indispensable for the functioning of the

Iron-Sulfur Proteins: Properties and Functions

65

Krebs cycle,55 and with anaerobic ribonucleotide reductase of Lactococcus lactis.5* One of the simplest uses of Fe-S clusters is observed if a certain function requires a distinct oxidation state, such as the alarm against oxidative damage. An example is the SoxR-SoxS system of E. coli, which simply depends on the oxidation state of a [2Fe-2S] cluster.36 The SoxR protein sounds the alarm against oxidative stress brought about by the superoxide anion, O". The SoxR protein occurs as a dimer with each monomer bearing a [2Fe-2S] cluster. SoxR is inactive when the cluster is in the 1+ state and becomes activated on oxidation to the 2+ state. SoxR is able to bind to DNA in both oxidation states and apparently even in the apoform, but only the oxidized form activates the gene coding for the SoxS protein, which in turn induces formation of a whole series of pro­ tective proteins and enzymes. A more drastic modification is used in other systems, namely complete dismantling of Fe-S clusters. This was first observed with glutamine phosphoribosylpyrophosphate amidotransferase of Bacillus subtilis, a key enzyme in the formation of pyrimidine nucleotides. 32 This enzyme has an oxygen sensitive 4Fe cluster, which is not used in any oxidation-reduction reaction. However, it stabilizes the protein against proteolytic attack: when the 4Fe cluster is destroyed by oxygen, the protein is then also rapidly degraded. One of the most interesting examples in this category is probably the bifunctional protein cytoplasmic aconitase, which is con­ verted to the "iron regulatory protein" (IRP) on complete removal of its [4Fe-4S] cluster; in this case, the protein is preserved34,35 and acquires a new function! Another well-studied example of a similar nature is the global transcription factor FNR of E. coli, which regulates the conversion of anaerobic to aerobic metabolism in this organism. Closely related proteins with similar functions occur in a great number of other micro­ organisms. 70 In the presence of oxygen, the 4Fe cluster of FNR is rapidly (within seconds) converted to a more stable 2Fe cluster, which then, within minutes or hours, depending on conditions, is converted to apoprotein. 47 Only the presence of the 4Fe cluster allows induction of the enzymes and transport proteins required for anaerobic metabolism. As with many tran­ scription regulators, FNR acts in its dimeric form of ~60kD. The monomeric form is inactive. The inactive 2Fe form is monomeric; how­ ever, it has been observed that in some mutants of FNR the monomeric form can have a 4Fe cluster, but such monomers are not transcriptionally active.58 Information so far available indicates that the Fe-S cluster of FNR is necessary for effective dimerization. 49 The influence of the cluster seems to be transmitted through the protein structure, as the cluster is not likely

66

Cellular Implications ofRedox Signalling

Active FNR dimer OOH

NH 3

DNA Binding and Transcription Activation

jo,

Fe

S

4Fo cluster

XK=2F« cluster

Inactive 2FeFNR monomer

Fig. 13. Scheme describing the pathway of FNR inactivation under aerobic conditions. (Courtesy of PJ Kiley, University of Wisconsin, Madison).

to be in the vicinity of the dimerization helix according to a synopsis of secondary structure data and the crystal structure of the closely related CAP protein. 73 It has been possible to follow the synthesis and conversion of the 4Fe cluster of FNR into the 2Fe cluster and its reconstitution in whole cells of £. coli by Mossbauer spectroscopy, which can easily dis­ criminate between the two cluster forms.67 By this technique and by the use of an oxygen-stable mutant it could also be ascertained that FNR con­ taining a 4Fe cluster is synthesized even in the presence of oxygen, but in the wild type protein the cluster is then obviously rapidly destroyed by oxygen. 8 Figure 13 shows a scheme that encompasses present ideas about the involvement of the Fe-S cluster in the function of FNR.

5. Conclusion and Outlook All these functions can be understood on the basis of the properties of Fe-S clusters, which were described at the outset. Of course, in the frame of this chapter it was only possible to give a glimpse of the iron-sulfur world, of which, just at the present time, we keep learning more practically every day that passes. It seems though that, while the biological aspects and implications are expanding in an unforeseen way, some of the basic chemical information is still lacking. We know preciously little about the

Iron-Sulfur Proteins: Properties and Functions

67

mechanisms of the reaction of Fe-S clusters with oxygen and with its reduction products, or with nitric oxide, while all clearly share the natural environment with each other. To a large extent this seems not so much for lack of trying, than for the complicated and multifaceted chemistry of not only iron and sulfur but also of oxygen and nitric oxide. The number of species that may be formed from each single one of these reactants, and consequently the number possible with all combined, is discouragingly complex, even if one only considers end products and does not attempt to follow the time course of the interactions. To me, as one who largely came from the (bio)chemical side into this field, progress on this front seems an impor­ tant goal for the near future. It clearly would benefit progress on the biological front considerably.

References 1.

Agar JN, Krebs C, Frazzon J, Huynh BH, Dean DR, et al. 2000. IscU as a scaffold for iron-sulfur cluster biosynthesis: Sequential assembly of [2Fe-2S] and [4Fe-4S] clusters in IscU. Biochemistry 39: 7856-7862 2. Agar JN, Yuvaniyama P, Jack RF, Cash VL, Smith AD, et al. 2000. Modular organization and identification of a labile mononuclear ironbinding site within the NifU protein. /. Inorg. Biol. Chem. 5:167-177 3. Arendsen AF, Hadden J, Card G, McAlpine AS, Bailey S, et al. 1997. The "prismane" protein resolved: X-ray structure at 1.7 A and multi­ ple spectroscopy of two novel 4Fe clusters. /. Biol. Inorg. Chem. 3: 81-95 4. Armstrong FA. 1997. Evaluations of reduction potential data in rela­ tion to coupling, kinetics and function. /. Biol. Inorg. Chem. 2:139-142 5. Armstrong FA, Williams RJP. 1999. Thermodynamic influences on the fidelity of iron-sulphur cluster formation in proteins. FEBS Lett. 451: 91-94 6. Band L, Bertini I, Ciurli S, Ferretti S, Luchinat C, et al. 1993. The elec­ tronic structure of [Fe4S4]3+ clusters in proteins. An investigation of the oxidized high-potential iron-sulfur protein II from Ectothiorhodospira vacuolata. Biochemistry 32: 9387-9397 7. Bates DM. 1999. Role of iron-sulfur cluster conversion in the oxygensensing mechanism of the Escherichia coli transcription factor FNR. Dissertation, University of Wisconsin, Madison 8. Bates DM, Popescu CV, Khoroshilova N, Vogt K, Beinert H, et al. 2000. Substitution of leucine 28 with histidine in the Escherichia coli

68

9.

10. 11. 12. 13. 14. 15. 16. 17.

18. 19.

20.

21. 22.

Cellular Implications ofRedox Signalling

transcription factor FNR results in increased stability of the [4Fe-4S]2+ cluster to oxygen. /. Biol. Chem. 275: 6234-6240. Battistuzzi G, D'Onofrio M, Borsari M, Sola M, Macedo AL, et al. 2000. Redox thermodynamics of low-potential iron-sulfur proteins. /. Biol. Inorg. Chem. 5: 748-760. Beinert H. 1973. Development of the field and nomenclature. In IronSulfur Proteins, ed. W Lovenberg, Academic Press New York, I: 1-36 Beinert H. 2000. Iron sulfur clusters: Ancient structures, still full of surprises. /. Biol. Inorg. Chem. 5: 2-15 Beinert H, Holm RH, Miinck E. 1997. Iron-sulfur clusters: Nature's modular, multipurpose structures. Science 277: 653-659 Beinert H, Kennedy MC. 1989. Engineering of protein bound ironsulfur clusters. 1989. Eur. /. Biochem. 186: 5-15 Bertini I. 1997. Determinants of reduction potentials in metalloproteins. /. Biol. Inorg. Chem. 2:108 Bertini I, Gori-Savellini G, Luchinat C. 1997. Are unit charges always negligible? /. Biol. Inorg. Chem. 2:114-118 Bertini I, Luchinat C, Rosato A. 1999. NMR spectra of iron-sulfur proteins. Adv. Inorg. Chem. ed. Sykes AG, Cammack R, 47: 251-282 Boll M, Albracht SPJ, Fuchs G. 1997. Benzoyl-CoA reductase (dearomatizing), a key enzyme of anaerobic aromatic metabolism. A study of adenosine triphosphatase activity, ATP stoichiometry of the reaction and EPR properties of the enzyme. Eur. }. Biochem. 244: 840-851 Bouton C, Raveau M, Drapier J-C. 1996. Modulation of iron regula­ tory protein function. /. Biol. Chem. 271: 2300-2306 Brazzolotto X, Gaillard J, Pantopoulos K, Hentze MW, Moulis J-M. 1999. Human cytoplasmic aconitase (iron regulatory protein-1) is converted into its [3Fe-4S] form by hydrogen peroxide in vitro but is not activated for iron-responsive element binding. /. Biol. Chem. 274: 21625-21630 Broderick JB, Henshaw TF, Cheek J, Wojtuszewski K, Smith SR, et al. 2000. Pyruvate formate-lyase-activating enzyme: Strictly anaerobic isolation yields active enzyme containing a [3Fe-4S]+ cluster. Biochem. Biophys. Res. Commun. 269: 451^56 Buckel W, Golding BT. 1998. Radical species in the catalytic path­ ways of enzymes from anaerobes. FEMS Microbiol. Rev. 22: 523-541 Butt JN, Fawcett SEJ, Breton J, Thomson AJ, Armstrong FA. 1997. Electrochemical potential and p H dependences of [3Fe-4S] <-> [M3Fe-4S] cluster transformations (M=Fe, Zn, Co, and Cd) in

Iron-Sulfur Proteins: Properties and Functions

23.

24.

25.

26. 27.

28.

29.

30. 31.

32.

33.

34.

69

Ferredoxin III from Desulfovibrio africanus and detection of a cluster with M = Pb. /. Am. Chem. Soc. 119: 9729-9737 Capozzi F, Ciurli S, Luchinat C. 1998. Determinants of electronic and functional properties of iron-sulfur proteins. In Structure and Bonding, Springer Verlag, Berlin, Heidelberg, 90: 127-160 Colbert CL, Couture MMJ, Eltis LD, Bolin JT. 2000. A cluster exposed: Structure of the Rieske ferredoxin from biphenyl dioxygenase and the redox properties of Rieske Fe-S proteins. Structure 8:1267-1278 Cosper NJ, Booker SJ, Ruzicka F, Frey PA, Scott RA. 2000. Direct FeS cluster involvement in generation of a radical in lysine 2,3aminomutase. Biochemistry 39:15668-15673 Craig EA, Voisine C, Schilke B. 1999. Mitochondrial iron metabolism in the yeast Saccharomyces cerevisiae. Biol. Chem. 380:1167-1173 Emptage MH, Dreyer J-L, Kennedy MC, Beinert H. 1983. Optical and EPR characterization of different species of active and inactive aconitase. /. Biol. Chem. 258:11106-11111 Fawcett SEJ, Davis D, Breton JL, Thomson AJ, Armstrong FA. 1998. Voltammetric studies of the reactions of iron-sulphur clusters [3Fe-4S] or [M3Fe-4S]) formed in Pyrococcus furiosus ferredoxin. Biochem. f. 335: 357-368 Fontecilla-Camps JC, Ragsdale SW. 1999. Nickel-iron-sulfur active sites: hydrogenase and CO dehydrogenase. Adv. Inorg. Chem., eds. Sykes AG, Cammack R, 47: 283-333 Gardner PR, Fridovich I. 1991. Superoxide sensitivity of the Escherichia coli aconitase. /. Biol. Chem. 266:19328-19333 Golinelli M-P, Chatelet C, Duin EC, Johnson MK, Meyer J. 1998. Extensive ligand rearrangements around the [2Fe-2S] cluster of Clostridium pasteurianum ferredoxin. Biochemistry 37: 10429-10437 Grandoni JA, Switzer RL, Makaroff CA, Zalkin H. 1989. Evidence that the iron-sulfur cluster of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase determines stability of the enzyme to degradation in vivo. ]. Biol. Chem. 264: 6058-6064 Hagen KS, Reynolds JG, Holm RH. 1981. Definition of reaction sequences resulting in self-assembly of [Fe4S4(SR)4]2" clusters from simple reactants. /. Am. Chem. Soc. 103: 4054-4063 Haile DJ, Rouault TA, Harford JB, Kennedy MC, Blondin GA, et al. 1992. Cellular regulation of the iron-responsive element binding protein: Disassembly of the cubane iron-sulfur cluster results in highaffinity RNA binding. Proc. Natl. Acad. Sci. USA 89:11735-11739

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35. Hentze MW, Kiihn LC. 1996. Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress. Proc. Natl. Acad. Sci. USA 93: 8175-8182 36. Hidalgo E, Ding H, Demple B. 1997. Redox signal transduction via iron-sulfur clusters in the SoxR transcription activator. Trends Biochem. Sci. 22: 207-210 37. Holm RH. 1992. Trinuclear cuboidal and heterometallic cubane-type iron-sulfur clusters: New structural and reactivity themes in chem­ istry and biology. Adv. Inorg. Chem., eds. Cammack R, Sykes AG, 38: 1-17 38. Howard JB, Rees DC. 1996. Structural basis of biological nitrogen fixation. Chem. Rev. 96: 2965-2982 39. Jensen LT, Culotta VC. 2000. Role of Saccharomyces cerevisiae ISA1 and ISA2 in iron homeostasis. Mol. Cell. Biol. 20: 3918-3927 40. Johnson MK. 1994. Iron-sulfur proteins. In Encyclopedia of Inorganic Chemistry, ed. King RB, John Wiley & Sons, England, 4: 1896-1915 41. Johnson MK, Duderstadt RE, Duin EC. 1999. Biological and synthetic [Fe3SJ clusters. Adv. Inorg. Chem., eds. Sykes AG, Cammack R, 47: 1-82 42. Kennedy MC, Antholine WE, Beinert H. 1997. An EPR investigation of the products of the reaction of cytosolic and mitochondrial aconitases with nitric oxide. /. Biol. Chem. 272: 20340-20347 43. Kennedy MC, Beinert H. 1988. The state of cluster SH and S2" of aconitase during cluster interconversions and removal. /. Biol. Chem. 263: 8194-8198 44. Kennedy MC, Emptage MH, Dreyer J-L, Beinert H. 1983. The role of iron in the activation-inactivation of aconitase. /. Biol. Chem. 258: 11098-11105 45. Kennedy MC, Kent TA, Emptage M, Merkle H, Beinert H, et al. 1984. Evidence for the formation of a linear [3Fe-4S] cluster in partially unfolded aconitase. /. Biol. Chem. 259: 14463-14471 46. Kent TA, Emptage MH, Merkle H, Kennedy, MC, Beinert H, et al. 1985. Mossbauer studies of aconitase. /. Biol. Chem 260: 6871-6881 47. Kiley PJ, Beinert H. 1999. Oxygen sensing by the global regulator, FNR: The role of the iron-sulfur cluster. FEMS Microbiol. Rev. 22: 341-352 48. Kispal G, Csere P, Prohl C, Lill R. 1999. The mitochondrial proteins A t m l p and Nfslp are essential for biogenesis of cytosolic Fe/S proteins. EMBO. J. 18: 3981-3989

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49. Lazazzera BA, Bates DM, Kiley PJ. 1993. The activity of the Escherichia coli transcription factor FNR is regulated by a change in oligomeric state. Genes Dev. 7: 1993-2005 50. Lee M, Arosio P, Cozzi A, Chasteen ND. 1994. Identification of the EPR-active iron-nitrosyl complexes in mammalian ferritins. Biochemistry 33: 3679-3687 51. Leibrecht I, Kessler D. 1997. A novel L-cysteine/cystine C-S-lyase directing [2Fe-2S] cluster formation of Synechocystis ferredoxin. /. Biol. Chem. 272:10442-10447 52. Lill R, Kispal G. 2000. Maturation of cellular Fe-S proteins: An essen­ tial function of mitochondria. Trends Biochem. Sci. 25: 352-356 53. Link TA. 1999. The structures of Rieske and Rieske-type proteins. Adv. Inorg. Chem., eds. Sykes AG, Cammack R, 47: 83-157 54. Liu A, Graslund A. 2000. Electron paramagnetic resonance evidence for a novel interconversion of [3Fe-4S]+ and [4Fe-4S]+ clusters with endogenous iron and sulfide in anaerobic ribonucleotide reductase activase in vitro. J. Biol. Chem. 275:12367-12373 55. Martius C, Lynen F. 1950. Probleme des Citronen-saurecyklus. Adv. Enzymol. 20: 167-222 56. Maskiewicz R, Bruice TC. 1977. Kinetic study of the dissolution of Fe4S^" cluster core ions of ferredoxins and high potential iron protein. Biochemistry 16: 3024-3029 57. Matsubara H, Saeki K. 1992. Structural and functional diversity of ferredoxins and related proteins. Adv. Inorg. Chem., eds. Cammack R, Sykes AG, 38: 223-280 58. Moore LJ, Kiley PJ. 2001. Characterization of the dimerization domain in the FNR transcription factor. / Biol. Chem., 276:45744-45750 59. Miihlenhoff U, Lill R. 2000. Biogenesis of iron-sulfur proteins in eukaryotes: A novel task of mitochondria that is inherited from bac­ teria. Biochim. Biophys. Ada 1459: 370-382 60. Nishio K, Nakai M. 2000. Transfer of iron-sulfur cluster from NifU to apoferredoxin. /. Biol. Chem. 275: 22615-22618 61. Noodleman L, Case DA. 1992. Density-functional theory of spin polarization and spin coupling in iron-sulfur clusters. Adv. Inorg. Chem., eds. Cammack R, Sykes AG, 38: 423^170 62. Oliveira L, Bouton C, Drapier J-C. 1999. Thioredoxin activation of iron regulatory proteins. /. Biol. Chem. 274: 516-521 63. Ollagnier-de Choudens S, Sanakis Y, Hewitson KS, Roach P, Baldwin JE, et al. 2000. Iron-sulfur center of biotin synthase and lipoate synthase. Biochemistry 39: 4165-4173.

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64. Pantopoulos K, Mueller S, Atzberger, A, Ansorge W, Stremmel W, et al. 1997. Differences in the regulation of iron regulatory protein-1 (IRP-1) by extra- and intra-cellular oxidative stress. /. Biol. Chem. 272: 9802-9808 65. Petering D, Fee JA, Palmer G. 1971. The oxygen sensitivity of spinach ferredoxin and other iron-sulfur proteins. /. Biol. Chem. 246: 643-653 66. Pierik AJ, Roseboom W, Happe RP, Bagley KA, Albracht SPJ. 1999. Carbon monoxide and cyanide as intrinsic ligands to iron in the active site of [NiFe]-hydrogenases. /. Biol. Chem. 274: 3331-3337 67. Popescu CV, Bates DM, Beinert H, Miinck E, Kiley PJ. 1998. Mossbauer spectroscopy as a tool for the study of activation/inactivation of the transcription regulator FNR in whole cells of Escherichia coli. Proc. Natl. Acad. Sci. USA 95:13431-13435 68. Schwartz CJ, Djaman O, Imlay JA, Kiley PJ. 2000. The cysteine desulfurase, IscS, has a major role in in vivo Fe-S cluster formation in Escherichia coli. Proc. Natl. Acad. Sci. USA 97: 9009-9014 69. Smith BE. 1999. Structure, function and biosynthesis of the metallosulfur clusters in nitrogenases. Adv. Inorg. Chem., eds. Sykes AG, Cammack R, 47:159-218 70. Van Spanning RJ, De Boer APN, Reijnders WNM, Westerhoff HV, Stouthamer AH, et al. 1997. FnrP and NNR of Paracoccus denitrificans are both members of the FNR family of transcriptional activators but have distinct roles in respiratory adaptation in response to oxygen limitation. Mol. Microbiol. 23: 893-907 71. Vanin AF, Stukan RA, Manukhina EB. 1996. Physical properties of dinitrosyl iron complexes with thiol-containing ligands in relation with their vasodilator activity. Biochim. Biophy. Ada 1295: 5-12 72. Warshel A, Papazyan A, Muegge I. 1997. Microscopic and semimacroscopic redox calculations: What can and cannot be learned from continuum models. /. Biol. Inorg. Chem. 2: 143-152 73. Weber IT, Steitz TA. 1987. Structure of a complex of catabolite gene activator protein and cyclic AMP refined at 2.5 A resolution. /. Mol. Biol. 198: 311-326 74. Yuvaniyama P, Agar JN, Cash VL, Johnson MK, Dean DR. 2000. NifSdirected assembly of a transient [2Fe-2S] cluster within the NifU protein. Proc. Natl. Acad. Sci. USA 97: 599-604 75. Zheng L, Dean DR. 1994. Catalytic formation of a nitrogenase ironsulfur cluster. /. Biol. Chem. 269:18723-18726

Chapter 4 The Ferredoxin Ferredoxin/Thioredoxin Thioredoxin System. A light-Dependent Redox Regulatory System in Oxygenic Photosynthetic Cells Peter Schurmann Laboratoire de Biochemie Vegetale, Universite de Neuchdtel, CH-2007 Neuchatel Switzerland [email protected]

Keywords: ferredoxin/thioredoxin system, ferredoxin, ferredoxinithioredoxin reductase (FTR), thioredoxin m, thioredoxin/, site-directed mutagenesis, signal transfer, target enzymes, fructose 1,6-bisphosphatase, NADPmalate dehydrogenase, redox potential, regulatory disulfides

1. Summary Redox signaling and the regulation via disulfide interchange reactions was first described for the activation of chloroplast enzymes by light. In recent years this type of regulation has gained a lot of interest since it appears to be involved not only in regulation of photosynthetic enzymes, but also in light harvesting, germination, transcription, translation, apoptosis and detoxification. The redox regulatory system of oxygenic photo­ synthetic organisms, known as the ferredoxin/thioredoxin system, links the activity of key enzymes to light, thereby regulating the carbon flow. Catalysts involved in carbon assimilation are activated by reduction in the light and deactivated by oxidation in the dark. In contrast an enzyme tunneling carbon intermediates into degradation is turned off by reduc­ tion in the light, but activated by oxidation in the dark. This lightdependent redox regulation avoids the concomitant operation of carbon assimilatory and degradative pathways and might also regulate the carbon flux depending on the light intensity. It operates as regulatory 73

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cascade involving several proteins: ferredoxin, ferredoxinrthioredoxin reductase (FTR) and thioredoxins modifying the activity of target enzymes by reduction of their regulatory disulfides. The electrons needed for the reductions are provided by the photosynthetic light reactions and transmitted by ferredoxin to FTR. This unique enzyme transforms the light signal, received in the form of electrons, into a thiol signal which is then transmitted through disulfide-dithiol interchanges involving thioredoxins to the target proteins. Several recent reviews have discussed various aspects of the ferredoxin/ thioredoxin system.8,15'26,39,56'65,69"71 This chapter will describe recent struc­ tural information obtained on the participating components of the system and advances in the understanding of its mechanism.

2. The Components of the Ferredoxin/Thioredoxin System All components of this regulatory system are rather small, soluble proteins containing either a Fe-S cluster or a redox-active disulfide bridge or both. They have been found in the chloroplasts of higher plants and algae as well as in cyanobacteria. The components have been purified and charac­ terized from different sources and their genes have been cloned and the proteins overexpressed. The availability of recombinant proteins finally enabled their structural analysis by X-ray crystallography. 2.1 Ferredoxin The first member of the system accepting the electron signal from the thylakoids is ferredoxin. The plant-type ferredoxins, involved in oxygenic photosynthesis, are small, acidic 2Fe-2S proteins of about 12 KDa. The Fe-S cluster is attached to the protein by 4 cysteine ligands, has a redox potential of around -400 mV and can carry one electron. The ferredoxins are well studied proteins and for many the primary structures are known showing that the positions of the four cluster liganding cysteines are present in a highly conserved cluster-binding motif. Several three- dimensional structures have also been obtained either by NMR or crystal- lography and they exhibit large similarities sharing the same fold.15 The structures reveal two patches of negative surface charges on either side of the Fe-S cluster, which have been shown to be essential for the interaction with other pro­ teins. Differential chemical modification of free and target bound ferredoxin indicates that interaction with positively charged FTR involves essentially only one such negative domain and that Glu92 in spinach ferredoxin is one of the important residues.19 This conclusion is supported by mutagenesis

The Ferredoxin/Thioredoxin System 75

Fig. 1. Sequence comparison of spinach and Synechocystis ferredoxin:thioredoxin reductase. The sequences were aligned using CLUSTALX77 and formatted with BOXSHADE (http://www.Ch.Embnet.org/software/BOX_form.html). The cysteine containing motifs are in bold type.

experiments in which the replacement of this C-terminal glutamate residue resulted in a protein incapable of reducing FTR.42 2.2 Ferredoxin:Thioredoxin Reductase FTR is the key enzyme of this regulatory system. It transmits the redox signal from ferredoxin to thioredoxins. FTR is a unique photosynthetic enzyme, different from the well-known NADP-dependent thioredoxin reductase, which is a flavoprotein, present also in the cytoplasm of plants. Purified FTR is a yellowish-brown protein with an apparent molecular mass of 20 to 25 KDa. It is composed of two dissimilar subunits, a catalytic subunit and a variable subunit. The catalytic subunit contains a 4Fe-4S cluster and a redox-active disulfide bridge, both essential for catalysis, whereas the variable subunit appears to have only structural function. The FTR has been isolated and characterized from different sources and a number of gene and protein sequences are known.15,70 In higher plants and green algae, the FTR is nucleus encoded and both subunits carry transit peptides which guide them after synthesis in the cytoplasm into the chloroplast. Interestingly, in the red alga Porphyra purpurea63 and the cryptomonad Guillardia theta21 the catalytic subunit is coded by the chloroplast genome suggesting that at least this subunit is of bacterial endosymbiotic origin. The catalytic subunits of FTR from different organisms have a constant size of about 13 kDa and a highly conserved primary structure. Among the strictly conserved residues are seven Cys, six of them organized in two CPC and one CHC motifs (Fig. 1). These six Cys are the functionally essential

76

Cellular Implications ofRedox Signalling

residues constituting the redox active disulfide bridge and ligating the Fe-S cluster. Cluster ligation does not follow the usual consensus motifs for 4Fe-4S centers,36 but shows a new arrangement with the following finger­ print: CPCX16CPCX8CHC (cluster ligands are in bold). In spinach FTR Cys54 and Cys84 form the active site disulfide. Cys54 is accessible to the solvent whereas Cys84 is protected. The four remaining cysteines, Cys52, Cys71, Cys73 and Cys82 are ligands to the iron center. This arrangement positions the redox-active disulfide bridge adjacent to the cluster.13 Two archaebacteria, Archaeoglobus fulgidusi6 and Methanobacterium thermoautotrophicum™ contain a gene coding for a protein with some strik­ ing resemblances to the catalytic subunit of FTR. The overall identities between the archaebacterial proteins and the photosynthetic FTRs are rather low (25-35%), but the CXC motifs, essential for the function of the FTR, are conserved at about identical positions. No functions are reported for those proteins in the archaea. However, the striking structural simi­ larities suggest that the catalytic subunit of photosynthetic FTR might be derived from such an ancient precursor protein whose function has been adapted during evolution. The variable subunits range in size from 8 to 13 kDa and show pro­ nounced sequence variability with only 46 to 60% identity within the eukaryotes and 33 to 40% between eukaryotes and prokaryotes. The size variability stems from a variable extension of the N-terminus present in all three known eukaryotic enzymes, but absent from the prokaryotic counterparts (Fig. 1). In spinach FTR, this N-terminal extension was found to be unstable, being degraded to discrete shorter peptides 78 which exhibit no functional differences. The FTRs from spinach and Synechocystis sp PCC6803 have been cloned and expressed in E. coif7,73 using a dicistronic construct containing the genes for both subunits in series in the same expression vector. Both recombinant proteins were perfectly active and produced in amounts large enough to initialize structural studies. Recombinant Synechocystis FTR crystallized as dark brown crystals,16 which diffracted very well and permitted structural resolution to 1.6 A (pdb ldj7). 1517 The FTR is a rather flat, disk-like molecule. The variable subunit is heart-shaped with a /J-barrel constituting the main body and with two loops forming the upper, outer part of the heart. The catalytic subunit, which sits on top of the variable subunit is an overall a-helical structure containing five helices. The Fe-S center and the active site disul­ fide bridge are both located in the catalytic subunit, in the center of the heterodimer, where the molecule is only 10 A across. The cubane 4Fe-4S cluster is situated on one side of the flat molecule close to the surface,

The Ferredoxin/Thioredoxin System 77

Fig. 2. Modeling of the interaction between ferredoxin (red, to the left), ferredoxin: thioredoxin reductase (variable subunit in green and catalytic subunit in blue, in the middle) and thioredoxin (yellow, to the right). The thin, disk-like structure of the FTR allows simultaneous docking of ferredoxin and thioredoxin on opposite sides of the molecule. The Fe-S centers and the disulfide bridges are shown in ball and stick representation. (Reproduced with permission from Ref. 15. Copyright Cambridge University Press).

which contains three positive charges. The redox active disulfide bridge is on the opposite surface, which has a more hydrophobic character. This arrangement with a positively charged docking site for the negatively charged ferredoxin on one side and a rather hydrophobic docking site for thioredoxins on the opposite side of the flat molecule is perfectly adapted for the transfer of electrons from ferredoxin to thioredoxin across its center (Fig. 2). These properties make the FTR a versatile thioredoxin reductase, capable of accepting electrons from diverse ferredoxins and reducing the disulfides of various thioredoxins.

2.3 Thioredoxins Plant cells contain at least four different types of thioredoxins/ 1 which display a certain specificity in their interaction with other proteins. Two types

78

Cellular Implications ofRedox Signalling

of thioredoxins, the/-type and the m-type, are located in the chloroplasts, one type, thioredoxin h, is present in the cytoplasm and mitochondria appear to house still another type. The thioredoxins involved in the ferredoxin/thioredoxin system transmitting the light-generated redox signal to target enzymes are the /- and m-type thioredoxins from the chloroplasts. These two types of thioredoxins can be clearly distinguished by their primary sequence and phylogenetic background. Both chloroplast thioredoxins are nucleus encoded with the exception of the m-type in red algae, where its gene was found on the chloroplast genome. 63

2.3.1 Thioredoxin m Thioredoxin m was originally described as activator protein for the NADPdependent malate dehydrogenase (NADP-MDH) of chloroplasts in C3 and C4 plants. This type of thioredoxin is found in chloroplasts of dicots, monocots and algae as well as in cyanobacteria and resembles strongly the thioredoxin from anoxygenic prokaryotes, both hetero-trophic and photosynthetic. Thioredoxins of the m-type are, therefore, also known as bacterialtype thioredoxins. Due to their structural relatedness the bacterial and m-type thioredoxins are functionally similar and can be used interchange­ ably. A comparison of thioredoxin m sequences from prokaryotes, eukaryotic algae and higher plants clearly demonstrates that they are related, however they display less sequence similarity than their /-type counter­ parts known to date. The greater diversity of the m-type thioredoxins even within higher plants can be seen in Fig. 3, where thioredoxins m and / sequences from the same five organisms have been aligned. The thiore­ doxin m sequences contain half as many conserved residues (23%) compared to the corresponding thioredoxin/sequences (46%).

2.3.2 Thioredoxin f Thioredoxin / has been discovered as the specific activator protein of chloroplast fructose 1,6-bisphosphatase (FBPase). In contrast to the m-type this thioredoxin is restricted to eukaryotic organisms. There are fewer sequences known for thioredoxin / but display a significantly higher homology than the m-type thioredoxins. The / thioredoxins are slightly longer than other types due to additional amino acids at their N-termini, and the C-terminal part of the sequences resemble classical animal thioredoxin in containing a third, strictly conserved Cys (Fig. 3).

The Ferredoxin/Thioredoxin System

79

Fig. 3. Sequence comparison between chloroplast thioredoxins m and/from the five same organisms with thioredoxin from E coli. The sequences were aligned from the redox active cysteines, which are given in bold type as is also the third cysteine in thioredoxin/. 2.3.3 Three-Dimensional

Structures of Thioredoxins

While extensive structural data has been available for thioredoxins from nonphotosynthetic organisms, such information has only recently been provided for plant thioredoxins. Crystal structures have been determined for a somewhat unusual thioredoxin from Anabaena (pdb ltxh) 66 and for the chloroplast thioredoxins / and m from spinach 9 and solution struc­ tures for thioredoxin m (pdb ldby) 5 1 and the cytosolic thioredoxin h (pdb ltof)59 from the green alga Chlamydomonas reinhardtii. The structure of spinach thioredoxin m has been solved for the oxidized and reduced protein at 2.1 and 2.3 A resolution, respectively (pdb lfb6, lfbO).9 The structure is very similar to that of E. coli thioredoxin,45 which corroborates the biochemical evidence showing the proteins are functionally interchangeable. The secondary structures of the m-type and E. coli thioredoxins are nearly identical and the surfaces around the active site Cys are largely similar. There is also no large conformational change between oxidized and reduced protein, thus confirming and extending

80

Cellular Implications ofRedox Signalling

observations reported for E. coli and human thioredoxins.43,80 However, some slight structural differences in the main chain conformation of the active site render the solvent-exposed Cys37 more accessible in the reduced protein. The structure of spinach thioredoxin/ 9 has been solved for two forms of recombinant protein, a "long form" (pdb lf9m) resembling closely the in vivo form1 and a N-terminus truncated "short form" (pdb lfaa). 20 Both structures are essentially identical aside from the N-terminus, which con­ tains an additional a-helix in the long form, and a difference in the con­ formation of their active site regions. Whereas the overall structure of thioredoxin/ does not differ markedly from a typical thioredoxin, its sur­ face topography is distinct from that of others. Thioredoxin/is more posi­ tively charged and some of these charges surround the active site where they must be instrumental in orienting thioredoxin/correctly with target proteins. The hydrophobic residues, also prominent in the contact area, may be more important in the less specific interaction with FTR, which reduces various thioredoxins efficiently. A striking difference is the pres­ ence of the third Cys exposed on the surface (Cys73 in spinach), 9.7 A away from the accessible Cys46 of the active site. As already mentioned, this third Cys is conserved in all/-type thioredoxins. The structural analy­ sis also shows that the active site Cys with the lower sequence number (Cys46 in spinach) is exposed whereas its partner is buried, confirming biochemical experiments which showed that Cys46 is the attacking nucleophile in the reduction of target disulfides. 6 Another, maybe important feature is the apparent flexibility of the active site region of thioredoxin/as evidenced by different conformations observed in its long and short forms. Trp45, which is part of the active site sequence (WCGPC), can flip its indole ring away from the active site. This is possible due to the absence of a hydrogen bond between the indole ring and the carboxyl group of a neighboring aspartate observed in thiore­ doxin m and E. coli thioredoxin. Trp45 of thioredoxin/cannot make such a hydrogen bond, because the residue corresponding to aspartate is Asn74, whose side chain points in the opposite direction and receives a hydrogen bond from the main chain nitrogen of Asn77. Asn74 in thiore­ doxin / is followed by the insertion of a Gln75 with respect to the other types of thioredoxins and this insertion appears to be a distinctive feature of /-type thioredoxins. It modifies the loop conformation (residues 74 to 77) keeping the Asn74 side chain away from Trp45. This deviating local conformation may represent an important structural factor contributing to the specificity of thioredoxin/ 9

The Ferredoxin/Thioredoxin System

2.3.4 Specificity of

81

Thioredoxins.

One of the puzzling facts is that chloroplasts contain two types of thioredoxins with practically identical redox potential 34 and catalyzing identical redox reactions. However, they display a certain selectivity in their interaction with target enzymes when tested under conditions approaching their in vivo situation. The Calvin cycle enzymes FBPase,29 sedoheptulose 1,7-bisphosphatase (SBPase),83 phosphoribulokinase (PRK)82 as well as Rubisco activase 84 and ATP synthase (CFj)72 are exclu­ sively or very efficiently activated by thioredoxin /. NADP-MDH, origi­ nally thought to be specifically light-regulated through thioredoxin m, was shown to be even more efficiently activated by thioredoxin f.29,35 Glucose 6-phosphate dehydrogenase (G6PDH), on the contrary, is modu­ lated specifically by thioredoxin m81 and appears to be so far the only enzyme responding exclusively to thioredoxin m. Recent reexamination of the interaction between the two chloroplast thioredoxins and PRK suggests that thioredoxin m might be somewhat more efficient in activating PRK than thioredoxin /. 28 In general these observations indi­ cate that, at least as far as carbohydrate metabolism is concerned, thio­ redoxin / functions primarily in enzyme activation (i.e. enhancing the rate of biosynthesis) whereas thioredoxin m acts mainly in enzyme deactivation (i.e. enhancing the rate of degradation). Thioredoxin / has also been reported to be an efficient activator of acetyl CoA carboxylase catalyzing the first committed step in fatty acid biosynthesis in chloroplasts68 as well as for ADP-glucose pyrophosphorylase catalyzing the first committed step in starch biosynthesis. 4 Thioredoxin m, however, has been suggested to be involved in processes like translation, 18,52 removal of reactive oxygen species 3 and N-metabolism activating ferredoxin: glutamate synthase. 53 The observed specificity of thioredoxins in their interaction with target proteins raises the question of which structural features could be respon­ sible for it. Answers have been sought by applying site-directed mutagenesis to thioredoxins. A sequence comparison based on the threedimensional structure of E. coli thioredoxin 24 revealed several residues, which in thioredoxin / are different from the consensus or from thiore­ doxin m. Such residues, especially if a change of charge is involved, could be, at least in part, responsible for the specificity. In spinach thioredoxin / s o m e of these residues have been replaced to make the protein more sim­ ilar to thioredoxin m and in E. coli thioredoxin50,60 and pea thioredoxin m54 residues typical for thioredoxin / have been inserted (see Fig. 4 and

82 Cellular Implications ofRedox Signalling

Fig. 4. Structure-based alignment9 of the sequences of the spinach chloroplast and E. coli thioredoxins. The positions of residues, which have been mutated, are indicated by T. Table 1. Summary of the various residues which have been mutated and their respective nonmutated counterparts in the three thioredoxins used to probe the interaction specificity. References are given in parentheses A = deletion mutant. Thioredoxin/ Q44 K58E (28, 29) C73S, C73A, C73G (20) N74A, N74D (28, 29) Q75D (28, 29) N77A (28, 29) E83 V89I/T105I (28) T105I (28, 29) K108

Thioredoxin E. coli

Thioredoxin m

E30K (60) E44 160 D61N (50) — N63 K69 I75/V91 V91 L94K (60)

P35 E49 T65 D66 — A68 K70E (pea (54) = Q74 spinach) 180/196 196 V100

Table 1). In general the analyses of the properties of the modified proteins point into the expected direction, i.e. conversion of thioredoxin / to a more m-like thioredoxin and, on the other hand, improvement of acti­ vation of FBPase by E. coli thioredoxin. Thioredoxin/contains more pos­ itively charged residues on its surface than E. coli thioredoxin or thioredoxin m. Replacement of positively charged or neutral amino acids by negatively charged residues reduces the affinity of thioredoxin/mutants for FBPase or PRK.28'29 Furthermore the replacement of the surface exposed

The Ferredoxin/Thioredoxin System

83

third Cys reduces the affinity for FBPase.20 In contrast introduction of positive charges in E. coli thioredoxin improves its capacity to activate FBPase, which however still requires thioredoxin concentrations well beyond any physiological level. It appears that electrostatic components play a crucial role in the interaction with the target proteins but are not the only important factors. Any mutation so far done on the thioredoxin/ has been counterproductive with respect to activation of FBPase, however beneficial with respect to activation of NADP-MDH where this has been tested.29 These results show that the interplay of several factors is respon­ sible for the specificity in the interaction of thioredoxins with their target proteins.

3. The Redox Signal Transfer through the Ferredoxin/Thioredoxin System The redox signal, originating in the thylakoid membranes in the form of electrons, has to be transmitted via ferredoxin, FTR and thioredoxins to the target proteins where it is received as a thiol signal. The transforma­ tion of the electron signal into a thiol signal is accomplished by FTR. That FTR is indeed capable of making this conversion has been clearly demon­ strated with isolated chloroplasts 14 and purified components. 23 Ferredoxin, the first soluble electron acceptor, carries the electrons, one at a time, from photosystem I to the FTR. Ferredoxin is a negatively charged protein which has been shown to form an electrostatically stabilized 1:1 complex with FTR.19,32 Although FTR can accept electrons from heterologous ferredoxins, as has been demonstrated by the use of spinach ferredoxin for the reduction of FTR from corn and Nostoc,22 Chlamydomonas,37 Synechocystis73 and soja (P. Schurmann, unpublished), the best electron donor appears to be the homologous ferredoxin, forming the most stable complex. This must be due to the arrangement of the neg­ ative charges on ferredoxin and the complementary positive charges on the FTR surface. The FTR contains a Fe-S center and a redox-active disulfide bridge. While all known disulfide reductions except the one by FTR are catalyzed by flavoproteins or by thiol/disulfide exchange reactions, the FTR uses its Fe-S center to cleave the active site disulfide. This appears to be possible not as a result of an unusual geometry of the Fe-S center, which is a normal cubane 4Fe-4S cluster, but due to the close proximity of active site disulfide and cluster. Of the two active site cysteines the one with the

84 Cellular Implications ofRedox Signalling [4Fe-4S]3*

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(cys)

t2_V ^ ^ V....J S(Cys)

2-

Fd

red

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S(Cys) Fe"' (His)

S

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( C y s ) s " ^* b (CVS)S

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•(Pro)

^(Cys)

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(Cys)s,

[4Fe-4S]3* S(Cys) (His) -Fe'

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[4Fe-4S]2* S(Cys) N N S Fe(His) Fe~pS I Fe-I

I rs(Cys) S V

(Cys)S \ 'S(Cys)'

_-
Fig. 5. Proposed reaction mechanism for reduction of thioredoxin by FTR. T stands for thioredoxin. (reprinted with permission from Ref. 76. Copyright American Chemical Society). lower sequence number (Cys57 in Synechocystis, Cys54 in spinach) is at the molecular surface acting as attacking nucleophile. The second cysteine (Cys87 in Synechocystis, Cys84 in spinach) is so close to the Fe-S center that it does not appear to exist as a free thiol until the final reduction step, but interacts during previous steps with the Fe-S center. The reduction of a disulfide is a two-electron process. However, ferre­ doxin is a one electron donor and only one ferredoxin molecule is able to dock at the FTR at one time. To acquire the two electrons necessary for the reduction of the active site disulfide bridge FTR has to mediate two con­ secutive one-electron transfers from ferredoxin and stabilize a one electron-reduced reaction intermediate (Fig. 5). The initial electron delivered by ferredoxin can be transferred via the Fe-S center directly to the disulfide that is in close contact with the cluster. 15 This cleaves the active-site disulfide of FTR producing a sol­ vent accessible sulfhydryl (Cys57 in Synechocystis) and a solventinaccessible cysteine-based thiyl radical (Cys87), which is stabilized by the covalent attachment of the cysteine to the cluster. It is proposed that

The Ferredoxin/Thioredoxin System

85

the Fe-S center donates an electron towards formation of a sulfur-sulfur bond between Cys87 and one of the inorganic sulfides of the cluster. 76 This results in an oxidation of the [4Fe-4S]2+ cluster, observed in the resting, EPR-silent enzyme, to a [4Fe-4S]3+ cluster.75'76 Based on the proximity between cluster atoms and active site Cys seen in the threedimensional model of the resting Synechocystis FTR it was suggested that in this intermediate stage the active-site cysteine coordinates an iron atom, which is closest, in a five coordinated cluster. 17 However, recent preliminary structural analyses of the reduced FTR (Dai et al., unpublished) favor a disulfide bridge to a sulfide ion in the cluster as originally proposed by Staples et al.76 This will have to be substantiated by further experiments. The surface exposed, nucleophilic cysteine of the one electron reduced intermediate is thought to attack the disulfide bridge of oxidized thioredoxin. In analogy with known mechanisms, the reduction of thioredoxin has been proposed to proceed via a mixed disulfide between thioredoxin and FTR.75,76 This intermediate mixed protein/protein disulfide covers up the thioredoxin docking site on one side of the flat FTR molecule. How­ ever, the ferredoxin docking site, on the opposite side of the FTR, stays free for an incoming ferredoxin to deliver the second electron needed for the complete reduction of thioredoxin. This electron will cleave the bond between the cluster and the active-site Cys87 thereby reducing the cluster back to its original 2+ oxidation state. Cys87, which becomes a nucle­ ophilic thiol can attack the intermolecular disulfide, releasing fully reduced thioredoxin and returning the active site of the FTR to the disul­ fide state. This proposed reaction mechanism, which is based on spectroscopic studies on native and active-site modified FTR is entirely compatible with the disk like structure of FTR, which allows simultaneous docking of thioredoxin and ferredoxin on opposite sides of the flat molecule (Fig. 2). An additional aspect of the mechanism of redox-mediated enzyme regulation concerns the oxidation-reduction properties of the different disulfides participating in the ferredoxin/thioredoxin system since they influence the activation state of the redox regulated enzymes. During the past several years these properties have been studied for all the compo­ nents of the regulatory chain and for several target enzymes by cyclic voltammetry, 67 the fluorescent probe mono-bromobimane and enzyme activity measurements.31'33,34,38,47'62 While the potentials measured by cyclic voltammetry were found to be overly positive, the values obtained by the other methods and by different groups are in reasonably good agreement.

86

Cellular Implications ofRedox Signalling

The redox potential of the disulfide of the FTR (Em= -320 mV, p H 7), which is significantly more negative than that of either thioredoxin m or /, insures that the two chloroplast thioredoxins are readily reduced in the light. On the other hand the potentials of the target enzymes vary from more positive to more negative than those of thioredoxins. Target enzymes involved directly or indirectly in the regeneration of the C 0 2 acceptor, PRK (-295 mV) and CFj (-280 mV), have the most positive potentials of the target enzymes. FBPase (-305 mV) and SBPase (-300 mV), which together control the entry of carbon into the regenerative phase of the Calvin cycle and into starch synthesis, have slightly more negative potentials than thioredoxin /, whereas NADP-MDH has a significantly more negative potential (-330 mV) keeping this enzyme inactive as long as there is no surplus of reducing equivalents. Since the members of the FTR system are located in the chloroplast stroma where the pH is increas­ ing from about 7.0 in the dark to 8.0 in the light and the redox potentials of the disulfide/dithiol couples are expected to be pH-dependent, 12 the p H dependency for several of the components has been determined.31,33'34 For all of them, except for PRK, the data obtained in the range of physio­ logical pHs could be fitted to a straight line with a slope of -59 m V / p H , a value expected for a process in which two protons are taken up per disulfide reduced. 12 The observed redox potential differences suggest that the chloroplast enzymes are light-activated sequentially with the most critical processes given priority and that the redox equilibria reached will result in differ­ ent degrees of activation. These equilibria may then be modified by enzyme effectors25 which would fine-tune the activities after the enzymes have been switched on by light.

4. Target Enzymes For a number of chloroplast enzymes a light-dependent increase in catalytic activity has been reported and in many cases this effect could be mimicked in vitro by sulfhydryl compounds like dithiothreitol. This was usually taken as evidence that a reduction by thioredoxin was involved. However, definite proof of a redox event involving thioredoxin requires the experimental evidence for an absolute requirement of thioredoxin and the demonstration of an accessible, redox-active disulfide bridge on the target enzyme structure. For most of the light-regulated chloroplast enzymes in carbon metabolism the presence of such a disulfide bridge has

The Ferredoxin/Thioredoxin System 87

Table 2. Regulatory site sequences of chloroplast target enzymes and their principal activating thioredoxin. The regulatory Cys are in bold and additional conserved residues are underlined. Target Enzyme Organism FBPase SBPase PRK ATP synthase NADP-MDH G6PDH Rubisco activase

Regulatory Site Sequence

Activator

DEC r JVX„ ORC r l VVNVC r „ Thioredoxin/ Thioredoxin/ ASC 52 GGIAC 57 VN SGC16GKX34VIC55LD Thioredoxin m/f EIC]99DINGKC205VD Thioredoxin/ Thioredoxin f/m KDC24FGVFC29TT KKC c:: VAHLTGEGNAYC™DV LTC119RIDKRENC15,DA Thioredoxin m Potato Thioredoxin/ Arabidopsis EGC392JDX14GTC41]VY Spinach Wheat Spinach Spinach Sorghum

been confirmed and its involvement in redox regulation demonstrated by site-directed mutagenesis (Table 2). The analysis of the primary structures of redox regulated target enzymes from various oxygenic photosynthetic organisms shows that they contain conserved Cys residues absent from counterparts not regu­ lated by thioredoxin. Some of these Cys have been shown to be linked in regulatory disulfides, which for certain enzymes, particularly those also occurring as cytosolic isoforms, are located on extra loops or extensions of the primary structure. The arrangement of the regulatory disulfide struc­ tures do not follow any consensus motif, as was proposed for some time, although in some of the enzymes a CXXXXC sequence is the responsible element or part of it (Table 2). The two redox-active cysteines are not necessarily close on the primary structure, but can be separated by many residues. The locations of the regulatory disulfides on extensions or inser­ tions of the primary structures of the different enzymes indicate that they have been added during adaptation to photosynthetic function and the absence of any consensus motif suggests that this adaptation to redox reg­ ulation arose multiple times during evolution and appears to be tailored to the mechanism of the particular enzyme. The reduction of the regulatory disulfides of the target enzymes by thioredoxin proceeds, like thioredoxin reduction, with the formation of a transient heterodisulfide complex between the two reaction partners. The reactive Cys, which is the solvent accessible one closest to the N-terminus

88

Cellular Implications ofRedox Signalling

of thioredoxin, cleaves the target disulfide by nucleophilic attack, thereby forming a covalently linked mixed disulfide. In a fast second step, the second sulfhydryl, which is inaccessible to solvent, attacks the mixed disulfide to produce oxidized thioredoxin and reduced target enzyme. For three target enzymes, PRK,7 NADP-MDH 30 and FBPase5 the formation of such a mixed disulfide has been demonstrated using mutants in which the inaccessible sulfhydryl of the regulatory site has been replaced dis­ abling the fast second step, thus conserving the stable covalent linkage between thioredoxin and target enzyme. In spinach PRK the two redox active sulfhydryls are Cysl6 and Cys55. Using mutant enzyme Cys55 has been identified as the residue pairing with Cys46 of thioredoxin/in an intermolecular disulfide. 7 With sorghum NADP-MDH a mixed disulfide was obtained between the internal Cys207 and thioredoxin using an enzyme devoid of its N-terminal disulfide bridge, which mimics a partial activation by loosening the interaction between the subunits. No heterodisulfide has so far been demonstrated for this enzyme between one of the terminal Cys and thioredoxin. 30 With spinach FBPase a heterodimer could be demonstrated between Cysl55 (corresponding to Cysl53 in pea) and Cys46 of thioredoxin/. 5 This reac­ tion proceeds very rapidly which would support the notion that the inter­ action between spinach FBPase and thioredoxin/is very specific. Further insights into possible structural changes occurring during reduction have recently been provided by the resolution of the threedimensional structures of two well-studied redox-regulated enzymes, the FBPase and the NADP-MDH. A model for the spinach chloroplast FBPase (pdb lspi) 79 has been known for some time, however, it showed no evidence for a regulatory disulfide, since the loop structure containing the putative disulfide was disordered. In view of the recent solution of the oxidized pea enzyme structure (pdb l d 9 q ) n in which the disulfide is clearly resolved on the loop structure, it appears probable that the earlier spinach model represents a reduced enzyme. For NADP-MDH the struc­ tures of the oxidized enzyme from sorghum (pdb 7mdh) 44 and Flaveria (pdb lciv) 10 have been reported, presenting entirely comparable features.

4.1 Fructose 1,6-bisphosphatase Chloroplast FBPase is a classical redox regulated enzyme and one of the first targets where an involvement of thioredoxin has been demonstrated. Plant cells contain two types of FBPases, one in the cytosol involved in

The Ferredoxin/Thioredoxin System

89

gluconeogenesis, and another one in the chloroplasts as member of the Calvin cycle. Both are homotetrameric enzymes of about 160 KDa. Their primary structures display extended homologies. However, the chloro­ plast isoform has an insert in the middle of the primary structure not pre­ sent in the cytosolic counterpart. 61 This insert contains three conserved Cys—C153X19C173IVNVC178 in pea—two separated by four hydrophobic residues and the third, Cysl53, by nineteen residues upstream toward the N-terminus. Although Cysl73 and Cysl78 are arranged in a CXXXXC motif, which was suggested as the redox active disulfide structure, 55 site-directed mutagenesis experiments40,41,64 (Y. Balmer et al. unpublished) and the three-dimensional structure 11 now indicate, that the redox-active disulfide is constituted by Cysl53 and Cysl73. It is located on a loop, which is extending out of the core structure making it accessible for thioredoxin and comprises the residues of the sequence insert. This loop is connected through two strands of a N-terminal-sheet with the active site, which is some 20 A away. A superposition of the active sites of the chloroplastic and gluconeogenic FBPases shows that most of the impor­ tant catalytic residues occupy similar positions except for Glul05, which is a critical ligand for Mg2+ ions essential in catalysis. In the oxidized enzyme this Glul05, which is on a loop connecting the two strands, is dis­ placed by Vall09 thereby preventing the coordination of Mg2+. Through reduction of the disulfide bridge the regulatory loop is destabilized allow­ ing the two strands to move some 8 A away from the active site. This shifts Glul05 in a catalytically favorable position thus restoring a functional active site.

4.2 NADP-Dependent Malate Dehydrogenase NADP-MDH is the best studied redox regulated chloroplast enzyme and appears to have the most complex regulatory mechanism. Several excellent recent reviews have summarized and discussed the results that have led to the present understanding of the regulation of this enzyme.2,57,58,65 Its acti­ vation requires the reduction of not only one, but of two regulatory disulfide bridges and a fifth Cys may participate by forming an intermediary disulfide with one of the N-terminal Cys. Compared to the perma­ nently active NAD-dependent MDH the chloroplast enzyme has a N- and a C-terminal extension each containing one regulatory disulfide bridge. The fifth Cys probably implicated, is at the interface of the two subunits of the dimeric enzyme. The functions of the five Cys had been rather well

90

Cellular Implications ofRedox Signalling

explored by using chemical modifications and site-directed mutagenesis. The determination of three-dimensional structure has confirmed the earlier biochemical results and placed the proposed activation mechanism on a structural basis. The two regulatory disulfides were found to exert differential effects on activation. Removal of the N-terminal disulfide yields no fully active enzyme, but considerably accelerates further activation by thioredoxin. Removal of the C-terminal disulfide does not change the activation kinetics, but yields an enzyme with a slight basal activity and which is no longer inhibited by NADP + , as is observed with the WT protein. However, removal of both disulfides provides a permanently active NADP-MDH. The three-dimensional structures confirm the presence of the two disulfides in the N-, respectively C-terminal extensions and show that both are on the surface and accessible to thioredoxins. The N-terminal extension with its disulfide is located at the interface between the two subunits and has interactions with the catalytic domain of one subunit and with the nucleotide-binding domain of the other subunit which might maintain them in a catalytically unfavorable position. Reduction of the disulfide loosens the N-terminal extension which might result in a more relaxed, catalytically efficient structure of the active site. A clearer picture emerges concerning the C-terminal extension. This extension, which is about 13 residues long, is held by the disulfide bridge against the core structure of the protein and its tip, carrying negative charges, is dipping into the active site obstructing access to the substrate by intrasteric inhibition.48 The negative charges mimic the substrate oxaloacetate and can, in addition, interact with NADP + if present in the active site, which explains the observed inhibition of activation by NADP + . Through reduc­ tion of the disulfide the C-terminal extension gains more mobility, as has been observed by NMR,49 liberating the active site and permitting access to the substrate.

5. Concluding Remarks Light provides not only the energy necessary for the assimilation of carbon dioxide in photosynthetic cells but it also controls the activity of several key enzymes involved. This control is exerted through the ferredoxin /thioredoxin system, which represents a mechanism perfectly adapted to the photosynthetic metabolism. It senses the "electron pres­ sure" built up in the stroma by the light reactions and transduces this

The Ferredoxin/Thioredoxin System

91

information into a dithiol signal, which is transmitted through disulfide/ dithiol interchanges to the target proteins. The reduction of the regulatory disulfides in the target proteins, which can only be accomplished by thioredoxins, probably due to some specific interaction, results in structural changes modifying the catalytic capacity of the enzymes. As a result of the structural studies on the components of the ferredoxin/thioredoxin system we start to understand how the signal is transferred through the redox cas­ cade and what are the potential structural changes on the target enzymes modifying their activity. Future structural studies should provide insights into the control of other redox regulated enzymes and studies on the reduced structures more details on the molecular events. The ferredoxin/thioredoxin system has been introduced with the advent of oxygenic photosynthesis in prokaryotes which, as a result of the new metabolic functions, had the need to be able to switch between assimilatory and dissimilatory pathways coexisting in the same cell com­ partment. With the invasion of cyanobacteria into the ancestral eukaryotic host this regulatory system arrived in the chloroplast where it has been adapted and extended. Whereas in cyanobacteria most of the carbon metabolism enzymes are not redox-regulated except PRK and G6PDH and the cells contain no /-type thioredoxins, in eukaryotes further enzymes, catalyzing key reactions, have been adapted to redox control by the introduction of redox sensitive structural elements. In addition a second thioredoxin, thioredoxin /, derived from the eukaryotic host has been added to take over the control over most of the carbon metabolism enzymes. The fact that chloroplasts have maintained two thioredoxins catalyzing the same reaction is puzzling and suggests that thioredoxin m has still other, probably specific functions. A future extended search for specific targets, especially for thioredoxin m, might solve this puzzle.

Acknowledgment The research in the author's laboratory is generously supported by the Schweizerischer Nationalfonds.

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Cellular Implications ofRedox Signalling

2. Ashton AR, Trevanion SJ, Carr PD, Verger D, Ollis DL. 2000. Struc­ tural basis for the light regulation of chloroplast NADP malate dehydro-genase. Physiol. Plant. 110: 314-321 3. Baier M, Dietz KJ. 1999. The costs and benefits of oxygen for photosynthesizing plant cells. Progress in Botany, Springer-Verlag, Berlin, Heidelberg, 60: 282-314 4. Ballicora MA, Frueauf JB, Fu Y, Schurmann P, Preiss J. 2000. Activa­ tion of the potato tuber ADP-glucose pyrophosphorylase by thioredoxin. /. Biol. Chem. 275:1315-1320 5. Balmer Y, Schurmann P. 2001. Heterodimer formation between thioredoxin / and fructose 1,6-bisphosphatase from spinach chloroplasts. FEBS Lett. 492: 58-61 6. Brandes HK, Larimer FW, Geek MK, Stringer CD, Schurmann P, Hartman FC. 1993. Direct identification of the primary nucleophile of thioredoxin/. /. Biol. Chem. 268:18411-1814 7. Brandes HK, Larimer FW, Hartman FC. 1996. The molecular path­ way for the regulation of phosphoribulokinase by thioredoxin /. /. Biol. Chem. 271: 3333-3335 8. Buchanan BB. 1991. Regulation of C 0 2 assimilation in oxygenic photosynthesis: The ferredoxin/thioredoxin system: Perspective on its discovery, present status, and future development. Arch. Biochem. Biophys. 288: 1-9 9. Capitani G, Markovic-Housley Z, del Val G, Morris M, Jansonius JN, Schurmann P. 2000. Crystal structures of two functionally different thioredoxins in spinach chloroplasts. /. Mol. Biol. 302:135-154 10. Carr PD, Verger D, Ashton AR, Ollis DL. 1999. Chloroplast NADPmalate dehydrogenase: Structural basis of light-dependent regula­ tion of activity by thiol oxidation and reduction. Structure 7: 461-^475 11. Chiadmi M, Navaza A, Miginiac-Maslow M, Jacquot JP, Cherfils J. 1999. Redox signalling in the chloroplast: Structure of oxidized pea fructose-l,6-bisphosphate phosphatase. EMBO }. 18: 6809-6815 12. Chivers PT, Prehoda KE, Raines RT. 1997. The CXXC motif: A rheo­ stat in the active site. Biochemistry 36: 4061-4066 13. Chow L-P, Iwadate H, Yano K, Kamo M, Tsugita A, et al. 1995. Amino acid sequence of spinach ferredoxin:thioredoxin reductase catalytic subunit and identification of thiol groups constituting a redox active disulfide and a [4Fe-4S] cluster. Eur. ]. Biochem. 231:149-156 14. Crawford NA, Droux M, Kosower NS, Buchanan BB. 1989. Evidence for function of the ferredoxin/thioredoxin system in the reductive

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94

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

Cellular Implications ofRedox Signalling

light/dark-modulated chloroplast enzymes and dithiothreitol: Fine-tuning by metabolites. Biochim. Biophys. Ada. 12^7:135-142 Follmann H. 2000. Light-dark and thioredoxin-mediated metabolic redox control in plant cells. In The Redox State and Circadian Rhythms, eds. Vanden Driessche T, Guisset J-L, Petiau-de Vries GM, Kluwer Academic Publishers Netherlands, pp. 59-83 Gaymard E, Franchini L, Manieri W, Stutz E, Schurmann P. 2000. A dicistronic construct for the expression of functional spinach chloro­ plast ferredoxin:thioredoxin reductase in Escherichia coli. Plant. Sci. 158:107-113 Geek MK, Hartman FC. 2000. Kinetic and mutational analyses of the regulation of phosphoribulokinase by thioredoxins. /. Biol. Chem. 275:18034-18039 Geek MK, Larimer FW, Hartman FC. 1996. Identification of residues of spinach thioredoxin / that influence interactions with target enzymes. /. Biol. Chem. 271: 24736-24740 Goyer A, Decottignies P, Lemaire S, Ruelland E, Issakidis-Bourguet E, et al. 1999. The internal Cys207 of sorghum leaf NADP-malate dehydrogenase can form mixed disulfides with thioredoxin. FEBS Lett. 444:165-169 Hirasawa M, Brandes HK, Hartman FC, Knaff DB. 1998. Oxidationreduction properties of the regulatory site of spinach phosphoribu­ lokinase. Arch. Biochem. Biophys. 350:127-131 Hirasawa M, Droux M, Gray KA, Boyer JM, Davis DJ, et al. 1988. Ferredoxin-thioredoxin reductase: Properties of its complex with ferredoxin. Biochim. Biophys. Acta. 935: 1-8 Hirasawa M, Ruelland E, Schepens I, Issakidis-Bourguet E, MiginiacMaslow M, Knaff DB. 2000. Oxidation-reduction properties of the regulatory disulfides of sorghum chloroplast nicotinamide adenine dinucleotide phosphate-malate dehydrogenase. Biochemistry 39: 3344-3350 Hirasawa M, Schurmann P, Jacquot J-P, Manieri W, Jacquot P, et al. 1999. Oxidation-reduction properties of chloroplast thioredoxins, ferredoxin: thioredoxin reductase and thioredoxin /-regulated enzymes. Biochemistry 38: 5200-5205 Hodges M, Miginiac-Maslow M, Decottignies P, Jacquot J-P, Stein M, et al. 1994. Purification and characterization of pea thioredoxin /expressed in Escherichia coli. Plant. Mol. Biol. 26: 225-234 Howard JB, Rees DC. 1991. Perspectives on non-heme iron protein chemistry. Adv. Protein Chem. 42:199-280

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37. Huppe HC, Lamotte-Guery FD, Jacquot J-P, Buchanan BB. 1990. The ferredoxin-thioredoxin system of a green alga, Chlamydomonas reinhardtii. Identification and characterization of thioredoxins and ferredoxin-thioredoxin reductase components. Planta 180: 341-351 38. Hutchison RS, Groom Q, Ort DR. 2000. Differential effects of low temperature induced oxidation on thioredoxin mediated activation of photosynthetic carbon reduction cycle enzymes. Biochemistry 39: 6679-6688 39. Jacquot J-P, Lancelin J-M, Meyer Y. 1997. Thioredoxins: Structure and function in plant cells. New Phytol. 136: 543-570 40. Jacquot J-P, Lopez-Jaramillo J, Chueca A, Cherfils J, Lemaire S, et al. 1995. High-level expression of recombinant pea chloroplast fructose1,6-bisphosphatase and mutagenesis of its regulatory site. Eur. }. Biochem. 229: 675-681 41. Jacquot JP, Lopez-Jaramillo J, Miginiac-Maslow M, Lemaire S, Cherfils J, et al. 1997. Cysteine-153 is required for redox regulation of pea chloroplast fructose-l,6-bisphosphatase. FEBS Lett. 401:143-147 42. Jacquot JP, Stein M, Suzuki K, Liottet S, Sandoz G, Miginiac-Maslow M. 1997. Residue Glu91 of Chlamydomonas reinhardtii ferredoxin is essential for electron transfer to ferredoxin-thioredoxin reductase. FEBS Lett. 400: 293-296 43. Jeng M-F, Campbell AP, Begley T, Holmgren A, Case DA, et al. 1994. High-resolution solution structures of oxidized and reduced Escherichia coli thioredoxin. Structure 2: 853-868 44. Johansson K, Ramaswamy S, Saarinen M, Lemaire-Chamley M, Issakidis-Bourguet E, et al. 1999. Structural basis for light activation of a chloroplast enzyme. The structure of sorghum NADP-malate dehydrogenase in its oxidized form. Biochemistry 38: 4319-4326 45. Katti SK, LeMaster DM, Eklund H. 1990. Crystal structure of thiore­ doxin from Escherichia coli at 1.68 A resolution. /. Mol. Biol. 212:167-184 46. Klenk HP, Clayton RA, Tomb JF, White O, Nelson KE, et al. 1997. The complete genome sequence of the hyperthermophilic, sulphatereducing archaeon Archaeoglobus fulgidus. Nature 390: 364-370 47. Knaff DB. 2000. Oxidation-reduction properties of thioredoxins and thioredoxin-regulated enzymes. Physiol. Plant 110: 309-313 48. Kobe B, Kemp BE. 1999. Active site-directed protein regulation. Nature 402: 373-376 49. Krimm I, Goyer A, Issakidis-Bourguet E, Miginiac-Maslow M, Lancelin J-M. 1999. Direct NMR observation of the thioredoxin-mediated

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Cellular Implications ofRedox Signalling

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61. Raines CA, Lloyd JC, Longstaff M, Bradley D, Dyer T. 1988. Chloro­ plast fructose-l,6-bisphosphatase: The product of a mosaic gene. Nucleic Acids Res. 16: 7931-7942 62. Rebeille F, Hatch MD. 1986. Regulation of NADP-malate dehydrogenase in C4 plants: Effect of varying NADPH to NADP ratios and thioredoxin redox state on enzyme activity in reconstituted systems. Arch. Biochem. Biophys. 249: 164-170 63. Reith M, MunhoUand J. 1997. Complete nucleotide sequence of the Porphyra purpurea chloroplast genome. Plant. Mol. Biol. Rep. 13: 333-335 64. Rodriguez-Suarez RJ, Mora-Garda S, Wolosiuk RA. 1997. Characteriza­ tion of cysteine residues involved in the reductive activation and the structural stability of rapeseed (Brassica napus) chloroplast fructose-l,6-bisphosphatase. Biochem. Biophys. Res. Commun. 232:388-393 65. Ruelland E, Miginiac-Maslow M. 1999. Regulation of chloroplast enzyme activities by thioredoxins: Activation or relief from inhibi­ tion? Trends Plant. Sci. 4:136-141 66. Saarinen M, Gleason FK, Eklund H. 1995. Crystal structure of thioredoxin-2 from Anabaena. Structure 3:1097-1108 67. Salamon Z, Tollin G, Hirasawa M, Knaff DB, Schurmann P. 1995. The oxidation-reduction properties of spinach thioredoxins / and m and of ferredoxin:thioredoxin reductase. Biochim. Biophys. Acta. 1230:114-118 68. Sasaki Y, Kozaki A, Hatano M. 1997. Link between light and fatty acid synthesis: Thioredoxin-linked reductive activation of plastidic acetyl-CoA carboxylase. Proc. Natl. Acad. Sci. USA 94:11096-11101 69. Scheibe R. 1994. Lichtregulation von Chloroplastenenzymen. Naturzvissenschaften 81: 443-448 70. Schurmann P, Buchanan BB. 2001. The structure and function of the ferredoxin/thioredoxin system in photosynthesis. In Advances in Photosynthesis, eds. Aro EM, Andersson B, and Kluwer Academic Publishers, Dordrecht, The Netherlands, 11: 331-336 71. Schurmann P, Jacquot J-P. 2000. Plant thioredoxin systems revisited. In Ann. Rev. Plant. Physiol. Plant. Mol. Biol, Annual Reviews, Inc., Palo Alto, USA, 51: 371-400 72. Schwarz O, Schurmann P, Strotmann H. 1997. Kinetics and thiore­ doxin specificity of thiol modulation of the chloroplast H + -ATPase. /. Biol. Chem. 272:16924-16927 73. Schwendtmayer C, Manieri W, Hirasawa M, Knaff DB, Schurmann P. 1998. Cloning, expression and characterization of ferredoxin:thioredoxin

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Chapter 5 Thioredoxin and Redox Regulation: Beginnings in Photosynthesis Lead to a Role in Germination and Improvement of Cereals Bob B. Buchanan Department of Plant and Microbial Biology, University of California, Berkeley, 471A Koshland CA 94720-3102, USA [email protected]

Keywords: Thioredoxin, redox regulation, food improvement, allergenicity, allergens, baking quality

1. Summary In this article, I trace the sequence of events that led to the discovery of the chloroplast ferredoxin/thioredoxin system and how that finding, in turn, led to elucidation of the function of the extraplastidic NADP-linked thioredoxin counterpart in cereals. The extension of the seed research to applications for improving foods is then described, including the new products currently under commercial development. Finally, I give a brief account of a strategy we have recently devised for the identification of thioredoxin target proteins. The article closes with a summary of lessons learned in this research that covered almost four decades. I shall give a personal perspective of the regulatory role of thioredoxin that was originally uncovered in studies on photosynthesis and show how this role has been extended to the germination of cereal grains. The application of these findings to the improvement of food is then discussed in the context of decreasing allergenicity, increasing digestibility and strengthening doughs. Pursuit of these applications has led to ongoing studies on transgenic cereals overexpressing thioredoxin in grain and the development of products. In coming full circle back to basis research, I describe a new strategy developed for the identification of thioredoxin 99

100

Cellular Implications ofRedox Signalling __ (ACETATE]

/ y >^-.ATP

ACETYL-CoA

CITRATE c5-ACONITATE

S*"

X

/

ISOCiTRATE N A D

/

vLy\^

-» G l u "" n o , < i

a-KETOGLUTARATE

/ / Cof""\h

/ / "

.. PYRUVATE i ' ' \ ,ATP

—

SUCClNYL-CoA ATP

1 \

FUVIN-Hj

PHOSPHOPYRUVATE

f

T J

\

FUMARATE

\ NADH* C

co,

SUCCINATE 0

~ ~ Z ^

X MALATE

OXALACETATE t

Asparlata

Fig. 1. Reductive carboxylic acid (Reverse citric acid) cycle of bacterial photo­ synthesis (from Ref. 25). target proteins in peanut that holds promise for investigating a broad spectrum of systems. The reader interested in a more comprehensive coverage of thioreodoxin should consult one of a number of reviews.

3,13,20,27,28,31,45,48,50,51,59

2. Ferredoxin-Thioredoxin System I shall start at the beginning. In the early 1960s, the finding of ferredoxinlinked carboxylation reactions in fermentative bacteria (1) (Eq. 1) led to the discovery of a cycle for the assimilation of carbon dioxide that was independent of the Calvin cycle — the mechanism that at the time was considered essential for growth of photosynthetic and other types of autotrophic cells. Acyl-CoA + C 0 2 + 2 Ferrdoxinreducii[1-> cc-Keto acid + CoA + Ferredoxin 0 ,

(1)

The new cycle, named the reductive carboxylic acid or reverse citric acid cycle,9,10,25 was found in photosynthetic bacteria — anaerobes believed to be early inhabitants of our planet. The cycle depended on reduced ferredoxin to promote the synthesis of the alpha-keto acids, pyruvate and

Thioredoxin and Redox Regulation 101

Fructose protein Ferredoxin — factor ; . . . „ »~ Diphosphatase t-

-i

•

e

CHLOROPHYLL hv Fig. 2. Activation of fructose diphosphatase in chloroplasts. A 1971 slide based on Ref. 14. alpha-ketoglutarate (from C 0 2 and acetyl-CoA and succinyl CoA, respec­ tively) and on an active citrate lyase to drive the citric acid cycle in the reverse direction (Fig. 1). After many years of controversy, the reverse citric acid cycle is now accepted by the scientific community.9,48'63 Following the bacterial work, we asked whether the new cycle occurs in oxygenic photosynthetic organisms — that is, chloroplasts of higher plants. We looked for the two ferredoxin-linked carboxylation enzymes as markers of the cycle, but our experiments were unsuccessful. We did, however, make an interesting observation which, it turns out, led to experiments that shaped my future career. Ferredoxin was found to activate fructose-l,6-bisphosphatase (then called fructose diphosphatase) in preparations from isolated chloroplasts.11 The ferredoxin-linked activation of this enzyme, a member of the Calvin cycle, was an unexpected finding in that the reaction does not involve oxidation or reduction (Eq. 2). Fructose 1,6-bisphosphate + H 2 0 -» Fructose 6-phosphate + P,

(2)

We proceeded to purify the crude cholorplast protein preparation and found that ferredoxin did not react with the enzyme directly but that an

102

Cellular Implications ofRedox Signalling

Fig. 3. The photosynthetic ferredoxin/thioredoxin system (from Ref. 55). additional protein fraction (termed the protein factor) was required (Fig. 2).14 It took a number of years to show that the protein factor actually consisted of three components functional in the regulation of photo­ synthesis — that is, two different thioredoxins ( n a m e d / a n d m) and the enzyme catalyzing their reduction by reduced ferredoxin, ferredoxinthioredoxin reductase (FTR).615-52'60 It also became clear that these three proteins functioned jointly in what became known as the ferredoxin/ thioredoxin system — a reductive mechanism that links light to the regu­ lation of enzymes of photosynthetic C 0 2 assimilation (Fig. 3). Since that time impressive progress has been made in elucidating the structural and physical properties of the components of the system as well as the enzymes of the different processes it regulates. Recently the structures of the components of the system have been published 21 as have the struc­ tures of two target enzymes, fructose biphosphatase 17,54 and NADPmalate dehydrogenase. 1636 The system has been shown to function widely and to control the activity of enzymes of multiple processes in oxygenic photosynthetic cells.3'6'20-31'48'50-51 The m- and/-type thioredoxins have also been found to have different evolutionary histories.30'49 2.1 NADP/Thioredoxin System About 15 years ago, when the overall picture was clear and the ferredoxin/ thioredoxin system began to be featured in standard textbooks, I decided to enter another field. In due course, my interest turned to the NADPlinked thioredoxin system that had been extensively studied in bacterial

Thioredoxin and Redox Regulation

y ^c Ferredoxin

103

Light FTR

Calvin Cycle >Thioredoxin f/m — > Related Enzymes

Carbon Substrates / NADPH

^ Thioredoxin h

Target Enzymes "^ Proteins of Cytosol, ER, Mitochondria

Fig. 4. Thioredoxin systems in plants. From a 1988 slide based on Ref. 44. and animals cells. While known to occur in plants5,56,57 neither the cellular location nor the properties of the protein members of the plant system were known at the time. Our experiments showed that the participating thioredoxin was of a new type (designated thioredoxin h with its own properties 37 ) that, as in bacterial and animal cells, was linked to the flavoprotein NADP-thioredoxin reductase (NTR).26,38,44 The plant N A D P / thioredoxin system can be summarized as shown in Eq. 3. The structures of NTR and thioredoxin h were determined recently.32,46 NADPH + Thioredoxin hm -> NTR -> Thioredoxin hred + NADP

(3)

Early cell fractionation experiments revealed that thioredoxin h was extraplastidic and occurred in the cytosol, endoplasmic reticulum and mitochondrion 44 (Fig. 4). Current evidence suggests that the N A D P / thioredoxin system is present in all plant cells, photosynthetic as well as nonphotosynthetic. As with i t s / a n d m counterparts, thioredoxin h has its own evolutionary history. 49 Following a description of the system, we set out to determine the func­ tion of thioredoxin h. Good fortune led us to cereals and to ask

104 Cellular Implications ofRedox Signalling

Fig. 5. Role of NADP/thioredoxin system in seed germination. A contemporary view.

several questions including, (1) whether the disulfide groups of seed storage proteins undergo redox change during grain formation, development and germination, and (2) whether thioredoxin was involved in germination and seedling development. Working with wheat in collaboration with K. Kobrehel, we found the answer to both questions to be yes.29,40 This and subsequent work provided evidence that thioredoxin h acts as an early wakeup call in germination and seedling development by facilitating the (a) mobilization of nitrogen and carbon through the reduction of storage proteins with disulfide groups (gliadins and glutenins); (b) inactivation of low-molecular-weight disulfide proteins that inhibit starch-degrading enzymes, and (c) activation of individual enzymes as occurs in chloroplasts4,12,33'34'35'41'42'53 (Fig. 5). A key feature turned out to be the specificity of thioredoxin in the reduction of mframolecular versus, mtermolecular disulfide bonds of the proteins studied.34,35'38'53'55 The observed proclivity of thioredoxin to reduce intramolecular disulfide bonds, confirmed in recent experiments, 63 has influenced our subsequent work.

Thioredoxin and Redox Regulation

105

3. Applications of Thioredoxin 3.1 Alleviation of Allergies The experiments discussed above revealed that four changes accompany the thioredoxin-linked reduction of low-molecular-weight proteins from plant as well as animal sources. • • • •

Loss (or gain) of biochemical activity. Increased susceptibility to proteolysis. Increased susceptibility to heat. Decrease in allergenicity.

The first three changes appear to be general features of low-molecularweight proteins containing intramolecular disulfide bonds. For example, on reduction by thioredoxin, the soybean Kunitz and Bowman-Birk trypsin inhibitors lose their ability to inhibit trypsin and show increased susceptibility to proteolysis and increased heat.34 The fourth change, decrease in allergenicity, may not be universal but has been observed with a number of allergens containing intramolecular disulfide bonds. We have studied the allergy problem using a colony of high IgE-producing dogs sensitized to specific foods.24 In our initial study with differentially soluble proteins from wheat (Osborne fractions),8 we used the hyper­ sensitive skin test response to (1) identify and rank the allergens according to their allergenicity (gliadins > glutenins > albumins > globulins); (2) show that the effect of the major allergens, gliadins and glutenins, was dimini­ shed on reduction by thioredoxin; (3) document that the results were statistically significant; and (4) show that reduced glutathione had no effect on allergenicity. The more we learn the better the dog seems to be as an allergy model for humans. 7 After the wheat work, we carried out a study on milk.23 We found that, as in humans, the major allergen is beta-lactoglobulin — a protein with a known human epitope 2 and with two disulfide bonds 47 — was reduced actively by thioredoxin. 23 In this case, the allergic response was shown to be mitigated by thioredoxin as measured by both skin tests and feeding challenges. In the latter experiments, we monitored allergenicity by both the immediate (vomit) and delayed (diarrhea/constipation) response of dogs fed untreated versus thioredoxin-treated beta-lactoglobulin. Thioredoxin is believed to mitigate the allergic response manifested by a wheal in skin tests8,23 and by the vomit response in feeding challenges, 23

106

Cellular Implications ofRedox Signalling

through changing the structure of the allergen (beta-lactoglobulin) so that it is less well recognized by the IgE of mast cells in the majority of animals and is much more readily digested in the stomach. These changes are reflected in the observed decrease in the immediate and delayed gastrointestinal responses. It appears that increased sensitivity to pepsin is a general feature accompanying the reduction of low-molecular-weight disulfide proteins by thioredoxin. Current data thus indicate that thioredoxin disarms food allergens in two ways: (1) by decreasing epitope accessibility to the IgE immune sys­ tem, thereby lowering the immediate vomit response, and (2) by increas­ ing sensitivity to pepsin, thereby facilitating digestion in the stomach and lowering the delayed gastrointestinal (diarrhea/constipation) response. As an extension of this work, we are currently testing the capability of thioredoxin to improve desensitization (immunotherapy) to ragweed pollen by shifting the immune response from IgE to IgG. Such a shift could make the desensitization process (immunotolerance) both safer and more effective. We have observed that a disulfide allergen of ragweed pollen (Amb t5) is largely inactivated on reduction by thioredoxin, thus making such a shift in the immune response conceptually feasible.22

3.2 Improved Dough Quality A second problem we are actively pursuing is the effect of thioredoxin on dough strength. Early studies in collaboration with K. Kobrehel showed that, when added to poor quality flour, components of the N A D P / thioredoxin system (NADPH, NTR and thioredoxin) strengthened dough products as determined by Farinograph measurements 61 and increased loaf volume and viscoelasticity.39 As seen below, these findings are currently being extended in experiments with transformed grain overexpressing thioredoxin h.

4. Thioredoxin-Enriched Grain 4.1 Cereal Transformations To make the application of thioredoxin economically feasible, we have transformed cereals to overexpress thioredoxin h. The overall goal is to determine whether the improvement in dough quality effected by thiore­ doxin in vitro can be obtained with thioredoxin h overexpressed in vivo. To

Thioredoxin and Redox Regulation

107

Fig. 6. DNA construct for transformation of cereals with wheat thioredoxin h Gene (from Ref. 18).

this end, in collaboration with P. G. Lemaux, we have developed a gene expression system designed to express proteins of interest specifically in the grain endosperm. 18 Using a barley Bj-hordein promoter, we have obtained maximal expression of thioredoxin h using a DNA construct with which the gene is linked to a signal sequence for targeting to the endosperm protein body. Here the wheat thioredoxin h gene (kindly pro­ vided by Dr Philippe Joudrier) is linked to the B a -hordein promoter and a protein body signal sequence (Fig. 6). Homozygous barley lines trans­ formed with this construct showed up to 30-fold enrichment in the con­ tent of thioredoxin h relative to null segregants. 19 A similar pattern of overexpression has recently been obtained with transformed wheat (unpublished findings).

4.2 Properties of Transgenic Cereals We have only recently begun to analyze transgenic cereals with increased levels of thioredoxin h. Our studies have corroborated earlier in vitro results and shown that transgenic grain grown either in the greenhouse or the field is enriched in starch debranching enzyme (also called limit dextrinase or pullulanase). 19 Based on spectrophotometric and gel assays, extracts from the homozygous transgenic lines showed up to 3-fold more starch debranching enzyme activity than corresponding null segregants. More recent analyses indicate that the transgenic barley also shows an acceleration in germination (radicle emergence) and oc-amylase biosyn­ thesis, both by up to a day.62 Other properties of the transgenic cereals are under investigation.

108 Cellular Implications ofRedox Signalling

Strategy

Target Protein

Chance

Chloroplast FBPase

Light activation

Chloroplast PRK + others

Dithiothreitol activation

Chloroplast NADP-MDH

mBBr / 1 D SDS-PAGE

Multiple suspected target proteins from seeds + other sources

Gene overexpression

Seed a-amylase

Cassette mutagenesis

Yeast periredoxin

mBBr/ 2D Electrophoresis

> 20 unknown proteins: 3 allergens + 2 proteins new to peanut

Fig. 7. Identification of new proteins targeted by plant thioredoxins.

5. A New Development One of the challenges of thioredoxin research is knowledge of its target pro­ teins. In the original studies on photosynthesis, target enzymes were identi­ fied by chance (e.g. fructose bisphosphatase) or by showing that an enzyme activated either by light or DTT in vitro (e.g. NADP-malate dehydrogenase) could be similarly activated by reduced thioredoxin. 6 (Fig. 7). The opportunity to label the sulfhydryl groups newly generated by thioredoxin in either known individual proteins34,35'38'41'53'55 or protein families40 with mBBr, in combination with one-dimensional gel elec­ trophoresis, led to the identification of a number of new targets. The proteins identified are extraplastidic and primarily serve a storage or pro­ tective function in seeds.3'4'40 In ongoing work with seeds, we have devel­ oped a new strategy for the identification of thioredoxin target proteins. 63 The approach is based on the application of mBBr to tag target proteins reduced in vitro by thioredoxin. The labeled proteins are isolated by electrophoresis [2D-isoelectric focusing/reducing SDS-PAGE or 2D-nonreducing/reducing SDS-PAGE] and identified by amino acid sequencing. When applied to extracts of peanut seeds we isolated at least 20 thioredoxin targets revealed by the fluorescent spots (Fig. 8) and identified 5, all with intramolecular disulfide bonds: 3 allergens (Ara h2, Ara h3, Ara h6) and 2 proteins not known to occur in peanut (desiccation-related and seed

Thioredoxin and Redox Regulation

109

Ul

o

Fig. 8. Strategy for identifying thioredoxin target proteins (from Ref. 63). maturation protein). 63 These findings open the door to the identification of proteins targeted by thioredoxin in a wide range of systems, thereby enhancing our understanding of its function and extending its technologi­ cal and medical applications. The present studies show how research initiated in the early 1960s on carbon dioxide fixation in fermentative bacteria led to the discovery of a carbon cycle in photosynthetic bacteria and then, sequentially to regulatory systems functional in oxygenic photosynthesis and seed germination. The seed research, in turn, has opened the door to emerging new technologies, including ones applicable to the improvement of major foods. Three lessons have been learned as this work has unfolded during the past four decades. The development of technologies and products from basic research — that is (1) the movement of results from the laboratory into the research and development pipeline — requires multiple talents and thus collabo­ ration with colleagues from diverse disciplines; (2) the trail from discovery to application is long; and (3) the trail is uncharted so that once a discovery is made, there is no way to predict whether it will result in a useful product or technology.

110

Cellular Implications ofRedox Signalling

Note added in proof The importance of the reverse citric acid cycle in chlorobium was recently confirmed in the determination of the complete genome sequence (J.A. Eisen et al. 2002. Proc. Natl. Acad. Sci. USA 99: 9509-9514).

References 1. Bachofen R, Buchanan BB, Arnon DI. 1964. Ferredoxin as a reductant in pyruvate synthesis by a bacterial extract. Proc. Natl. Acad. Sci. USA 51: 690-694 2. Ball G, Shelton MJ, Walsh BJ, Hill D, Hosking CS, Howden MEH. 1994. A major continuous allergenic epitope of bovine /J-lactoglobulin recognized by human IgE binding. Clin. Exp. Allergy 24: 758-764 3. Besse I, Buchanan BB. 1997. Thioredoxin-linked plant and animal processes: The new generation. Bot. Bull. Acad. Sin. (Taipei) 38: 1-11 4. Besse I, Wong JH, Kobrehel K, Buchanan BB. 1996. Thiocalsin: A thioredoxin-linked substrate-specific protease dependent on calcium. Proc. Natl. Acad. Sci. USA 93: 3169-3175 5. Berstermann A, Vogt K, Follman H. 1983. Plant seeds contain several thioredoxins of regular size. Eur. }. Biochem. 131: 339-344 6. Buchanan BB. 1980. Role of light in the regulation of chloroplast enzymes. Ann. Rev. Plant Physiol. 31: 341-374 7. Buchanan BB. 2001. Genetic engineering and the allergy issue. Plant Physiol. 126: 5-7 8. Buchanan BB, Adamidi C, Lozano RM, Yee BC, Momma M, Kobrehel K, Ermel RW, Frick OL. 1997. Thioredoxin-linked mitigation of aller­ gic responses to wheat. Proc. Natl. Acad. Sci. USA 94: 5372-5377 9. Buchanan BB, Arnon DI. 1990. A reverse KREBS cycle in photo­ synthesis: Consensus at last. Photosyn. Res. 24: 47-53 10. Buchanan BB, Evans MCW, Arnon DI. 1967. Ferredoxin-dependent carbon assimilation in Rhodospirillum rubrum. Arch. Microbiol. 59:32-40 11. Buchanan BB, Kalberer PP, Arnon DI. 1967. Ferredoxin-activated fructose diphophatase in isolated chloroplasts. Biochem. Biophys. Res. Commun. 29: 74-79 12. Buchanan BB, Lozano RM, Wong JH, Jiao J, Yee BC, Kobrehel K, Nimbona C. 1994. Thioredoxin linked reduction of wheat storage proteins. I. Physiological consequences. In Gluten Proteins, Associa­ tion of Cereal Research, Detmold, Germany, pp. 369-380 13. Buchanan BB, Schurmann P, Decottignies P, Lozano RM. 1994. Thioredoxin: A multi-functional regulatory protein with a bright

Thioredoxin and Redox Regulation

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future in technology and medicine. Arch. Biochem. Biophys. 314: 257-260 Buchanan BB, Schurmann P, Kalberer PP. 1971. Ferredoxin-activated fructose diphosphatase of spinach chloroplasts: Resolution of the system, properties of the alkaline fructose diphosphatase component, and physiological significance of the ferredoxin-linked activation. /. Biol. Chem. 246: 5952-5959 Buchanan BB, Wolosiuk RA. 1976. Photosynthetic regulatory protein found in animal and bacterial cells. Nature 264: 669-670 Carr PD, Verger D, Ashton AR, Ollis DL. 1999. Chloroplast NADPmalate dehydrogenase: Structural basis of light-dependent regula­ tion of activity by thiol oxidation and reduction. Structure 7: 461^75 Chiadimi M, Navaza A, Miginiac-Maslow M, Jacquot JP, Cherfils J. 1999. Redox signaling in the chloroplast structure of the oxidized pea fructose-1, 6-biphosphatase. EMBO }. 18: 6809-6816 Cho M-J, Choi HW, Buchanan BB, Lemaux PG. 1999. Inheritance of tissue-specific expression of hordien promotor-uicLA fusions in transgenic barley plants. Theor. Appl. Genet. 98:1253-1262 Cho M-J, Wong JH, Marx C, Jiang W, Lemaux PG, Buchanan BB. 1999. Overexpression of thioredoxin h leads to enhanced activity of starch debranching enzyme (pullulanase) in barley grain. Proc. Natl. Acad. Sci. USA 96:14641-14646 Dai SD, Schwendtmayer C, Johansson K, Ramaswamy S, Schurmann P, Eklund H. 2000. How does light regulate chloroplast enzymes? Structure-function studies of the ferredoxin/thioredoxin system. Q. Rev. Biophys. 33: 67-108 Dai SD, Schwendtmayer C, Schurmann P, Ramaswamy S, Eklund H. 2000. Redox signaling in chloroplasts: Cleavage of disulfides by an iron-sulfur cluster. Science 287: 655-658 del Val G, Yee BC, Buchanan BB, Frick OL. 1999. Disulfide bond reduction by thioredoxin alleviates the allergenicity of ragweed pollen. /. Aller. Clin. Immunol. 103: S139 del Val G, Yee BC, Lozano RM, Buchanan BB, Ermel RW, Lee YM, Frick OL. 1999. Thioredoxin treatment increase digestibility and lowers allergenicity of milk. /. Aller. Clin. Immunol. 103: 690-697 Ermel EW, Kock M, Griffey SM, Reinhart GA, Frick OL. 1997. The atopic dog: A model for food allergy. Lab. Animal Sci. 47: 40^49 Evans MCW, Buchanan BB and Arnon DI. 1966. A new ferredoxindependent carbon reduction cycle in a photosynthetic bacterium. Proc. Natl. Acad. Sci. USA 55: 928-934

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26. Florencio FT, Yee BC, Buchanan BB. 1988. A NADP/thioredoxin system in green leaves: Purification and characterization of NADPthioredoxin reductase and thioredoxin h from spinach. Arch. Biochem. Biophys. 266: 491-507 27. Follmann H, Haberlein I. 1996. Thioredoxin: Universal, yet specific thiol-disulfide redox cofactors. BioFactors 5: 147-56 28. Fridlyand LE, Scheibe R. 1999. Regulation of the Calvin cycle for C 0 2 fixation as an example for general control mechanisms in metabolic cycles. Biosystems 51: 79-93 29. Gobin P, Ng PKW, Buchanan BB, Kobrehel K. 1997. Sulfhydryldisulfide changes in proteins of developing wheat grain. Plant Physiol. Biochem. 35: 777-783 30. Hartman H, Syvanen M, Buchanan BB. 1990. Contrasting evolution­ ary histories of cholorplast thioredoxins / and m. Mol. Biol. Evol. 7: 247-254 31. Jacquot JP, Lancelin JM, Meyer Y. 1997. Thioredoxins: Structure and function in plant cells. New Phyto. 136: 543-570 32. Jacquot J-P, Rivera-Madrid R, Marinho P, Kollarova M, Le Marechal P, Miginiac-Maslow M, Meyer Y. 1994. Arabidopsis thaliana NAPHP thioredoxin reductase. cDNA characterization and expression of the recombinant protein in Escherichia coli. }. Mol. Biol. 235:1357-1363 33. Jiao J, Yee BC, Buchanan BB. 1992. Thioredoxin-linked changes in properties of protease inhibitors of seed. Plant Physiol. 99S: 57 34. Jiao J, Yee BC, Kobrehel K, Buchanan BB. 1992. Effect of thioredoxinlinked reduction on the activity and stability of the Kunitz and Bowman-Birk soybean trypsin inhibitor proteins. /. Agric. Food Chem. 40: 2333-2336 35. Jiao J, Yee BC, Wong JH, Kobrehel K, Buchanan BB. 1993. Thioredoxinlinked changes in regulatory properties of barley a-amylase/subtilisin inhibitor protein. Plant Physiol. Biochem. 31: 799-804 36. Johansson K, Ramaswamy S, Saarinen M, Lemaire-Chamley M, Issakidis-Bourguet E, Miginiac-Maslow M, Eklund H. 1999. Structural basis for light activation of a chloroplast enzyme. The structure of Sorghum NADP-malate dehydrogenase in its oxidized form. Biochemistry 38: 4319-4326 37. Johnson TC, Cao RQ, Kung JE, Buchanan BB. 1987. Thioredoxin and NADP-thioredoxin reductase from cultured carrot cells. Planta 171: 321-331 38. Johnson TC, Wada K, Buchanan BB, Holmgren A. 1987. Reduction of purothionin by the wheat seed thioredoxin system and potential

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function as a secondary thiol messenger in redox control. Plant Physiol. 85: 446-451 Kobrehel K, Buchanan BB, Bergmann CJ, Wong JH, Yee BC. 1994. Thioredoxin-linked reduction of wheat storage proteins II. Techno­ logical consequences. In Gluten Proteins 1993, Association of Cereal Research, Detmold, Germany, pp. 381-392 Kobrehel K, Wong JH, Balough A, Kiss F, Yee BC, Buchanan BB. 1992. Specific reduction of wheat storage proteins by thioredoxin h. Plant Physiol. 99: 919-924 Kobrehel K, Yee BC, Buchanan BB. 1991. Role of the N A D P / thioredoxin system in the reduction of a-amylase and trypsin inhibitor proteins. /. Biol. Chem. 266: 16135-16140 Lozano RM, Wong JH, Yee BC, Peters A, Kobrehel K, Buchanan BB. 1996. New evidence for a role for thioredoxin h in germination and seedling development. Planta 200:100-106 Madigan MT, Martinko JM, Parker J. 1997. Brock Biology of Micro­ organisms, 8th edn., Prentice Hall, Upper Saddle River, New Jersey, pp.650-651 Marcus F, Chamberlin SH, Chu C, Masiarz FR, Shin S, Yee BC, Buchanan BB. 1991. Plant thioredoxin h: An animal like thioredoxin occurring in multiple cell compartments. Arch. Biochem. Biophys. 287: 195-198 Meyer Y, Verdoucq L, Vignols F. 1999. Plant thioredoxins and glutaredoxins: Identity and putative roles. Trends Plant Sci. 4: 388-394 Mittard V, Blackledge MJ, Stein M, Jacquot J-P, Marion D, Lancelin JM. 1997. NMR solution structure of an oxidised thioredoxin h from the eukaryotic green alga Chamydomonas reinhardtii. Eur. ]. Biochem. 243: 374-383 Papiz MZ, Sawyer L, Eliopoulos EE, North AC, Findlay JB, Sivaprasadarao R, Jones TA, Newcomer ME, Kraulis PJ. 1986. The structure of (3-lactoglobulin and its similarity to plasma retinolbinding protein. Nature 324: 383-385 Ruelland E, Miginiac-Maslow M. 1999. Regulation of chloroplast enzyme activities by thioredoxins: Activition or relief from inhibi­ tion? Trends Plant Sci. 4: 136-141 Sahrawy M, Hecht V, Lopez-Jaramillo J, Chueca A, Meyer Y. 1996. Intron position as an evolutionary marker of thioredoxin and thiore­ doxin domains. /. Mol. Evol. 42: 422^31 Schurmann P, Buchanan BB. 2001. The structure and function of the ferredoxin/thioredoxin system in photosynthesis. In Regulation of

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Photosynthesis. Advances in Photosynthesis, eds. Andersson B, Aro E-M, Kluwer Academic Publishers. Dordrecht, The Netherlands, 11:331-361 Schurmann P, Jacquot J-P, 2000. Plant thioredoxin systems revisited. Ann. Rev. Plant Physiol. Plant Mol. Biol. 51: 371-400 Schurmann P, Wolosiuk RA, Breazeale VD, Buchanan BB. 1976. Two proteins function in the regulation of photosynthetic C 0 2 assimila­ tion in chloroplasts. Nature 263: 257-258 Shin S, Wong JH, Kobrehel K, Buchanan BB. 1993. Reduction of castor seed 2S albumin protein by thioredoxin. Planta 189: 557-560 Villeret V, Huang S, Zhang Y, Xue Y, Lipscomb WN. 1995. Crystal structure of spinach chloroplast fructose-1, 6-biphosphatase at 2.8 A resolution. Biochemistry 34: 4299^306 Wada K, Buchanan BB. 1981. Purothionin. A seed protein with thiore­ doxin activity. FEBS Lett. 124: 237-240 Wagner W, Follmann H. 1977. A thioredoxin from green algae. Biochem. Biophys. Res. Commun. 77:1044-1051 Wagner W, Follmann H, Schmidt A. 1978. Multiple forms of thioredoxins. Z Naturforsch Teil C. 33: 517-520 Wahlund TM, Tabita FR. 1997. The reductive tricarboxylic acid cycle of carbon dioxide assimilation: Initial studies and purification of ATP-citrate lyase from the green sulfur bacterium Chlorobium tepidum. J. Bacteriol. 179: 4859-4867 Wolosiuk RA, Ballicora MA, Hagelin K. 1993. The reductive pentose phosphate cycle for photosynthesis C 0 2 assimilation: Enzyme modu­ lation. FASEB J. 7: 622-37 Wolosiuk RA, Buchanan BB. 1977. Thioredoxin and glutathione reg­ ulate photosynthesis in chloroplasts. Nature 266: 565-567 Wong JH, Kobrehel K, Nimbona C, Yee BC, Balough A, Kiss F, Buchanan BB. 1993. Thioredoxin and bread wheat. Cereal Chem. 70: 113-114 Wong JH, Ren P-H, Cai N, Cho M-J, Lemaux PG, Buchanan BB. 2000. Transgenic barley grain over-expressing wheat thioredoxin h shows improved germination properties. Ann. Meeting Amer. Soc. Plant Physiol, Abstract No. 187 Yano H, Wong JH, Lee YM, Cho M-J, Buchanan BB. 2001. A strategy strategy for the identification of proteins targeted by thioredoxin. Proc. Natl. Acad. Sci. USA 98: 4794^799

Chapter 6 The Role of Thioredoxin in Regulatory Cellular Functions Junji Yodoi/ Hajime Nakamura, Hiroshi Masutani, Yumiko Nishinaka, and Itaro Hattori Department ofNeurosurgery, Graduate School of Medicine, Kyoto University, Kyoto, 606-8507 Japan '[email protected]

Keywords: thioredoxin, oxidative stress, thioredoxin transgenic mice

1. Summary Increasing evidence has indicated that oxidative stress mediates various cellular responses, although continuous and excessive stress is threaten­ ing life on the earth. Regulation of reduction/oxidation (redox) is funda­ mentally important to maintain homeostasis of life. Thioredoxin (Trx) is a 12 kD protein with redox-active dithiol in the active site. Human thiore­ doxin has been cloned as adult T cell leukemia derived factor produced by HTLV-I transformed cells. Thioredoxin is one of the major components of the thiol-reducing system and plays multiple roles in cellular processes such as proliferation, apoptosis and gene expression. Thioredoxin is induced by a variety of stresses including viral infection.1,2 The promoter sequences of the Trx gene contain a series of stress-responsive elements except for heat shock element. Thioredoxin promotes DNA binding of transcription factors such as NF-kappaB, AP-1 and p53.3,4 Thioredoxin has been already demonstrated to be directly associated with target proteins and activate those proteins by dithiol-dependent reduction. The impor­ tance of the Trx catalytic site has also been shown in the interaction between Trx and Trx-binding proteins such as Trx-binding protein-2/ vitamin D3 up-regulated protein-1 (TBP-2/VDUP1) 5 and apoptosis signal-regulating kinase-1 (ASK-1).6 We have identified Trx-binding 115

116

Cellular Implications of Redox Signalling

protein-2 (TBP-2), which was identical to vitamin D3 up-regulated protein-1 (VDUPl). TBP-2/VDUP1 suppressed the reducing activity of Trx. Treatment of HL-60 cells with vitamin D3 caused an increase of TBP2/VDUP1 expression, suggesting that the Trx-TBP-2/VDUPl interaction may be an important redox regulatory mechanism in cellular processes, including differentiation of myeloid/macrophage lineage. Potential action of TBP-2/VDUP1 as a redox-sensitive tumor suppressor will be discussed. The biological functions of thioredoxin may be strictly regu­ lated by its enzymatic reaction and/or structure-dependent interaction with the target. 7 We will also discuss our recent data on the anti-apoptotic activity of mitochondoria-specific thioredoxin-2 (Trx-2) based on in vitro knock out system (in cooperation with Spyrou G.). Redox regulation by thioredoxin plays a crucial role in biological responses against oxidative stress. Transgenic mice overexpressing thioredoxin show resistance against ischemic and excitotoxic neuronal injury.8,9 In addition, thiore­ doxin transgenic mice exhibit u p to 30% extension of median life span and one-third of maximum life span. Overexpressing thioredoxin may have protected mice from oxidative stress-induced tissue damage during aging process. Thioredoxin-Tg mice are useful to investigate the biological functions of thioredoxin in vivo. Thioredoxin is also secreted from the activated cells as a redox-sensitive cytokine with cytokine-like and chemokine-like activities.10 Understanding of thioredoxin-dependent redox regulation will give us a new strategy for preventing diseases related to oxidative stress. Reactive oxygen species (ROS) are generated in eukaryotic cells from oxygen during respiration for energy metabolism, or in response to vari­ ous stimuli, such as UV irradiation, X-ray, ischemia/reperfusion, inflam­ matory cytokines and chemical carcinogens. ROS can alter or disrupt the balance of redox potential in cells, which may cause various cellular dysfunction and diseases. 1112 Redox regulation is fundamentally impor­ tant to maintain homeostasis of life. Eukaryotic cells have acquired sev­ eral regulatory systems to maintain intracellular redox status by scavenging ROS in evolution. Those systems basically include the glutathione (GSH)13 and the thioredoxin systems 14 based on mono- and di-thiol reaction respectively. In addition to this basic function to cope against oxidative stress, recent evidence has accumulated indicating that reducing molecules such as thioredoxin play important roles in cellular signaling through not only the reduction of cysteine residues of, but rather the interaction with, various important components of signal transduction pathways. Thioredoxin physiologically has cytoprotective

The Role of Thioredoxin in Regulatory Cellular Functions

117

effects against oxidative stress by scavenging ROS together with peroxiredoxin (thioredoxin-dependent peroxidases) system and is induced by various oxidative stresses through the activation of responsive elements in its promoter sequence. In addition, thioredoxin is quickly translocated from the cytoplasm into the nucleus upon oxidative stress and various stimuli, physically interacting with Ref-1 (redox factor 1)/APEX, an endoexonuclease located in the nucleus. Several reports showed that thioredoxin and/or Ref-1 enhance the DNA binding activity of AP-1, polyoma enhancer binding protein-2 (PEBP2), NF-kappaB, p53, and other transcription factors. Thioredoxin is considered as a unique redoxsensitive regulator/modulator of cellular signaling. In this chapter we focus on thioredoxin and its associated molecules and discuss the role of thiore­ doxin-dependent redox regulation in cellular functions.

2. Cytoprotective Effects of Thioredoxin Thioredoxin has been shown to play crucial roles in cytoprotection against a variety of oxidative stress. Recombinant thioredoxin can pro­ tect cells from anti-Fas antibody-induced apoptosis and cytotoxicity induced by TNF-alpha, hydrogen peroxide and activated neutrophils. 1516 Thioredoxin is also a potent costimulator of various cytokine expression. 1718 Recently, Nilsson et al. reported that thioredoxin induces the secretion of TNF-alpha and maintains the expression of Bcl-2, whereby prolongs survival of B-LCL.19 Overexpression of thiore­ doxin has been observed in a wide variety of oxidative conditions such as viral infection, diabetes, ischemic/reperfusion and malignant tis­ sues.20"22 During viral infection, considerable amount of ROS is gener­ ated, causing tissue damage and DNA breaks. As thioredoxin was first purified from HTLV-1 transformed cells,21 thioredoxin is induced and/or secreted from transformed cells related to infection of viruses such as HTLV-1, EBV,23 hepatitis C virus and papilloma virus. Elevated thioredoxin level in serum was also reported in late stage HIV patients. 24 Recently, Sono et al. have reported that thioredoxin suppresses lytic replication of EBV induced by 12-0-tetradecanoylphorbol-13-acetate (TPA) and prevented the cell death evoked by the lytic induction. 1 These observations suggest that thioredoxin is closely involved in both the process of virus infection and the prognosis of the infected patients. In vivo study showed that recombinant thioredoxin attenuated ischemia/ reperfusion lung injury in rat.25

118

Cellular Implications of Redox Signalling

NADPH + H+ 4 TRX-R-S2

TRX-(SH)2

oxidized protein

NADP*

TRX-S2

reduced protein

TRX-R-(SH)2

TRX-R: thioredoxin reductase TRX: thioredoxin Fig. 1. Reducing cycle of thioredoxin.

3. Thioredoxin and its Related Molecules Thioredoxin was first discovered in 1964 by Peter Reichard et al. in Sweden as a co-enzyme of proton-donor from NADPH to ribonucleotide reductase. 26 Later it has been studied intensively by Arne Holmgren et al. Thioredoxin is a small protein having oxidoreductase activity via its redox-active disulfide/dithiol site within the conserved active sequence, -Cys-Gly-Pro-Cys-.14 Reduced thioredoxin can reduce protein disulfide bonds and oxidized thioredoxin is reduced by NADPH and thioredoxin reductase cascade (Fig. 1). Thioredoxin appears to be present in essentially all living cells including prokaryotes as well as plant.14 It is considered as a more primitive redox regulating molecules than GSH, because it exists in the life lacking GSH. We identified an active cytokine-like principle named adult T-cell leukemia (ATL)-derived factor (ADF) from HTLV-I positive cell line ATL-2. After purification of ADF and cloning of the cDNA, ADF was found to be a human homologue of thioredoxin.27 Several cytokine-like factors proved to be identical or closely related to thioredoxin, indicating that thioredoxin has multiple functions in extracellular as well as intracellular, environment.20 We will mention about them later in the "Extracellular function of thioredoxin". Thioredoxin reductase has a selenium-containing active center in the C-terminals and there exist several isoforms of thioredoxin reductase. 28 In the past years, new members of thioredoxin-related molecules in the mammalian system have been identified. They share the similar active sites: -Cys-X-Y-Cys- and they are called thioredoxin superfamily. Table 1 summarizes the members of human thioredoxin superfamily. Glutaredoxin (GRX) was discovered as another proton-donor for ribonucleotide reductase in the Escherichia coli lacking thioredoxin 14 GRX

Table 1. TRX superfamily.

Thioredoxin Thioredoxin 2 TRX related protein (TRP32) Glutaredoxin (GRX) Nucleoredoxin Protein disulfide isomerase (PDI) Ca binding protein-1 (CaBPl) Ca binding protein-2 (ERp72) Phospholipase C E{

kDa

Localization

Active Site Sequence

12 12 32 12 48 55 49 72 61

Cytosol Mitochondria Cytosol Cytosol Nucleus Endoplasmic reticulum Endoplasmic reticulum Endoplasmic reticulum Endoplasmic reticulum

-Cys-Gly-Pro-Cys-Cys-Gly-Pro-Cys-Cys-Gly-Pro-Cys-Cys-Gly-Tyr-Cys-Cys-Gly-Pro-Cys-[Cys-Gly-His-Cys] -[Cys-Gly-His-Cys] -[Cys-Gly-His-Cys] -[Cys-Gly-His-Cys] '

2232-

^ i3 |L o HTO* ^ B" g55 |. §• 3' S' 3" #

a* a* 3"

3" <3 .3

n SSL. O

c4 a* -t

K 3 rs

3* 3

Vi h-*

120

Cellular Implications ofRedox Signalling

• uv Chemokine- and/ cytokinc-likc, effect moa

Oxidative Stress

• X-ray • ishemia/reperfusion • viral infection • chemical carcinogen

——.

Transcription) / [ Factors J J l L ^ » AP-1

TRX gene

X active x active

apoptosis -1--1--

/->. M™?

CNJMS^T)

* PEBP2 PEBP2 / P53 I //-►GeneExpressioi T » / • differentiation ' • growth

Fig. 2. Intracellular a n d extracellular activities of thioredoxin.

known as thioltransferase, has GSH-disulfide oxidoreductase activity with redox-active site, -Cys-Pro-Tyr-Cys-.29 GRX reduces low molecular weight disulfides and proteins in concert with NADPH and GSH reductase. There is accumulating evidence that GRX as well as thioredoxin plays an important role in redox regulation of signal transduction. Grx regulates the activation of transcription factors such as nuclear factor I,30 OxyR31 and PEBP-2.32 We detected differential expressions of GRX and thioredoxin in the differentiation of macrophage 33 and mouse embryos. It is also reported that GRX is detected within the HIV-1 virus and regulates the activity of glutathionylated HIV-1 protease. 34 Mammalian thioredoxin 2 (Trx2) has high homology with thioredoxin and has an active site Cys-Gly-Pro-Cys with thiol-reducing activity.35 It has mitochondrial insertion signal and is specifically localized in mito­ chondria. Recent studies have shown that mitchondorial thioredoxin reductase, Trx2 and mitchondorial peroxiredoxin III compose one of the mitochondorial antioxidant system as well as manganese-superoxide dismutase (Mn SOD) and mitchondorial GSH/GPx system.36,37 To studys the function of Trx2, we cloned the chicken Trx2 cDNA and generated conditional Trx2 deficient cells expressing a tetracycline-repressible Trx2

The Role of Thioredoxin in Regulatory Cellular Functions 121

Table 2. Peroxiredoxin family in human, (modified from the Refs. 38 and 85).

Prxl : PrxII : Prx III : Prx IV : PrxV : Prx VI :

Name

Length in Amino Acids

Localization

PAG, NKEFA TSA, NKEFB AOP-1 AOE372, TRANK AOEB166 ORF6

199 198 256 271 214 224

Cystosol and nucleus Cytosol Mitochondria Cytosol / secreted Mitochondoria / microsome Cytosol

transgene, using a DT 40 cell line.86 The growth of Trx2 deficient cells was significantly retarded and most of Trx2 deficient cells fell into apoptosis. And intracellular ROS levels increased in Trx2 deficient cells. Thioredoxin 2 deficient cells were more sensitive to exogenous hydrogen peroxide and GSH depletion. Moreover, cytochrome c was released into the cyto­ plasm and caspase-9 was activated in Trx2 deficient cells. These results indicate that Trx2 not only regulates the generation of ROS through Trx2/ peroxiredoxin system in mitchondoria but also plays a crucial role in the mitchondorial apoptotic signal pathway. However, the biological func­ tions of these members have not yet been fully clarified. Peroxiredoxins are considered to be members of a new family for intracellular hydrogen peroxidase 38 (Table 2). Six members of peroxire­ doxin family have been identified in human, all of which utilizes thiore­ doxin as the electron donor except peroxiredoxin VI. The features and functions of peroxiredoxin family were well described in a recent review elsewhere. 39 Thus, the thioredoxin system is composed of several related molecules forming a network of recognition and interaction through its active site cysteine residues.

4. Thioredoxin Knock Out and Transgenic Mice To analyze the biological functions of thioredoxin, we developed thiore­ doxin knock out mice. Taketo et al. characterized the mouse genome, which contain one active thioredoxin gene on chromosome 1 and one processed pseudogene on chromosome 4.40 The thioredoxin gene extends over 12 kb and contains five exons separated by four introns. 41 To develop thioredoxin knock out mice, a part of the mouse thioredoxin gene including

122 Cellular Implications ofRedox Signalling

Fig. 3. Thioredoxin overexpressing transgenic mice (modified from Ref. 8) (a) Design of transgene of thioredoxin. (b) Expression of thioredoxin in various tissues of transgenic mice.

the translation start codon was deleted by homologous recombination in embryonic stem (ES) cells. Heterozygotes are viable, fertile and appear normal. Thioredoxin hetero-knock out mice are now available and under investigation for stress sensitivity. In contrast, homozygous mutants die shortly after implantation at the egg cylinder formation stage.42 One pos­ sible explanation for the early lethality of thioredoxin homo-knock out embryos is impaired DNA replication after maternal thioredoxin is lost in the embryo. Interestingly, Ref-1 deficient mice also die shortly after implantation at day 5.43 Since Ref-1 and thioredoxin operate coordinately in the redox-sensitive activation of transcription factor such as AP-1 or in the DNA repair/replication, both Ref-1 and thioredoxin may be essential for early embryonic development. Then, we have developed thioredoxin overexpressing transgenic mice (Trx-Tg mice) where human thioredoxin is overexpressed in C57/BL6 strain mice systemically by using beta-actin promoter. 8 Human thiore­ doxin cDNA was inserted between the beta-actin promoter and the betaactin terminator and used to generate the transgenic mice [Fig. 3(a)]. The apprearance and behavior of Trx-Tg mice are normal. Thioredoxin-Tg mice contain several fold larger amounts of h u m a n thioredoxin protein in the most organs compared with endogenous mouse thiore­ doxin protein level [Fig. 3(a), 4].

The Role of Thioredoxin in Regulatory Cellular Functions 123

Fig. 4. Expression of human thioredoxin (hTrx) in the brain of transgenic mice. Immunohistochemical study of thioredoxin in Tg mice hTrx was observed in cortex (A, C) and hippocampus (B, D) of Tg mice but not of WT mice (E, F). A hTrx signal was shown in only Tg mice, (modified from Ref. 8.)

5. Characteristics of Trx-Tg Mice 5.1 Resistance Against Focal Cerebral Ischemic Injury8 Focal cerebral ischemia was induced by the occlusion of the middle cerebral artery using the intraluminal filament technique in mature male mice under general anesthesia. Twenty four hours later, the animal was sacrificed and the brain section was analyzed. The infarcted areas and volume in Trx-Tg mice were significantly smaller than in wild type C57BL/6 mice (Fig. 5). Since oxidative modification of proteins is accompanied by the gener­ ation of protein carbonyl derivatives, the protein carbonyl contents of the soluble fraction of crude brain cortical extract preparations were analyzed at 24 hrs after ischemia. The protein carbonyl contents in Trx-Tg mice were significantly less than in wild type mice.

5.2 Resistance Against Excitotoxic Hippocampal Injury9 Thioredoxin-Tg mice also showed a resistance against kainic acid-induced excitotoxicity, in which the oxidative stress is involved. Mice were injected intraperitoneally with 20 m g / k g kainic acid. The mice were observed for seizure incidence for 1 hr after the injection. Although seven of ten Trx-Tg

124

Cellular Implications ofRedox Signalling

(mm3) 120 100 "

n=each9, *p<0.01 Fig. 5. Infarct volumes in Tg and WT mice. Infarct volume was smaller in Tg mice than in WT mice (n = 9 each), (modified from Ref. 8) mice and all ten wild type mice exhibited severe seizure, the mean seizure score described elsewhere was significantly lower in Trx-Tg mice than in wild type mice at 30 min and 45 min after treatment. Seven days after kainic acid treatment, hippocampal neuronal damage was assessed by cresyl violet staining. Thioredoxin-Tg mice showed more undamaged neu­ rons in the hippocampal CA1 and CA3 regions than wild type mice.

5.3 Resistance Against Oxidative Stress and Elongated Survival Our recent data shows that Trx-Tg mice are more resistant to oxidative stress than wild type C57BL/6 mice. Fasting-induced lipid peroxidation measured by thiobarbituric acid-reactive substances (TBA-RS) formation is reduced in the liver of Trx-Tg mice as compared with wild type mice. Bone marrow cells from Trx-Tg mice are more resistant to UV light exposure-induced cytocide than those from wild type mice. Moreover, Trx-Tg mice exhibited extended life span. Relative to wild type C57BL/6 mice, the percent increase in the Trx-Tg mice was 22% in the maximum life-span and 35% in the median life-span. Telomerase activity in spleen tissues in Trx-Tg mice was higher than that in wild type mice (Mitsui et al., submitted). We also have found that Trx-Tg mice are more resistant to

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125

bleomycin-induced lung fibrosis and cytokine-induced lethal interstitial pneumonia than wild type mice (Hoshino et al., in preparation). Moreover, when wild-type E7.5 mouse embryos are cultivated in vitro under a high oxygen concentration, their growth is retarded and developmental abnor­ malities are frequently produced (Kobayashi et ah, submitted). In contrast, embryos from Trx-Tg mice grow normally. These results suggest that Trx-Tg mice are resistant to a variety of oxidative stresses.

5.4 Pancreatic Beta Cell Specific Overexpression of Thioredoxin 44 Several reports have shown that locally produced ROS are involved in the destruction of pancreatic beta cell, resulting in the development of diabetes mellitus. Non-obese diabetic (NOD) mice spontaneously develop autoimmune diabetes with remarkable similarity to human insulin-dependent DM. In cooperation with Miyazaki et al., NOD transgenic mice that overexpress thioredoxin in the pancreatic beta cells by using human insulin promoter was produced to elucidate the effect of thioredoxin on the ROS-induced development of DM. The Ins-thioredoxin transgene containing a human thioredoxin cDNA under the human insulin promoter was microinjected into fertilized eggs of NOD mice to generate NOD transgenic mice. Human thioredoxin mRNA was exclu­ sively detected in the pancreas by RT-PCR. Western blotting using antihuman thioredoxin antibody showed a high level of human thioredoxin expression in the lysate of pancreatic islets from transgenic mice. There was no difference of insulin secretion capacity between NOD thioredoxin-transgenic mice and thioredoxin-negative littermates. To examine the effect of thioredoxin expression on the development of DM, female NOD thioredoxin-transgenic mice and thioredoxin-negative litter­ mates were monitored for glucosuria u p to 32 weeks of age. The onset of glucouria was significantly retarded and the cumulative incidence of dia­ betes was significantly reduced in NOD thioredoxin-transgenic mice compared with thioredoxin-negative littermates. Next, the protective role of thioredoxin was examined in another diabetes model induced by streptozocin, an ROS-inducing agent. Male NOD thioredoxin-transgenic mice were mated with female C57BL/6J mice to produce (NOD X B6) Fl mice. There was no difference of fasting blood glucose levels between (NOD X B6) Fl thioredoxin-transgenic mice and their non-transgenic littermates. Streptozocin (250 mg/kg) was injected intraperitoneally in 8-week-old (NOD X B6) Fl thioredoxin-transgenic mice and their non-transgenic

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littermates. Seven days after streptozocin injection, the blood glucose levels were elevated more than 500 m g / d l in seven of nine non-transgenic mice and only two of nine thioredoxin-transgenic mice. Pancreatic insulin contents were significantly higher in thioredoxin-transgenic mice than those in thioredoxin-negative littermates. These results clearly indicate that thioredoxin plays a crucial role to prevent oxidative stress-induced development of diabetes mellitus.

6. Extracellular Function of Thioredoxin Human thioredoxin is secreted from various types of cells including acti­ vated normal B-lymphocytes and virus-transformed cells, and secreted thioredoxin exhibits several cytokine-like activities.20,22 In 1985, human thioredoxin was first purified from the culture supernatant of an adult T-cell luekemia (ATL) cell line ATL-2 transformed with HTLV-I, as ATLderived factor (ADF).4S It induces IL-2 receptor alpha-chain (IL-2Ralpha) expression on human large granular lymphocyte (LGL) cell line YT and thus it was considered a novel cytokine with IL-2Ralpha inducing activity.14 In 1989, the cDNA sequence revealed that ADF is a human homologue of thioredoxin. 27 Expression and production of thioredoxin are markedly enhanced in Epstein-Barr virus (EBV)-infected lymphoblastoid B-cell lines as well as in HTLV-I-infected T-cell lines. During the study of B-cell transformation by EBV, Wakasugi and co-workers independently reported an IL-1-like soluble factor produced by EBV transformed B-lymphoid cell named 3B6-IL-1, which was later found to be identical to ADF/Trx.17,27'46 This factor has a co mitogenic activity on thymocytes and human HSB-2 cell line, despite the complete lack of the pyrogenic activity that is a char­ acteristic nature of macrophage-derived IL-ls. The producer cell 3B6 uses this factor as an autocrine growth factor. Later on, several other cytokinelike factors previously reported were shown to be identical to or related to thioredoxin. These include MP6-BCGF,47 B-cell growth factor derived from the T-cell hybridoma MP6, that is comitogenec with IL-4 and other cytokines; eosinophil cytotoxicity enhancing factor (ECEF),48 produced by activated U937 cells; one component of early pregnancy factor (EPF),49 originally defined as an immunosuppressive serum factor; and surface associated sulfhydryl protein (SASP).50 While thioredoxin is predominantly localized in cytosol and play a pivotal role in the maintenance of redox status in cells, it is secreted by lymphocytes, hepatocytes, fibroblasts, virus infected cells and cancer cells

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in response to a variety of stimuli, and act as a cytokine.17'47,51'52 Because the protein has no classical secretory signal sequence, thioredoxin is con­ sidered to be released through a unique thiol-related mechanism. The secretion of thioredoxin as well as IL-lbeta is enhanced by drugs that block transport by the classical secretory pathway, 52 suggesting that thioredoxin and IL-lbeta use a novel leaderless pathway for secretion. Although the mechanism of extracellular thioredoxin action has not been fully understood, the enzymatic activity of thioredoxin seems to be cru­ cial for the most of its biological functions. Indeed, a catalytic site mutant thioredoxin (C32S/C35S), in which the two active cysteines are substi­ tuted to serine residues, has no growth promoting activity.53 The require­ ment of the catalytic site has also been shown for other biological functions including chemokine-like effects (see below) and regulation of transcription factors.

7. A Truncated Form of Thioredoxin A truncated form of thioredoxin lacking the C-terminal 16 or 24 amino acids was first described as eosinophil cytotoxicity enhancing factor (ECEF) with 10-kDa molecular weight. 48 It shows significant cytotoxicityenhancing activity on eosinophils and U937 cells at concentrations as low as 10 pM, whereas full-length thioredoxin shows no such activity. Although the truncated thioredoxin retains the conserved active site, it has no dithiol-reductase activity. These data indicate that the enzymatic activity do not correlate with the ECEF activity. The cleavage of exo­ genous recombinant thioredoxin by uninfected and HIV-infected macrophages was described in the earlier report.54 However, the proteolytic cleavage mechanism of thioredoxin has not been clarified. More recently, it was shown that endogenous truncated thioredoxin is pro­ duced and released from normal monocytes and other cell lines such as MP6, 3B6 and U937 by physiological stimuli and oxidative stress.55'56 It should be noted that all these cells produce and release full-length thiore­ doxin as well. Previous studies on the MP6-derived thioredoxin, which was a mixture of thioredoxin and truncated thioredoxin, showed that its B-cell stimulatory activity was more than 100-fold higher than purified placental derived thioredoxin. 57 To the contrary, opposing effects of thioredoxin and truncated thioredoxin have been shown on the develop­ ment of HIV-I infection.54 A recent report by Pekkari et al. showed that a recombinant truncated form of thioredoxin (1-80 aa) by itself, which is a

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dimer in solution, is a potent mitogenic cytokine stimulating growth of resting human peripheral blood mononuclear cells (PBMC), whereas fulllength thioredoxin at the same dose is not.58 The cytokine-like effects of the two proteins may be mediated via different mechanisms. Thioredoxin as a co-stimulatory molecule of cytokine action; thiore­ doxin is also a potent co-stimulus in the expression and release of cytokines.18 The expression of cytokines is redox regulated, and thiore­ doxin can augment the expression and release of several cytokines including IL-1, IL-2, IL-6. IL-8 and TNF-alpha.18 Thioredoxin exhibits growthpromoting activity in combination with IL-2. In addition, thioredoxin augments the growth promoting effects of other cytokines or growth fac­ tors on lymphocytes as well as on non-lymphoid cells.17,51 Cytoprotective activity of thioredoxin is mainly explained by its scavenging activity for ROS.22 As ROS are considered signal messengers, the scavenging effect of thioredoxin may contribute to the modulation of immune responses by cytokines and growth factors through ROS-mediated signals.

8. Chemokine-Like Activity of Thioredoxin In 1993, the first report about chemokine-like activity of thioredoxin pro­ vided evidence that thioredoxin can induce migration of eosinophils from patients with hyper-eosinophilia, although thioredoxin exhibited little activity on eosinophils from healthy donors. 59 Thioredoxin also shows enhancing effects on both chemotactic and chemokinetic activity of the complement anaphylatoxin peptide C5a on eosinophil migration. In con­ trast, thioredoxin shows no modulation of migratory behavior of human eosinophils by IL-3, IL-5, or granulocyte-macrophage colony-stimulating factor. A catalytic site mutant thioredoxin (C32S/C35S), shows neither migration activity nor enhancing effect. More recently, chemokine activ­ ity of thioredoxin towards polymorphonuclear leukocytes (PMNs), monocytes, and T-lymphocytes from normal individuals was clearly demonstrated by Bertini et al. in a standard in vitro chemotaxis assay with micro Boyden chambers. 60 The potency of the chemotactic action of thioredoxin was comparable with that of known chemokines such as IL-8 for PMNs, MCP-1 for monocytes, and RANTES for T-cells, respectively, at the optimal concentrations (0.1-2.5 nM; 1-30 ng/ml). The chemokine activity of thioredoxin was also demonstrated in vivo using murine air pouch model. As mutant thioredoxin (C32S/C35S) is not chemotactic, the chemokine activity is related to its enzymatic action like most of the

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biological effects of thioredoxin. While the family of chemokines are G-protein dependent, the chemokine action of thioredoxin is G-protein independent, 30 suggesting that it is not mediated via known chemokine receptors. The prerequisite of the enzymatic activity suggest that thiore­ doxin may modulate membrane target proteins or receptors through the interaction with their thiols to transduce its signal. Neither specific pro­ teins nor reservoirs on cell membrane have been discovered yet.

9. Circulating Thioredoxin Levels in Human Plasma Circulating thioredoxin can be detected in plasma of healthy donors. The plasma levels of full-length thioredoxin and truncated thioredoxin were determined as the median value of 16-55 ng/ml and 2-175 ng/ml, respec­ tively, by sandwich ELISA.24,58 The levels of thioredoxin or truncated thioredoxin in plasma are within the physiological range in terms of their cytokine- or chemokine-like activities. These data suggest that plasma thioredoxin levels may reflect a variety of states of inflammatory reac­ tions and immune responses against extracellular stimuli such as virus infection. Actually, thioredoxin levels are increased in plasma of HIV-infected individuals. 24 In addition, the association between elevated plasma thioredoxin and decreased survival in HIV-infected patients was recently reported.10 An increase of plasma or serum thioredoxin has also been described in other diseases such as hepatocellular carcinoma (HCC),61 and rheumatoid arthritis. 62 Considering the two forms of thioredoxin (full-length and truncated thioredoxin) have different biological functions as described above, levels of these proteins in plasma samples are prefer­ ably distinguished and analyzed.

10. Thioredoxin Binding Proteins Several thioredoxin-binding proteins have been isolated. MAP kinase is involved in one of signaling pathways activated by various oxidative stresses.63 Apoptosis signal-regulating kinase (ASK-1) described by Ichijo et al. mediates apoptosis signal by activating the c-Jun N-terminal kinase (JNK) and p38 MAP kinase pathways. It was shown to become inactive by binding to reduced thioredoxin. 64 When thioredoxin is oxidized under oxidative stress, it dissociates from ASK-1 and apoptosis signal is transduced (Fig. 2). Nishiyama et al. have reported another thioredoxin

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binding protein, TBP-2, which is identical to vitamin D3-upregulated protein-1 (VDUP1).5 In 1 alpha, 25-dihydroxyvitamin D3-treated HL-60 cells, TBP-2/VDUP1 expression was enhanced, whereas thioredoxin expres­ sion and the reducing activity were down-regulated. 1 alpha, 25-dihydroxyvita­ min D3 is important for regulation of calcium homeostasis and hormone secretion65 and is a potent inducer of myeloid cell differentiation. It is likely that Trx-TBP-2/VDUPl interaction plays an important role in the redox regulation of growth and differentiation of the cells sensitive to a variety of inducers, including 1 alpha, 25-dihydroxyvitamin D3 responses. The redox active site in thioredoxin is required for the binding to both TBP-2 and ASK-1. The conformation of thioredoxin adjacent to the active site may be critical for the binding between these molecules. A phagocyte oxidase component, p40 phox was also reported to have thioredoxin binding property, although the physiological role of Trx-p40 phox interaction is still unclear.66 These three proteins were all cloned by yeast two-hybrid system. Watson et al. identified thioredoxin as a protein kinase C (PKC)-interacting protein using phage display system and showed that exogeneouly added thioredoxin inhibits auto-phosphorylation of PKC and PKC-mediated phosphorylation of histone in vitro.67 In future other new thioredoxin binding proteins would be cloned and understanding of novel roles of thioredoxin would be given to us.

11. Redox Regulation of Transcription Factors by Thioredoxin Transcription factors are important sensing and signaling components of oxidative signaling. Redox regulation appears to be involved in various steps of activation of transcription factors. Several important transcription factors have been shown to be modu­ lated by reducing agents.712,68 Thioredoxin is one of key mediators that modulate activities of transcription factors (Fig. 1). Ref-1, AP endonuclease (APEX), enhances DNA-binding activities of many nuclear transcription factors such as AP-1, NF-kappaB, ATF/CREB, Myb, HIF-lalpha 69 and p53 through redox-dependent mechanism. The reduction of Ref-1 by thiore­ doxin is required for its activity.3,6 Thus, thioredoxin regulates many transcriptional events thorough Ref-1. Thioredoxin can also directly enhance the DNA binding activities of several factors. Ueno et al. reported that thioredoxin facilitates p53-mediated p21 activation through both Ref-1-independent and -dependent mechanisms. 4 The enhancement of

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DNA binding activity by thioredoxin was also reported in nuclear receptors such as glucocorticoid receptor70 and estrogen receptor. 71 Regulation of NF-kappaB by thioredoxin has been extensively investigated.72"74 Treatment of NF-kappaB with oxidants such as diamide or hydrogen peroxide renders the protein incapable of DNA binding, while reduction of the protein by thioredoxin as well as thiol reductants such as DTT enhances its DNAbinding activity. This action is associated with the reduction of Cys62 in the DNAbinding loop of NF-kappaB p50 by thioredoxin. 74 Interestingly, studies on targeted-over-expression of thioredoxin either in cytosol or in nucleus have indicated that thioredoxin has dual and opposing roles depending on its localization in the regulation of NF-kappaB. 75 In the cytosol, thiore­ doxin interferes with the activities of NF-kappaB by blocking the dissoci­ ation of I-kappaB from NF-kappaB, whereas thioredoxin enhances its DNA binding activities in nucleus. Thioredoxin translocation from cytosol to nucleus is induced by a wide variety of oxidative stresses including UV irradiation, 75 hydrogen peroxide, 15 or hypoxia, 76 treatment with CDDP. 4 Therefore, it is assumed that thioredoxin is translocated to the nuclear compartment upon oxida­ tive stress and/or appropriate stimulation to interact with Ref-1. Nuclear localization of thioredoxin is often observed in pathological tissue. In cervical tissue, thioredoxin expression is observed in human papilloma virus-infected cells and thioredoxin is localized in the nucleus. 77 In the renal proximal tubules, thioredoxin is induced and translocated to nuclei by oxidative damage mediated by Fe-nitrilotriacetate. 78 Although further investigation is needed, thioredoxin translocation may be related to the cell activation, cyto-protection and pathogenesis of oxidative stressrelated disorders. Structural analysis of thioredoxin complexed with Ref-1 or NF-kappaB was performed by Qin et al. and revealed that these complexes represent kinetically stable mixed disulfide intermediates in the same binding loca­ tion of thioredoxin, but in opposing orientations.79,80 This indicates thioredoxin has the potential to target a wide range of proteins by balancing versatility in substrate recognition.

12. Redox Regulation of Apoptosis by Thioredoxin Apoptosis can be considered as a finely regulated mechanism to main­ tain genome stability by eliminating cells with severe DNA damage

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caused by oxidative stress. Jun kinase/SAP kinase and p38 kinase play important roles in oxidative stress-induced apoptosis. 81 Thioredoxin binds to ASK-1, a MAPKKK, and inhibits the apoptosis process in the specificity of the cell type. 64 When cytosolic thioredoxin is oxidized by oxidative stress, ASK-1 is dissociated from oxidized thioredoxin and activated to induce an apoptosis signal. Indeed, we previously showed that thioredoxin prevents apoptosis induced by TNF, 81 or L-cystine and glutathione depletion. 82 In addition, over-expression of thioredoxin negatively regulates p38 Map kinase activation and IL-6 production by TNF-alpha-stimulated cells. Recently, Hirota et ah have shown that thiore­ doxin blocks increase of ROS in response to epidermal growth factor treatment and inhibits p38 MAP kinase activation. (Hirota et ah, paper under revision) These results indicate that thioredoxin plays a criti­ cal role for p38 Map kinase activation. 83 Interestingly, peroxiredoxin II is reported to be an inhibitor of apoptosis with a mechanism distinct from that of Bcl-2.84

13. Conclusion As we described in this chapter, thioredoxin, a redox regulating protein, and its network play important roles in regulating cellular signaling and gene expression. Further analysis of the role of thioredoxin in the oxida­ tive stress response and the mechanism of the thioredoxin gene regula­ tion by oxidative stress should help to elucidate how cells link the oxidative stress response to gene regulation. Because oxidative stress is involved in various diseases as well as cachexia and aging process, increasing knowledge in the redox regulation will lead us to plan a new strategy for diagnosis and treatment of various diseases including pathological aspects of aging.

14. Acknowledgements We thank A. Mitsui (Ajimomoto Co. Ltd,), K. Hirota (Kyoto University), T. Hoshino (Kurume University), and M. Kobayashi (Kyoto University) for providing unpublished observations.

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59.

60.

61.

62.

63. 64.

65. 66.

67.

68. 69.

Cellular Implications of Redox Signalling

blood mononuclear cells and is present in human plasma. /. Biol. Chem. 275: 37474-37480 Hori K, Hirashima M, Ueno M, Matsuda M, Waga S, Tsurufuji S, Yodoi J. 1993. Regulation of eosinophil migration by adult T-cell leukemia-derived factor. /. Immunol. 151: 5624-5630. Bertini R, Howard OM, Dong HF, Oppenheim JJ, Bizzarri C, Sergi R, Caselli G, Pagliei S, Romines B, Wilshire JA, Mengozzi M, Nakamura H, Yodoi J, Pekkari K, Gurunath R, Holmgren A, Herzenberg LA, Herzenberg LA, Ghezzi P. 1999. Thioredoxin, a redox enzyme released in infection and inflammation, is a unique chemoattractant for neutrophils, monocytes, and T-cells. /. Exp. Med. 189:1783-1789 Miyazaki K, Noda N, Okada S, Hagiwara Y, Miyata M, Sakurabayashi I, Yamaguchi N, Sugimura T, Terada M, Wakasugi H. 1998. Elevated serum level of thioredoxin in patients with hepatocellular carcinoma. Biotherapy. 11: 277-288 Maurice MM, Nakamura H, van der Voort EA, van Vliet AI, Staal FJT, Tak PP, Breedveld FC, Verweij CL. 1997. Evidence for the role of an altered redox state in hyporesponsiveness of synovial T-cells in rheumatoid arthritis. /. Immunol. 158: 1458-1465 Ichijo H. 1999. From receptors to stress-activated MAP kinases. Oncogene 18: 6087-6093 Saitoh M, Nishitoh H, Fujii M, Takeda K, Tobiume K, Sawada Y, Kawabata M, Miyazono K, Ichijo H. 1998. Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK-1). EMBO } . 17: 2596-2606 Suda T, Shinki T, Takahashi T. 1990. The role of vitamin D in bone and intestinal cell differentiation. Ann. Rev. Nutr. 10: 195-211 Nishiyam A, Ohno T, Iwata S, Matsui M, Hirota K, Masutani H, Nakamura H, Yodoi J. 1999. Demonstration of the interaction of thioredoxin with p40phox, a phagocyte oxidase component, using a yeast two-hybrid system. Immunol. Lett. 68:155-159 Watson JA, Rumsby MG, Wolowacz RG. 1999. Phage display identifies thioredoxin and superoxide dismutase as novel protein kinase C-interacting proteins: Thioredoxin inhibits protein kinase C-mediated phosphorylation of histone. Biochem. }. 343 (Part 2): 301-305 Arrigo AP. 1999. Gene expression and the thiol redox state. Free Radic. Biol. Med. 27: 936-944 Ema M, Hirota K, Mimura J, Abe H, Yodoi J, Sogawa K, Poellinger L, Fujii-Kuriyama Y. 1999. Molecular mechanisms of transcription acti­ vation by HLF and HIF-lalpha in response to hypoxia: Their stabilization

The Role of Thioredoxin in Regulatory Cellular Functions

70.

71.

72.

73.

74.

75.

76.

77.

78.

139

and redox signal-induced interaction with CBP/p300. EMBO } . 18: 1905-1914 Makino Y, Okamoto K, Yoshikawa N, Aoshima M, Hirota K, Yodoi J, Umesono K, Makino I, Tanaka H. 1996. Thioredoxin: A redoxregulating cellular cofactor for glucocorticoid hormone action. Cross talk between endocrine control of stress response and cellular antioxidant defense system. /. Clin. Invest. 98: 2469-2477 Hayashi S, Hajiro-Nakanishi K, Makino Y, Eguchi H, Yodoi J, Tanaka H. 1997. Functional modulation of estrogen receptor by redox state with reference to thioredoxin as a mediator. Nucleic Acids Res. 25: 4035^040 Schulze-Osthoff K, Schenk H, Droge W. 1995. Effects of thioredoxin on activation of transcription factor NF-kappaB. Meth. Enzymol. 252: 253-264 Okamoto T, Sakurada S, Yang JP, Merin JP. 1997. Regulation of NFkappaB and disease control: Identification of a novel serine kinase and thioredoxin as effectors for signal transduction pathway for NF-kappaB activation. Curr. Top. Cell Regul. 35:149-161 Matthews JR, Wakasugi N, Virelizier JL, Yodoi J, Hay RT. 1992. Thioredoxin regulates the DNA binding activity of NF-kappaB by reduction of a disulphide bond involving cysteine 62. Nucleic Acids Res. 20: 3821-3830 Hirota K, Murata M, Sachi Y, Nakamura H, Takeuchi J, Mori K, Yodoi J. 1999. Distinct roles of thioredoxin in the cytoplasm and in the nucleus. A two-step mechanism of redox regulation of transcrip­ tion factor NF-kappaB. /. Biol. Chem. 274: 27891-27897 Ema M, Mimura J, Abe H, Yodoi J, Sogawa K, Poellinger L, FujiiKuriyama Y. 1999. Molecular mechanisms of transcription activation by HLF and HIF-1 alpha in response to hypoxia: Their stabilization and redox signal-induced interaction with CBP/p300. EMBO /. 18: 1905-1914 Fujii S, Nanbu Y, Nonogaki H, Konishi I, Mori T, Masutani H, Yodoi J. 1991. Coexpression of adult T-cell leukemia-derived factor, a human thioredoxin homologue, and human papillomavirus DNA in neoplastic cervical squamous epithelium. Cancer 68: 1583-1591 Tanaka T, Nishiyama Y, Okada K, Hirota K, Matsui M, Yodoi J, Hiai H, Toyokuni S. 1997. Induction and nuclear translocation of thioredoxin by oxidative damage in the mouse kidney: Indepen­ dence of tubular necrosis and sulfhydryl depletion. Lab. Invest. 77: 145-155

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79. Qin J, Clore GM, Kennedy WM, Huth JR, Gronenborn AM. 1995. Solution structure of human thioredoxin in a mixed disulfide inter­ mediate complex with its target peptide from the transcription factor NF-kappaB. Structure 3: 289-297 80. Qin J, Clore GM, Kennedy WP, Kuszewski J, Gronenborn AM. 1996. The solution structure of human thioredoxin complexed with its target from Ref-1 reveals peptide chain reversal. Structure 4: 613-620 81. Ichijo H, Nishida E, Irie K, Dijke PT, Saitoh M, Moriguchi T, Takagi M, Matsumoto K, Miyazono K, Gotoh Y. 1997. Induction of apoptosis by ASK-1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science 275: 90-94 82. Iwata S, Hori T, Sato N, Ueda TY, Yamabe T, Nakamura H, Masutani H, Yodoi J. 1994. Thiol-mediated redox regulation of lym­ phocyte proliferation. Possible involvement of adult T-cell leukemiaderived factor and glutathione in transferrin receptor expression. /. Immunol. 152: 5633-5642 83. Hashimoto S, Matsumoto K, Gon Y, Furuichi S, Maruoka S, Takeshita I, Hirota K, Yodoi J, Horie T. 1999. Thioredoxin negatively regulates p38 MAP kinase activation and IL-6 production by tumor necrosis factor-alpha. Biochem. Biophys. Res. Commun. 258: 443-447 84. Zhang P, Liu B, Kang SW, Seo MS, Rhee SG, Obeid LM. 1997. Thiore­ doxin peroxidase is a novel inhibitor of apoptosis with a mechanism distinct from that of bcl-2. /. Biol. Chem. 272: 30615-30618 85. Koops B, Clippe A, Bogard C, Arsalane K, Wattiez R, Hermans C, Duconseille E, Falmagne P, Bernard A. 1999. Cloning and characteri­ zation of AOEB166, a novel mammalian antioxidant enzyme of the peroxiredoxin family. /. Biol. Chem. 274: 30451-30458 86. Tanaka T, Hosoi F, Yamaguchi-Iwai Y, Nakamura H, Masutani H, Ueda S, Nishiyama A, Takeda S, Wada H, Spyrou G, Yodoi J. 2002. Thioredoxin-2 (TRX-2) is an essential gene regulating mitochondriadependent apoptosis. EMBOJ. 21: 1695-1703

Chapter 7 Protein S-Thiolation, S-Nitrosylation, and Irreversible Sulfhydryl Oxidation: Roles in Redox Regulation James A Thomas* and Robert Mallis Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University Ames, Iowa 50011-3260 "[email protected] Helmut Sies Heinrich-Heine- University Institutfur Physiologische Chemie I Postfach 101007 D-40001-Duesseldorf, Germany [email protected]

Keywords: S-thiolation, S-nitrosylation, Sulfhydryl oxidation, protein cysteine

1. Summary Protein S-thiolation, S-nitrosylation, and irreversible oxidation are oxidative modifications of protein sulfhydryls that may significantly modify cellular metabolism. The first two forms of oxidation are fully reversible in intact cells. They generate interest as regulatory events that potentially modify a large number of processes in response to both oxida­ tive and nitrosative stress. Examination of nearly any metabolic or signal transduction pathway produces a number of candidate proteins that might be altered by oxidative modification of this kind. Thus, the signifi­ cance of the combined effects of oxidation on many different proteins may produce cellular metabolic changes necessary for survival during short term oxidative or nitrosative stress. It may be necessary to understand 141

142

Cellular Implications ofRedox Signalling

these processes by combining many individual protein modifications into a global metabolic plan in order to fully understand the importance of oxidative protein modifications. Irreversible oxidation, in which protein sulfhydryls are oxidized to states that are not normally reversible by metabolic processes, is less appreciated as an important biological event and little data exist concerning the extent to which it occurs in vivo. Thus, the rate of degradation and turnover of irreversibly oxidized proteins in which the only damage is to a reactive sulfhydryl is largely speculative at the present time, and accumulation of proteins containing damaged sulfhydryls is a hypothetical event suitable for discussion. Intracellular proteins contain a variety of reactive cysteines that have diverse roles in the biology of eukaryotic cells. The surface exposure of these cysteines is a functional necessity that imposes a restrictive redox state on the intracellular environment. For the most part, normal cellular homeostasis would seem to be designed to maintain a fully reduced state of these cysteines in order to maintain normal biological function. The chemistry associated with reduced protein thiols seems to provide many essential roles in catalysis, regulation, electron transfer, and protein struc­ ture. Thus, oxidative or nitrosative stress is particularly threatening to the function of proteins whose role depends on the nucleophilic or coordinate bonding properties of a protein cysteine. This chapter will discuss the functions of reactive cysteines, the protein structural elements relevant to those functions, and evidence that both oxidation and nitrosative stress alter the oxidation status of these protein sites. Finally we will discuss the potential for irreversible damage to these sulfhydryls and suggest the possibility that various cellular functions are compromised by a popula­ tion of unusable proteins that have irreversibly oxidized cysteines.

1.1 Roles of protein cysteines in cells Cysteine contains a thiol functional group that provides sites for formation of coordinate bonds to metal ions, a nucleophilic atom that can readily add to electrophilic sites on small molecules, and an easily reversible site for oxidation/reduction chemistry. In addition it provides a site for covalent attachment of materials that can modify protein properties significantly. Intracellular proteins contain a variety of zinc and iron binding sites that utilize the coordinate bonding properties of the cysteine sulfur. These include sites where the role seems to be entirely structural, i.e. a zinc atom is coordinately bound with tetrahedral chemistry to four ligands includ­ ing two, three, or four protein sulfhydryls. These sites are prominent in

Monothiol Modification in Redox Regulation

143

DNA binding proteins where structures that interact with DNA are frequently dependent on zinc binding sites.1 However, such sites also occur in other enzymes and proteins, as exemplified by alcohol dehydrogenase and aspartate transcarbamylase. Apparently the tight complex of cysteine and zinc is used as an alternative and more complex variant to the structural protein disulfides that are found abundantly in extracellu­ lar proteins. Since oxidized cysteines may be only transiently permitted in the reduced environment inside cells, the complex formed with a struc­ tural zinc is a very feasible alternative to disulfide protein cross-links. In proteins containing iron-sulfur centers, the participating cysteine probably has a direct role in the redox chemistry associated with the center. In many of these proteins the cysteines are quite buried but in the case of aconitase, it has become clear that enough surface exposure exists to permit oxidative modification of the site.2 Another chapter in this book discusses this protein in detail. Such a site is also thought to be important in the regulation of the prokaryote transcription factor, SoxR.3 The nucleophilic role of sulfhydryls is well documented and it is clear that active site cysteines are important catalytic entities in both transfer/ addition reactions (S-transferases, glyceraldehyde 3-P dehydrogenase, glutathione reductase, thioredoxin/glutaredoxin), and in peptidase (caspase, calpain, papain) activity. These cysteines may be among the most acidic (lowest pKa) since the nucleophilicity is enhanced by ionization of the thiol to the thiolate anion. The protein chemistry leading to the ionization of the thiol usually depends on the charge and electron with­ drawing properties of the surrounding protein structures. 4 It appears that all acidic cysteines are not directly involved in catalysis, however, as exemplified by the enzyme, creatine kinase. 5 Covalent adducts to protein cysteines, i.e. lipids, ADPribose, and protoporphyrin rings, may be essential to function. When the adduct is linked by a thioester bond, it may be transitory and have transitory regu­ latory effects on the function of protein. Palmitoylation of proteins results from thioester protein links with reactive cysteines, and it is well known that this modification is dynamic. 6 ADPribose is attached to a protein cysteine by an S-glycoside bond and this addition is also known to be reversible.7 On the other hand, adducts linked through thioether bonds, i.e. prenyl anchors and protoporphyrin rings, are not known to be reversible. When a farnesyl or geranyl isoprenoid is attached to the C-terminal cysteine, it is thought to produce membrane association of the protein. 8 Cytochrome c is a good example of a thioether-linked protoporphyrin ring. Finally, many reactive cysteines found on protein surfaces may have no known metabolic role other than a postulated antioxidant or protective

144

Cellular Implications ofRedox Signalling

function.8 Additional functions of such reactive cysteines may yet be elucidated by new experimental approaches to the study of protein function. It is postulated that protein cysteines involved in each of the biologi­ cal roles described above, may also react with oxidative molecular species that are the subject of this chapter. One of the following sections of this chapter will provide examples of proteins that are known to be potential oxidation sites in cellular metabolism. 1.2 Molecular Mechanisms of S-thiolation and S-nitrosylation Both S-thiolation and S-nitrosylation may result from the action of reactive oxygen or nitrogen species. Thus, each may represent a protein modifica­ tion that is primarily effective as a response to oxidative or nitrosative stress, but the oxidative nature of both protein modifications makes is likely that they may have much in common. Both are reversible in intact cells, suggesting that they may play important regulatory roles that modify cell metabolism during periods of increased oxidative or nitrosative stress.

1.2.2

S-thiolation

Resting levels of S-thiolated proteins in cells are low (less than 1% of the total protein is S-thiolated). In order to perturb this state, a rather substan­ tial oxidative stress seems to be required. 910 The best-studied examples demonstrated that S-thiolation of neutrophil 10 or monocyte 11 pro­ teins occurs within minutes after stimulating the oxidative burst. Actin was identified as the most abundant protein involved in the neutrophils, while glyceraldehyde 3-P dehydrogenase was identified in monocytes. In hepatocytes that were co-cultured with stimulated neutrophils 9 a similar rapid response was observed and carbonic anhydrase III was the most abundant protein that was affected. These experiments suggest that tran­ sient S-thiolation occurs in cells which are proximate to immune cells. In cells exposed to a hyperactivated immune response, or which have hypoactive reduction systems, the magnitude and duration of S-thiolation could be increased. Thus there exists a set of conditions in which the steady state is likely to be perturbed by normal functions of the immune system in vivo. Disease states in which immune cells are hyperactivated or activated against self-antigens may produce increased protein S-thiolation in both non-immune and immune cells. It has also been found that other oxidants such as menadione, may induce significant protein S-thiolation, if present at toxic concentrations. 9

Monothiol Modification in Redox Regulation

145

Two mechanisms of S-thiolation have been discussed extensively. Protein disulfide-exchange has long been thought to provide a workable mechanism for oxidation [Reaction (1) below] since oxidative stress frequently increases cellular GSSG concentrations significantly. protein-S" protein-S" protein-SH protein-SH

protein-S' (+ 0 2 )

+ + + +

+

RSSR ROO' R'OH' HOOH

~*

► >► ►

protein-S-SR protein-S' protein-S* protein-SOH

+ + + +

RS" ROO" RH/HOH H20

protective reaction after (2) or (3) leading to S-thiolation: glutathione-SH ► [protein-S-S*" - glutathione] >~ protein-S-S-glutathione + 02'+ H+ (5) protective reaction after (4) leading to S-thiolation: glutathione-SH > - protein-S-S-glutathione + H 2 0

protein-SOH

+

protein-S'

oxidative reaction after (2) (3) or (4) leading to damage: . (0 2 ) ► protein-SOOH/protein S 0 3 H

+

(1) (2) (3) (4)

(6)

(7)

However, thiol disulfide exchange is a rather slow reaction that requires high GSSG concentrations that seem unlikely in intact cells.4 In addition, experiments such as those with neutrophils and hepatocytes cited above have shown that protein S-thiolation can occur without significant increases in GSSG. Thus, a glutathione-dependent trapping mechanism in which oxi­ dized protein sulfhydryls are generated as either a thiyl radical or sulfenic acid [Reactions (2) - (4)] may be a primary mechanism for formation of S-thiolated proteins. These activated protein intermediates react with the pool of cellular glutathione to produce a mixed disulfide adduct of protein and glutathione [Reactions (5) and (6)]. Such a mechanism depends on a substantial supply of reduced glutathione to effectively trap partially oxidized protein cysteines (thiyl radical or sulfenic acid forms). This pro­ posed mechanism also leads to the hypothesis that in the absence of suffi­ cient glutathione, partially oxidized forms of protein cysteine may react with oxygen or other oxidants to produce extensively oxidized species such as sulfinic and sulfonic acids [Reaction (7)]. The effectiveness of glutathione in this role may be compromised by some oxidative event that depletes the glutathione pool significantly. This suggestion accounts for the importance of a large cellular pool of glutathione and provides a model for protein sulfhydryl damage that is related to the concentration of cellular glu­ tathione. In general, researchers have assumed that GSH is more reactive towards oxidants than proteins, while in fact, the opposite seems to be true.12 The model for protein S-thiolation has been extensively studied with pure proteins.12"14 Superoxide, H 2 0 2 , and peroxynitrite 15 have been shown

146

Cellular Implications ofRedox Signalling

to directly cause S-thiolation in protein model systems and in cells. Secondary reactions of these molecules result in formation of various radicals (peroxyl, alkyl, thiyl and others), some of which can be shown to cause protein S-thiolation in protein model systems (unpublished results).

2.2.2.

S-nitrosylation

The protein oxidative effects of nitrogen-based reactive species are not as well understood, but analogies to S-thiolation suggest that the molecular mechanisms may have much in common. Attempts to understand both forms of protein modification draw heavily on the chemistry of low molecular weight thiols. This rationale assumes that the complex chemistry of protein thiols with all the potential modifications that result from the protein environment, and that of low molecular weight thiols, is similar. This model has not always provided explanations for observations that include the fact that nitroso glutathione may S-thiolate some cysteines while S-nitrosylating others, 1617 the fact that some cysteines do not form S-glutathiolated species as a consequence of charge interactions, 18 and the fact that protein S-nitrosylation apparently occurs in the presence of a very large excess of glutathione in intact cells.19 First, the mechanism of formation of protein-NO adducts may involve several fundamentally different mechanisms reminiscent of those proposed for protein S-thiolation (see reactions below). If cellular responses to nitrosative stress are similar to responses for oxidative stress, protein S-nitrosothiols will occur to an extent greater than or equal to S-nitrosylation of the glutathione pool. It has been suggested that transnitrosation of proteins from low molecular weight S-nitrosothiols is a feasible mecha­ nism for protein S-nitrosylation [Reaction (8)]. This reaction is quite remi­ niscent of thiol/disulfide exchange and probably is significant only when high concentrations of S-nitrosoglutathione are manifested in cells. Transnitrosation could therefore be significant in causing S-nitrosylation of intracellular proteins if scavenging of NO occurs to a significant extent in the extracellular space. Data indicate that low levels of S-nitrosothiols occur in vivo (approximately 10 uM in plasma, 23 less than 100 pmol/mg of cellular protein in NIH-3T3 cells19). However, in order to cause significant modification of S-nitrosothiols in cells, external concen­ trations of S-nitrosothiols such as S-nitrosocysteine or S-nitroso­ glutathione must be in millimolar concentrations or greater.

Monothiol Modification in Redox Regulation + + + + + + NA + Protein-SH Protein-S-N"-Or][ +

Protein-SH or Protein-SH Protein-SH Protein-S* 4NO"

(Protein-SH Protein-SH

+ +

RSNO -M RSNO < ONOO" NO"

°2

► ► >. >-

►

Protein-SH NO-

—^~ ^=*~

o2

—►

NO" RSNO

+

electron

Protein-SNO Protein-SSR Protein-S" Protein-SNO 2N203 HNO : Protein-S-N"-OH Protein-SNO

+ +

+ + +

RSH NO"

Protein-SNO

o- 2 -

147 (8)

+

H* (9) (Ref 20) (10) (Ref 21) (11) (Ref 22)

- ► Protein-SNO + reduced acceptor) acceptor + NO". -► Protein-SSR

Thus, in order for S-nitrosylation to occur in vivo, S-nitrosothiol concen­ trations must be at least 100-fold greater than at resting levels. Thus, transnitrosylation for the formation of S-nitrosylated proteins and thioldisulfide exchange for the formation of S-thiolated proteins may suffer from the same limitation, i.e. the concentration of available low molecular weight reactants seems too low to account for significant protein sulfhydryl modification. However, there are two important differences between S-nitrosylation and S-thiolation systems. First, GSSG may S-thiolate sites relatively slowly because of its net negative charge (-2) and steric interference by the bulk of a glutathione molecule regardless which sulfur is attacked by the thiolate nucleophile. Thus, direct oxidation of proteins [Reactions (2) - (6)] should have a greater contribution to S-thiolation than would thiol-disulfide exchange. However, GSNO has a less negative charge (-1) without the bulk of the glutathione moity to interfere with a nucleophilic attack on the nitrogen of the N O [Reaction (8)]. The orientation of the S-nitrosothiol during the nucleophilic attack (i.e. attack on the nitrogen of the N O or the sulfur atom of the glutathione) may be influenced by the charge characteristics of the site in question. 1617 Positively charged residues in close proximity to the thiolate may be able to orient the glutathione mol­ ecule in some cases so that the nucleophilic attack is on the sulfur atom and an S-glutathiolated protein is produced [Reaction (8)] as is the case for creatine kinase. Alternatively, GSH may associate with some protein sites and an S-glutathiolated end product may result via an S-nitrosylated protein intermediate. Thus charge and steric factors may account for the reported occurrence of both S-thiolation and S-nitrosylation of proteins in cells treated with exogenous S-nitrosocysteine18 and may ultimately deter­ mine the relative contribution of transnitrosylation versus radical medi­ ated mechanisms for S-nitrosylation of protein thiols. Secondly, GSSG is normally not able to reach very high concentrations in cells because of the efficacy of the glutathione disulfide reductase

148 Cellular Implications ofRedox Signalling

enzymatic system and because any disulfides produced in the extracellular space do not readily cross the cellular membrane. In contrast, S-nitrosothiols are able to cross cellular membranes, apparently by transport processes that have not been completely elucidated.19,24 Thus, cells exhibit selectivity for uptake of low molecular weight S-nitrosothiols and S-nitrosocysteine is more readily taken up than S-nitrosoglutathione.19,25 The uptake of low molecular weight S-nitrosothiols may be cell-type specific. Protein disulfide isomerase (PDI), which exhibits differential expression with cell type, has also been implicating in transporting NO into cells via trans-nitrosation.24 The inability of cells to take up S-nitrosothiols may be a mechanism by which the nitrosative effects are blunted in the cells proximal to immune cell activation. To date, no quantitative measure of the nitrosative stress that results from the oxidative burst has been made, although relative lev­ els have been assessed in cell culture conditions.26 Measurements of intracellular S-nitrosothiols have not been made in cells proximal to immune cell activation. Thus, even if S-nitrosothiols are scavenged in the extracellular space, there is the potential for their entering nearby cells. It is possible that concentrations of GSNO do not have to be as high for S-nitrosylation and S-thiolation to occur as GSSG concentrations would need to be for signifi­ cant S-thiolation to occur. However, transnitrosation is most likely to be a mechanism for redistribution of S-nitrosothiols among protein thiols and for denitrosation of protein S-nitrosothiols. A second mechanism similar to that proposed for S-thiolation may require formation of either a reactive protein intermediate [thiyl radical— Reactions (2), (3) or (9)] or some reactive low molecular weight species other than S-nitrosothiol [Reaction (9) and (10)]. Because nitrosative stress occurs in an environment of oxidative stress during immune cell activation, and may itself cause oxidative events [Reaction (11)], mechanisms of this type may ultimately play a very large role in modification of protein thiols.

2.2.3. Interaction of S-nitrosylation Irreversible Oxidation

and S-thiolation

or

The potential for interchange between the S-nitrosylated state and the disulfide state of the cysteine is significant. S-thiolated proteins and low molecular weight disulfides form rapidly inside cells that are treated with extracellular S-nitrosothiols.19 Disulfide formation may be a physiologi­ cally important step in the degradation of S-nitrosothiols, since disulfides are a measured end product of nitrosothiol degradation 27 and free thiols seem to be requisite in S-nitrosothiol degradation in vivo.28 A recent report

Monothiol Modification in Redox Regulation

149

has suggested that glutathione disulfide S-oxide, also a degradation pro-duct of GSNO, is a much more effective thiolating agent than GSSG,29 but the study does not investigate the formation of a mixed proteinglutathione S-oxide, which may form just as readily in mixtures of proteins, GSH and GSNO. Reaction (8) is a simplification of what has proven to be a complex reaction path, 30 but in vivo studies bear out the overall stoichiometry remarkably well. 19 In purified protein systems, GSNO S-nitrosylates all four available cysteines on H-ras with only minimal S-glutathiolation,18 while it has recently been shown that S-nitrosothiols can selectively S-glutathiolate one cysteine on creatine kinase without formation of the protein S-nitrosothiol.17 It has been suggested that S-nitrosothiols are converted to irreversible oxidation products of thiols although scant physical evidence exists of these reactions. This lack of evidence may, however, be due to the lack of methods for measuring these modifications easily (see Sec. 2 of this review). The oxidized product of Reaction (11) can react with N O to form the protein sulfenic acid and nitrous oxide:27 Protein-SN'OH

+

NO

►

Protein-SOH

+

N20.

(12)

Since protein sulfenic acids may be intermediate to protein S-thiolation, combining rapidly with intracellular GSH [Reaction (6)], this reaction could be responsible for protein denitrosylation in vivo. Additionally, oxidative events apparently convert S-nitrosylated cysteine to sulfinic or sulfonic acid,30,31 although the reaction path for this is not well understood. Protein-SNO

+

electron acceptor

► Protein-Sox

(x = 2,3).

(13)

Studies have shown complex reaction pathways in which NO and a reduced thiol can be oxidized to an S-nitrosothiol. Because NO can cross cellular membranes, it remains uncertain whether NO generated extracellularly reacts with thiols in the extracellular space or whether S-nitrosothiols form largely inside the cell. If S-nitrosothiol formation occurs mainly in the extracellular space, then one would expect that much of the S-nitrosothiol would not be able to enter cells, particularly the protein S-nitrosothiols such serum albumen, reportedly the most abundant S-nitrosothiol in plasma. 32 Cells also exhibit selectivity for uptake of low molecular weight S-nitrosothiols. Data indicate that S-nitrosocysteine is more readily taken up than S-nitrosoglutathione. 1925 Because amino acid transporters may be involved in this transport, the uptake of low mole­ cular weight S-nitrosothiols may be cell-type specific. Protein disulfide isomerase (PDI), which exhibits differential expression with cell type, has

150

Cellular Implications ofRedox Signalling

also been implicating in transporting N O into cells via transnitrosation. 24 The inability of cells to take up S-nitrosothiols may be a mechanism by which the nitrosative effects are blunted in the cells proximal to immune cell activation. To date, no quantitative measure of the nitrosative stress that results from the oxidative burst has been made, although relative levels have been assessed in cell culture conditions 33 and measurements of intracellular S-nitrosothiols have not been made in cells proximal to immune cell activation. While there are many studies showing S-nitrosylation of proteins in vitro, few studies show S-nitrosylation of specific proteins isolated from cellular systems. The cardiac calcium release channel was found to be S-nitrosylated on one thiol per subunit (each subunit contains 21 cysteine residues) in isolated canine hearts.34 Methionine adenosyltransferase was significantly S-nitrosylated in rat hepatocytes treated with S-nitrosoglutathione monoethyl ester, resulting in enzyme inhibition. 35 Caspase-3, which has a reactive cysteine residue required for activity, was immunoprecipitated from three different human B- and T-cell lines and found to be S-nitrosylated constitutively 36 Fas stimulation decreased caspase-3 S-nitrosylation. Most recently, H-ras found to be S-nitrosylated in immunoprecipitates from NIH-3T3 cells treated with S-nitrosocysteine.18 Since the hypothetical control of metabolic and signaling pathways depends upon modification of thiol groups, these studies represent a start to research that will undoubtedly increase significantly in the future.

1.3 Molecular Mechanism of Dethiolation and Denitrosylation Since both of these mild oxidative events are reversible in intact cells, the reversal reactions may also contribute significantly to the importance of each modification. Both glutaredoxin and thioredoxin proteins are poten­ tially involved as reductants in dethiolation. 37-39 Other chapters in this book provide details on the cellular roles of these proteins, but it should be emphasized that S-glutathiolated proteins are probably uniquely sensitive to glutaredoxin. This aspect of glutaredoxin's action was recently highlighted when the structure of S-glutathiolated carbonic anhydrase III became available. Subsequent modeling studies with both glutaredoxin and thioredoxin showed that glutaredoxin could form a productive com­ plex with the S-glutathiolated protein without significant protein-protein interactions between the glutaredoxin and carbonic anhydrase III. The figure shows the complex that can be formed between these two proteins

Monothiol Modification in Redox Regulation

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Fig. 1. A molecular model of the complex between S-glutathiolated carbonic anhydrase III and glutaredoxin. This model was developed from the published struc­ tures of carbonic anhydrase III (1FLJ) and glutaredoxin (1GRX). The bond angles for the three sulfur intermediate between these two molecules were optimized.

in which the carbonic anhydrase Ill-bound glutathione acts as a docking site for the glutaredoxin molecule. A productive 3-sulfur complex (left figure) is easily demonstrated with this model. On the other hand, thioredoxin could not be modeled into a complex without considerable overlap between the two proteins. It seems likely that the action of thioredoxin would have less specificity and require higher concentrations since it did not easily form a reductive complex. Experiments in two laboratories have supported this probability. Denitrosation of proteins has not been studied as thoroughly and relevant information must again be obtained from experiments with low molecular weight S-nitrosothiols. Recent experiments in intact cells showed that denitrosation of intracellular proteins occurs at nearly the same rate as dethiolation of glutathiolated proteins. 19 It is interesting that the removal of high molecular weight thiols, but not low molecular weight thiols, significantly diminished the ability of plasma to degrade GSNO.32 Similar studies have demonstrated the critical role of thiols in nitrosothiol degradation as well as a minor role for divalent cations.28 To date evidence is inconclusive with resect to an enzymatic system for

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Cellular Implications ofRedox Signalling

Table 1. Methods used to study protein cysteine oxidative modification Method

Detection Limit Advantages

Disadvantages

Gel Electrofocusing

Micrograms of protein

Techniques for method require some practice. Issues unresolved for detecting proteins by immunoblot.

Capture/Release

Micrograms of protein (pmol of adduct)

Radioactive Detection

Micrograms of protein

MAL-PEG

Nanograms of protein

MAL-Biocytin

Nanograms of protein

Readily Quantitated Adduct can be identified. Multiple Samples per determination. A single method can be used for S-nitrostylation, S-thiolation, and irreversible oxidation. Adduct can be identified. Quantitation possible. Individual assays available for S-nitrosylation and S-thiolation. Identification of modified protein.

Very sensitive. Quantitaion is possible. Multiple samples per determination. Differentiates between reversible and irreversible oxidation. Very sensitive. Multiple samples per determination. Can differentiate between reversible and irreversible oxidation.

Extensive sample preparation protocol. No method for irreversible oxidation. Assay is specific. Protein synthesis must be inhibited. Adduct identity not possible. Not method for S-nitrosylation or irreversible oxidation. Cannot differ­ entiate between possible reductionsensitive adducts.

Quantitation is questionable. cannot differentiate between possible reductionsensitive adducts.

Monothiol Modification in Redox Regulation

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Table 1. Continued Method

Detection Limit Advantages

Sulfinic / Sulfonic Acid Analysis

Micrograms of protein

Protein Activity/Function

Below nanograms of protein

Disadvantages

Unambiguous Extensive sample identification of preparation. protein modification. Multiple samples increase labor. Very sensitive Cannot identify methods. protein Activity correlates modification. with cellular Each protein function. requires specific methodology.

denitrosylation of protein or low molecular weight S-nitrosothiols, but degradation of S-nitrosothiols may be linked mechanistically to the reduc­ tion of protein and low molecular weight thiols (see above and Ref. 40).

1.4 Effects of Oxidative Modification on Protein Function The importance of post-translational covalent modification has a long history that places it in a central position with regard to cellular regulation. Most often the concepts that evolved to understand allosteric regulation have been incorporated into our understanding of the mechanisms for these protein effects. Thus, effects of protein phosphorylation can clearly be understood in terms of a two or more state protein model in which modification either favors some protein conformation or where it occurs only on a particular conformation of the affected protein. The structural elucidation of the glycogen phosphorylase model demonstrated that phosphorylation may cause very specific protein conformation changes that result in dramatic changes in protein function. To date, there is no similar model or concept to explain the importance of oxidative modifi­ cation in metabolic regulation. It seems clear that exposed protein sulfhydryls can be modified almost randomly, lacking the specificity that seems inherent in the phosphorylation system. In addition oxidative events such as S-glutathiolation, S-nitrosylation, or irreversible oxidation have generally been shown to have similar effects on the modified protein. Each of these modifications produces distinct chemistry where the addition product is negatively charged (S-glutathiolation and irre­ versible oxidation), or even uncharged (S-nitrosylation). The specific

154

Cellular Implications ofRedox Signalling

Table 2. Model for oxidation of H-ras Cysteine Modified 118 181,184 186

Type of Modification

Effect of Modification

S-nitrosylation S-nitrosylation, S-glutathiolation S-nitrosylation, S-glutathiolation

Activation of GTP turnover Inhibition of palmitoylation, Inactivation Inhibition of farnesylation, Inactivation

protein changes generated by these modifications have been documented only for carbonic anhydrase III. In that case the S-glutathiolation seems to have no effect on the protein, and the attached glutathione molecules do not perturb the protein structure in the least. The generation of structural information on other proteins is one of the fundamental needs that must be undertaken in the future in order to support a regulatory role for protein sulfhydryl modification.

2. Analytical Methods for Protein Oxidation The single most important limitation to understanding the biology of protein cysteine oxidative modification is the availability of sensitive methods for each specific protein modification. The following is a brief overview of methods which have been used successfully to date, empha­ sizing sensitivity, selectivity, and applicability to experiments with intact cells and tissues.

2.1 Protein Separation by Charge 2.1.1 Gel Isoelectric Focusing The most versatile method for the study of oxidation of protein cysteines is gel isoelectric focusing (IEF). Proteins differing by as little as one charge can be separated by IEF, providing both quantitative and qualitative information about protein oxidation.12'13,30 Detection of IEF-separated pro­ teins is similar to SDS-PAGE (i.e. Coomassie blue staining), and analysis of 1 (Xg or less are routine. Coomassie blue stained band densities are proportional to protein content over approximately 5-fold range, and multiple dilutions of samples are beneficial. Qualitative information can

Monothiol Modification in Redox Regulation

155

include the number of sites modified, which sites are modified, and the type of oxidative modification, i.e. S-glutathiolation, irreversible oxidation, or S-nitrosylation. While IEF is most readily implemented on proteins with only one or two reactive cysteines, when used in combination with point mutants, proteins with as many as four sites can be studied effec­ tively. The method depends on appropriate chemical modification of protein samples including reducing agents such as dithiothreitol (DTT) to identify irreversible modifications such as sulfinic or sulfonic acids, and alkylating agents such as iodoacetamide (uncharged) and iodoacetic acid (single negative charge). Thus, uncharged adducts (S-nitroso or S-cysteine groups) can be detected by alkylation with a charged agent, and charged adducts (S-glutathionyl groups) require an uncharged agent. Multiple samples can be separated on a single IEF gel, routinely up to twenty samples can be applied per gel. Native gel IEF is commonly used, but urea-based denaturing IEF gels have been of some use as well. Native IEF has been useful for some cytosolic proteins, however IEF has pro­ duced mixed results when separations of membrane-associated proteins or protein sub-domain constructs were attempted. An advantage which may seem at first to be a disadvantage is that proteins which appear pure by other methods such as SDS-PAGE separate as multiple bands with IEF. Even when other methods will be used to assay protein oxidation, IEF is an useful tool for determining the purity of the protein substrate. IEF has been used to study a single protein in cellular extracts in combi­ nation with Western blotting techniques. The sensitivity for Western blotting appears to be much less than for SDS-page Western blotting. The decreased sensitivity results from unidentified interference with antibody binding to the transferred protein. Theoretically, this methodology will detect picogram quantitities of proteins in complex mixtures, making IEF-Western blotting potentially very powerful if the technical issues are resolved.

2.1.2 Capture/Release - Measurement of Adducts from Isolated Proteins

Released

Glutathione may be released from protein with DTT and the glutathione may be measured by various methods such as HPLC, mass spectrometry, scintillation counting (35S-glutathione), or a combination of these. There are several attractive features shared by these methods regardless of the ultimate means of detection. Among the advantages is the identifica­ tion of the adduct by either molecular weight, comigration with standard

156

Cellular Implications ofRedox Signalling

compounds in HPLC separations, or by incorporation of radioactive label. Quantitation is achieved by normalizing the results of the assay for protein content, which is trivial for pure proteins, but does introduce significant error into the assay. When measuring modification of proteins from cellular extracts, protein content may be normalized against known amounts of pure protein separated by SDS-PAGE and detected by Western blotting. In both purified and mixed protein systems, the protein must be separated from any low molecular weight thiols in the reaction mix­ ture. This may be accomplished by dialysis, precipitation with trichloroacetic acid (TCA), or by immunoprecipitation (IP). Great care must be taken to ensure that all non-covalently bound thiols are removed from the protein (proteins may have to be dialyzed up to five days to remove non-covalently bound thiols in cellular extracts) before release of covalently bound thiols with DTT. Detection limits for HPLC methodology vary, but, typically, picomoles of GSH can be detected. If cells are radioactively labeled with 35S methionine/cysteine mixture, the maxi­ mum specific activity which can be expected is ~250 cpm/pmol placing the lower limit of detection at the picomole level. Thus, if one expects 1-10% (mole/mole) modification of the protein, ~1 ug of protein is required to make an accurate determination of S-glutathiolated protein. While mass spectrometry is not quantitative, approximately 1 picomole of compound is needed per assay, making detection limits similar to HPLC and radiolabeling methodologies. S-nitrosylation can be detected by releasing the adduct from the protein. If Hg 2+ is added to the S-nitrosothiol under acidic conditions, nitrous acid (nitrite at higher pH) is formed. Nitrite is readily quantified by a variety of assays including the Greiss reagent assay for which a con­ servative detection limit is on the order of 100 picomoles.41 Fluorometric assay systems such as the diamino-naphthalene (DAN) assay reliably detect as little as 10 picomoles of nitrite. 42 Measurement of photolysisreleased NO using chemiluminescence provide quantitation of as little as 1 picomole of NO, 36 placing the sensitivity for this methodology around the same level as that for S-glutathiolation. Again, great care must be taken to ensure that proteins are separated from any low molecular weight contaminants which may interfere with the assay. These methodologies may also be applied to protein mixtures (e.g. total cytosolic proteins) and in this case sensitivity is not an issue. Because a wide variety of proteins may be immunoprecipitated from cells routinely, these methodologies are suited for most proteins.

Monothiol Modification in Redox Regulation

2.1.3 Radioactive

157

Methods

Cells can be incubated with 35S methionine/cysteine to label the cellular glutathione pools and proteins from these cells may be separated by SDS-PAGE and the dried gel may be exposed to autoradiography film.10 This method is advantageous in that it provides a direct link between the radiolabeled adduct and the protein in question and so may be used in conjunc­ tion with the release and detect methodology described above to help confirm the results. A disadvantage of the technique is that cycloheximide must be used in order to prevent incorporation of 35S-cysteine into proteins. Incubation with the labeling medium must be short to minimize alterations in cellular metabolism during labeling. This short incubation time prevents complete incorporation of label into the cellular glutathione pool, resulting a cellular glutathione specific activity which is about 1/10 that of labeling cysteine. For rapid turnover proteins, the need for cycloheximide may be critical. As a general rule, to be able to detect S-thiolation of a given protein, there should be ~1 ug of protein per lane on an SDS-page. Thus, this tech­ nique is about as sensitive as the DTT-release methodologies above. Although glutathione accounts for >90% of all protein bound thiols, the iden­ tity of the protein adduct cannot be ascertained directly. Theoretically, it should be possible to quantify the amount of adduct if one is able to normalize for amount of protein as described above, provide a radioactive standard for use with the gel, and determine the specific activity of the glutathione pool. However, in practice, this methodology remains qualitative.

2.1.4 Maleimide-Derivatized

Polyethylene

Glycol

(Mal-PEG)

Detection of oxidatively modified cysteines on proteins. Cellular proteins with reactive cysteines are substrates for reaction by with maleimidederivatized polyethylene glycol (Mal-PEG).43 The resulting protein adducts have an increased size that is easily detected by SDS-PAGE. In conjunction with Western blotting, the detection limits for this method are considerable better than those already described. For each reactive sulfhydryl that reacts with Mal-PEG, the apparent molecular weight of the protein increases by the size of the PEG adduct. Thus, multiple sites produce several bands of increased size. When a specific reactive cysteine is oxidized, the reaction with Mal-PEG is blocked. If all reactive cysteines are blocked, Mal-peg has no affect on the molecular size of the protein.

158 Cellular Implications ofRedox Signalling

This method can be used to detect either reversible oxidative/ nitrosative modification of a protein (modifications that are readily reversed by addition of dithiothreitol such as S-thiolation, S-nitrosylation, or protein disulfide formation), or irreversible modification of a protein (the oxidative modification is not affected by the addition of di-thiothreitol). This technique provides two distinct advantages over other cellular protein methods. First, the sensitivity is equivalent to that of SDSPAGE/Western blotting, i.e. protein amounts can be easily detected in the low nanogram range. Thus this technique is about 1000 times more sensi­ tive than any of the other techniques. Second, the number of adducts per mole of protein can be quantified using densitometry. Thus, no external standards are needed. Using DTT to remove all reducible sulfhydryl modifications, one is left with irreversibly oxidized sulfhydryls that will not react with Mal-PEG. The primary disadvantage of this technique is that there is no way to determine the identity of any oxidative adduct. While it is useful to determine the exact nature of modification in cells, the ability to monitor the kinetics of modification of the protein in concert with activation and inactivation in situ makes this technique extremely valuable for future research.

2.1.5 Maleimide-derivatized Biocytin (MAL-biocytin) Fluorescent Detection of Oxidatively Modified Cysteines on Proteins The use of biocytin-conjugated maleimide (MAL-Biocytin) was first introduced some years ago,44 this technology has recently been adapted for use in determining surface exposed loops in membrane proteins. 45 Like the MAL-PEG assay, the sensitivity is essentially the same as SDSPAGE/Western blot. Modification can be quantified as a percentage of total thiols for each protein, but determining the stoichiometry of reac­ tive thiols is not trivial. This technology could also be used to measure total protein thiols in an ELISA-type of assay using an automated plate reader.

2.2.6 Irreversible

Oxidation

Most recently, our laboratory has developed a robust method for deter­ mination of both sulfinic and sulfonic acid in purified proteins. 46 The

Monothiol Modification in Redox Regulation

159

method requires protection of reduced sulfhydryls and subsequent acid hydrolysis of the protein and separation by reverse phase HPLC. Treatment of replicate samples with sodium hypochlorite before hydrolysis oxidizes any protein sulfinic acid to a sulfonic acid. The method utilizes other amino acids in the protein to normalize the extent of modification for any protein with a known amino acid sequence. The detection limit for this assay is in the low picomolar range, typically requiring between 5 and 10 |J.g of protein. Thus, the sensitivity is within an order of magnitude of the detection of S-nitrosylated or S-glutathiolated species.

2.1.7 Protein Activity

and Binding

Assay

Because thiols are critical for the activity of some proteins, protein activity may be used to indirectly measure oxidative damage to these proteins. However, kinetic analysis of reversible modification of sites on these proteins is complicated, since stopping oxidation/reduction of reactive sites requires either addition of reducing agents or alkylating agents. With the careful use of controls it should be possible to measure recovery of activity in previously alkylated samples upon reduction with DTT of reversibly oxidized proteins and loss of activity due to irreversible oxida­ tion by comparison of DTT treated samples compared with activities of untreated samples. However, great care must be taken to insure that reac­ tions at secondary sites have no effect on protein activity. Obviously, correlation of oxidative events in cells with protein activity is of great value, but characterization of modification by some of the other method should be carried out as well.

3. Oxidative Modification of Reactive Cysteines in Selected Proteins We will divide the following discussion into abundant proteins and those of less abundance. In general the data available for abundant proteins provides a basis for understanding potential changes in less abundant proteins, but it seems clear that the most important regulatory effects of oxidative modification may occur on the less abundant proteins involved in signal-transduction and gene regulation.

160

Cellular Implications ofRedox Signalling

3.1 Abundant Proteins Much of the basic information about protein sulfhydryl oxidation has been derived from proteins that are found at relatively high concentration in vivoF^ For some proteins, the only data about oxidative modification has been obtained by assessment of either enzyme or binding activity. These types of experiments have relied heavily on establishing that the oxidative modification of interest affected the purified protein in a manner consistent with activity or binding changes that occur in intact cells. On the other hand, several studies have examined specific molecular changes in pro­ teins associated with oxidative or nitrosative stress of intact cells. The combination of these two types of experiments, i.e. assessment of mole­ cular modification of a specific protein in vivo correlated with changes in the activity of that protein, have not often been achieved.

3.1.1 Glyceraldehyde 3-P Dehydrogenase GAPDH has a reactive cysteine that is directly involved in the catalytic mechanism of the protein since it forms a covalent intermediate with the substrate during catalysis. Any oxidative modification of this cysteine produces a completely inactive enzyme. Since it is an abundant protein in many cells, it has been the subject of many reports describing oxidative modification during both oxidative and nitrosative stress. In cultured monocytes, the extent of S-glutathiolation correlated with the initiation of the oxidative burst, while dethiolation correlated with the cessation of the oxidative burst. 11 S-glutathiolation of the protein has also been observed in endothelial cells,50"52 and it was suggested that the protein might be oxidized to the S-glutathiolated form even in NO-treated cells. The propensity of this protein to form S-glutathiolated species may be related to the acidic nature of the reactive cysteine. In experiments with the yeast, two different isoforms of GAPDH were studied. Surprisingly, only one of these isoforms was regulated by S-thiolation during oxidative stress.53 S-ADP-ribosylation of the protein was reported in NO-treated cells.54,55

3.2.2 Carbonic anhydrase III CAIII is one of many isoforms of carbonic anhydrase and it was discovered that it contained two reactive cysteines several years ago.9 Interestingly, no

Monothiol Modification in Redox Regulation

161

function has yet been determined for these reactive cysteines. Since the protein is expressed at high levels in some cell types, it has been relatively easy to study the oxidative modification of the protein. Recently the struc­ ture of S-glutathiolated form of this protein was published. 56 The reactive cysteines183186 both reside in areas of negative surface charge density. One cysteine183 is less reactive and has at two conformations, one of which is clearly more buried. The protein has been reported to be S-glutathiolated 9 and possibly even irreversibly oxidized in both cultured cells and whole animals. Recent evidence suggests that its expression is related to oxida­ tive stress.57iS8 Molecular mechanisms of both S-glutathiolation and dethiolation of CAIII have been studied extensively. It has been suggested that the protein is S-glutathiolated by direct oxidation 1213 and that glutaredoxin is a very efficient catalyst of the dethiolation reaction.14 The kinetics of S-glutathiolation and dethiolation in vivo correlated with the amount and duration of added oxidants. S-glutathiolated carbonic anhydrase has been found in aged rats suggesting that the oxidation state of protein cysteines is altered with aging.59

3.1.3 Creatine Kinase The cytoplasmic form of CK has one reactive cysteine per subunit and although the cysteine is not a part of the catalytic mechanism, it is clearly important for enzyme activity.5 The protein is very abundant in a number of muscle cells and it is available from commercial sources at high purity. It has been used for a number of model studies in which S-thiolation, S-nitrosylation, dethiolation, and irreversible oxidation have been explored. Oxidative modification of the reactive cysteine completely inhibits enzyme activity. It is one of the most acidic protein cysteines and is prob­ ably completely ionized at neutral pH. Recent publication of the structure of this protein 60 has made it possible to understand oxidation experiments at the molecular level. The reactive cysteine clearly resides directly between an area of surface positive charge density and an area of surface negative charge. The cysteine is important for substrate binding in the active site. Early experiments showed that the protein was inhibited by S-thiolation, and that oxidative inactivation of the enzyme in cardiac cells could be explained by this mechanism. 13 Recently, it has been demon­ strated that the acidic cysteine in this protein reacts in a unique manner

162

Cellular Implications ofRedox Signalling

with S-nitroso glutathione, forming S-glutathiolated creatine kinase in preference to the S-nitrosylated protein.61 This property may be related to the surface charge properties of creatine kinase, changing the nucleophilic character of the thiolate anion necessary for reaction with appropriate molecules. This protein is easily S-glutathiolated by several reactive oxygen species if a pool of reduced glutathione is present.13'62 When the glu­ tathione concentration is inadequate, such reactions produce irreversibly oxidized forms of the protein that recently were identified as the sulfinic acid and sulfonic acid species.46 Surprisingly, the sulfinic acid form of the protein was present in abundance. Recent experiments indicate that cellular proteins may contain little sulfonic acid but significant amounts of the sulfinic acid.

3.1.4 Glycogen

Phosphorylase

Glycogen metabolism utilizes several enzymes that contain reactive sulfhydryls, i.e. glycogen synthase, glycogen phosphorylase, protein phosphatases and kinases. Enzymes involved in glycogen metabolism were some of the first in which oxidative mechanisms were thought to represent important regulatory mechanisms. 63 ' 64 Phosphoryase b (dephosphorylated) has been used as a model protein for study of protein sulfhydryl oxidation because commercially available protein of high purity is suitable for definitive studies on the mechanism of both S-thiolation and dethiolation.13 It has two reactive cysteines per subunit and oxidative modification by either S-thiolation or S-nitrosylation of these cysteines does not cause any apparent activity change, although the protein may have less affinity for the glycogen particle in the oxidized state (unpublished observations). The protein is easily studied by gel electrofocusing and by other molecular techniques. 30 It has been used as a substrate protein to study the enzymology of protein dethiolation. 37

3.1.5 Glutathione

S-Transferase

mGST (micorsomal glutathione S-transferase) is a rather unusual form of this enzyme that is closely associated with a number of membranes. It has an unusual trimeric structure and it is the only protein known to be acti­ vated by formation of an S-glutathiolated form. 65 Although other

Monothiol Modification in Redox Regulation

163

enzymes including glucose 6-P dehydrogenase, 66 and APS reductase from plants 67 are activated under conditions that might lead to S-glutathiolation, mGST remains the only protein in which activation by S-glutathiolation has been demonstrated by molecular techniques.

3.1.6 Actin Actin is a cytoskeletal protein that has a single reactive cysteine. It has been suggested that S-glutathiolation,10 S-nitrosylation,68 and S-ADPribosylation69 modifications can alter the biological function of the protein. Since actin is found as both a soluble pool of protomers and a polymer­ ized filament in cells, it has been suggested that oxidative modification of the cysteine may have regulatory effects on the polymerization/ depolymerization process.70 Actin's role in neutrophil function has been of particular interest since these cells produce copious amounts of superoxide anion, nitric oxide, and hypochlorite on stimulation. It has also been suggested that oxidation of actin monomers may lead to the generation of covalently linked dimers or even higher oligomers. 71

3.2.7 Hemoglobin Hemoglobins from several eukaryotes including man, are known to have at least two reactive cysteines per tetramer. 72 The protein can be either S-glutathiolated or S-nitrosylated in vitro, and this reaction has been implicated in both transport of protein-bound NO, 73 and in oxidative regulation of red blood cell function during oxidative stress.74 It has been shown that hemoglobin is probably attack by oxidative mechanisms that result in formation of thiyl radicals that may be trapped by added spin traps. 75 Thus, the abundance of hemoglobin in red blood cells makes it a target for several different kinds of oxidative modification.

3.2 Less Abundant Proteins These proteins are generally present at low concentrations in cells and consequently, less is known about molecular events that lead to their oxidative modification in cells.

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Cellular Implications of Redox Signalling

3.2.1 Protein

phosphatases

The effect of oxidative and nitrosative stress on protein phosphorylation may occur by oxidative modification of either protein kinases, protein phos­ phatases, or the phosphoprotein substrate for this modification.76 Evidence for a direct role of oxidative stress as regulators of both protein kinases and protein phosphatases has been published. 77 Recently, it was reported that protein kinase C-a was easily S-thiolated and inactivated, and subsequent experiments with NIH3T3 cells confirmed that it could be S-thiolated in vivo.™ Additionally, S-glutathiolated protein tyrosine phosphatase IB was demonstrated in A431 cells after hydrogen peroxide treatment.79

3.2.2 NF-kappaB and c-Jun/AP-1 Much research effort has been designed to understand the "redox" regu­ lation of the NF-kappaB and c-Jun transcription factors. Recent reviews have treated the subject extensively,48,49 pointing to the very strong possi­ bility that redox regulatory effects are complex. In an important molecular study with c-Jun protein, it was recently reported that S-glutathiolation may be an important aspect of this process. 80 When an NO-generating agent was incubated with the protein in the presence of glutathione, c-Jun became S-glutathiolated. It was suggested that this modification inhibited DNA binding activity of the protein.

3.2.3 p53 The tumor supressor, p53, plays a major role in the transcription ("reading") of DNA, in cell growth and proliferation, and in a number of metabolic processes. Because p53 suppresses abnormal cell proliferation (it acts like an "emergency brake" in the cell cycle), it may represent an important mechanism for protection against cancer. It also appears to be involved in programmed cell death, or apoptosis. When a mutation in the p53 gene results in the substitution of one amino acid for another, p53 loses its abil­ ity to block abnormal cell growth. Indeed, some mutations produce a p53 molecule that actually stimulates cell division and promotes cancer. Almost 50% of human cancers contain a p53 mutation — including cancers of the breast, cervix, colon, lung, liver, prostate, bladder, and skin — and these cancers are more aggressive, more apt to metastasize, and more often fatal. p53 is a potent transcription factor and once activated, it

Monothiol Modification in Redox Regulation

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represses transcription of one set of genes (several of which are involved in stimulating cell growth) while stimulating expression of other genes involved in cell cycle control. It is a phosphoprotein of about 390 amino acids which can be sub­ divided into four domains: a highly charged acidic region of about 75 to 80 residues, a hydrophobic proline-rich domain (position 80 to 150), a central region (from 150 to about 300), and a highly basic C-terminal region. The sequence of p53 is well conserved in vertebrate species, but there have been no proteins homologous to p53 identified in lower eukaryotic organ­ isms. p53 is phosphorylated at many sites by casein kinases I and II, JNK1, cdk's, DNA-PK, and these sites reside in the N- and C-terminal domains of the protein. Recently, it has become clear that oxidative mechanisms can also regulate p53 function. The central region of the protein, whose struc­ ture is shown in the accompanying figure is responsible for DNA-binding activity. It is the only domain of p53 that contains cysteine residues and it does not contain phosphorylation sites. Mutations observed in human tumors and malignancies almost always map to this region of the protein. Apparently, three of the cysteines 176,238, and 242 are essential for a zinc binding site as labeled in the figure. Cys277/182 are clearly the most surface exposed cysteines that are located in the DNA/protein interface. Although mutation of these residues to serine had no affect on biological activities of p53, adducts to these two cysteines would probably impede proper DNA binding. Cysteine 277 is a highly conserved cysteine in p53 as divergent as human and squid, and it is also found in p53-like proteins p51/63 and p73. Mutational studies have shown that cys/ser conversion of cysl76, 238, and 242 affects DNA binding, and transactivation and transformation suppression activity of p53. Similar changes to cys 124, 135,141, and 275 affect both transactivation and transformation suppres­ sion activities. Recent work on the effects of PDTC (pyrrolidine dithiocarbamate) on cellular p53, has suggested that oxidative modification of p53 can indeed be responsible for altered expression of p53-related gene products. Oxidation of p53 was detected by a specific protocol that depends on a mobility shift of p53 when modified by a sulfhydryl reactive form of polyethylene glycol.81 Further experiments with this method have shown that PDTC produces 25% oxidation of p53 suggesting that at least one sulfhydryl on p53 was sensitive to oxidation. Oxidation correlated with a decrease in the activation of p53 downstream effector genes and altered subcellular localization of the protein. 82 The oxidative modification was reversible and additional studies showed that Ref-1 and thioredoxin were

166

Cellular Implications ofRedox Signalling

Fig. 2. Molecular models of p53 interaction with a DNA as depicted in the file 1TUP. effective reductants of the oxidized protein. 83 The site of oxidation has not yet been clarified.

3.2.4 H-ras H-ras (p21hras, Ha-ras) is a low molecular weight (~19 kDa protein) G-protein which is critical to activation of several signal transduction pathways, including the extracellular signal-regulated kinases (Erk-1 and Erk-2). These pathways are activated when several different cell types are exposed to reactive oxygen or reactive nitrogen species.84,85 In most stud­ ies, H-ras was an essential component for the activation of Erk-1/2 by ROS or RNS. H-ras has two types of lipid modifications that are directly bound to reactive cysteines on the protein.86,87 These modifications were thought to be the only mechanisms for covalent regulation of the function of this protein. Recently we reported that H-ras is S-thiolated in NIH-3T3 cells which are exposed to diamide and both S-glutathiolated and S-nitrosylated in cells exposed to S-nitrosocysteine.18 H-ras has four potentially reactive cysteine residues (118,181,184, and 186), the latter three of which reside near the C-terminal prenylation site of the protein. Published structures of H-ras lack information about the three C-terminal cysteines, presumably because this part of the protein has much freedom of movement.88,89 Both X-ray crystal and NMR structures

Monothiol Modification in Redox Regulation

167

show that Cysll8 is surface exposed near the beginning of a critical loop for binding the guanine nucleotide di- or tri-phosphate. It resides in a region of surface negative charge density. Oncogenic forms of H-ras have mutations that result in a loss of the ability of the protein to hydrolyze GTP, thus leaving the protein in a continuously active, GTP-containing state. It has been suggested that S-nitrosylation of Cysll8 can activate H-ras by increasing the turnover of the guanine nucleotide by an unknown mechanism which presumably involves changing the conformation of this loop and affecting the bound guanine nucleotide. S-glutathiolation of this site could not be demonstrated, 18 and it is suggested that either steric restraints around Cysll8 or, more likely, charge repulsion prevents the addition of negatively charged glutathione molecules. Potentially, activa­ tion of H-ras in cells by low levels of H 2 0 2 could occur by S-thiolation with an uncharged thiol like cysteine or by oxidation to a sulfenic, sulfinic or sulfonic acid. The remaining three reactive cysteine residues at the C-terminal of the protein must be lipidated for H-ras to function properly in cells. Cysl86 is farnesylated while Cysl81 and Cysl84 are palmitoylated. Farnesylation is a prerequisite for palmitoylation, and both farnesylation and palmitoylation are presumed to be essential for activation of H-ras, since mutating any of the three C-terminal cysteines interferes with membrane localization and transformation of cells by oncogenic forms of the protein. Because oxidative modification of any of these three cysteines might incur the same loss of function as a Cys mutation by blocking lipid modification reactions, oxidation of these residues is likely to inactivate H-ras. Farnesylation of Cysl86 is an irreversible modification, so oxidation of this residue will only occur on newly synthesized H-ras. Approximately 10% of Cysl86 is available for oxidation in NIH-3T3 cells, since 90% or more of H-ras is farnesylated in these cells. Thus, in cases of acute oxidative insult, oxidative modification of Cysl86 would only occur on the fraction of the cellular H-ras that was not yet farnesylated. Chronic oxidative stress has the potential to modify a larger fraction of Cysl86 and could affect H-ras activity by trapping newly synthesized H-ras in the cytosol. Cysl81 and 184 are normally palmitoylated in cells, and may be more important targets for oxidation. Palmitoylation is a transient modification, with significant rates of turnover of palmitate during the life of the protein. It is known that the palmitates of H-ras turn over more rapidly when cells are incubated with S-nitrosocysteine.90 The mechanism of this increased turnover is speculative at present, but it is tempting to suggest that modification of Cysl81/184 might be involved. Because other signal

168

Cellular Implications ofRedox Signalling

transduction proteins such as the trimeric G-proteins are normally palmitoylated, oxidative events may also affect other signal transduction systems for which H-ras may be a model. All three C-terminal cysteines of H-ras react with S-nitrosoglutathione, generating S-nitrosylated forms of these cysteines. Two of the cysteines are also readily S-glutathiolated. N O adducts have a small size and are neutral while glutathione adducts are considerably more bulky and have a negative charge. Evidence suggests that glutathione is more likely to form stable adducts with neutral or positively sites. The positively charged residues in close proximity to the C-terminus cysteines of H-ras are thought to be important for palmitoylation of a protein. Palmitoylated cysteines may thus represent a subset of cysteine residues which are susceptible to S-glutathiolation. Because surface exposure is a strong determinant for oxidative modification, the localization of palmitoylated and farnesylated cysteines at the C-termini of proteins, which are often unstructured and solvent exposed, makes competition between lipidation and oxidation a likelihood in cells. The following table summarizes the potential modification of specific H-ras cysteines. Minimal nitrosative events may modify any of the four cysteines. If only a small amount of H-ras is activated by S-nitrosylation of Cysll8, it may be sufficient to activate the ERK-1/2 and other pathways. Inactivation of a small fraction of H-ras by blocking of lipidation would have little or no effect on the pathway, since a greater fraction of the protein remains unmodified. At higher levels of ROS or RNS, oxidation of H-ras becomes extensive enough to act as an effective competitor for lipidation. Such events would drastically reduce the participation of H-ras in signal transduction. Thus, H-ras in oxidant-treated cells should become resistant to activation by extracellular ligands such as TNF. A recent study has shown just such an inactivation when cells are exposed to high levels of S-nitrosocysteine,90 although the exact mechanism of inactivation of the pathway was not elucidated. Further studies in this system should explore the interaction between lipidation and oxidation of each cysteine as well as the membrane localization of H-ras and the over­ all activation state of pathways in which H-ras is a participant.

4. Perspective — Questions in Need of Answers The fundamental principles for the oxidative modification of the large pool of exposed and reactive protein cysteines in intact cellular proteins

Monothiol Modification in Redox Regulation

169

are largely untested, but enough progress has been made to suggest the following overall concepts. Protein oxidation probably results from any number of different oxidizing molecules that abstract an electron from various protein locations. Subsequently, the electronic complexity of the protein structure produces electron deficient sulfur atoms at exposed cysteines. These become the reactive sites most likely to be modified by further chemical events. The protein sulfhydryls may be oxidized by a variety of mechanisms including reversible oxidative addition of a cellular metabolic product such as glutathione or nitric oxide. These adducts result in S-glutathiolated or S-nitrosylated proteins. In some cases ADP-ribosylation may be included as a reversible modification. S-thiolation and S-nitrosylation appear to be in direct competition with irreversible oxidation that results from incomplete protection. The attack of readily available oxygen or derived molecules further oxidizes protein cysteine to sulfinic acids and possibly even sulfonic acids. Protein sulfhydryls that are susceptible to these oxidative mechanisms may have several roles in the affected protein. They may be simply antioxidants, residing on surface sites that have no biological role other than reversible oxidation (carbonic anhydrase III). They may be involved directly in catalysis, or in binding substrate to an enzyme (GAPDH, creatine kinase, caspases). They may be necessary for attachment of lipids or other important protein modifications (H-ras). They may be integral to binding sites that are important in transcriptional regulation (p53, Jun-1, NF-kappaB). They are part of internal structures such as iron-sulfur centers or zinc binding sites (aconitase, p53, alcohol dehydrogenase). The functional differences between an S-glutathiolated, S-nitrosylated, or irreversibly oxidized protein cysteine may be very subtle. It is not clear that such differences exist. The different forms of reversible protein sulfhydryl modification are the direct result of different protein surface chemistries, or simple abundance of a particular protein adduct. Although it is important to study these phenomena, the interpretation of the experimental observations will undoubtedly change considerably as we learn more about the metabolic principles that affect these processes. Important progress in this aspect of metabolism will only come with improved methods for detecting protein modifications of interest in unique biological model systems. Methods for detecting protein S-nitrosylation are considerable less effective at present than those for detecting either S-glutathiolation or irreversible oxidation. The use of gel electrofocusing for study of the S-nitrosylation of specific proteins would considerably

170

Cellular Implications ofRedox Signalling

improve this problem. Thus, it is important to consider this and other innovative methods for improving experimentation on S-nitrosylation. Either Mal-PEG of some other reagent of a similar nature seems to pro­ vide a general method for at least detecting the extent of protein sulfhydryl oxidation for low abundance proteins in model systems. Used in conjunction with studies on purified proteins, this method could pro­ vide the first quantitative information on protein sulfhydryl modification of signal-transduction and transcription factor proteins during oxidative or nitrosative stress. Importantly, this same method (Mal-PEG) may also provide valuable information about irreversible damage to specific pro­ teins under these same conditions. Some of the most interesting aspects of protein sulfhydryl modifica­ tion for future study include: (1) Assessing each form of protein modifi­ cation in a model system responsive to several different types of oxidative or nitrosative stress. (2) Assessing the extent and ramifications of irreversible damage to protein sulfhydryls. Indeed, it will be interesting to determine whether protein sulfinic and sulfonic acids are "irreversible" in the biological sense. (3) By direct studies of a single protein one could assess the effectiveness of S-glutathiolation, S-nitrosylation, and irreversible oxida­ tion as modifiers of a specific biological function. (4) It will be important to determine whether proteins containing cysteines of different functional properties are actually modified by either S-glutathiolation or S-nitrosylation in vivo. There is still much to be done to understand the important role of protein sulfhydryls in the normal progression of oxidative and nitrosative stress.

Acknowledgment "H.S. is a Fellow of the National Foundation for Cancer Research (NFCR), Bethesda, MD. Support by Deutsche Forschungsgemeinschaft (SFB 575/B4) is gratefully acknowledged."

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84. 85. 86. 87. 88. 89. 90.

Chapter 8 Radical Scavenging by Thiols: Biological Significance and Implications for Redox Signaling and Antioxidant Defense Christine C Winterbourn Free Radical Research Group, Department of Pathology, Christchurch School of Medicine and Health Sciences, PO Box 4345 Christchurch, New Zealand [email protected]

1. Summary The ability of thiols to undergo reversible oxidation and reduction enables them to contribute to many cell functions. Thiol groups are involved in the activity of numerous enzymes and have a major role in the antioxidant defenses of the cell. Additionally, there are a large number of cell proteins containing reduced thiol groups that are not known to parti­ cipate in enzyme catalysis. It is becoming clear that many of these are bind­ ing or regulatory proteins, the function of which can be modified by oxidation of the sulfhydryl group. Thus, sulfhydryl oxidation can affect cell function not only through inactivating thiol enzymes, but also by altering the binding characteristics of molecules involved in signaling pathways.1"4 Cells undergo a number of responses when exposed to oxidative stress. 5 Thiols are considered to be prime targets for oxidation. On the one hand, the redox state of the cell (which reflects the relative concentrations of oxidized and reduced thiols) influences the sensitivity of the cell to reactive oxidants. On the other, the behavior of different thiols dictates what that response will be. Cells encounter a range of oxidants that differ considerably in their chemical properties. These include peroxides and hypochlorous acid which undergo predominantly two electron reactions with thiols, and free radical species which prefer one electron pathways. Although frequently grouped together as reactive oxygen species, or ROS, it is important to recognize that this is a generic term rather than an entity 175

176 Cellular Implications ofRedox Signalling

Table 1. Potential radical sources in the cell. Mitochondrial respiration Metal catalysed oxidations Peroxidase-mediated oxidations Redox cycling or autoxidation of xenobiotics UV and y-radiation Oxidoreductases e.g. NADPH oxidases, xanthine oxidase, lipoxygenase, nitric oxide synthase Lipid peroxidation

and reactive oxidants vary considerably in reactivity and selectivity. They are likely to undergo quite different reactions in the cell and it should not be assumed that they will all have the same influences on regulatory pathways. The importance of GSH plus glutathione peroxidase as an antioxidant defense system against peroxides is well recognized and understood. 5 The antioxidant role of the thioredoxin peroxidases (peroxiredoxin) systems, which are linked to thioredoxin/thioredoxin reductase, has more recently become apparent.3,6,7 These enzymatic systems act through nonradical mechanisms and are ultimately dependent on NADPH for reducing equivalents. Both may also be involved in redox regulation. Activity of the GSH/glutathione peroxidase system, by altering the GSH : GSSG ratio, has the ability to influence the overall redox state of the cell. However, as discussed elsewhere in this book, there is increasing evidence that more specific redox changes may be more critical. The thioredoxin peroxidase system, by causing selective oxidation of thiore­ doxin and the thiol proteins that it controls, could provide such specificity. How cells control free radicals is less well characterized, at least for mammals. Cells are continually exposed to free radicals from a variety of sources (Table 1). Superoxide dismutase is ubiquitously present to remove superoxide radicals enzymatically, but it is generally considered that other radicals are scavenged chemically by low molecular weight antioxidants such as glutathione, ascorbic acid, a-tocopherol, and dietary components such as the carotenoids and polyphenolics. 5,8 Vitamin E is important in the lipid phase where it is a good scavenger of peroxyl radicals and inhibitor of lipid peroxidation. GSH and ascorbate are aqueous antioxidants. Scavenging by vitamin E generates the tocopheroxyl radical, which must be recycled if vitamin E is to retain its antioxidant capacity. This can occur through reaction with ascorbate and

Radical Scavenging by Thiols 177

Table 2. Examples of oxidation of GSH to its thiyl radical. Compound or Class of Compound

System

Ref.

Tyrosine Phenols Sugars DNA bases Nitrogen dioxide and peroxynitrite Aromatic amines Phenothiazines Ethanol (hydroxyethyl radical)

Peroxidase Peroxidase, radiolysis Radiolysis Radiolysis Direct reaction Peroxidase, autoxidation Peroxidase Thermal decomposition

28,57 26, 30, 57-59 60 61 62 26, 30, 58, 63 58 64

possibly glutathione, 9 resulting in radical transfer from the lipid to the aqueous phase. Ultimately, therefore, protection against lipid peroxyl rad­ icals requires effective aqueous phase scavenging systems. This article con­ siders the radical scavenging properties of GSH and ascorbate and their roles in the antioxidant defenses of the cell. It also considers thiol proteins as potential radical scavengers and whether radical reactions could be involved in regulating redox-sensitive cell functions.

2. Radical Scavenging by GSH GSH reacts with a wide range of radical species. These include hydroxyl, phenoxyl, alkoxyl, arylamino, peroxyl, semiquinone and carbon centred radicals 1011 as exemplified in Table 2. Some of the parent compounds that give rise to these radicals occur physiologically, others are drugs or envi­ ronmental chemicals. Some, such as the flavonoids, are themselves radical scavengers and of interest for their potential health benefits as antioxidants. It is possible that an abilty to channel radicals to physiologi­ cal antioxidants such as GSH may be an important factor in this regard. GSH is typically present inside cells at millimolar concentrations. It is theoretically possible, therefore, for it to scavenge a large proportion of the radicals generated within a cell. For this to be the case, GSH must react sufficiently rapidly with the radicals it encounters to outcompete other potential targets. Furthermore, if it is to provide antioxidant protection, then products of the scavenging reaction must be benign. A characteristic of radical scavenging reactions is that they generate another radical, in this case the thiyl radical, GS". As described in more detail elsewhere,12"14

178

Cellular Implications ofRedox Signalling

there are features of thiyl radical chemistry that are critical for GSH and other thiols to act as effective scavengers and antioxidants. Scavenging by GSH is reversible (Reaction (1), where R" is a geneiic radical), and in many cases, the equilibrium lies far to the left (e.g. for acetaminophen K = 3 x 10"4). R"

+

GSH <

»

GS"

+

GS"

+

GS" - * = * " GSSG-

GSSG~

+

02

RH

(1) (2)

► GSSG

+

02-

(3)

Furthermore, GS" is an oxidizing species that can react with hydro­ gen donating molecules including NADH, polyunsaturated fatty acids, retinol, and ferrocytochrome c.15"17 On these grounds, GSH would be expected to be poor both as a scavenger and as a protective antioxidant. Yet in experimental systems acetominophen and similar radicals are efficiently scavenged by GSH.18 This is because Reaction (1) is kinetically driven in the forward direction by removal of GS" through reactions with the thiolate (GS") and oxygen (Reactions (2) - (3).1419,20 Dimerization of GS" radicals to give the disulfide (GSSG) is of limited significance at the low steady state radical concentrations that are likely to be present physiologically. The other pathways are favored by at least three orders of magnitude even under relatively hypoxic conditions and will dominate radical decay. GS"

+

02

<

>

GSOO.

(4)

A key feature of thiyl radical chemistry is the equilibrium between GS" and the strongly reducing radical, GSSG*" [Reaction (2)]. GSSG"" is prob­ ably the strongest reductant produced in biological systems. 8 The position of equilibrium 2 depends on the thiolate ion concentration, which is dependent on the pH, the pX of the thiol (8.8 for GSH) and the GSH concentration. 14 At pH 7.4 and 5 mM GSH, the ratio of GS" to GSSG"" is 2:1. However, because the equilibrium is established rapidly, reactions of GSSG"" may dominate even though its concentration is relatively low. Both radicals react with oxygen [Reactions (3) and (4)]. Reaction (3) is very fast (k = 2 x 108 M"1 s"1) and irreversible. It provides the driving force for displacing equilibrium 1 and the combination of Reactions (1) to (3) account for the good scavenging ability of GSH. Although Reaction (4) is fast, it is also reversible and contributes less to GS" removal than Reaction (3). However, it is the most likely route to the higher oxidation state forms of glutathione (such as the sulfonic acid) that are minor products in some radical systems.21"23 Reaction (4) may be more significant at lower pH

Radical Scavenging by Thiols

179

GSH + R

lbRH GS

-Jt GSSG °2 [—*■ GSSG SOD I °2

+ H

2 °2

Fig. 1. Concerted action of reduced glutathione (GSH) and superoxide dismutase (SOD) in free radical scavenging. (R* is a generic radical, GS" glutathionyl radical, GSSG oxidized glutathione, GSSG" glutathione disufide radical anion.) where less thiolate is present. GSSG*~ may undergo alternative reacions to Reaction (3) (e.g. with quinones, heme proteins and phenoxyl radicals) when oxygen is limiting. As shown in Fig. 1, consequences of radical scavenging by GSH are oxy­ gen consumption and superoxide production. The sequence could, there­ fore, be regarded as a generator of oxidative stress. However, from another perspective, this mechanism enables GSH to act as an intermediary for channeling radicals to superoxide. Thus, superoxide acts as a radical sink and, provided superoxide dismutase is present, the sequence provides an elegant mechanism for a single enzyme to control the effects of radical generation.19,24 GSH therefore needs the concerted action of superoxide dismutase to function as a radical scavenging antioxidant. 25 As hydrogen peroxide is produces from the dismutation of superoxide, it must also be removed enzymatically for full antioxidant protection. There is a plethora of evidence that superoxide and hydrogen peroxide are produced during radical scavenging by GSH.10,26"31 Examples include systems where phenoxyl radicals are generated from tyrosine or dietary flavonoids by peroxidases or during the autoxidation of hydroquinones and hydroxypyrimidines such as dialuric acid in the presence of GSH.32"34 These latter compounds undergo superoxide-dependent autoxi­ dation via a semiquinone intermediate. GSH alone, by reducing the semiquinone and generating superoxide, enhances autoxidation and the resultant hydrogen peroxide production, and but with superoxide

180

Cellular Implications ofRedox Signalling

dismutase also present, the whole process is inhibited. These are good examples of where both GSH and superoxide dismutase are required for effective antioxidant protection.

3. Reaction of Superoxide with Thiols The superoxide generated as a result of radical scavenging by GSH could potentially react with more GSH and set up a chain reaction. If this reaction were fast, then large amounts of GSH could be oxidized for each initial radical generated. Data from a number of sources indicate that superoxide does react with GSH, but published values for the rate constant range from a very low value of 15 M"1 s_1 to more than 105 M"1 s_1(21).35"37 There are methodological problems with many of these studies, as regeneration of superoxide was not considered. These have recently been overcome. Our studies, both with GSH and other low molecular weight thiols, indicate that the reaction occurs but is relatively slow, so that at physiological thiol concentrations there is a short chain.21,38 Using a rigorous kinetic approach for N-acetylcysteine, Benrahmoune et al.37 have measured a rate constant of 68 M"1 s"1. This agrees well with our estimate of 100-1000 M_1 s"1 for GSH,21 which reacts 2-3 times faster than N-acetylcysteine.38 These reactions are several orders of magnitude slower than the reaction of superoxide with ascorbate, which has a rate constant of about 105 M"1 s-1.39 The reaction between GSH and superoxide should play a minimal role in superoxide removal or GSH oxidation in the cell.

4. Relative Scavenging Roles of GSH and Ascorbate Although GSH is an effective scavenger in many experimental systems, the question arises as to whether this is important physiologically or whether there are other more effective radical traps. Ascorbate is an obvi­ ous alternative.814,40'41 It is a better one electron reductant than GSH8 and scavenges a wide range of radicals including those listed in Table 2. It could act either by scavenging other radicals directly or by intercepting the glutathione thiyl radical, as shown in Fig. 2. The ascorbate radical is relatively stable, does not react with oxygen, and decays primarily by dismutation. Thus scavenging by ascorbate could bypass superoxide production from GSH and the need for superoxide dismutase. Wardman 14 has considered radical reactions involving ascorbate and GSH from a thermodynamic perspective, at different pO z and pH, and with

Radical Scavenging by Thiols GSH + RH

»-

\.

181

GS + RH

ascorbate

/ ascorbyl radical

i

ascorbate + dehydroascorbate

Fig. 2. Ascorbate as a radical sink. various ascorbate and GSH concentrations in the physiological range. These calculations show that with GSH in the millimolar range and 10-100 fold lower ascorbate concentrations, thiyl radicals would react mostly with ascorbate, with between 2 and 15% giving rise to superoxide. Thus, this simple calculation implicates ascorbate as the more favored radical sink. Sturgeon and coworkers41 have addressed the question experimentally with a tyrosine/peroxidase system by measuring oxygen uptake and thiyl radical formation by spin trapping. With 8 mM GSH, they showed pro­ gressive inhibition of both by ascorbate in the 25-100 uM range. It can be concluded from both approaches that ascorbate should dominate over GSH as a radical scavenger under typical intracellular conditions. However, conditions will vary between cell types and within cell compartments, and will also change with time of exposure to an oxidative stress. Unless cultured cells are supplemented, ascorbate concentra­ tions are almost undetectable. 42 There are a number of experimental studies in which thiyl radicals have been trapped in cells subjected to free radical stress.43,44 Therefore, it is reasonable to assume that there will be conditions where scavenging by GSH is important, and that relative concentrations of GSH and ascorbate will influence the extent to which GSH undergoes radical-mediated oxidation with resultant superoxide generation.

5. Radical Scavenging by Vicinal Thiols It is possible that other thiols could be much more effective radical scavengers than GSH. As Reaction (2) is critical in the scavenging pathway, a low pK or presence of a vicinal thiol group should facilitate formation of the disulfide bond and accelerate the reaction. The vicinal thiols,

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[DTT]nM Fig. 3. A: Stopped flow analysis of loss of A610 due to the Wurster's blue radical (3 flM) with 13 \lM dithiothreitol (DTT). B: Plot of first order rate constants obtained by fitting exponential curves to stopped flow data at different DTT concentrations. Each point is the mean and S.D. of at least 5 determinations. dihydrolipoic acid and thioredoxin, have both been s h o w n to scavenge phenoxyl radicals and generate superoxide in the process. 45 In contrast to

Radical Scavenging by Thiols

183

GSH, it was not possible to trap the thiyl radical with these compounds. This implies a rapid reaction of the radical with the vicinal thiol group that should enhance their scavenging efficiency. We have compared the scavenging ability of a mono and vicinal thiol by measuring rate con­ stants for reaction of GSH and dithiothreitol with the stable tetramethylphenylenediamine (Wurster's blue) radical. Whereas the GSH reaction was slow enough to follow with conventional spectrophotometry, the dithiothreitol reaction was much faster and required stopped flow. Rate measurements (Fig. 3) indicate a rate constant for dithiothreitol of 1.4 x 106 M"1 s"1, which is 5000 times higher that the 300 M"1 s"1 we mea­ sured for GSH. Within the constraints of extrapolating results with a stable radical to short-lived species, this observation suggests that there may be physiological vicinal thiol compounds that are much more effi­ cient radical scavengers than GSH. However, concentration as well as reactivity is important when making such an assessment. As thiol pro­ teins are likely to be in the micromolar range compared with millimolar concentations of GSH, a difference in reactivity of at least 3-4 orders of magnitude is required for such a mechanism to be relevant. Glutaredoxin (thiol transferase), the 12 kDa thiol protein that catalyses glutathionyl-disulfide interchange reactions specifically,46'47 may also act as an intracellular radical scavenger. Mieyal and coworkers,48 (personal communication) have recently observed that generating glutathionyl radicals in the presence of glutaredoxin leads to oxygen-dependent acceler­ ated formation of GSSG. They propose an initial reaction between the glu­ tathionyl radical and the thiolate of glutaredoxin [the equivalent of Reaction (2)], which is favored not only because of the low pX of the thiolate49 but also because of the affinity of the enzyme for the glutathionyl moiety.50 They have interpreted the oxygen requirement as indicative of oxygen reacting with the enzyme anion radical to give superoxide and the typical glutaredoxin-SSG intermediate, which is then turned over by GSH to regenerate the enzyme thiolate and GSSG. If further experiments can establish that this reaction sequence indeed produces superoxide in the equivalent of Reaction (3), and that it is fast enough to be physiologically relevant, then it has the potential to be a significant radical scavenging pathway and source of superoxide in the cell. As proposed by Mieyal and coworkers, 48 it also could provide a mechanism for inducing S-thiolation of proteins, a process that may be critical in regulating redox sensitive signalling pathways.51-52

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Cellular Implications ofRedox Signalling

6. Possible Convergence of Ascorbate and GSH Scavenging Pathways An intriguing possibility, although at this stage highly speculative, is that radical scavenging pathways involving thiols, ascorbate and superoxide may be linked by the mammalian selenoenzyme, thioredoxin reductase. Although in plant antioxidant defense, it is well established that ascorbyl radicals are handled enzymatically,53 in mammals ascorbyl radical reductases are ill defined and it has generally been considered that the radicals break down chemically. Thioredoxin reductase and glutaredoxin have both been shown to have dehydroascorbate reductase activity.54,55 However, in addition to this, thioredoxin reductase has recently been reported by May and coworkers56 to function as an ascorbyl radical reductase. They proposed that there is an initial reaction between the radical and the low pK selenocysteinyl residue at the active site of the enzyme, and subsequent disulfide formation with the neighboring thiol. Although not proven, it would be expected [by analogy with Reactions (2) and (3)] that a selenosulfide radical cation intermediate would be formed and that it would react with oxygen to give superoxide. Such a mechanism for enzymatic removal of ascorbyl radicals were established, it does have a certain elegance in providing a route for transmitting radical character via ascorbate to sulfur/selenium and thence to superoxide. It would enable radicals to be controlled enzymati­ cally, regardless of whether ascorbate or a thiol was the initial scavenger, and in each case superoxide would be the ultimate radical sink.

Acknowledgments I am grateful to Rex Munday for his collaboration during the early stages of this work and for supplying the Wurster's blue, to Alexander Peskin for assistance with the stopped flow experiments, to Carlos Gitler for stimulating discussion on vicinal thiol compounds and John Mieyal for sharing his unpublished results. This work was supported by a grant from the Health Research Council of New Zealand.

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3. Sun QA, Wu Y, Zappacosta F, Jeang KT, Lee BJ, Hatfield DL, et al. 1999. Redox regulation of cell signaling by selenocysteine in mammalian thioredoxin reductases. /. Biol. Chem. 274: 24522-24530 4. Allen RG, Tresini M. 2000. Oxidative stress and gene regulation. Free Radic. Biol. Med. 28: 463-499 5. Halliwell B, Gutteridge JMC. Free Radical Biology and Medicine. Oxford, Oxford University Press, 1999 6. Powis G, Mustacich D, Coon A. 2000. The role of the redox protein thioredoxin in cell growth and cancer. Free Radic. Biol. Med. 29: 312-322 7. Kang SW, Chae HZ, Seo MS, Kim K, Baines IC, Rhee SG. 1998 Mammalian peroxiredoxin isoforms can reduce hydrogen peroxide generated in response to growth factors and tumor necrosis factoralpha. /. Biol. Chem. 273: 6297-6302 8. Buettner GR. 1993. The pecking order of free radicals and antioxidants. Lipid peroxidation, oc-tocopherol, and ascorbate. Arch. Biochem. Biophys. 300: 535-543 9. Niki E, Tsuchiya J, Tanimura R, Kamiya Y. 1982. Regeneration of vitamin E from oc-chromanoxyl radical by glutathione and vitamin C. Chem. Lett. 789-792 10. O'Brien PJ. 1988. Radical formation during the peroxidase catalyzed metabolism of carcinogens and xenobiotics. The reactivity of these radicals with GSH, DNA, and unsaturated lipid. Free Radic. Biol. Med. 4:169-183 11. D'Aquino M, Bullion C, Chopra M, Devi D, Devi S, Dunster C, et al. 1994. Sulfhydryl free radical formation enzymatically by sonolysis, by radiolysis, and thermally. Vitamin A, curcumin, muconic acid, and related conjugated olefins as references. Meth. Enzymol. 233: 34-46 12. Asmus KD. 1990. Sulfur-centered radicals. Meth. Enzymol. 186: 168-180 13. Wardman P, von Sonntag C. 1995. Kinetic factors that control the fate of thiyl radicals in cells. Meth. Enzymol. 251: 31—45 14. Wardman P. 1995. Reactions of thiyl radicals. In Biothiols in Health and Disease, eds. Packer L, Cadenas E, Marcel Dekker Inc., New York, pp. 1-20 15. Schoneich C, Bonifacic M, Asmus KD. 1989. Reversible H-atom abstraction from alcohols by thiyl radicals. Determination of absolute rate constants by pulse radiolysis. Free Radic Res. Commun. 6: 393-^05 16. Schoneich C, Dillinger U, von Bruchhausen F, Asmus KD. 1992. Oxidation of polyunsaturated fatty acids and lipids through thiyl

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31. Galati G, Chan T, Wu B, O'Brien PJ. 1999. Glutathione-dependent generation of reactive oxygen species by the peroxidase-catalyzed redox cycling of flavonoids. Chem. Res. Toxicol. 12: 521-525 32. Winterbourn CC, Munday R. 1989. Concerted action of reduced glutathione and superoxide dismutase in preventing redox cycling of dihydroxypyrimidines, and their role in antioxidant defense. Free Radic. Res. Commun. 8: 287-293 33. Winterbourn CC, Munday R. 1989. Glutathione-mediated redox cycling of alloxan. Mechanisms of superoxide dismutase inhibition and of metal-catalyzed OH* formation. Biochem. Pharmacol. 38: 271-277 34. Munday R. 2000. Autoxidation of naphthohydroquinones: Effects of pH, naphthoquinones and superoxide dismutase. Free Radic. Res. 32: 245-253 35. Bielski BHJ, Shiue GG. 1978. Reaction rates of superoxide radicals with the essential amino acids. Ciba Found. Symp., 65: 43-56 36. Dikalov S, Khramtsov V, Zimmer G. 1996. Determination of rate con­ stants of the reactions of thiols with superoxide radical by electron paramagnetic resonance: Critical remarks on spectrophotometric approaches. Arch. Biochem. Biophys. 326: 207-218 37. Benrahmoune M, Therond P, Abedinzadeh Z. 2000. The reaction of superoxide radical with N-acetylcysteine. Free Radic. Biol. Med. 29: 775-782 38. Winterbourn CC, Metodiewa D. 1999. Reactivity of biologically rele­ vant thiol compounds with superoxide and hydrogen peroxide. Free Radic. Biol. Med. 27: 322-328 39. Cabelli DE, Bielski BHJ. 1983. Kinetics and mechanism for the oxida­ tion of ascorbic acid/ascorbate by hydroperoxyl/ superoxide radi­ cals. A pulse radiolysis and stopped flow photolysis study. /. Phys. Chem. 87:1809-1812 40. Kagan VE, Yalowich JC, Day BW, Goldman R, Gantchev TG, Stoyanovsky DA. 1994. Ascorbate is the primary reductant of the phenoxyl radical of etoposide in the presence of thiols both in cell homogenates and in model systems. Biochemistry 33: 9651-9660 41. Sturgeon BE, Sipe HJJ, Barr DP, Corbett JT, Martinez JG, Mason RP. 1998. The fate of the oxidizing tyrosyl radical in the presence of glutathione and ascorbate. Implications for the radical sink hypothesis. /. Biol. Chem. 273: 30116-30121

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42. Tu B, Wallin A, Moldeus P, Cotgreave I. 1995. The cytoprotective roles of ascorbate and glutathione against nitrogen dioxide toxicity in human endothelial cells. Toxicol. 98:125-136 43. Kwak H-S, Yim H-S, Chock PB, Yim MB. 1995. Endogenous intracellular glutathionyl radicals are generated in neuroblastoma cells under hydrogen peroxide oxidative stress. Proc. Natl. Acad. Sci. USA 92: 4582^586 44. Stoyanovsky DA, Goldman R, Jonnalagadda SS, Day BW, Claycamp HG, Kagan VE. 1996. Detection and characterization of the electron paramagnetic resonance-silent glutathionyl-5,5-dimethyl-l-pyrroline N-oxide adduct derived from redox cycling of phenoxyl radi­ cals in model systems and HL-60 cells. Arch. Biochem. Biophys. 330: 3-11 45. Goldman R, Stoyanovsky DA, Day BW, Kagan VE. 1995. Reduction of phenoxyl radicals by thioredoxin results in selective oxidation of its SH-groups to disulfides. An antioxidant function of thioredoxin. Biochemistry 34: 4765-4772 46. Holmgren A. 1989. Thioredoxin and glutaredoxin systems. /. Biol. Chem. 264:13963-13966 47. Mieyal JJ, Srinivasan U, Starke DW, et al. 1995. Glutathionyl specificity of thioltransferases: Mechanistic and physiological implications. In Biothiols in Health and Disease., eds. Packer L, Cadenas E, Marcel Dekker Inc., New York, pp. 305-372 48. Starke DW, Mieyal JJ. 1999. Catalysis of glutathione-thiyl radical transfer reactions by thioltransferase (glutaredoxin). FASEB }. 13A: 481 (Abstract No. 397.8) 49. Srinivasan U, Mieyal PA, Mieyal JJ. 1997. p H profiles indicative of rate-limiting nucleophilic displacement in thioltransferase catalysis. Biochemistry 36: 3199-3206 50. Yang Y, Jao S, Nanduri S, Starke DW, Mieyal JJ, Qin J. 1998. Reactivity of the human thioltransferase (glutaredoxin) C7S, C25S, C78S, C82S mutant and NMR solution structure of its glutathionyl mixed disulfide intermediate reflect catalytic specificity. Biochemistry 37: 17145-17156 51. Cotgreave IA, Gerdes R. 1998. Recent trends in glutathione bio­ chemistry — Glutathione-protein interactions: A molecular link between oxidative stress and cell proliferation? Biochem. Biophys. Res. Commun. 242: 1-9

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52. Thomas JA, Poland B, Honzatko R. 1995. Protein sulfhydryls and their role in the antioxidant function of protein S-thiolation. Arch. Biochem. Biophys. 319:1-9 53. Asada K. 2000. The water-water cycle as alternative photon and electron sinks. Philos. Trans. Roy. Soc. Lond. B. Biol. Sci. 355: 1419-1431 54. May JM, Mendiratta S, Hill KE, Burk RF. 1997. Reduction of dehydroascorbate to ascorbate by the selenoenzyme thioredoxin reductase. /. Biol. Chem. 272: 22607-22610 55. Washburn MP, Wells WW. 1999. The catalytic mechanism of the glutathione-dependent dehydroascorbate reductase activity of thioltransferase (glutaredoxin). Biochemistry 38: 268-274 56. May JM, Cobb CE, Mendiratta S, Hill KE, Burk RF. 1998. Reduction of the ascorbyl free radical to ascorbate by thioredoxin reductase. /. Biol. Chem. 273: 23039-23045 57. Nakamura M, Yamazaki I, Ohtaki S, Nakamura S. 1986. Characterization of one- and two-electron oxidations of glutathione coupled with lactoperoxidase and thyroid peroxidase reactions. /. Biol. Chem. 261:13923-13927 58. Subrahmanyam W , McGirr LG, O'Brien PJ. 1987. Glutathione oxi­ dation during peroxidase catalyzed drug metabolism. Chem. Biol. Interact. 61: 45-59 59. D'Arcy Doherty M, Wilson I, Wardman P, Basra J, Patterson LH, Cohen GM. 1986. Peroxidase activation of 1-naphthol to naphthoxy or naphthoxy-derived radicals and their reaction with glutathione. Chem. Biol. Interact. 58:199-215 60. Baker MZ, Badiello R, Tamba M, Quintiliani M, Gorin G. 1982. Pulse radiolytic study of hydrogen transfer from glutathione to organic radicals. Int. J. Radic. Biol. 41: 595-602 61. Willson RL. 1983. Free radical repair mechanisms and the interac­ tions of glutathione and vitamins C and E. In Radioprotectors and Anticarcinogens, eds. Nygaard OF, Simic MG, Academic Press, New York, pp. 1-22 62. Quijano C, Alvarez B, Gatti RM, Augusto O, Radi R. 1997. Pathways of peroxynitrite oxidation of thiol groups. Biochem. }. 322: 167-173 63. Bonini MG, Augusto O. 2001. Carbon dioxide stimulates the produc­ tion of thiyl, sulfinyl, and disulfide radical anion from thiol oxidation by peroxynitrite. /. Biol. Chem. 276: 9749-9754

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Chapter 9 Ascorbate and Glutathione Metabolism in Plants: H 2 0 2 -Processing and Signalling Christine H Foyer1 Crop Performance and Improvement Division Rothamsted Research Harpenden, Herts AL5 2JQ, UK [email protected]

Keywords: Vitamin C, signal transduction, photosynthesis catalase, defense responses, glutathione biosynthesis

1. Summary Ascorbate and glutathione are versatile molecules in plant cells with many diverse and important functions in the regulation of growth and develop­ ment as well as in defense. With one or two notable exceptions, ascorbate and glutathione are the most abundant low molecular weight antioxidants in plant tissues being present in millimolar concentrations. Plant transfor­ mation and associated technologies have allowed partial elucidation of the factors regulating the homeostasis of the ascorbate and glutathione pools, particularly biosynthesis. In the ascorbate-glutathione cycle these anti­ oxidants function in a coupled sequence to remove active oxygen species (AOS), and most notably H2Oz. However, recent evidence shows that the reduced ascorbate/oxidized ascorbate (DHA) redox pair and the reduced glutathione (GSH)/glutathione disulphide (GSSG) redox pair are not always coupled and that the degree of coupling may vary between the different intracellular compartments. As a result, the ascorbate and glutathione pools may become oxidized independently in different cellular compart­ ments due to local increases in H 2 O z production, for example in the chloroplast, peroxisome or apoplast. This may add specificity to the oxidative signal and allow differential defense responses to prevent disease or damage. Ascorbate and glutathione are here considered as signal transducing molecules conveying information on cellular redox state. 191

192 Cellular Implications of Redox Signalling

(a)

(b)

Fig. 1. The roles of ascorbic acid (Asa) and glutathione (GSH) in removing H 2 0 2 in the chloroplast. The Meher-peroxidase (water-water cycle) reaction is shown in (a) and the ascorbate-glutathione cycle is depicted in (b).

2. Introduction Plants have evolved to exploit the oxidative potential of oxygen while preventing uncontrolled oxidation. Accordingly, they contain a diverse array of constitutive and inducible defense mechanisms. Perhaps, one of the best characterized of these is the ascorbate-glutathione cycle that is found in most compartments of plant cells. The ascorbate-glutathione cycle functions either alongside or together with superoxide dismutases (SOD), non-specific peroxidases (PX) and catalases (CAT) to control AOS concentrations. In addition to ascorbate and glutathione the cycle consists of several enzymatic components (Fig. 1). Of particular note is the central role of ascorbate-specific peroxidases (APX) in the elimination of H 2 O z . Glutathione peroxidases (GPX) are also found in plant cells but they do not play a predominant role in H 2 0 2 detoxification but rather together with the peroxiredoxins, they remove lipid and alkyl peroxides generated in the vicinity of the chloroplast thylakoid membranes. Hydrogen per­ oxide is a product of primary metabolism in plants. 17 It is produced at high flux rates by processes associated with photosynthesis. 17 In addition, there are a number of other processes in leaves that are capable of producing AOS particularly H 2 0 2 at high rates. Perhaps the most important of these is the pathogen-induced oxidative burst that is a key feature of the

Ascorbate and Glutathione Metabolism in Plants 193

Fig. 2. The production of superoxide and hydrogen peroxide by reactions associated with photosynthesis. The photosynthetic electron transport chain is composed of two photosystems (PSI and PSII) acting in series. Light driven superoxide production occurs by the donation of single electrons to molecular oxygen at the reducing side of Photosystem I (PSI). Hydrogen peroxide (H202) is then produced in the chloroplasts from superoxide as a result of the action of superoxide dismutase. Oxygen competes with C0 2 for assimilation in photo­ synthesis, into the substrate ribulose-1, 5-bisphosphate (RuBP) at the level of primary enzyme of carbon fixation, ribulose-1, 5-bisphosphate carboxylase/ oxygenase (Rubisco). The fixation of oxygen, to produce phosphoglycollate, is the first step of the pathway known as photorespiration. H 2 0 2 is produced in this pathway from glycollate exported from the chloroplasts to the peroxisomes. hypersensitive response. 42 It is becoming increasing clear that an early general response to environmental stresses such as wounding, drought, extremes of temperature or physical and chemical shocks is AOS accumu­ lation.17 The concept that H 2 0 2 is a toxic metabolite whose destruction is desirable in all situations has been replaced by the notion that H 2 0 2 is an important and useful metabolite in plants. While local changes in the rate of H 2 0 2 production appears to be involved in the regulation of growth and develop-ment in plants, 37 high levels of H 2 0 2 accumulation trigger pro­ grammed cell death in defense reactions. The role of H 2 0 2 in defense against pathogens is, perhaps the best characterized of all its functions in plants. Much evidence has accumulated in support of a role for H 2 0 2 as a signal-transducing molecule in activating defense responses. H 2 0 2 has been shown to mediate both intra- and inter-cellular communication during plant defense reactions.15,29 The signaling properties of AOS are probably

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Cellular Implications ofRedox Signalling

not exclusive to H 2 0 2 . Superoxide, for example, induces distinct sets of defense proteins in bacteria and yeast. Plant cells have evolved to tolerate comparatively high steady-state concentrations of H 2 O z . The pool size of this oxidant in leaves is tightly controlled by the antioxidant system. This essentially determines the lifetime of H 2 0 2 within the cellular environ­ ment. Ascorbate (vitamin C) and glutathione, are key to the antioxidant defenses. These antioxidants function in concert with antioxidant enzymes to provide an effective system for the control of H 2 0 2 . Rather than a system designed to completely eliminate AOS, the leaf antioxidant system appears to be one that permits control of the redox state of various cellular components. The interaction between H 2 0 2 production and the antioxidant system provides not only an effective mechanism for defense against uncontrolled oxidation but also facilitates signal transduction in a compartment specific manner to add to the network of metabolic information on cellular redox status.

3. Hydrogen Peroxide Production Associated with Photosynthesis Two processes associated with photosynthesis have a very high capacity for H 2 0 2 production even under optimal conditions. (Fig. 2). These are the Mehler reaction and photorespiration.17'36 In the first process HjC^ is pro­ duced within the chloroplast but in the second H 2 0 2 is liberated in the peroxisome from a product exported from the chloroplast. (Fig. 2). This means that the oxidative perturbation occurs at different sites in the two processes and information associated with photosynthesis is transmitted to different organelles. Thus, photorespiratory flux provides a potential mechanism for signaling information concerning photosynthesis away from the chloro­ plast.36 This location is also distinct from that of H 2 0 2 produced during an oxidative burst by the plasmalemma. In the latter case H J O J is produced in the apoplast. In contrast to the chloroplasts and peroxisome which have robust antioxidant defenses the apoplast contains little or no glutathione and no reducing power to re-cycle ascorbate. In the cytoplasm the oxidative sig­ nal (H202) is rapidly destroyed but in the apoplast it is much more long lived. 3.1 Peroxisomal Hydrogen Peroxide Production Associated with Photorespiration During photosynthesis the primary enzyme of photosynthetic carbon assimilation, ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco)

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uses C 0 2 from the atmosphere to carboxylate the substrate, ribulose-1, 5-bisphosphate (RuBP). In plants with the C3 pathway of photosynthesis, however, there is competition between C 0 2 and 0 2 at the active site of Rubisco as this enzyme can also use oxygen to oxygenate RuBP36. The oxygenation reaction initiates the photorespiratory pathway that results in a substantial loss of fixed carbon as C0 2 . It also produces H 2 0 2 at high rates through the action of peroxisomal glycollate oxidase. To give an idea of the oxidative load that photorespiration places on the leaf of an average C 3 plant in air in optimal growth conditions the following calculations can be made. 36 An average net rate of photosynthetic C 0 2 assimilation for a C 3 leaf is about 200 umol h"1 (mg Chi)"1, with a ratio of carboxylation to oxygenation by Rubisco of 2.5. This would yield a rate of H 2 O z formation in the peroxisome of about 100 umol h"1 (mg Chi) -1 . Assuming that the peroxisome is about one quarter of the size of the chloroplast, having a volume of about 7 uL (mg Chi)' 1 , this would cause an increase in the peroxisomal H 2 0 2 of about 250 mM per minute. 36 Photorespiratory C 0 2 loss would be much higher if catalase were not effective in preventing chemical decarboxylation of keto-acids such as glycoxylate and hydroxypyruvate in the peroxisome. 5 Three unlinked structural catalase genes (Catl, Catl, and Cat3f6 have been found in C3 plants (such as Nicotiana plutnbaginifolia) and in C4 plants (such as maize). These genes encode biochemically distinct catalase isoenzymes (CAT-1, CAT-2, and CATS). Two of these are expressed in mature tobacco leaves. CAT-1 comprises about 80% of the leaf catalase activity and is considered to fulfil the role of H 2 0 2 -scavenging during photorespiration while CAT-2 accounts for the remaining 20% and is localized in the phloem.46 Trans­ formed tobacco lines deficient in either CAT-1 or CAT-2 (or both) develop necrotic lesions on their leaves when exposed to high light because the production of H 2 O z through photorespiration is increased in these con­ ditions.5'6,40 Similarly, catalase deficiency in barley was lethal when plants were grown in air, but this could be avoided by growth under nonphotorespiratory conditions.28 In contrast, in maize, a C4 plant with a markedly decreased photorespiratory flux, catalase deficiency had no marked effect. These data not only confirm the importance of catalase in photorespiration but also implicate photorespiratory H 2 O z production in the induction of systemic defence responses that appear to share some fea­ tures with systemic acquired resistance induced by pathogen attack.40,50 Leaf catalase activities are known to decline under certain stress conditions. This could lead to both local and systemic responses to photorespiratory H 2 O z as observed in transformed plants and mutants lacking catalase.

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Cellular Implications ofRedox Signalling

3.2 Chloroplastic Hydrogen Peroxide Production Associated with the Mehler Reaction The univalent reduction of oxygen by the photosynthetic electron transport chain is called the "Mehler reaction". Electron carriers within the photosynthetic electron transport chain having electrochemical potentials commensurate with the reduction of molecular oxygen exist in both PSII and PSI. The principal site of oxygen reduction is on the reducing side of PSI (Fig. 1). All the electron transport components on the reducing side of PSI, from the iron-sulphur centers to reduced thioredoxin, are autooxidizable, and i.e. they can donate electrons to oxygen and produce superoxide. Superoxide can hence be generated within the thylakoid membrane or at the membrane surface. Ferredoxin, ferredoxin, NADPoxidorectase (FNR) and other dehydrogenases, such as monodehydroascorbate reductase (MDHAR; Refs. 32 and 24) and glutathione reductase (GR), can all generate superoxide at the membrane surface. Oxygen reduction may serve a useful function in preventing over-reduction of the electron transport chain. The Mehler reaction is considered to "poise" the electron carriers for more efficient functioning. Superoxide produced by the thylakoid membranes is converted to molecular oxygen and H 2 0 2 by the action of SOD. To give an idea of the oxidative load that the Mehler reaction places on an average C3 plant in air in optimal growth conditions the following calculations can be made. For average net rate of photosynthesis of about 200 |imol h _1 (mg Chi) -1 , with about 10% of the electrons flowing through the photosynthetic elec­ tron transport chain used for oxygen reduction, the rate of H 2 0 2 forma­ tion in the chloroplast would be about 39 |J,mol h"1 (mg Chi) -1 . Assuming that the chloroplast volume is about 30 (iL (mg Chi)"1, this would increase in the chloroplast H 2 0 2 by about 22 mM per minute. H 2 0 2 is a strong oxidant and a potent inhibitor of photosynthetic C 0 2 assimilation 25 because it can rapidly oxidize key protein thiol groups on several enzymes of the Benson-Calvin cycle such as fructose-1, 6-bisphosphatase and sedohepulse-1, 7-bisphosphatase. H 2 0 2 will also inactivate other chloroplast enzymes such as Cu/ZnSOD and APX. Chloroplasts contain both membrane (thylakoid)-bound and soluble forms of APX that have a very high affinity for both ascorbate and H202P The sequential production and destruction of superoxide and H 2 0 2 in the chloroplast is called the "Mehler-peroxidase cycle" or the "water-water cycle". This cycle consists of (a) electron transfer from water through the photosynthetic electron transport chain to oxygen producing superoxide

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at PSI, (b) the dismutation of the superoxide radical by SOD to form H 2 0 2 , (c) the reduction of H 2 0 2 to water by APX, and (d) the regeneration of ascorbate from monodehydroascorbate (MDHA). At the thylakoid membrane MDHA radicals are reduced directly to ascorbate by reduced ferredoxin produced by PSI.32 The Mehler-peroxidase cycle on the constitutes a thylakoid-bound scavenging system for superoxide and H 2 0 2 and may form a relatively closed system acting within a 5-10 n m layer on the surface of the membrane. 32 Superoxide and H 2 O z escaping the thylakoid membrane antioxidant defenses or arriving in the chloroplast from the cytosol are effectively scav­ enged in the stroma by stromal C u / Z n or Fe SODs and APX isoformsv (sAPX). These enzymes together with the other enzymes of the ascorbateglutathione cycle [MDHAR, dehydroascorbate reductase (DHAR) and GR] protect stromal enzymes from oxidation using NADPH produced by the electron transport chain. It should be noted also that the ascorbate — glutathione cycle not only functions in the chloroplast stroma but has also been found in the cytosol, peroxisomes, mitochondria of photosynthetic and non-photosynthetic tissues.49 It is interesting to note that the reduction of DHA by GSH is catalyzed by several types of enzyme in plant tissues.27,41

4. Ascorbic Acid Ascorbate is a primary antioxidant in leaves, directly reducing hydroxyl radicals, superoxide and singlet oxygen. 22 It is also an important secondary antioxidant, regenerating the oxidized forms of tocopherols. In addition, ascorbate is an important enzyme co-factor, for example, in the violaxanthin de-epoxidase reaction of the xanthophyll cycle.33 Ascorbate is a major metabolite of chloroplasts from higher plants where it is found at very high concentrations (10-50 mM; Refs. 14 and 18). In green leaves there can be as much ascorbate as chlorophyll and it can represent about 10% of the total soluble carbohydrate pool. 33 Leaf ascorbate contents can, however, vary markedly as ascorbate biosynthesis is regulated by inter­ nal (developmental) stimuli and external (environmental) cues, parti­ cularly light. 71819 The ascorbate redox system consists of reduced ascorbate, MDHA, and DHA. MDHA radicals have a short lifetime and disproportionate spontaneously at neutral pH values to DHA and ascorbate. DHA is unstable at p H values above 7.0 and rapidly undergoes hydrolytic ring

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cleavage. DHA can be recycled to ascorbate via the reactions of the ascorbate-glutathione cycle or it can be cleaved to yield tartrate and oxalate. Ascorbic acid influences plant cell growth by effecting cell elongation and cell division37 but the mechanisms that afford this regulation remain to be elucidated. Ascorbate is required for the synthesis of hydroxyprolinerich glycoproteins (HRGP). It is a cofactor for prolyl hydroxylase, an enzyme that hydroxylates the prolyl residues of HRGP proteins. These proteins are involved in cell wall synthesis and assembly in mitotic cells. The oxidation of these proteins results in enhancement of wall-strength, a defense mechanism against pathogen attack. Ascorbate also regulates lignification, a process that involves the oxidation of a wide range of aro­ matic and phenolic compounds by apoplastic peroxidases using extra­ cellular H 2 O z . Apoplastic ascorbate inhibits this process because of reduction of hydroxyl radicals and quinones in addition, apoplastic ascor­ bate, together with ascorbate oxidase and MDHA, are considered to be key factors regulating cell elongation. Conversely, the reduction state of the cytoplasmic antioxidant pools is thought to be an important compo­ nent of cell cycle regulation. Ascorbate is undetectable in the quiescent center's of maize roots, where the cells do not divide and the mitotic cycle is interrupted in the Gl phase. 33 In view of the many and diverse functions of ascorbate in plants, it is remarkable that the pathway of ascorbate biosynthesis has only recently been resolved. A very useful tool in the elucidation of this pathway was the ascorbate-deficient Arabidopsis thaliana, mutant, vtcl.7~9A5 The mutant which was initially identified via its sensitivity to elevated ozone concen­ trations, 7 possesses decreased GDP-mannose pyrophosphorylase activity, and as a result accumulates much less (30%) ascorbate than the wild-type.7,9 With the marked exception of the last step, the ascorbate bio­ synthesis pathway is cytosolic45 involving the conversion of D-glucose-6P to GDP-D-mannose and GDP-L-galactose, followed by the hydrolysis to L-galactose. The existence of this pathway is supported evidence from transgenic potato plants expressing antisense GDP-mannose pyrophos­ phorylase, the enzyme catalyzing the synthesis of GDP-mannose from D-mannose-1-P. These transgenic potato plants show a reduction in ascorbate content of between 44-72% compared with untransformed controls. The last step of ascorbate biosynthesis involves the oxidation of L-galactono-1, 4-lactone by L-galactono-y-lactone dehydrogenase, an enzyme localized in the inner mitochondrial membrane. 4 This is the only step in the pathway to have been characterized in any detail.

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The vtcl mutant has similar rates of photosynthesis and energy dissipation to the wild type but whole plant growth is decreased. 43 No change in total leaf H 2 0 2 contents was observed in the vtcl mutant but total peroxidase activity was significantly increased. An intriguing obser­ vation is that the distribution of APX activity between chloroplast and cytosol was modified in vtc-1 leaves,43 suggesting that the leaf ascorbate concentration regulates the compartmentation of the antioxidant system in A. thaliana. This effect is not linked to the ascorbate redox state which is similar in the wild type and mutant, 7,43 In other A. thaliana plants, trans­ formed with an antisense construct to have a low chloroplastic peroxiredoxin content the ascorbate redox state (but not the total ascorbate content) was modified. 3 In this case the transcripts for thylakoid APX, stroma-soluble APX, and stromal MDHA reductase were increased in the transformants and the activities of respective enzymes were increased in leaf extracts. 3 These changes were correlated with an increased oxidation of leaf ascorbate in the absence of any change in the leaf glutathione pool. 3 Since the final step of ascorbate synthesis takes place in the mitochondrial intermembrane space,4 ascorbate must be transported from the mitochondria to all other cellular compartments and to the apoplast. Plant cells contain multiple t r a n s p o r t e r s for ascorbate and DHA. 2 2 Carrier-mediated movement of ascorbate in the direction of the electro­ chemical gradient has been suggested for the chloroplast envelope, 1 but active transport against an electrochemical gradient may occur on the plasma lemma. The plasma membrane contains at least three different mechanisms of ascorbate transport and could facilitate ascorbate-mediated transport of reducing equivalents between the cytosol and apoplast. A highly specific b-type cytochrome transferring electrons from cytosolic ascorbate to extracellular acceptors, including MDHA, has been found on the plasma membrane together with MDHAR. Other ascorbate carriers selectively transport L-ascorbate and DHA between the cytosol and apoplast. 22

5. Glutathione Glutathione (y-glu-cys-gly) has a broad spectrum of functions in plants.33-34 It is a major reservoir of non-protein reduced sulfur being involved in both the storage and transport of reduced sulfur. Glutathione is a general protector of cell function. It is antioxidant, linked to the

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detoxification of H 2 0 2 via GR in the ascorbate-glutathione cycle and organic peroxides via GPX. GSH protects proteins against oxidation of protein thiol groups during stress and is involved in general cellular redox regulation and buffering. It is also central to the elimination of xenobiotics from the cytoplasm and in modification and transport of hor­ mones and other endogenous compounds, via formation of glutathione-Sconjugates. Gluathione is an important regulator of gene transcription and translation, as well as enzyme activity.34,35 Glutathione cannot be functionally replaced, except perhaps by one of its homologues which are present in some plants. In these homologues another amino acid replaces the carboxy-terminal glycine. The A. thaliana rmll mutant is deficient in the first enzyme of the glutathione biosynthetic pathway, y-glutamylcysteine synthetase (y-ECS) and contains no detectable glutathione. This mutant shows a very poor root development and a small shoot system, and can survive only in tissue culture supplied with GSH. Glutathione homeostasis is tightly controlled. Homeostatic regulation involves a complex interplay between synthesis, degradation, transport, storage, oxidation-reduction, further metabolism and catabolism as plants respond to environmental, developmental and nutritional triggers. In recent years much information on the control network that regulates glutathione biosynthesis and homeostasis in plant cells has come from the study of transformed plants modified in GSH biosynthesis and regeneration. This regulation involves coarse regulation of de novo synthe­ sis of the enzymes of GSH biosynthesis and fine control of the flux capac­ ity of the pathway by feedback inhibition.33,35 The pathway of glutathione biosynthesis is similar in plants and animals (Fig. 3) consisting of two ATP-dependent steps, catalyzed by y-ECS and glutathione synthetase (GS). This reaction sequence occurs in both the chloroplasts and cytosol of plant cells and occurs in both photosynthetic and non-photosynthetic tissues.33 Pea chloroplasts contained about 70% of the total leaf y-ECS activity and about 50% of leaf GS activity while in spinach about 60% of /-ECS and 50% GS were found in the chloroplasts. 33 These enzymes are present in plant cells at very low protein contents and activities. In addition the procedures for enzyme extraction and assay are far from trivial. Hence these enzymes have not been extensively puri­ fied or characterized. Much of the understanding of their structure, regulation and function has been obtained from the application of molecular techniques and plant transformation.

Ascorbate and Glutathione Metabolism in Plants 201

Fig. 3. The pathway of glutathione biosynthesis in plants. Glutathione is synthesized in two steps, catalyzed by y-glutamyl cysteine synthetase (y-ECS) and glutathione synthetase (GS). The glutathione pool is maintained in the reduced form by the action of glutathione reductases. Glutathione S-transferases (GST) catalyse conjugation of xenobiotics to GSH.

The gene encoding y-ECS, (gshl) was originally cloned from A. thaliana by complementation of an £. coli mutant deficient in this enzyme. Heterologous expression of the Arabidopsis y-ECS in a yeast mutant recov­ ered only about 10% of the wild-type yeast GSH but complementation confirmed that this gene encodes a protein with y-ECS activity. Functional complementation of an E.coli mutant deficient in GS activity was also used to clone the A. thaliana gene for this enzyme, gshl. Plant species that make GSH homologues have synthetase isoforms with modified amino acid substrate specificity. Legume species have GS isoforms that use either glycine to form GSH or /J-alanine to form homoglutathione. In Medicago truncatula separate genes encode GS and homoglutathione synthetase (hGS). The two genes are very homologous and are found on the same fragment of genomic DNA. The change in specificity appears to have arisen by gene duplication after the evolutionary divergence of the Leguminaceea.

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5.1 Regulation of Glutathione Biosynthesis The regulation of GSH biosynthesis is controlled at multiple levels49,5051. As well as transcriptional and translational controls, post-translational regulation via the action of protein factors or covalent modification is also suggested. Plant y-ECS is susceptible to inhibition by GSH, which competes with glutamate. 33 This regulation may be an important homeostatic constraint that prevents excessive GSH accumulation. However, by far the most important factors controlling GSH accumulation in plants are the activity of y-ECS and the availability of cysteine. The expression of both gshl and gshl is enhanced in the presence of heavy metals such as cadmium and copper, 50 and by the application of jasmonic acid. By contrast expression is not modified by either GSH or GSSG,50 or by the oxidative stress imposed by the application of H 2 0 2 . However, oxidative stress appears to be essential for the translation of the gshl and gshl transcripts in stress conditions. This has led to the concept that post-transcriptional regulation gshl and gshl transcripts provides an additional level of control of GSH synthesis.49 In this scenario H 2 0 2 (or low GSH/GSSG ratios) enhances translation of the existing gshl and gshl transcripts.47 Studies in other systems such as cancer cells have shown that transcription of the y-ECS gene is regulated by protein factors when the cells are challenged with chemotherapeutic agents. This involves conserved antioxidant response elements upstream of the coding y-ECS sequence. Post-translational regulation of y-ECS is also indicated in animals where protein phosphorylation may be involved in y-ECS regulation. 33 A smaller regulatory subunit acts to increase the catalytic potential of the larger catalytic subunit in the rat enzyme by increasing the Kt for GSH and decreasing the Km for glutamate. This would serve to alleviate any feedback controls and allow the enzyme to operate under in vivo condi­ tions. The large catalytic subunit of the animal y-ECS, is capable of cataly­ sis. Overexpression of this polypeptide alone yielded increased GSH levels in transfected cells. Highest GSH levels were, however, obtained by dual over-expression of both subunits. 33 This type of control has not yet been reported for plant y-ECS. The activity of y-ECS clearly limits the rate of glutathione synthesis in most conditions in both plants and animals. The evidence that y-ECS is responsible for maintaining the GSH concentration in plant cells is as fol­ lows: (1) The cad-2 A. thaliana mutant, which has a mutation in the gshl gene, has only one-third of the tissue GSH contents of the wild type. (2) Increases in glutathione contents accompany increases in y-ECS in tissues

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treated with cadmium. (3) Overexpression of an E. coli y-ECS but not GS, in poplar or tobacco substantially increases the glutathione pool. 33 (4) Over-expression of the A. thaliana gshl gene, in either the sense or antisense orientation, was used to produce a range of transformed plants with leaf GSH contents ranging from 2 to 150% compared to the untransformed controls.51 Tissue glutathione contents have also been modified in poplar, mustard and tobacco by the introduction of the bacterial genes encoding y-ECS and GS.33 Enhanced leaf y-ECS activity was achieved by overexpression of the bacterial y-ECS gene, whether the protein was targeted to the choloroplast or cytosol. Overexpression of y-ECS (but not GS) led to constitutive increases in leaf glutathione of up to 400%. The leaf cysteine pool was slightly enhanced in response to increased y-ECS activities, sug­ gesting co-ordinate regulation of cysteine synthesis and glutathione synthesis. However, incubation of leaf discs with cysteine always increased glutathione contents, particularly in the light. However, the effect was less marked in plants overexpressing yECS. 33 In untransformed plants the dipeptide produced by the yECS reaction, y-EC, is present in very low amounts. In the poplars overexpress­ ing yECS, however, y-EC was greatly increased. The marked increase in y-EC reflected a shift in control from y-ECS to GS, whether the bacterial y-ECS was present in the cytosol or chloroplast. This suggested that overexpression of both enzymes together would increase the potential for constitutive enhancement of tissue glutathione contents even further than that achieved by y-ECS overexpression alone. This effect was observed when tobacco lines expressing each of the biosynthetic enzymes were crossed to produce hybrids over-producing both enzymes. Transformed Brassica juncea and poplar plants overexpressing the bacterial yECS or GS genes showed normal phenotypes and displayed enhanced tolerance to cadmium and other heavy metals.

6. Coupling Between Ascorbate and Glutathione Pools Coupling between the ascorbate (Ascorbate/DHA) and glutathione (GSH/GSSG) redox pairs was first described in plants and only later recognized in animals.12,31 The relative redox potentials of the two antioxidant couples favour net electron flow from reduced glutathione to DHA.33 This type of glutathione redox cycling was first described as a necessary part

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of H 2 O z removal in the chloroplast but is now recognized as an important feature of ascorbate recycling in all cellular compartments. 12,33 The chemical reduction of DHA by GSH occurs at significant rates even in the absence of an enzyme catalyzing the reaction, particularly at alkaline pH. 48 In conditions of high APX activity, however, DHAR activity may be essential to ensure effective maintenance of the reduced form of ascorbate. DHAR's have been purified from several plant species.10,13,27 The amino acid sequence of the purified rice enzyme suggests that it is a specific DHAR,27 containing domains that match those encoded by several EST's from A. thaliana.16 Gene sequences encoding specific chloroplastic isoforms remain to be identified. Other proteins (such as glutaredoxins and protein disulphide isomerases 44 and certain types of trypsin inhibitor 41 can also catalyze DHAR-like activity. DHAR capacity varies widely between the different compartments of the plant cell. Differences in DHAR activity mean that the tightness of coupling between the glutathione and ascorbate pools might differ considerably the various cellular compartments. 34 In particular, recent evidence sug­ gests that the two antioxidants are coupled less tightly coupled in the chloroplast than in other cellular compartments. 34,35 The chloroplast perhaps has less of a requirement for DHAR than the cytosol as it is able to regenerate ascorbate from MDHA through ferredoxin via a photo­ chemical reaction. While chloroplasts contain millimolar amounts of GSH and have substantial GR activity, the primary function of these may be in processes other than ascorbate regeneration. There is no doubt that some degree of redox coupling between the ascorbate and glutathione pools occurs in the chloroplast. For example, addition of exogenous H 2 0 2 to spinach chloroplasts causes turnover of both the ascorbate and glutathione pools 1 and enhanced GR activity increases the ascorbate pool in transformed tobacco. In plants with decreased catalase, an increase in oxidative load occurs in the peroxisomes.36,40 In this situation, where the increase in H 2 0 2 flux is outside the chloroplasts, the glutathione pool is preferentially oxidized suggesting close coupling of GSH turnover and ascorbate peroxidation. 3439 In contrast to the effects observed when cata­ lase activity is decreased, a deficiency in 2-cys peroxiredoxin in the chloroplast caused specific effects on the ascorbate pool and in particular, an increased ascorbate oxidation state. 3 In this case the glutathione pool remained highly reduced, even though the lower redox potential of the glutathione redox pair compared to the ascorbate/DHA pair would favour oxidation if these pools were coupled. The redox equilibration of

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the glutathione-ascorbate couples is restricted by kinetic limitations that prevent a sufficiently fast reaction between DHA and glutathione in the 2-cys peroxiredoxin transformants. 34 This might suggest that redox cou­ pling between ascorbate and glutathione in the chloroplast is limited by low DHAR activity. In the cytosol, DHAR activity is higher allowing tighter coupling of the two antioxidants. This facilitates preferential oxidation of the glutathione pool under conditions of enhanced oxidation. Imperfect ascorbate-glutathione redox coupling in the chloroplast could also be explained by the existence of a pool of ascorbate to which glu­ tathione does not have access. One possibility is that an intrathylakoidal pool of ascorbate becomes oxidized when 2-cys peroxiredoxin activity is insufficient. Similarly the apoplastic ascorbate pool can be completely oxidized without any change in tissue glutathione rexox state.

7. Ascorbate and Glutathione in Signal Transduction Gene expression is responsive to changes in cellular redox status, particularly increases in oxidative load (Fig. I).34 The concept that H 2 0 2 is a trigger for both local and systemic defense responses is widely accepted. H 2 0 2 is a diffusible molecule but it has a relatively short half-life (1 ms) and this limits its effectiveness as a long distance mobile signal. One way that this might be overcome is via relay or amplification mechanisms to facilitate signal transduction. An intriguing possibility is that specificity may be accorded to the signal, or indeed that the signal may be trans­ duced by interaction with ascorbate and/or glutathione. These antioxi­ dants may be important signal transducing molecules. [Fig. 4(b)]. The ascorbate and glutathione pools are crucial redox components of plant cells.33 Changes in the intracellular ascorbate or glutathione concentrations have important consequences for plant growth and development. While ascorbate is implicated in the regulation of cell growth and division, the role of this antioxidant in gene expression has received less attention. Glutathione has been implicated in the control of gene expression in several studies. 2 ' n ' 21 ' 30 ' 47 Recent data suggest that both ascorbate contents and redox state could also influence gene expression in A thaliana.34,43 Decreased ascorbate in the vtcl mutant correlates with increased cytosolic APX activity, while the perturbation of the ascorbate redox state in plants with decreased 2-cys peroxire­ doxin is linked to enhanced abundance of transcripts for chloroplastic APX and MDHAR.3'34

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Cellular Implications of Redox Signalling

(b) Fig. 4. The roles of oxidants and antioxidants in the induction of defense responses, (a) Changes in oxidative load can either strengthen the plant by induc­ tion of defense genes or kill by induction of cell death. Antioxidants attenuate the cell death response (b) Differential regulation of gene expression by the ascorbate and glutathione pools. The coupling of these antioxidants redox pairs depends on the activity of dehydroascorbate reductase (DHAR).

Some oxidative stress responsive elements h a v e b e e n identified in the p r o m o t e r s of p l a n t genes. 1 5 H 2 O z -sensitive translation factors are also

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present 48 . The multiple roles of GSH within the cell, together with the stability of GSSG, make this redox couple ideally suited to information transduction. In animal cells redox regulation of the transcription factor NF-kappa B involves glutathione. Glutathione augments the activity of T-cell lymphocytes and is thus important for T-cell function. In plants, the GSH /GSSG ratio is likely to be more influential in the regulation of gene expression than the absolute size of the glutathione pool, as this is depen­ dent on several factors such as sulfur nutrition, transport and storage as well as oxidative stress. GSH and GSSG may themselves potentiate the signal but a second putative mechanism involves thiolation. This occurs by spontaneous oxidation of protein sulphydryl groups to form disulphides with low molecular weight thiols (such as GSH). The formation of such intramolecular disulphide bonds within proteins alters their config­ uration and biological activity and may be a crucial signaling event. There are many examples of proteins that undergo thiolation in animals but relatively few have been described in plants. In plants lacking catalase, transfer from high C 0 2 to air causes a rapid increase of leaf H 2 0 2 and a marked accumulation of the glutathione pool,36 accompanied by a dramatic shift in the GSH/GSSG ratio.6,35'43 Futhermore, the GSH/GSSG ratio decreased and sustained oxidation of the glutathione pool preceded the large accumulation in total leaf glutathione. This increase in total leaf gluathione pool is probably linked to upregulation of enzyme synthesis. 51 In contrast, little or no perturbation of the ascorbate redox state was found in catalase-deficient plants.36,43 The accumulation of glutathione and the net oxidation of the pool occur in both chloroplastic and extra-chloroplastic compartments. 35 Such observa­ tions implicate glutathione and the GSH/GSSG ratio in the genetic responses to increased peroxisomal oxidative load (Fig. 2) It is important to note that the symptoms that are observed in catalase-deficient plants under photorespiratory conditions, are not simply result of chemical damage. 6 Rather, they resemble pathogen-associated cell death responses or precocious senescence.36 This suggests that cell death in these plants is regulated involving common signal transducing components to those employed in the hypersensitive response to pathogens. 6,36 Compartment-specific variations in the ascorbate/DHA and GSH/GSSG ratios may have considerable significance for redox signaling. Firstly, the activity of the antioxidant system determines cellular H 2 0 2 concentrations and therefore influences the known roles of this oxidant in signal transduction. The rapid movement of H 2 0 2 through plant

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Cellular Implications ofRedox Signalling

membranes is primarily due to the presence of aquaporins. The interaction between aquaporins and H 2 0 2 decomposition will determine intercompartmental flux and concentrations.20 Cellular gradients in H 2 0 2 concentra­ tion are important, particularly across the plasma lemma. Localized accumulation of H 2 0 2 in the apoplast, for example, is essential in acclimatory responses to pathogens and pollutants. In the intracellular environment sustained differences in local H 2 0 2 concentrations must occur. Very low H 2 0 2 concentrations, for example, are sufficient to inactivate C 0 2 fixation in isolated chloroplasts and these are far below the global leaf concentrations. Changes in the ascorbate/DHA and GSH/GSSG ratios and total antioxidant concentrations can be more pronounced than changes in leaf H 2 0 2 contents and effective co-ordination of gene expres­ sion might require that these changes be compartment-specific.34 A change in oxidative load in the chloroplast (as observed, for example, in plants with low 2-cys peroxiredoxin activity) influenced the transcription of nuclear genes.3'26 The chemical identity of the signal(s) responsible for transmitting information on chloroplast redox state and antioxidant status to the nucleus is unresolved, but much evidence implicates electron trans­ port components such as plastoquinone. 17

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30. Link G, Tiller K, Baginsky S. 1997. Glutathione: A regulator of chloroplast transcription. In Regulation of Enzymatic Systems Detoxify­ ing Xeno-Biotics in Plants. NATO ASI Series ed. Hatzios KK, Kluwer Academic, Dordrecht, Boston, London, pp. 125-137 31. Meister A. 1994. Glutathione-ascorbic acid antioxidant system in animals. /. Biol. Chem. 269: 9397-9400 32. Miyake C, Asada K. 1994. Ferredoxin-dependent photoreduction of the monodehydroascorbate radical in spinach thylakoids. Plant Cell Physiol. 35: 539-549 33. Noctor G, Foyer CH. 1998. Ascorbate and glutathione: Keeping active oxygen under control. Ann. Rev. Plant. Physiol. Plant. Mol. Biol. 49:249-279 34. Noctor G, Veljovic-Jovanovic S, Foyer CH. 2000. Peroxide processing in photosynthesis: antioxidant coupling and redox signalling. Roy. Soc. Philos. Trans. 355:1465-1475 35. Noctor G, Gomez L, Vanacker H, Foyer CH. 2000. Glutathione homeostasis and signalling: the influence of biosynthesis, compartmentation and transport. /. Exp. Bot. 53:1283-1304 36. Noctor G, Veljovic-Jovanovic S, Novitskaya, L, Foyer CH. 2002. Drought and oxidative load in wheat leaves: a predominant role for photorespiration? Ann. Bot. 89: 841-850 37. Schopfer P, Plachy C and Frahry G. 2001. Release of reactive oxygen intermediates (superoxide radicals, hydrogen peroxide and hydroxyl radicals) and peroxidase in germinating radish seeds controlled by light, gibberellin and abscisic acid. Plant Physiol. 125: 1591-1602 38. Smith IK, Kendall AC, Keys AJ, Turner JC, Lea PJ. 1984. Increased levels of glutathione in a catalase-deficient mutant of barley. Hordeum vulgare L. Plant Sci. Lett. 37: 29-33 39. Smith IK, Kendall AC, Keys AJ, Turner JC, Lea PJ. 1985. The regula­ tion of the biosynthesis of glutathione in leaves of barley. Hordeum vulgare L. Plant Sci. 41:11-17 40. Takahashi H, Chen Z, Du H, Liu Y, Klessig DF. 1997. Development of necrosis and activation of disease resistance in transgenic tobacco plants with severely reduced catalase levels. Plant}. 11: 993-1005 41. Trumper S, Follmann H, Haberlein 1.1994. A novel dehydroascorbate reductase from spinach chloroplasts homologous to plant trypsin inhibitor. FEBS Lett. 352:159-162 42. Vanacker H, Carver TLW, Foyer CH. 1998. Pathogen-induced changes in the antioxidant status of the apoplast in barley leaves. Plant Physiol. 117:1103-1114

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43. Veljovic-Jovanovic S, Pignocchi C, Noctor G, Foyer CH. 2001. Low ascorbic acid in the vtc-1 mutant of Arabidopsis is associated with decreased growth and intracellular redistribution of the antioxidant system. Plant Physiol. 127: 426-435 44. Wells WW, Xu DP, Yang Y, Rocque PA. 1990. Mammalian thioltransferase glutaredoxin and protein disulfide isomerase have dehydroascorbate reductase activity. /. Biol. Chan. 265:15361-15364 45. Wheeler GL, Jones MA, Smirnoff N. 1998. The biosynthetic pathway of vitamin C in higher plants. Nature 393: 365-369 46. Willekens H, Chamnongpol S, Davey M, Schraudner M, Langebartels C, Van Montagu M, Inze D, Van Camp W. 1997. Catalase is a sink for H 2 0 2 and is indispensable for stress defense in C 3 plants. EMBO }. 16: 4806^816 47. Wingate VPM, Lawton MA, Lamb CJ. 1988. Glutathione causes a massive and selective induction of plant defense genes. Plant Physiol. 87: 206-210 48. Winkler BS. 1992. Unequivocal evidence in support of the nonenzymatic redox coupling between glutathione/glutathione disul­ fide and ascorbic acid/dehydroascorbic acid. Biochim. Biophys. Ada 1117: 287-290 49. Xiang C, Bertrand D. 2000. Glutathione synthesis in Arabidopsis: Multilevel controls coordinate responses to stress. In Sulfurnutrition and Sulfur Assimiulation in Higher Plants, ed. Brunold C et ah, Paul, Haupt, Bern, Switzerland, pp. 409^12 50. Xiang, Oliver DJ. 1998. Glutathione metabolic genes coordinately respond to heavy metals and jasmonic acid in Arabidopsis. Plant Cell. 10: 1539-1550 51. Xiang C, Werner BL, Christensen EM, Oliver DJ. 2001. The biological functions of glutathoine revisited in Arabidopsis trangenic with altered glutathione levels. Plant Physiol. 126: 564-574 52. Zhang H, Wang J, Nickel U, Allen RD, Goodman HM. 1997. Cloning and expression of an Arabidopsis gene encoding a putative peroxisomal ascorbate peroxidase. Plant. Mol. Biol. 34: 967-971

Chapter 10 Disulfide Bond Formation in the Periplasm and Cytoplasm of Escherichia Coli Jon Beckwith Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA jon [email protected]

1. Summary Disulfide bonds contribute to the structure of a large number of important proteins. These disulfides play a role in the folding pathways of these proteins and lend stability to their final folded structure. It has been com­ mon wisdom for some time that most or all proteins with structural disul­ fide bonds are found in extracellular compartments. In gram-negative bacteria, such proteins are found only among those secreted through the cytoplasmic membrane. Secreted toxins, appendages such as pili, periplasmic proteins and some cytoplasmic membrane proteins contain disulfide bonds. In eukaryotic cells, proteins with stable disulfide bonds are often found among the proteins that pass through the endoplasmic reticulum. They include secreted proteins as well as a large number of membrane receptors. For many years, the accepted explanation for the specialized subcellular location of proteins with disulfide bonds was based on a simplistic view of the process of disulfide bond formation. The explanation went as fol­ lows: the periplasm of bacteria, because it is exposed to oxygen, and the lumen of the endoplasmic reticulum, perhaps because of the presence of oxidized glutathione, are oxidizing environments. The formation of disul­ fide bonds in proteins, an oxidative step, takes place spontaneously in such environments. In contrast, the cytoplasms of both eukaryotic and prokaryotic cells are reducing environments; there is net generation of electrons yielding reduced molecules such as NADH and NADPH. Disulfide bonds either cannot form under reducing conditions or, if they do, they are con­ verted to free cysteine residues by the reducing environment. 213

214

Cellular Implications ofRedox Signalling

The assumption that disulfide bonds form spontaneously in an oxidizing compartment derived directly from the important experiments of Anfinsen and coworkers on protein folding.1 Their demonstration that reduced and denatured ribonuclease could reassemble into its active structure in the test tube, in the presence of oxygen, and in the absence of any catalysts suggested to many, that no catalyst for the oxidative folding process was necessary. In retrospect, the kinetics of disulfide bond for­ mation in ribonuclease in these experiments was very slow, incommen­ surate with the rapid kinetics that is observed in vivo. As a result of these assumptions, two questions about disulfide bond formation remained unexplored for many years: (1) How do disulfide bonds form in proteins in their specialized compartments? (2) Why do they not form in cytoplasmic proteins? The answer to the first question was thought to be "spontaneously" and the answer to the second ques­ tion was "because of the reducing environment in the cytoplasm". In the last decade, these questions have been reopened and unexpected answers have emerged. We became interested in the issue of disulfide bond formation, while studying protein secretion in E. coli. One of the proteins we used as a model for this process was alkaline phosphatase (AP). AP is a homo dimeric non-specific phosphomonoesterase found in the periplasmic space. Each of its monomers contains two disulfide bonds that are essen­ tial for AP to fold into an stable, active enzyme. 46 The protein is synthe­ sized initially with an amino-terminal signal sequence that directs it to the protein translocation machinery in the cytoplasmic membrane. We devised schemes for obtaining mutations that altered the cellular location of AP from the periplasm to the cytoplasm.30 These mutations were all character­ ized and shown to alter the signal sequence. We then discovered that when AP was localized to the cytoplasm in these mutants, it no longer exhibited enzymatic activity.29,30 Based on our knowledge of the role of disulfide bonds in the functioning of AP and aware of the lore that the reducing environment of the cytoplasm would not allow disulfide bond formation, we suspected that the lack of activity of cytoplasmically-localized AP was due to these factors. Subsequently, we went on to demonstrate that the cytoplasmic AP had all of its cysteines in the reduced form.13 Despite this gratifying explanation for our results, we were not satis­ fied. First, we were not immersed enough in the field of protein folding to realize that the process of disulfide bond formation was thought to be a spontaneous one. In fact, I assumed that there must be an enzyme

Disulfide Bond Formation in the Peri-plasm and Cytoplasm ofE. coli 215

involved. This line of thought caused us to try to devise schemes for isolating mutations that would be defective in the process of disulfide bond formation, thus allowing us to identify such enzymes. At the same time, the explanation for the lack of activity of alkaline phosphatase local­ ized to the cytoplasm— "the reducing environment of the cytoplasm"— seemed to avoid an important question: what is it specifically about the "reducing environment" that prevents disulfide bond formation in the cytoplasm? As a result of posing these two questions, we initiated studies of disulfide bond formation on both sides of the cytoplasmic membrane. Studies in our and other labs have resulted in surprises. First, there are enzymes both in eukaryotic and prokaryotic cells that are essential for the efficient formation of disulfide bonds. Further, elaborate pathways of electron transfer are required for the maintenance of activity of these enzymes and of enzymes involved in disulfide bond isomerization. Second, the absence of disulfide bonds in cytoplasmic proteins is not simply the consequences of a "reducing" cytoplasm. Rather, it is due to the absence of catalysts that promote the formation of these bonds, cata­ lysts like those found in the periplasm and endoplasmic reticulum. We have been able to generate bacterial strains in which such catalysts (oxidized thioredoxins) accumulate in the cytoplasm and promote rela­ tively efficient disulfide bond formation in the cytoplasm. Altogether, these studies have revealed that E. coli expresses an impressive array of thiol-disulfide oxdioreductases. These proteins play an important role in numerous cellular processes (see Fig. 1).

2. Disulfide Bond Formation in Extracytoplasmic Proteins While we did develop some ideas for obtaining mutations of E. coli that were defective in disulfide bond formation, the actual discovery of such mutations came to us by accident.5 We had constructed a strain for entirely different purposes that proved to be a source of these mutations. A gene fusion strain, in which the cytoplasmic enzyme /J-galactosidase was fused to a periplasmic domain of the cytoplasmic membrane protein MalF, exhibited a Lac phenotype due to the absence of /J-galactosidase activity. In this strain, the /J-galactosidase was dragged by the export sig­ nals of MalF into the membrane where it could not assemble into an active enzyme. An amino-terminal portion of /J-galactosidase was protruding into the periplasm. We expected that mutants defective in the process of

216 Cellular Implications ofRedox Signalling | Cytochrome c ! Biogenesis | Reduced /Oxidized Periplasmic Substrates

DsbA

X Respiratory Chain

±. DsbC

CcmG

DsbG Periplasm

DsbD

DsbB

Cytoplasm

Oxidized Substrates: Arsenate-Reductase

\ Glutaredoxins (grxA, grxB, grxC)

Oxidized Substrates: OxyR, Hsp33 Ribonucleotide-Reductase PAPS Reductase Met-Sulfoxide-Reductase_ J

Glutathione (gshA, gshB)

!

t {901)

Tpx

\ Thioredoxins (tncA, trxC)

t Glutathione Oxido-reductase

H,0,, R-OOH

Thioredoxin Reductase (trxB)

\ NADPH

t

KatG

AhpC

t AhpF

t NADH

Fig. 1. Thiol-disulfide redox proteins of Escherichia coli. Those components not referred to in the text include KatG, catalase, Tpx, a thioredoxin peroxidase, CcmG, a component of a pathway necessary for maintaining the cysteines of apo-cytochrome c's reduced, and DsbG, a DsbC homologue which has yet to be assigned an in vivo function. assembling the protein in the membrane would allow /}-galactosidase to reside in the cytoplasm where it would be active. These strains would be Lac+. Expecting mutations affecting the membrane insertion process, we instead found mutations (dsbA',) that interfered with disulfide bond formation. Apparently, DsbA was able to introduce disulfide bonds in the portion of /J-galactosidase that was exposed to the periplasm. This part of the protein contains several cysteine residues. These disulfide bonds stabilized the transmembrane structure of the hybrid protein, preventing its proper folding. Eliminating DsbA allowed the /3-galactosidase portion of the protein to undergo translocation back into the cytoplasm where it was active. At the same time, a number of other laboratories, by equally inadver­ tent approaches, also detected mutations in the dsbA gene.22,35 With these and other selections, a second gene, dsbB, required for disulfide bond formation was discovered.4,6,11,32

Disulfide Bond Formation in the Periplasm and Cytoplasm ofE. coli 217

Fig. 2. A pathway for protein disulfide bond formation. The reoxidation of DsbA aerobically occurs by passage of electrons to DsbB, then to quinones (Q), then to cytochrome bd or bo oxidases, and finally to oxygen. DsbA is a small periplasmic protein which is a member of the thioredoxin superfamily. This family is characterized by a conserved "thioredoxin fold" and a common active site motif: cys-x-x-cys.27 The active form of DsbA has the two cysteines joined in a disulfide bond. The process of disulfide bond formation begins with a disulfide exchange between this oxidized form of DsbA and reduced cysteine residues of substrate proteins. This exchange results in the passage of electrons to DsbA which then becomes reduced. The protein must be reoxidized in order to restore its activity. The reoxidation step is performed by DsbB; in dsbB mutants, DsbA accumulates in the reduced form (Fig. 2).4 DsbB is a cytoplasmic membrane protein with 4 transmembrane segments and 2 periplasmic loops each containing a pair of essential cysteine residues. A likely reoxi­ dation intermediate between DsbA and DsbB has been identified as a mixed disulfide heterodimer containing a disulfide bond between cysteine 30 of DsbA and cysteine 104 of the second periplasmic domain of DsbB.3839 DsbB is relatively specific in its choice of substrates; it appears unable to promote disulfide bond formation in the normal substrates of DsbA.

218

Cellular Implications of Redox Signalling

However, when cytoplasmic thioredoxin is converted to a periplasmic protein by attaching a signal sequence to its amino-terminus, it is oxi­ dized by DsbB.12'20 Further, eukaryotic protein disulfide isomerase (PDI), when expressed in the periplasm of E. coli is also oxidized by DsbB.34,50 PDI is also a member of the thioredoxin family. These results suggested that DsbB is specific in its oxidizng activity, reacting only with members of the thioredoxin family. Even there, its activity is limited to certain members of the family, being unable for instance to oxidize the protein DsbC in its native state (see below). For the pathway leading to disulfide bond formation to be functional, at least one more oxidative step is required: the oxidized form of DsbB must be regenerated in order for continuous reactivation of DsbA. At the time of the discovery of DsbB, quinones and other components of the membrane electron transport systems of E. coli were suggested as likely recipients of electrons from DsbB, restoring the latter protein to the oxi­ dized state. 4 Subsequently, Ito's group showed that depleting cells of quinones or of cytochromes resulted in defects in reoxidation of DsbB (and, therefore, DsbA). 25 More recently, Bader et al.2 provided in vitro evidence for the role of membrane electron transport components in the reoxidation of DsbB. Furthermore, they are able to specify which cytochromes and quinones can function in this pathway. Their report describes the reconstitution of a highly purified in vitro system that repli­ cates the in vivo phenomena. In particular, the reoxidation of DsbB is shown to be dependent on the presence of either cytochrome bd or bo terminal oxidases and of either a menaquinone or ubiquinone electron acceptor. These findings provide a satisfying explanation for yet another unresolved question about disulfide bond formation. It is known that the Dsb system still functions efficiently to promote disulfide bond formation under anaerobic growth conditions. 7 What is the source of oxidation potential when oxygen is not present? Now, with the identifi­ cation of menaquinone as an effective recipient of electrons from DsbB, a pathway via menaquinone to final electron acceptors other than oxygen appears likely and is supported by their data. Thus, DsbB would switch its use of primary electron acceptors depending on the degree of aerobiosis. We have described a mutation in the dsbB gene which results in such a strong defect in the interaction between DsbB and menaquinone, that anaerobic disulfide bond formation is drastically reduced. 21

Disulfide Bond Formation in the Peri-plasm and Cytoplasm ofE. coli 219

3. Protein Disulfide Bond Isomerization While the early studies of Anfinsen's group appeared to obviate the need for a disulfide bond forming enzyme, their results did focus attention on the need for a protein disulfide bond isomerase (PDI). Finding that the spontaneous oxidation of ribonuclease often resulted in the formation of incorrect disulfide bonds, Anfinsen suggested that an enzyme was neces­ sary to correct these errors. Anfinsen and his coworkers proceeded to identify such an activity in cell extracts.16 In this case, the discovery of PDI preceded the detection of a protein with a similar activity in bacteria, DsbC, by over three decades.33'45 Studies over the last several years in both prokaryotes and eukaryotes have illuminated aspects of the pathway leading to disulfide bond isomerization (Fig. 3). Like DsbA, protein disulfide bond isomerases, including DsbC, contain thioredoxin domains. Via the reduced form of their cys-x-x-cys active site, they are able to attack disulfide bonds of misoxidized proteins and promote shuffling of non-native disulfide bonds to obtain the properly oxidized protein. The mixed disulfide bond intermediate formed between DsbC and its substrate during this process might be resolved in two different ways: (1) attack of another cysteine in the substrate protein on the disulfide bond would lead to the formation of a new disulfide bond in the substrate protein and release of DsbC in the reduced state or (2) attack by the second cysteine of active site of DsbC on the disulfide bond would lead to transfer of the disulfide bond to DsbC and restoration of the reduced form of the substrate protein. 49 In the latter case, the substrate protein would be reoxidized by DsbA giving the system another chance to form the correct disulfide bond. How does DsbC recognize proteins with incorrectly formed disulfide bonds and promote formation of the correct ones? Recent studies on the structure and function of DsbC shed light on these questions. DsbC is a homodimer that contains two domains in each monomer — a dimerization domain and a thioredoxin-like domain. 28 Determination of the structure of the dimeric DsbC by X-ray crystallography reveals that the thioredoxin like domains surround a large cleft in which substrate proteins may sit. This structure, in conjunction with the finding that DsbC is a very effective chaperone for protein folding 9 suggests a model for its action. As a chaperone, DsbC may recognize exposed hydrophobic surfaces on the misoxidized substrate protein, which is then incorporated into the cleft.

220 Cellular Implications of Redox Signalling

Fig. 3. A pathway for disulfide bond isomerization. The reduction of DsbC occurs by passage of electrons from cytoplasmic thioredoxins first to the membrane-imbedded domain of DsbD, then to the carboxy-terminal thioredoxinlike domain of DsbD, then to the amino-terminal periplasmic domain of DsbD and finally to DsbC. The dark circles with numbers inside them represent the cysteine residues of DsbD involved in this electron transfer.

There, the reactive cysteine of the active site can attack the inappropriate disulfide bond, generating the reduced protein, which then may be released from the cleft. Thus exposed again to the periplasmic milieu, the protein may be acted on by DsbA to join the free cysteines to the correct partners in the polypeptide chain. At this point in the history of the fold­ ing pathway of the substrate protein, some of its domains may have folded properly, and regions of it may now be more appropriately placed for the formation of the correct disulfide bonds. By what may be a repeated iterative process of reduction and oxidation, assisted by progressively proper folding, the protein may rapidly be oxidized to its correct final form. DsbC requires a dedicated reductant, DsbD, to maintain its active site cysteines in the reduced state (Fig. 3). In dsbD mutants, oxidized DsbC accumulates. 40 DsbD may be necessary either because DsbC acts mainly as a reductant in the isomerization process and, therefore becomes oxidized, or because DsbC is oxidized by some other component of the periplasm. DsbD is a cytoplasmic membrane protein, providing a striking

Disulfide Bond Formation in the Periplasm and Cytoplasm ofE. coli 221

parallel to the DsbB-DsbA system. However, in contrast to the DsbB-DsbA pathway which uses intra-membraneous electron transfer components, DsbD derives its electrons from cytoplasmic proteins. The cytoplasmic thioredoxin pathway passes electrons to DsbD to maintain the latter's DsbC-reducing activity.40,41 The topological arrangement of DsbD in the membrane indicates that it has two large periplasmic domains, one at its amino-terminus and one thioredoxin-like domain at its carboxy-terminus.10,17,47 The central section of the protein is highly hydrophobic, containing eight transmembrane segments. Each of these three domains includes a pair of cysteines that are essential for the protein's function as a reductant of DsbC. 47 The presence of these essential cysteines suggested that the mechanism of transfer of electrons from cytoplasmic thioredoxin across the membrane and ulti­ mately to DsbC might involve a cascade of disulfide bond reduction steps. While significant progress has been made in working out the steps in this pathway, the mechanism for this electron transfer pathway has yet to be fully described. However, our finding that the three domains speci­ fied above can be separately cloned and then expressed together in cells to reconstitute a functional DsbD has facilitated the studies on the process of electron transfer.24 Results from our laboratory and those of Missiakas and coworkers indicate that electrons are transferred directly from thio­ redoxin to a cytoplasmically oriented cysteine in the membrane-imbedded domain of DsbD.10,24 From there, electrons are transferred to the carboxyterminal thioredoxin-like domain and then to the amino-terminal hydrophilic domain. It is this last component of DsbD that is responsible for the transfer of electrons to oxidized DsbC, reducing it and thus regen­ erating DsbC as a protein disulfide isomerase. Each of these transfer steps utilizes the essential cysteines in the participating domains and appears to involve intermediates in which those domains are bonded together as mixed disulfides. There are still major questions remaining about the transfer of elec­ trons through DsbD. If a cascade of disulfide bond reductions is respon­ sible for this transfer, one of these disulfides should be found within the membrane domain of DsbD. Yet, the two cysteines in this domain appear to be too far apart to form such a bond. The proposed distance between the cysteines is based on the topological analysis of DsbD and the knowl­ edge that thioredoxin in the cytoplasm and the thioredoxin-like domain of DsbD must each interact with one of the two cysteines. These contra­ dictory pictures of the molecule lead us to speculate that either (1) there

222

Cellular Implications ofRedox Signalling

is movement of transmembrane domains of DsbD during its cycles of electron transfer changing the relative positions of the two cysteines, (2) the cysteines are in fact close to each other in the membrane and par­ tial channels provide accessibility to the "thioredoxin" proteins on both sides of the membrane, and (3) that there is never a disulfide bond between these two cysteines. The periplasm contains both oxidative systems for making disulfide bonds (DsbA-DsbB) and reductive systems for breaking them. How are the components of these systems, protected from each other? One might have expected, for example, that DsbB would find DsbC a willing sub­ strate as DsbB appears to oxidize any thioredoxin-like protein that is presented to it. Recent studies on DsbC provide an explanation for the apparent shielding of its active site from oxidation by DsbA or DsbB.3 The presence of the cleft in DsbC formed in its dimeric structure protects it from oxidation by DsbB. Mutations that disrupt the dimerization domain now allow oxidation of DsbC.

4. Disulfide Bond Formation in the Cytoplasm As in the studies that revealed DsbA and DsbB, we used a genetic approach to understand why disulfide bonds did not form in cytoplasmic proteins. We sought mutants of E. coli that altered the environment of the cytoplasm so that disulfide bonds could form, for instance, in the cytoplasmically localized alkaline phosphatase. 15 Starting with a form of AP that was missing its signal sequence, we devised a genetic selection that demanded the presence of an active AP in the cytoplasm. Given the con­ cept of a reducing cytoplasm that prevented disulfide bonds from accu­ mulating in proteins, we imagined that we were looking for mutations in genes that coded for the responsible reducing enzymes. That is, if we could eliminate the enzyme that kept cysteines reduced in proteins, we would accumulate oxidized AP. Curiously, our perception of the goal of our mutant hunt persisted well after we had discovered the DsbA protein in our laboratory. It took us a while to realize that if the periplasm needed a protein that was actively catalyzing disulfide bond formation, then certainly we would need such a catalyst to observe effective disulfide bond formation in the cytoplasm. We obtained a collection of mutations that allowed AP to become active in the cytoplasm, all of which mapped to the gene (trxB) for thio­ redoxin reductase. 15 In these mutant strains, thioredoxin could no longer be

Disulfide Bond Formation in the Periplasm and Cytoplasm ofE. coli 223

reduced. It accumulated in the oxidized (disulfide-bonded) form as a result of its action in reducing enzymes such as ribonucleotide reductase. Working from our perception that we were looking for the appropriate reductant, we assumed we had found it. i.e. thioredoxin was the protein that maintained the cysteines of cytoplasmic proteins (AP in this case) reduced. When we knocked out thioredoxin reductase, there was no longer any reduced thioredoxin around to carry out this function. But we then found that mutations in the gene for thioredoxin (trxA) did not have the same phenotype; they allowed very little disulfide bond formation in AP. It was at this point that we began to realize that it might be the accu­ mulation of oxidized thioredoxin rather than the elimination of reduced thioredoxin that resulted in an active AP. However, our attempts to verify this alternative hypothesis failed. For example, a double mutant, trxB, trxA, which according to this latter hypothesis should no longer catalyze disulfide bond formation in alkaline phosphatase, still exhibited signifcant AP activity. These experiments and others 37 led us hypothesize that there was a second thioredoxin in E. coli that was contributing to the phenotype we observed in the trxB mutants. In fact, our group and the group of Spyrou in Sweden identified a second thioredoxin, thioredoxin 2, the product of the trxC gene.31,43 We showed that in the trxB mutant, thioredoxin 1 accumulates almost entirely in the oxidized form, while thioredoxin 2 accumulates largely in the oxidized form. We could eliminate the phenotype of the trxB mutants — the activation of alkaline phosphatase — by introducing into trxB strains both the trxA and trxC mutations. In contrast, simply eliminating thio redoxins 1 and 2 in a trxA, trxC double mutant did not allow alkaline phos­ phatase to be active in the cytoplasm. Thus, our data led to the conclusion that the disulfide bond formation we observed was due to the action of oxidized thioredoxins on the cysteines of AP (Fig. 4). These two proteins were essentially acting just as DsbA did in the periplasm. The thio­ redoxins are acting as effective oxidants even though the redox potential of these proteins is much lower than that of DsbA; they are much more active as reductants than as oxidants. The slower kinetics of disulfide bond formation we observed may have been due to this lower redox potential or to the absence of domains that DsbA contains that might facili­ tate its action as an oxidant of cysteines in substrate proteins. So, the explanation for the absence of disulfide bonds in cytoplasmic proteins is not "the reducing environment" of the cytoplasm per se. It is the absence of an oxidant. Perhaps if a cell could contain both high con­ centrations of oxidized and reduced thioredoxins, the reduced thioredoxins

224

Cellular Implications of Redox Signalling

Active AP

TrxATl

i

TrxC

Thioredoxin

NADPH Fig. 4. The formation of disulfide bonds in cytoplasmic alkaline phosphatase in a trxB mutant. In the trxB mutant missing thioredoxin reductase, oxidized forms of the two thioredoxins accumulate. These oxidized thioredoxins can now transfer their disulfide bonds to the reduced alkaline phosphatase, generating the oxidized active form of the enzyme.

Disulflde Bond Formation in the Periplasm and Cytoplasm ofE. coli 225

might interfere with the oxidation process, but we have no evidence for this. Furthermore, the now "oxidizing" cytoplasm still has the glutathione pathway which provides both reduced glutathione and reduced glutaredoxins as reductants. One of the implications of these findings is that there may be condi­ tions of growth or responses to environmental stresses where formation of disulfide bonds in the cytoplasm becomes possible and plays a physio­ logical role. In recent years, several examples have been reported of proteins that are activated by disulfide bond formation under conditions of oxidative stress.1819,23'S1 Earlier, we had found that in E. coli cultures sitting at 0°C, cytoplasmic alkaline phosphatase activity accumulates. 14 Our data were consistent with an explanation in which the cells were becoming depleted of NADPH, the source of electrons for the thioredoxin reductase/thioredoxin pathway, thus allowing oxidized thioredoxins to accumulate. Recent findings on eukaryotic viral protein assembly lead to similar conclusions about the cytosol of eukaryotic cells. In two cases, it appears that viruses carry their own machinery for disulfide bond formation, a machinery that functions in the cytosol.26,44 The fact that this oxidative system works efficiently in a cytosol that maintains its reducing path­ ways provides additional support for the importance of an oxidative system in disulfide bond formation rather than the absence of reducing pathways.

5. An Alternative Disulfide Reducing Pathway as an Environmental Switch Flexibility of E. coli in dealing with challenges to its disulfide reducing systems has been further exemplified by our recent studies on mutants that eliminate the requirement for NADPH to provide electrons for the two major reductive pathways. We have constructed gor, T trxB double null mutants that are missing the enzymes glutathione oxidoreductase and thioredoxin reductase. 37 These mutants grow extremely slowly, prob­ ably because of the absence of the major sources of electrons for reducing the essential enzyme, ribonucleotide reductase. The growth defect is reversed by the addition of the reductant dithiothreitol to the growth medium. However, these mutants throw off suppressors that restore normal growth at a very high frequency, approximately 1 in a thousand bacteria. We have characterized these suppressor mutations.

226

Cellular Implications ofRedox Signalling

The gor, trxB strains carrying the suppressor mutation grow with nearnormal generation times. 8 The suppressor mutations must somehow be restoring the ability of cells to reduce ribonucleotide reductase. Surprisingly, therefore, the strains now exhibit a highly oxidizing cyto­ plasm. Not only is signal sequenceless alkaline phosphatase efficiently oxidized, but when complex eukaryotic proteins with multiple disulfide bonds, such as tissue plasminogen activator are cloned into the suppres­ sor strains, they are also assembled into their active forms in the cyto­ plasm. These activities were greatly enhanced when an isomerase acitvity was introduced into the periplasm by expressing a signal sequenceless DsbC. 8 All of the suppressor mutations obtained at this very high frequency map to a single gene, ahpC."'2 The AhpC protein is a component of the alkylhydroperoxidase system, important in E. coli and many other organ­ isms for reducing hydrogen peroxide and alkyl hydroperoxides (see Fig. I). 36 This system functions reductively in the following way: elec­ trons are transferred from NADH via a protein-bound flavin to a pair of cysteines in the carboxy-terminal domain of AhpF, the other component of the system. These electrons are then transferred to a thioredoxin-like domain of AhpF, also with two redox-active cysteines and thence to the separate polypeptide, AhpC, which also has redox-active cysteines. AhpC then uses its free cysteines to reduces peroxides. In collaboration with biochemist Leslie Poole, we have combined genetic and biochemical analysis to reveal aspects of the functioning of this suppressor. 42 First, from both in vivo studies and in vitro studies on purified enzyme, we find that the mutation has lost its peroxidase activ­ ity. At the same time, we find in vivo that the suppressing activity of the ahpC mutation (ahpC*) depends on the presence of glutathione and glutaredoxin-1. The simplest conclusion at this stage of the work is that the alteration of the protein has converted it from a peroxidase to an enzyme that channels electrons into the glutathione pathway, perhaps by reducing oxidized glutathione. NADH is now used to feed electrons into the glutathione pathway rather than NADPH. Just as striking as the biochemical consequences of this mutation is the nature of the mutation itself. We have found that the mutational change in all of the ahpC* mutations we have analyzed is identical. The mutations result from an amplification of a triplet repeat located close to one of the active site cysteines of AhpC. A TCT sequence repeated four times in the wild-type genes is amplified to a repeat of five TCT's. This amplification results in a single amino acid addition to the protein with the profound

Disulfide Bond Formation in the Peri-plasm and Cytoplasm ofE. coli 227

effects described above. Not surprisingly this amplified repeat is itself unstable and reverts to the wild-type sequence at high frequency. The repeated sequence found in the wild-type E. coli ahpC gene is also found in several other gram-negative bacteria. We are led by these find­ ings to suspect that this ability to switch the function of this gene at such a high frequency serves some adaptive function for the bacteria. Other mechanisms, such as phase variation of flagellar antigens and switching back and forth between the presence and absence of the ability to utilize /3-glucosides involve similar forward and backward mutation frequen­ cies. If this reversible mutational switch is, in fact, an evolutionary mechan­ ism conserved for adaptive reasons, two possible reasons for this mechanism might be considered. First, it may be that the bacteria have conserved this switch mechanism to allow them to alternate between defending against peroxidative stress and defending against oxidative stress that generates disulfide bonds. Second, the mechanism may have persisted because it allows the bacteria to change survival strategies under conditions where either NADH or NADPH become limiting.

6. Conclusion A number of themes are becoming prominent in the field of disulfide bond metabolism. (1) The thioredoxin proteins can serve a variety of functions and many of them can perform either oxidative or reductive functions depending on the environment. A recent striking example of this flexibil­ ity comes from the findings of and Tsai et al.4S They showed that, in addi­ tion to acting as an isomerase and as an oxidase, PDI of eukaryotic cells can also act as a reductase in vivo. (2) Cascades of electron transfer via disul­ fide bonds are becoming apparent. The transfer of electrons in the DsbBDsbA pathway, the thioredoxin-DsbD-DsbC pathway and within and between the subunits of alkyl hydroperoxidase involve anywhere from three to five pairs of cysteines that are required for sequential electron transfer. (3) In these disulfide cascade pathways, in no case are electrons transferred directly between thioredoxin-like domains of proteins. In the pathway from thioredoxin reductase in the cytoplasm to DsbC in the periplasm, thioredoxin-like proteins are only used at alternating steps in the process. The same is true for the AhpCF system and the DsbB-DsbA system. (While DsbB does not have a domain with a thioredoxin fold, it does have a cys-X-X-cys motif whose redox potential behaves in muta­ tional analysis much like that of other thioredoxin molecules.)

228

Cellular Implications ofRedox Signalling

Finally, as so often occurs in science, dogma which is important in the genesis of a field can also have its restraining effects on new ways of look­ ing at problems. The concept of the presence or absence of disulfide bonds being simply due to the "reducing" and "oxidizing" environments of different compartments limited thinking about the posssibility of specific enzyme systems being responsible for these "environments".

Acknowledgments This work was supported by grants from the National Institute of General Medical Sciences, #'s 55090 and 41883. The author is an American Cancer Society Research Professor. He thanks Federico Katzen, Dani Ritz and Hiroshi Kadokura for helpful comments on this manuscript.

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8. Bessette PH, Aslund F, Beckwith J, Georgiou G. 1999. Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cyto­ plasm. Proc. Natl. Acad. Sci. USA 96:13703-13708 9. Chen J, Song JL, Zhang S, Wang Y, Cui DF, Wang CC. 1999. Chaperone activity of DsbC. /. Biol. Chem. 274: 19601-19605 10. Chung J, Chen T, Missiakas D. 2000. Transfer of electrons across the cytoplasmic membrane by DsbD, a membrane protein involved in thiol-disulphide exchange and protein folding in the bacterial periplasm. Mol. Microbiol. 35: 1099-1109 11. Dailey FE, Berg HC. 1993. Mutants in disulfide bond formation that disrupt flagellar assembly in Escherichia coli. Proc. Natl. Acad. Sci. USA 90: 1043-1047 12. Debarbieux L, Beckwith J. 2000. On the functional interchangeability, oxidant versus reductant, of members of the thioredoxin superfamily. /. Bacteriol. 182: 723-727 13. Derman AI, Beckwith J. 1991. Escherichia coli alkaline phosphatase fails to acquire disulfide bonds when retained in the cytoplasm. /. Bacteriol. 173: 7719-7722 14. Derman AI, Beckwith J. 1995. Escherichia coli alkaline phosphatase localized to the cytoplasm slowly acquires enzymatic activity in cells whose growth has been suspended: A caution for gene fusion stud­ ies. /. Bacteriol. 177: 3764-3770 15. Derman AI, Prinz WA, Belin D, Beckwith J. 1993. Mutations that allow disulfide bond formation in the cytoplasm of Escherichia coli. Science 262:1744-1747 16. Goldberger RF, Epstein CF, Anfinsen CB. 1963. Acceleration of reac­ tivation of reduced bovine pancreatic ribonuclease by a microsomal system from rat liver. /. Biol. Chem. 238: 628-635 17. Gordon EHJ, Page MD, Willis AC, Ferguson SJ. 2000. Escherichia coli DipZ: Anatomy of a transmembrane protein disulphide reductase in which three pairs of cysteine residues, one in each of three domains, contribute differentially to function. Mol. Microbiol. 35:1360-1374 18. Gostick DO, Green J, Irvine AS, Gasson MJ, Guest JR. 1998. A novel regulatory switch mediated by the FNR-like protein of Lactobacillus casei. Microbiology 144: 705-717 19. Jakob U, Muse W, Eser M, Bardwell JCA. 1999. Chaperone activity with a redox switch. Cell 96: 341-352 20. Jonda S, Huber-Wunderlich M, Glockshuber R, Mossner E. 1999. Complementation of DsbA deficiency with secreted thioredoxin

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33. Missiakas D, Georgopoulos C, Raina S. 1994. The Escherichia coli dsbC (xprA) gene encodes a periplasmic protein involved in disulfide bond formation. EMBO /. 13: 2013-2020 34. Ostermeier M, De Sutter K, Georgiou G. 1996. Eukaryotic protein disulfide isomerase complements Escherichia coli dsbA mutants and increases the yield of a heterologous secreted protein with disulfide bonds. /. Biol. Chem. 271:10616-10622 35. Peek JA, Taylor RK. 1992. Characterization of a periplasmic thiol/disulfide interchange protein required for the functional matu­ ration of secreted virulence factors of Vibrio cholerae. Proc. Natl. Acad. Sci. USA 89: 6210-6214 36. Poole LB, Godzik A, Nayeem A, Schmitt JD. 2000. AhpF can be dis­ sected into two functional units: Tandem repeats of two thioredoxin-like folds in the N-terminus mediate electron transfer from the thioredoxin reductase-like C-terminus to AhpC. Biochemistry 39: 6602-6615 37. Prinz WA, Aslund F, Holmgren A, Beckwith J. 1997. The role of the thioredoxin and glutaredoxin pathways in reducing protein disulfide bonds in the Escherichia coli cytoplasm. /. Biol. Chem. 272:15661-15667 38. Raina S, Missiakas D. 1997. Making and breaking disulfide bonds. Ann. Rev. Microbiol. 51:179-202 39. Rietsch A, Beckwith J. 1998. The genetics of disulfide bond formation. Ann. Rev. Genet. 32: 163-184 40. Rietsch A, Belin D, Martin N, Beckwith J. 1996. An in vivo pathway for disulfide bond isomerization in Escherichia coli. Proc. Natl. Acad. Sci. USA 93:13048-13053 41. Rietsch A, Bessette P, Georgiou G, Beckwith J. 1997. Reduction of the periplasmic disulfide bond isomerase, DsbC, occurs by passage of electrons from cytoplasmic thioredoxin. /. Bacteriol. 179: 6602-6608 42. Ritz D, Lim J, Reynolds CM, Poole L, Beckwith J. 2001. Conversion of a peroxiredoxin into a disulfide reductase by a triplet repeat expan­ sion. Science 204: 158-160. 43. Ritz D, Patel H, Doan B, Zheng M, Aslund F, Storz G, Beckwith J. 2000. Thioredoxin 2 is involved in the oxidative stress response in Escherichia coli.}. Biol. Chem. 275: 2505-2512 44. Senkevich TG, White CL, Koonin EV, Moss B. 2000. A viral member of the ERV1/ALR protein family participates in a cytoplasmic path­ way of disulfide bond formation. Proc. Natl. Acad. Sci. USA 97: 12068-12073

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45. Shevchik VE, Condemine G, Robert-Baudouy J. 1994. Characterization of DsbC, a periplasmic protein of Erwinia chrysanthemi and Escherichia coli with disulfide isomerase activity. EMBO J. 13: 2007-2012 46. Sone M, Kishigami S, Yoshihisa T, Ito K. 1997. Roles of disulfide bonds in bacterial alkaline phosphatase. /. Biol. Chem. 272: 6174-6178 47. Stewart EJ, Katzen F, Beckwith J. 1999. Six conserved cysteines of the membrane protein DsbD are required for the transfer of electrons from the cytoplasm to the periplasm of Escherichia coli. EMBO } 18: 5963-5971 48. Tsai B, Rodighiero C, Lencer WI, Rapoport TA. 2001. Protein disul­ fide isomerase acts as a redox-dependent chaperone to unfold cholera toxin. Cell 104: 937-948 49. Walker KW, Gilbert HF. 1997. Scanning and escape during protein-disulfide isomerase-assisted protein folding. /. Biol. Chem. 272: 8845-8848 50. Zhan XM, Schwaller M, Gilbert HF, Georgiou G. 1999. Facilitating the formation of disulfide bonds in the Escherichia coli periplasm via coexpression of yeast protein disulfide isomerase. Biotechnol. Program. 15: 1033-1038 51. Zheng M, Aslund F, Storz G. 1998. Activation of the OxyR transcrip­ tion factor by reversible disulfide bond formation. Science 279: 1718-1721

Chapter 11 The Thiol Redox Paradox in the Requirement for Disulfide Isomerization in the Eukaryotic Endoplasmic Reticulum Melissa Schwaller, Anton Soloyvov, Ruoyu Xiao, Johanna Lundstrom-Ljung and Arne Holmgren* Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77, Stockholm, Sweden * [email protected] H. F. Gilbert Verna and Mans McLean Department of Biochemistry Baylor College of Medicine, Houston, Texas 77030 [email protected]

Keywords: secreted proteins, protein disulfide isomerase (PDI), endoplasmic reticulum (ER), Grxl, thioredoxin, structural disulfides, disulfide isomeri­ zation, chaperone, redox buffer, oxidase activity, isomerase activity, disulfide isomerization, Oxidative Protein Folding, Thioreoxin Family, Kox, Scanning and Escape, substrate inhibition, Secretion in Yeast, domains of PDI

1. Summary The formation of the correct disulfide bonds in secreted proteins requires protein disulfide isomerase (PDI), a 55 kDa member of the thioredoxin family. PDI is constructed from four tandem thioredoxin structural domains. The end domains are catalytic; each has an active site sequence, CGHC, that contributes to the oxidation, reduction and isomerization activity. The cen­ ter domains have no redox active sites and are thought to bind unfolded protein substrates and enhance the isomerase activity. Mechanistic studies in vitro show that the initial formation of disulfide bonds is error prone, 233

234

Cellular Implications of Redox Signalling

requiring disulfide isomerization to achieve the correct structure. For the refolding of reduced substrate proteins in vitro, where both oxidation and isomerization is needed, the formation of native RNase is optimal in a glutathione redox buffer composed of 1 mM GSH and 0.2 mM GSSG ([GSH]2/[GSSG] = 5 mM), near the estimated redox state of the endoplasmic reticulum (ER). The redox requirements for isomerization, overall a redox neutral reaction, are more complex. As long as the redox buffer does not become too reducing and the misfolded substrate concentration does not become too high, there is no requirement for a redox buffer, but only if PDI is maintained in a reduced state. PDI is not a very efficient catalyst of isomerization, with turnover numbers near 1 min -1 . Deletion and sitedirected mutagenesis shows that efficient in vitro isomerization requires a catalytic domain along with a catalytically inactive, protein binding domain, and some mechanism for providing a reasonable concentration of both reduced and oxidized active sites. In vivo, PDI is an essential gene in yeast, and a number of experiments suggest that the isomerase activity is the essential feature. PDI is also a substrate for the ER oxidase, EROl which maintains PDI in a predomi­ nantly oxidized state, although a small amount (10-20%) of reduced PDI is present. When we initiated experiments to determine the structural features of PDI that could complement the essential activity of PDI in yeast, we were surprised to find that the catalytic domains, which have almost undetectable isomerase activity (~5% of wild-type), were able to rescue the lethal deletion of the yeast PDI1 gene, even when expressed from single copy plasmids with expression partially suppressed by glucose. Catalytically inactive domains do not rescue. Even more surpris­ ing, we find that other thioredoxin family members, Grxl and thioredoxin, can rescue lethality although growth rates are low. However, growth can be restored to near wild-type levels by providing the exogenous oxidant, diamide. The ER thiol redox paradox is... "If the disulfide isomerase activ­ ity of PDI is the essential feature in vivo, why do molecules with no detectable in vitro isomerase activity complement the PDI1 deletion and support near wild-type growth rates after adding an exogenous oxidant?"

2. Disulfides and Protein Structure and Stability In both eukaryotes and prokaryotes, secreted protein are often stabilized by structural disulfides that chemically crosslink two cysteine residues.1 These chemical crosslinks are added as the protein folds into its native conforma­ tion so that the pairing of the proper cysteine residues depends on the for-

Disulfide lsomerization in the Eukaryotic Endoplasmic Reticulum

235

mation of non-covalent interactions involving other residues of the protein. The eukaryotic endoplasmic reticulum is a compartment specialized for forming native disulfides. It provides catalysts of oxidation and disulfide isomerization along with a quality control apparatus that assures the faith­ ful and rapid formation of disulfides. Our purpose in this review is to describe the enzymology of native disulfide formation in secreted proteins. We will emphasize how the redox potentials of protein disulfides and the redox potentials of the catalysts for disulfide formation and isomerization are linked in vitro and in the eukaryotic endoplasmic reticulum.

3. Quality Control in the Endoplasmic Reticulum 3.1 General Quality Control and Secretion For extracellular proteins, disulfides that stabilize the structure of secreted proteins are introduced by and oxidative folding pathway present in the lumen of the endoplasmic reticulum (ER). This specialized compartment contains catalysts to introduce disulfides into proteins as they fold. A quality control system is in place to ensure that disulfide formation and protein fold­ ing lead to the correct structure through the retention and degradation of misfolded proteins.2 Because oxidative folding can be error-prone in its initial states,3 the ER must provide a mechanism to introduce disulfides into pro­ teins and a mechanism to correct those disulfides that may form incorrectly. 3.2 Formation of Disulfides in the Endoplasmic Reticulum Not all of the components of the ER oxidative folding pathway are known, and our understanding of the regulation of this pathway is even less complete. Secreted proteins are introduced into the ER cotranslationally, with their cysteines in the reduced state. For some proteins, disulfide formation begins even before the completion of protein synthesis 4 ; how­ ever, for other proteins, disulfide formation is posttranslational. 5 EROl, a recently discovered sulfhydryl oxidase provides oxidizing equivalents to protein disulfide isomerase (PDI), which, in turn, introduces disulfides into substrate proteins (Fig. I).6,7 PDI also serves as a chaperone and provides a quality control step through catalysis of the isomerization of incorrect disulfides that may have formed during oxidation. 8 The ER also provides a redox buffer of glutathione and its disulfide.9 Other oxidative pathways may contribute, including a flavoprotein oxidase that couples the formation of cystamine

236 Cellular Implications ofRedox Signalling NEW PROTEINS

ISOMERASE HS SH

n

-a

HS SH

tf HS SH

HS SH

GSH

GSSG

NATIVE PROTEINS

Fig. 1. Components of the oxidative folding apparatus in the endoplasmic reticulum. EROl oxidizes PDI, which in turn introduces disulfide bonds into newly translated and translocated proteins. When mistakes are made in cysteine pair­ ing, the isomerase activity of PDI catalyzes the rearrangement of the disulfides to their native pairing. The redox state of the ER may also be mediated by a glutathione redox buffer. A quality control apparatus retains and eventually degrades misfolded proteins so that only native proteins exit the ER. (mercaptoethylamine disulfide) to the reduction of oxygen.10 In addition to PDI, there are also other thioredoxin family members present in the ER, although they are expressed at low levels compared to PDI.11 At least some of these other family members may be present to facilitate the fold­ ing of specific secreted proteins. EROl, the subject of another chapter in this volume, is an essential protein in yeast that has recently been identified in higher eukaryotes. 6,7 It is a membrane-associated protein, most likely a flavoprotein 12 that receives electrons from PDI and transfers them to another, unknown acceptor. EROl has been trapped in a covalent complex with mutant PDI molecules with a single active site cysteine, suggesting that EROl normally serves to oxidize PDI, which, in turn, introduces the disulfides into proteins. 13 Overexpression of PDI cannot complement the EROl-null strain, nor can overexpression of EROl rescue strains that have no PDI. However, an EROl deletion can be rescued by including the organic oxidant, diamide, in the growth medium. 6 One of the most abundant proteins in the ER, PDI is an essential protein of the disulfide forming pathway. 14 It is a 57 kDa protein that may approach concentrations of near mM in the ER lumen. In vitro, PDI can introduce disulfides into substrate proteins (oxidase activity) and catalyze

Disulfide Isomerization in the Eukaryotic Endoplasmic Reticulum

237

the isomerization of incorrect disulfides, facilitating the attainment of the native disulfide configuration (isomerase activity).15 A redox role for PDI is clear from the inability of mutants of PDI lacking the redox active cysteines to complement the null mutation. 16 Mutants of PDI with immea­ surably low in vitro oxidase activity also rescue the lethal PDI deletion, albeit with a slow growth rate, suggesting that the essential activity of PDI is its isomerase activity. Lodish and coworkers have shown that the ER contains glutathione. 9 Using a cysteine-containing peptide with an N-linked glycosylation site they were able to show that the peptide visited the ER and was glycosylated. When it was reisolated from cultured mammalian cells, a portion of the peptide was present as a glutathione mixed disulfide. From indepen­ dent measurements of the thiol/disulfide redox potential of the peptide, the GSH/GSSG ratio of the ER was estimated to be 1:1 to 3:1. Taniyama et al. also showed that a mutant lysozyme in which one cysteine of a native disulfide was mutated to serine was secreted from yeast as a glu­ tathione mixed disulfide. 17 The role of glutathione in the ER is not entirely clear. The tripeptide is not essential for PDI activity in vitro.18 Whether GSH is essential for protein secretion is uncertain. Knockout of the GSH1 gene, responsible for the first step in glutathione biosynthesis, has given a mixed phenotype. Cuozzo and Kaiser report that it is not essential for growth or ER secretion.19 Lodish et al. showed that the ER could prefer­ entially support the import of GSSG; however, influx was slow and required very high concentrations of GSSG relative to the normally low concentration in the eukaryotic cytosol. Interestingly, one of the muta­ tions that can suppress the lethal deletion of EROl is GSH1. Kaiser has interpreted this to mean that GSH is imported as a reductant into the ER and opposes the action of EROl. 19 While the normal balance of the redox state of the ER may depend on EROl and GSH, backup mechanisms for supplying oxidizing equivalents to the ER are clearly in place. The redundancy in ER oxidative folding pathways is also apparent from thioredoxin family members that are present in the yeast ER. Only one of these, MPD1, can rescue the lethal PDI1 deletion and then only when overexpressed. However, some of these PDI homologs are neces­ sary when PDI mutants with single active site cysteines replace yeast PDI.11 Robertus and Ziegler have described a flavin monooxygenase in yeast that catalyzes the oxygen-dependent oxidation of thiols to disul­ fides. Cystamine is a particularly good substrate of this enzyme and they have suggested that the import of cystamine into the ER could provide oxidizing equivalents. 10

238 Cellular Implications of Redox Signalling

Fig. 2. Oxidative protein folding. The formation of disulfide bonds (oxidation) early in the folding process is often error-prone. To reach the native configuration, incorrect disulfides must be rearranged into a native structure (isomerization). 3.3 Constraints Imposed by the Folding Environment of the ER The redox environment in the ER places constraints on the ability to form and rearrange disulfides during protein folding. Disulfide formation requires an oxidation, either direct or mediated through a small-molecule thiol/disulfide redox buffer. However, disulfide isomerization requires breaking incorrect disulfide bonds which requires free thiols. In vitro, this balance can be maintained by a redox buffer.20 The disulfide component of the redox buffer provides oxidizing equivalents for disulfide formation while the thiol component enables the isomerization of incorrect disulfides through thiol/disulfide exchange.

4. Oxidative Protein Folding Oxidative protein folding couples the formation of structural disulfides to the development of the folded structure of the protein (Fig. 2). The thermodynamic linkage between disulfide formation and protein stability means that non-covalent interactions that stabilize the native fold of the protein make it easier to form a native disulfide within that structure. 1 Eventually, it is the non-covalent interactions within the folding protein that specify which disulfides will pair most effectively. When disulfide formation occurs early in folding, before the correct non-covalent

Disulfide Isomerization in the Eukaryotic Endoplasmic Reticulum

239

structure is developed, cysteine pairing is more likely to be incorrect. Although there are examples in which disulfide formation proceeds by the direct and orderly pairing of only native disulfides,21 the initial forma­ tion of disulfides is more often error prone because of the incomplete formation of the correct, folded structure. 22 Efficient folding requires mechanisms for correcting these mistakes in cysteine pairing. When disulfide formation is incorrect, reaching the native structure requires disulfide isomerization (Fig. 2). There is no net utilization of redox equi­ valents during disulfide isomerization. However, isomerization is initi­ ated by thiol/disulfide exchange reactions that require a free thiol. Consequently, isomerization can occur only when the environment is not so oxidizing as to make free thiols unavailable. The driving force for the formation of correct disulfides is the stability of the native structure. Isomerization will continue until further isomeri­ zation is kinetically or thermodynamically unfavorable. For many proteins this also represents the most thermodynamically stable struc­ ture. However, the biologically active, native structures of some disulfidecontaining proteins represent kinetic traps that are prevented from further rearrangement by a kinetic barrier. 23

5. Protein Disulfide Isomerase Anfinsen and his colleagues realized that spontaneous oxidative folding was too slow to be biologically relevant. They succeeded in isolating an activity that would catalyze the formation of native RNase from the scrambled form. Named protein disulfide isomerase (PDI), this ERlocalized protein is required for the formation and secretion of disulfidecontaining proteins. 24

5.1 Structural Organization — The Thioreoxin Family PDI is a member of a large family of oxidoreductases, characterized by an active site with a CXXC sequence and a a/(i-fold similar to thioredoxin. 25 PDI is composed of four structural domains and an acidic C-terminal tail, a-b-b'-a'-c (Fig. 3).26'27 The sequences of domains a and a' are similar as are the sequences of domains b and b'. The catalytic sites, CGHC, are located in the a and a' domains. Domains a and a' also display sequence similar­ ity to thioredoxin while b and b ' do not. NMR structures of a and b

240

Cellular Implications ofRedox Signalling

Fig. 3. Domain structure of PDI. PDI contains two pairs of internally similar domains. The a and a' domains contain the two active site sequences (CGHC) and show sequence similarity to thioredoxin. The b and b' domains also have a thio­ redoxin fold but show no sequence similarity to thioredoxin. From the structure of individual domains and the sequence, PDI is composed for four tandem thio­ redoxin domains coupled to a C-terminal acidic tail that also contains the KDEL, ER retention signal. The initial sequence of the rat PDI gene and definition of domain boundaries is described in Ref 26. Domain boundaries are shown accord­ ing to those define by Darby et ah by protease mapping and sequencing.

domains show that both have thioredoxin folds although the CXXC motif is missing from the b domains.28,29 There are a number of different thioredoxin family members in the endoplasmic reticulum. In mammalian cells, these include erp57,30 erp72,31 and pancreas-specific isozyme of PDI (PDIp).32 In yeast, the family members MPD1, MPD2, EUG1, and EPSI are expressed in the ER, yet in very low amounts compared to yeast PDI.11 Only MPDI and EUG1 will rescue a yeast strain that has had its PDI gene deleted, but their ability to rescue the PDI deletion also depends on the presence of other of the yeast ER PDI-like proteins.

5.2 Active Site Properties The redox properties of the PDI active site dithiol/disulfide can be defined on the thiol/disulfide redox scale which defines the oxidation potential by the equilibrium with GSH and GSSG (Fig. 4). Lundstrom and Holmgren estimated the overall equilibrium for full-length, wild-type through equilibrium between PDI and thioredoxin. The value of Kox they measured was 3 mM with no indication that the two active sites behaved differently.33 Darby and Creighton 34 measured Xox directly on the isolated a and a' domains expressed independently. The Km value was 1 mM for

Disulfide Isomerization in the Eukaryotic Endoplasmic Reticulum TAX

Fig. 4. Definition of Kox. The equilibrium between a glutathione redox buffer (GSH and GSSG) and a dithiol/disulfide site is used to define Kox, an equilibrium constant with units of M. Km represents an oxidation potential (higher values imply it is easier to form the disulfide).

Table 1. Complementation of the lethal PDI1 deletion by the individual domains of PDI.

Expressed Protein Wild-type PDI a' a' b b' Thioredoxin Thioredoxin (P34H) Glutaredoxin-1

Isomerase Stimulation Activity Growth by (% of wt) Viability Rate Diamide 100 3 1 <1 <1 <1 10% <1

Inhibition by Diamide

+++ +++ +++

+++ +++ +++

+ ++ ++

+++ +++ +++

+ ++ +

+++ +++ +++

both. In addition, they d e t e r m i n e d the e q u i l i b r i u m constant for forming a PDI-glutathione m i x e d disulfide. Interestingly, this e q u i l i b r i u m constant is unfavorable. For typical cysteine r e s i d u e s , the e q u i l i b r i u m constant for reaction w i t h g l u t a t h i o n e is expected to b e n e a r one. Its redox potential places PDI a m o n g the m o r e oxidizing active site disulfides of the thioredoxin family. W i t h the exception of PID a n d the bac­ terial DsbA 3 5 (a periplasmic oxidase), other m e m b e r s of the thioredoxin-PDI

242 Cellular Implications ofRedox Signalling

Table 2. Redox potentials of thioredoxin family members.

K, (mM) PDF PDI-a domain PDI-a' domain P34H-thioredoxin Glutaredoxin 1 Thioredoxin (£. coli)

3.1 0.7 1.9 96 400 1400

^SSG

% Reduced (GSH7GSSG) = 5 mM

0.01 0.05 6

62 88 72 5 1.2 0.4

Ref. 33 34 34 36 36

a

Kox is the equilibrium constant for oxidation of the active site dithiol by GSSG. KSSG is the equilibrium constant for the formation of a mixed disulfide with glutathione and the more N-terminal active site cysteine. 'measured for the intact molecule in which both active sites bocome oxidized. b

family are much poorer oxidizing agents. In fact, the redox potential of the CXXC motif in PDI and related structures varies from a low of about 1 mM for PDI or its domains to a high of 1400 mM for thioredoxin and glutare­ doxin,36 over a 103 range in redox potential (Table 2). Part of this variation is due to the nature of the two residues between the active site cysteines. In addition, a low pKa due to stabilization of the thiolate anion of the more N-terminal active site cysteine is correlated to the redox potential. 35

5.3 Catalytic Properties of PDI and its Mutants PDI is, by far, the best catalyst of the oxidative folding (oxidation and isomerization) of all the thioredoxin-PDI family members. In the presence of a glutathione redox buffer, it catalyzes rapid disulfide formation fol­ lowed by rate-limiting isomerization. By contrast, E. coli thioredoxin and glutaredoxin have no isomerase activity in an ER-like redox buffer at neutral pH. Interestingly, glutaredoxin is an excellent oxidase and will catalyze disulfide formation quite effectively but it fails to accelerate the subsequent disulfide isomerization. 37 Similarly, the isolated a and a' domains of PDI display near wild-type oxidase activity but fail to display significant isomerase activity. Deletion mutants of PDI and mutants in which the domain order is rearranged suggest that full-length PDI is essential for high isomerase activity (Table 1). The only constructs with an incomplete number of PDI domains that display high isomerase activity have the combination of domains b'a'c. Moving the b ' domain relative to a'

Disulfide Isomerization in the Eukaryotic Endoplasmic Reticulum

243

destroys isomerase activity as does deleting the c domain. The b ' domain has been implicated as a peptide/protein binding site for PDI; however, other sites in the molecule are also likely to contribute to substrate binding and isomerization.38 In addition, simply having an appropriate active site redox potential is insufficient to provide for good isomerase activity since the a and a' domains exhibit very low isomerase activity.

6. Redox Optimum for Oxidative Folding 6.1 Redox Dependence of PDI If we begin with a reduced substrate molecule, oxidation and isomeriza­ tion are both required to reach the native, folded structure. To catalyze transfer of oxidizing equivalents to substrate, the PDI active site must be oxidized as an intramolecular disulfide (active site mutants with a single cysteine are not able to catalyze substrate oxidation). 39 To catalyze isomerization, the PDI active site must be reduced in order to break incor­ rect disulfides and facilitate their rearrangement. Consequently, the active site of PDI must be balanced between oxidized and reduced states to effectively catalyze oxidative folding. In vitro, a glutathione redox buffer can be used to maintain the solution redox state at a constant value.40 A glutathione redox buffer is present in the ER; however, its essential role in oxidative folding has been questioned.

6.2 Redox Potential of Substrates The variation of the velocity of disulfide formation and isomerization with the GSH and GSSG concentrations of the glutathione redox buffer arise from effects on redox state on both the substrate and PDI. During oxidative folding, the disulfides that form very early in folding are not particularly stable, having glutathione redox potentials that are esti­ mated in the mM range. However, the disulfides in the folded, native proteins are considerably more stable with oxidation potentials in the range of 1 M to 105 M.1 The redox buffer must be sufficiently oxidizing to support the formation of the early disulfides in folding. In addition, the redox buffer must be sufficiently reducing to support isomerization without completely reducing the substrate disulfides that are essential to subsequent folding. The net result is that an optimum GSH and GSSG concentrations are expected for both catalyzed and uncatalyzed

244 Cellular Implications of Redox Signalling

0.6

I 0.4 1 0.2 |

4 8 [GSH] (mM)

12

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— • —/• >.

f I 0.4 — 3.

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% >

n

T

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«"■"•"■•-.■.

N

i , r-r

4 8 [GSH] (mM)

12

(C)

4 8 [GSH] (mM)

(e)

Fig. 5. Optimum redox buffer for PDI-catalyzed and uncatalyzed oxidative folding of reduced RNase. The optimum rate of native RNase formation is observed at a [GSH] = 1 mM and [GSSG] = 0.2 mM (taken from Ref. 18 with permission). oxidative folding. 41 The PDI-catalyzed folding of reduced, denatured RNase occurs with the highest velocity at a GSH concentration of 1.0 mM with a GSSG concentration of 0.2 mM (Fig. 5). This redox optimum, represents a glutathione redox potential of [GSH]2/[GSSG] = 5 mM, close to the redox potentials of the early RNAse disulfides that form and the redox state of PDI. If we assume that the redox effects on uncatalyzed refolding represent constraints imposed by the substrate, the ratio of the rate of the

Disulfide Isomerization in the Eukaryotic Endoplasmic Reticulum

•

•

—

a a. Q a. +

245

•

3

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•J

o JO

2

0

p

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1

1

0.2

1 0.4

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1 0.6

[GSH] 2 /[GSSG] Fig. 6. Ratio of PDI catalyzed to uncatalyzed RNase isomerization as a function of the redox state of the glutathione redox buffer. To remove contributions from effects of the redox buffer on the redox state of the substrate, the rate of the PDI catalyzed isomerization of reduced RNase is divided by the rate of the uncatalyzed isomerization (adapted from Ref. 18 with permission). PDI-catalyzed reaction to that of the uncatalyzed reaction should reveal the redox requirements for PDI during isomerization coupled to disulfide oxidation. As can be seen from Fig. 6, PDI exhibits half of its maximal isomerase activity at a [GSH]2/[GSSG] of 0.05 mM. This suggests that the redox requirements for efficient isomerization by PDI may be quite different from what is required for optimum disulfide formation. At a [GSH] 2 / [GSSG] of 5 mM, PDI with a Kox of 1 mM would be 80% reduced while the active site would be only 5% reduced at a [GSH]2/[GSSG] of 0.05 mM.

7. Mechanism of Isomerization 7.1 Scanning and Escape Disulfide isomerization does not change the redox state of the substrate protein. However, the mechanism by which incorrect disulfides are

246 Cellular Implications ofRedox Signalling

Fig. 7. Scanning and escape mechanism for catalysis of disulfide isomerization by PDI. After forming a covalent intermediate with the substrate, the partitioning of this intermediate is governed by the relative rates of subsequent thiol/disulfide exchange reactions. If intramolecular isomerization of the substrate does not occur in a timely fashion, PDI can escape by using the second active site cysteine to reduce the substrate disulfide and form oxidized PDI (adapted from Ref. 44 with permission). broken and new disulfides are formed requires a free thiol. Disulfide isomerization is initiated by PDI attacking one of the disulfides of the substrate (Fig. 7). The partitioning of this covalent PDI-substrate complex can proceed in three directions, depending on the effective molarity of the nucleophilic sulfur. Reversing the initial attack reforms the original disul­ fide. This fate will be favored when the original protein disulfide is stable and there is a high tendency to form it by intramolecular displacement of PDI. Consequently, native (more stable) disulfides should be harder to reduce although PDI can attack them. 42 The second possibility is that the substrate thiol will attack another disulfide of the substrate and initiate an intramolecular rearrangement. Such intramolecular rearrangements are observed during the uncatalyzed isomerization of bovine pancreatic trypsin inhibitor,43 but it is a very slow reaction and it is difficult to see how PDI would facilitate it since PDI does not seem to be able to unfold its substrates. If an intramolecular rearrangement forms a very stable disulfide with high effective molarity, it

Disulfide Isomerization in the Eukaryotic Endoplasmic Reticulum

247

will be favored. However, if intramolecular isomerization and reversal of the attack are both slow PDI can extricate itself (escape) by using the sec­ ond (C-terminal) cysteine. Escape provides a way to prevent PDI from becoming entangled with slowly rearranging substrates and it also reduces the substrate disulfide, allowing it the possibility of oxidizing in a different way. The dominance of the escape pathway would mean that PDI catalyzes isomerization by cycles of reduction and reoxidation requiring the presence of both oxidized and reduced PDI to affect isomerization.44 Mutation of the more C-terminal cysteine should disable the escape route, and this is confirmed experimentally. Active site mutants of PDI that have only one cysteine, CGHS (the second active site is inactivated by mutation of both cysteines to serine), accumulate in mixed disulfide inter­ mediates with protein substrates.44 In a redox buffer, the isomerase activity of these mutants is measurable, but relatively low compared to the appro­ priate PDI mutant with only one active site. In addition, the isomerase activity is absolutely dependent on the presence of a redox buffer suggest­ ing that GSH, albeit less efficiently, can provide an alternative escape mech­ anism that involves intermolecular displacement of PDI by GSH.39,44

8. Substrate Inhibition and Recycling of Oxidized PDI 8.1 Substrate Inhibition Using purified substrates and PDI, a glutathione redox buffer is not essen­ tial for catalyzing isomerization. If PDI is reduced and then gel-filtered to remove all of the low molecular weight thiol, it catalyzes the isomerization of scrambled RNase (sRNase) just as effectively as in the presence of a redox buffer.44 However, under these conditions, the system is very sensitive to inhibition by the addition of excess scrambled substrate. At high substrate concentrations, the rate of isomerization falls and becomes significantly slower than the rate observed in the presence of redox buffer (Fig. 8). This "substrate inhibition" does not occur when a redox buffer is present. It is difficult to account for substrate inhibition if intramolecular isomerization were the dominant mechanism of catalysis. However, catal­ ysis of isomerization through cycles of reduction and reoxidation would provide two alternative mechanisms that could account for the substrate inhibition. Without a redox buffer, reduction of a substrate disulfide through the escape mechanism would produce oxidized PDI. To regenerate, reduced PDI and initiate new rounds of isomerization (reduction), the

248

Cellular Implications ofRedox Signalling

2.0

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+ GSH/GSSG

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I

1.5

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/ D)

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z

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£

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-1 10

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1— 50

[sRNase] (|iM) Fig. 8. Substrate inhibition of PDI. At high concentrations of sRNase the substrate inhibits isomerization in the absence of a redox buffer. The redox buffer relieves this substrate inhibition (adapted from Ref. 44 with permission). oxidized PDI must be recycled to reduced PDI while the reduced sub­ strate must be reoxidized in an alternative configuration of disulfides (Fig. 7). By this mechanism, substrate inhibition would be arise when the excess scrambled substrate binds to oxidized PDI and prevents it from reoxidizing the substrate. Alternatively, excessive rounds of escape that occur much faster than subsequent reoxidation would deplete reduced PDI and inhibit the initiation of reduction events. To distinguish between these alternatives (depletion of oxidized PDI or depletion of reduced PDI), we added extra oxidized PDI and found that it had little effect on the rate of isomerization. By contrast, adding a small amount of GSH (GSSG is removed with excess NADPH and glutathione reductase) relieves the substrate inhibition by excess sRNase. This suggests that depletion of reduced PDI is the major reason for substrate

Disulfide Isomerization in the Eukaryotic Endoplasmic Reticulum

249

inhibition. If the amounts of oxidized and reduced PDI must be maintained to promote both oxidation and reduction, adding too much GSH should inhibit refolding and promote substrate reduction. Such seems to be the case in the absence of PDI where it takes about 1 mM GSH to inhibit refolding by half. However, when a catalytic amount of wild-type PDI is present, refolding persists to a significantly higher concentration of GSH (1 mM) suggesting that the PDI active site preserves oxidizing equivalents and can donate them back to substrate much better than glutathione itself. In the presence of glutathione reductase and NADPH, the equilibrium position for the system of GSH, PDI and sRNase is obviously far toward fully reduced RNase and PDI. However, under these conditions, the rate of formation of native RNase is sufficiently fast to reach the native struc­ ture and become kinetically resistant to GSH reduction. GSH relieves substrate inhibition by excess sRNase by maintaining the PDI redox state in a steady-state that is far removed from equilibrium.

9. Secretion in Yeast The in vitro catalytic properties of PDI are sufficient to provide sufficient catalysis of disulfide formation and isomerization in vivo; however, there must be some mechanism to provide oxidizing equivalents and to regu­ late disulfide formation during protein secretion. Bardwell and Beckwith have defined the elements of disulfide formation in the periplasm of E. coli.45 In this pathway, which will be described in detail in another chap­ ter in this volume, oxidizing equivalents are transferred to the DsbA pro­ tein by components of the bacterial electron transport chain using oxygen as a terminal electron acceptor. Oxidized DsbA, another member of the thioredoxin superfamily, inserts disulfides into secreted proteins. DsbA has little isomerase activity, this activity being provided by another thioredoxin family member, DsbC. Interestingly, reducing equivalents are introduced into the periplasm through a separate pathway using DsbD, an integral membrane protein. Recently, the laboratories of Kaiser6,19 Weissmann 712 and Sitia46 have begun to elaborate the essential components of the disulfide formation apparatus of yeast and higher eukaryotes (Fig. 1). The immediate source of oxidizing equivalents in yeast is the essential protein, EROl. This membrane-associated flavoprotein can be trapped in a covalent complex

250

Cellular Implications of Redox Signalling

with mutants of PDI with one active site cysteine suggesting that PDI is normally oxidized by EROl in yeast.13 Whether EROl can directly oxidize protein substrates is not known, but there is a stimulation of oxidation by flavin.12 PDI is an essential protein in yeast; deletion of the PDI1 gene is lethal. It is clear that the oxidoreductase activity of PDI is the essential feature because a mutant with no active site cysteines does not restore viability. 14 Strains in which EROl has been deleted can be rescued if the growth m e d i u m contains the small-molecule oxidant diamide. However, diamide cannot rescue strains that are missing PDI, suggest­ ing that PDI has an essential function other than substrate oxidation. 6 Complementation of the lethal deletion with mutants of PDI that have low, but measurable isomerase activity but no measurable oxidase activ­ ity suggests that the isomerase activity is the essential feature of the molecule. 16 In addition to the insertion of newly synthesized proteins into the ER in their reduced form, the ER may have other mechanisms for setting the thiol/disulfide redox state. Lodish and coworkers found glutathione in the ER and estimated the ratio of GSH/GSSG to be approximately 1:1 to 3:1.9 A GSSG transporter in the ER was also reported, but this transport mechanism was relatively slow and exhibited a Km for GSSG that was considerably higher than the cellular concentration. Kaiser and his labo­ ratory have recently shown that deletion of the GSH1 gene and interrup­ tion of glutathione biosynthesis in yeast is not lethal; however, a low concentration of GSH (or DTT) in the medium seems to be required. 19 Surprisingly, disruption of the GSH1 gene will rescue the lethal EROl deletion. Kaiser has suggested that GSH may be transported into the ER to oppose the oxidative action of EROl and to maintain PDI and other ER oxidoreductases in an appropriate redox state to catalyze reduction or isomerization. 19 PDI in the yeast ER is mostly oxidized as shown by labeling with AMS. 13 This is consistent with a role for PDI in substrate oxidation; how­ ever, the predominance of the oxidized will limit the ability of PDI to serve as an isomerase. However, recent experiments in mammalian cells suggests that PDI is predominantly reduced (see Sitia's chapter in this volume), implying a more important role for PDI-dependent isomeriza­ tion. Whether this is due to an intrinsic difference between yeast and higher eukaryotes or a regulatory phenomenon that depends on the specific growth conditions of the experiments that were performed is not yet clear.

Disulfide Isomerization in the Eukaryotic Endoplasmic Reticulum

251

9.1 Redox and Structural Requirements for Yeast Growth Both rat and human PDI will rescue the lethal deletion of yeast PDI and support growth rates that are comparable to the parental strain. To deter­ mine what part(s) of PDI are essential, we transformed yeast with a vector expressing individual PDI domains with an N-terminal signal sequence and a C-terminal HDEL for ER retention. Despite the fact that they have very low isomerase activity, the a and a' domains of PDI are sufficient to not only rescue the lethal PDI1 deletion but to support wildtype growth rates, even when expressed from single-copy plasmids (Table 2). Vectors containing b or b ' were ineffective. Consequently, only one catalytically active domain is absolutely essential for supporting yeast ER secretion, at least under the conditions of laboratory growth in defined media. Titrating in increasing glucose suppresses growth of all wild-type and a or a'-expressing strains at a similar glucose concentration suggesting that the amounts of expression of wild-type, a and a' are com­ parable. In addition, expression of the a and a' domain from single copy plasmids is also able to support wild-type growth. Since species with very low isomerase activity support yeast viability, we were interested in the ability of other species with very different redox potentials to substitute for PDI. Raines has previously reported that the P34H mutation of thioredoxin will support yeast growth but that thio­ redoxin will not.47 We find that both P34H thioredoxin and wild-type E. coli thioredoxin will support yeast growth when expressed with an ER signal sequence (Table 1). The growth rate of thioredoxin is lower than that of P34H-Trx, but both will support growth. Although thioredoxin and P34H-Trx have been reported to have isomerase activity, the activity of P34H thioredoxin is <10% of wild-type PDI and thioredoxin supports even less isomerase activity.48 Similarly, E. coli Grxl when expressed with a signal sequence and an HDEL retention signal, will complement the PDI deletion mutant although the growth rate is relatively slow compared to wild type. In vitro, even high concentrations of Grxl exhibit no detectable isomerase activity; however, Grxl will accelerate substrate oxidation through the formation of protein-glutathione mixed disulfides under standard assay redox conditions. 37 There is no correlation between the ability of a molecule to substitute for PDI in the yeast ER with its ability to catalyze substrate isomerization in vitro (Table 2). The Kox values span a range of 1000-fold, corresponding to a difference in E°' of 90 mV. If the redox state of the ER were maintained with a glutathione redox buffer set at the redox optimum for oxidative

252

Cellular Implications of Redox Signalling

folding, PDI would be predominantly reduced while thioredoxin and glutaredoxin would be even more oxidized. For example, at equilibrium in a redox buffer with a [GSH]7[GSSG] of 1 mM, PDI would be 50% reduced but only one molecule in 2000 would be reduced with thio­ redoxin. The unavailability of the reduced protein would seriously limit catalysis of disulfide isomerization and account for our inability to observe isomerase activity with these proteins in vitro. The organic oxidant, diamide, 49 will support the formation of protein disulfides. The lethal EROl null mutation can, in fact, be rescued if diamide is included in the growth medium. 13 When we examined the growth rates of APDI1 mutants containing a plasmid expressing various PDI mutants, domains, and thioredoxin or Grxl, we find that low con­ centrations of diamide stimulate the growth of Grxl but not wtPDI, a, a' or thioredoxin. With Grx substituting for PDI, diamide stimulates the growth of this strain to near wild-type growth rates. This suggests that all of the molecules other than Grx can support the oxidase activity required for growth but Grx cannot, requiring supplemental oxidation to achieve wild-type growth. If the disulfide isomerase activity of PDI is the essential feature in vivo, why do molecules with no detectable in vitro isomerase activity comple­ ment the PDIl deletion and support near wild-type growth rates after adding an exogeneous oxidant? The observation that the PDI a and a' domains along with Grxl and thioredoxin from E. coli will rescue the lethal deletion of yeast PDI although all of these molecules have minimal isomerase activity could mean that the isomerase activity of PDI is not its essential feature and that there is some other essential function. Another option is that the ER maintains the redox state of these catalysts by a mechanism that involves independent mechanisms of oxidation and reduction so that the redox state of the catalyst is maintained in a steady state that is far away from equilibrium. The system suggested by Kaiser19 for regulating the ER redox state could account for the relative indepen­ dence of catalysis of isomerization on the redox potential of the catalyst. In his model, EROl injects oxidizing equivalents into the ER while reduced glutathione is imported into the ER to bring in extra reducing equivalents. If equilibration with the redox buffer is slow compared to catalysis of disulfide isomerization, the steady-state redox state of the ER may allow these thioredoxin family members to exist in redox states that will facilitate isomerization, but which cannot be maintained in vitro with a redox buffer alone. An ER mechanism to independently regulate oxidation

Disulfide lsomerization in the Eukaryotic Endoplasmic Reticulum

253

and reduction mechanisms may provide a sensitive control mechanism or it may allow the oxidative folding of a wider variety of substrates.

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Chapter 12 Mechanisms Controlling Redox Balance in Cells. Inhibition of Thioredoxin and of Thioredoxin Reductase

Carlos Gitler, Batia Zarmi, a n d Edna Kalef Department of Biological Chemistry Weizmann Institute of Science, Rehovot, Israel [email protected]

Keywords: glutathione, Protein Thiol Labeling, N-iodoacetyl-3-iodotyrosine IAIT, DTT, cetiltrimethyammonium bromide (CTABr), Arsenical, Affinity Chromatography, arsines, post NEM-DTT procedure, N-ethylmaleimide NEM, phenylarsine oxide PAO, phosphotyrosine, phosphatases, NADPH-thioredoxin reductase-thioredoxin system, lymphoblast cell extract, glutathione-S-(5-thio-2-nitrobenzoic acid GSSNB, oxCEDTNB, oxCEGSSNB, protein-S-S-glutathione mixed disulfides, NADPH-Dependent Oxidoreductases, pyridine nucleo-tide dependent disulfide oxidoreductases, Sepharose-GSH, Growth Initiation, covalent regulatory process, redox ratio, VTP/IPD, Growth Factors

1. Summary Redox regulation is a covalent metabolic regulatory process analogous to phosphorylation. The conformation of a protein is switched between two states by the oxidation to form a disulfide of the vicinal thiols of proximal cysteinyl-side chains present on the protein surface. In the case of an enzyme, dithiol/disulfide conversion at a site other than the active site (allosteric site), can function to alter the affinity for the substrates. In some proteins, notably thioredoxin, dithiol/disulfide conversion can alter the protein's capacity to bind to other proteins and therefore, to function as a regulatory subunit. To be able to study the subject, we had to develop methodology with suf­ ficient sensitivity to be able to selectively identify monothiol and vicinal 257

258

Cellular Implications of Redox Signalling

thiol proteins in cells and affinity chromatography procedures to selectively enrich vicinal and monothiol proteins. Another essential method involved the quantitative determination of the distribution of thiol proteins between the reduced (dithiol or monothiol) and oxidized (intraprotein disulfide or mixed disulfide) forms. Once the methods were available, we addressed the following questions. Firstly, what is the nature and distribution of thiol pro­ teins in cells? Secondly, how can redox regulation occur in cells that have high concentrations of glutathione (GSH) and a very efficient oxidized glutathione (GSSG) reducing system? Thirdly, what is the role of the nonactive site thiols in mammalian thioredoxin? Fourthly, how are the vicinal thiols in different proteins selectively oxidized to the disulfides? Some of the answers that we have found to these questions are discussed in this chapter.

2. Methods Developed to Study Protein Thiols 2.1 Selective Radioactive Protein Thiol Labeling Reagent N-iodoacetyl-[125I]-3-iodotyrosine(IAIT) Initially, we developed a method that could label protein thiols with sufficient sensitivity to detect minor cellular components. 1 We made a radioactive thiol-selective iodoalkylating reagent N-iodoacetyl-[ 125 I]-3-iodotyrosine (IAIT). This reagent can be made with very high specific radioactivity. We routinely use it at 20 Ci/mmole but can prepare it at 1000 Ci/mmole. One unusual and attractive aspect of IAIT is that it reacts preferentially with protein thiols. Thus, IAIT selectively labels protein sulfhydrils even in the presence of up to 20 rnM of a small dithiol such as DTT. This allows labeling of cellular thiol proteins in the completely reduced state. This is important because it dispenses with the necessity to remove the reductant prior to labeling avoid­ ing the problem of re-oxidation of the thiol proteins. In the absence or pres­ ence of a non-ionic detergent, IAIT labels protein thiols as a function of their apparent pK's. Since the thiolate is the labeled species, lower pK's result in greater reactivity. The proximity of cationic side chains to the cysteine sulf­ hydrils represents the main contributor to lowering pX's of protein thiols. When we IAIT labeled protein thiols in the presence of a cationic deter­ gent like cetiltrimethyammonium bromide (CTABr), the bound detergent lowered the pK's of the protein thiols and made them highly reactive. For example, in a human erythrocyte lysate, labeling without and with triton X-100 results in little label incorporated into hemoglobin. In the presence of CTABr, the major IAIT labeled protein was hemoglobin. The presence of CTABr also increases the reactivity of the thiols of DTT. Therefore, prior to labeling, DTT levels have to be lowered to below 0.1 mM.

Mechanisms Controlling Redox Balance in Cells 259 protein

protein

c s

r C y s 5s .

h y ^sH (X)n

+ O=AS-/~S — ►

^Cys- SH (X)y |-Cys-SH (a)

pc)n

y^f~\

f-Cys-S ^ = / (X)y |~Cys- SH

^ ^

(b)

H,0

(c) Scheme 1

2.2 Arsenical-Based Affinity Chromatography of Vicinal Thiol Proteins Vicinal thiol proteins (a) react covalently with trivalent arsines (b, phenylarsine oxide) to form dithioarsine derivatives (c) as shown in Scheme 1. The pharmacological effects of arsenic and arsenicals derive from this interaction. Protein dithioarsines are stable as long as other dithiols are not present. Dithioarsines cannot be labeled with IAIT. The protein-dithioarsines complexes are stable so that they can be separated by SDS-PAGE, in the absence of thiols. However, addition to a protein-dithioarsine, of a 2X molar excess (with regards to the arsenical concentration) of a dithiol such as 1,2-dithioglycerol (British anti-lewisite, BAL) will result in the com­ plete removal of the arsenical from the protein. This reversibility prompted our use of Sepharose-bound arsines in affinity chromatogra­ phy to selectively enrich VTPs. We prepared Sepharose 4B-linked to aminohexanoyl-4-aminophenylarsine oxide (As-Sepharose). Proteins before passage through the affinity medium were reduced with 2 mM DTT. The DTT was lowered to 20 uM by dialysis or a Sephadex G-25 spin column. Passage of proteins through an As-Sepharose column resulted in selective binding of VTPs but not of monothiol proteins. After removal of unbound proteins, the bound proteins could be eluted batch-wise or by gradients with /3-mercaptoethanol or with DTT.

2.3 Thiol/Disulfide Exchange to Purify Monothiol Proteins Monothiol proteins can be selectively purified by first converting them into the 5-thio-2-nitrobenzoic acid-mixed disulfides by treatment with DTNB. On removal of excess DTNB, the monothiol protein-TNB mixed disulfides can be selectively bound to a column containing a sufficiently

260

Cellular Implications ofRedox Signalling

long bridge and a free thiol at the end. As shown in Scheme 2 , vicinal thiol proteins do not form mixed disulfides on treatment with DTNB. This converts the procedure into a monothiol-selective purification method.

2.4 Determination of the Redox State of Thiol Proteins in Intact Cells A method (post NEM-DTT procedure) was developed to determine, in intact cells, the fraction of a given thiol-protein present as the disulfide. It involves quenching of free thiols with high concentrations of cell perme­ able N-ethylmaleimide (NEM). Disulfides are not modified by NEM. After NEM removal, the disulfides are reduced with 2 mM DTT and labeled with IAIT without removal of the DTT, or enriched by affinity chromatography with As-Sepharose. Following this latter procedure, proteins are sufficiently pure after gel electrophoresis to sequence.

3. Nature and Redox State of Thiol Proteins in Cell Extracts Surprisingly, the major fraction of the thiol proteins in cells are VTPs. This can be shown by the fact that labeling with IAIT is progressively reduced on addition of phenylarsine oxide (PAO), to intact cells or to cell extracts. As shown in Fig. 1, protein vicinal thiols react with the trivalent arsenical of PAO to form stable dithioarsines that are not labeled by IAIT. Monothiols do not form stable thioarsines. Interestingly, on addition of PAO to cells, acid phosphatase activity is reduced in parallel to IAIT labeling. This strongly suggests that the majority of the cellular phosphotyrosine phosphatases are VTPs. Almost all cellular protein thiols are found in the reduced state. To study the systems reducing IPDs, cellular extract proteins were oxidized by reaction with small active disulfides. Our results show that IPDs are reduced to the dithiols mainly by the activity of the NADPH-thioredoxin reductase-thioredoxin system. GSH, even in the presence of glutaredoxin(s) cannot reduce the majority of IPDs to VTPs. Thioredoxin can form mixed disulfides which can inhibit completely its activity as an IPD reductant. 3.1 The Majority of Cell Proteins are Present in the Reduced State Comparison of cell extracts labeled immediately on cell lysis with IAIT in the absence and presence of DTT, showed that the majority of the cellular proteins are reduced. 2 Treatment of intact cells by the NEM-DTT procedure

Mechanisms Controlling Redox Balance in Cells 261

100

80 o H 60 <4-l

PTPase activity

c P 40 S-f

20

■IAIT labeling

TXN 0 25 12 6 3 PAO (^M)

(a)

10 20 30 40 50 60 Phenylarsine oxide (fiM) (b)

Fig. 1. Effect of addition of phenylarsine oxide to L1210 lymphoblasts on the thiol proteins labeled by IAIT. Phenylarsine oxide was added to cells at the indi­ cated concentrations and after incubation at 37° for 15 min, cells were lysed with 0.5 % triton X-100-50 mM HEPES buffer, pH 7.4-1 mM EGTA-1 mM MgCl2 and protease inhibitors. The nuclei were removed by centrifugation. (a) 10 uCi of IAIT was added to 0.1 ml of the extract and after labeling for 30 min reaction was stopped with sample buffer and proteins were examined by SDS-PAGE. (b) after labeling with IAIT and stopping the reaction with sample buffer, the labeled proteins were precipitated with methanol1 and the total [125I]- IAIT incorporaton was measured. In another fraction of the triton extract, phosphatase activity was measured at pH 5.5 using p-nitrophenylphosphate as substrate. to identify proteins present in cells as disulfides further showed that the majority of cell proteins are reduced. Few protein disulfides were observed in contrast to the large number of reduced proteins.2 However, proteins are not reduced because of the high cellular concentrations of GSH, which as shown in Table 1 and Fig. 2, cannot reduce the majority of the cell IPDs. These are continuously reduced by the NADPH-dependent thioredoxin reductase-thioredoxin system. On inhibition of thioredoxin reductase, or in the presence of an oxidative stress, disulfide-containing proteins are formed that can persist in the cell over a significant time. Protein

262

Cellular Implications ofRedox Signalling

dithiol/disulfide\conversion can thus play a role in redox regulation (see section on growth factors below). 3.2 The Majority of the Cell Thiol Proteins Contain Surface-Localized Vicinal Thiols When extracts of L1210 lymphoblasts are prepared by neutral detergent cell lysis followed by immediate labeling with IAIT, a large number of proteins were labeled [Fig. 1(a), lane 0 uM PAO]. Addition of increasing concentrations of phenylarsine oxide to the cells 15 min prior to the preparation of the extracts, gave the IAIT labeling patterns shown in [Fig. 1(a)] and the quantitative values shown in [Fig. 1(b)]. A progressive blocking of IAIT labeling of thiol proteins was observed. Equivalent decreased thiol protein labeling was recorded when PAO was added directly to the cell lysates followed immediately by IAIT labeling. Consequently, dithioarsine formation is rapid and stable derivatives are formed that do not release the thiols for alkyaltion during the 30 min incu­ bation with IAIT. Some 50% reduction in the labeling of thioredoxin was observe all ready at 3 uM PAO. The PAO titration shows [Fig. 1(b)] that 20 uM PAO blocks about 60% of the label incorporation into cell thiol proteins. Thus, some 2 / 3 of the IAIT-labeled thiol proteins contain vicinal thiols. The proteins not inhibited by ~25 uM PAO are mainly monothiol proteins. Affinity chromatography of the same extracts on As-Sepharose showed that the major fraction of the extract thiol proteins bound to the resin arsine groups. The non-adsorbed proteins were the same monothiol proteins seen in [Fig. 1(a)], lane 25 uM PAO the labeling of which was not blocked by PAO. Of the selectively bound proteins, about 20% could be eluted with ^-mercaptoethanol, while the major fraction was only eluted with high con­ centrations of DTT. The differences in affinity may be due to geometry of the vicinal thiols. Alternatively, interactions with the amino acid side chains present in the protein proximal to the thiols might affect the affinity for the arsine. This is an interesting aspect worth examining because it may allow specific interactions between the arsenicals and distinct proteins. The phosphatase activity reported in [Fig. 1(b)] was assayed with p-nitrophenylphosphate, an analog of phosphotyrosine. The parallel inhibition of vicinal thiol protein labeling and the inhibition of phos­ phatase activity indicate that many of the cell phosphotyrosine phosphatases are vicinal thiol-containing proteins that may be sensitive to redox regulation (see Sec. 4). However, removal of the arsenical by use of BAL, did not restore phosphatase activity. This suggests that

Mechanisms Controlling Redox Balance in Cells 263

PTPases may utilize the vicinal thiols to form metal complexes. Iron-iron and zinc-iron complexes have been shown to be present in purple phosphatases. 3 One surprising result of arsenical-based affinity chromatography was the finding4 that some 4-5% of the total cell extract proteins bound selec­ tively to the As-Sepharose. This again is evidence that many cell proteins contain vicinal thiols. The high concentration found might derive from the fact that structural proteins such as tubulin can interact with PAO. The bridge used in the As-Sepharose was not very long. Thus, proteins that bound to the arsine contain their dithiols on or close to the protein surface. This surface localization allows VTPs to be oxidized and reduced by protein-protein interaction. Resent evidence suggests that protein sulfhydril oxidases like ERV1/ALR can oxidize protein dithiols to disulfides in native proteins. As will be shown in Fig. 2, thioredoxin can reduce intraprotein disulfides almost as effectively as the small dithiol DTT. Contrast this finding with the fact that the 17 buried disulfides of BSA cannot be reduced by TXN. Surface localization of the dithiols is thus critical for redox regulation. 3.3 Properties of Thiol Proteins Oxidized by Reaction with Active Disulfides To determine which cellular reducing systems are responsible for keeping thiol proteins reduced, we prepared a total cell extract substrate where the thiols were converted to disulfides by thiol/disulfide exchange with small reactive disulfides. protein

H

CyS

protein

protein l-Cys^

-SH

hcys- S H (X)y

^ + 2R-S-S--R—>•

Ucys-- S (X)y

Ucys-SH (a)

Wn

| 3R-SH

"*

|-cys-s-s-R

(X)y |-Cys-S-S-R

(b)

(c)

Scheme 2

The reactions of symmetrical reactive disulfides R-S-S-R (such as G-S-S-G or DTNB) with proteins containing monothiols and vicinal thiols are shown in Scheme 2.

264

Cellular Implications ofRedox Signalling

Stable mixed disulfides of the type protein-Cys-S-S-R are formed with monothiols by thiol/disulfide exchange (b, c blue). Reaction with one of the vicinal thiols will also form a mixed disulfide (b). However, its lifetime will be very short because the proximal thiol is present at a very high local concentration (calculated to be 0.6-1 x 104 M)5,6. Thus, the vicinal thiol will attack the nascent mixed disulfide to expel RSH and form the intraprotein disulfide (b - » c red). For the same reason, the reverse reaction is highly unfavorable. For example, GSH, even at the high concentrations present in cells and organelles (1-10 mM) cannot reduce IPDs. On attack by GSH on the IPD in (c), the same mixed disulfide-vicinal thiol interme­ diate will be formed (b). The vicinal thiol effectively competes with a second GSH molecule required for further reduction of the mixed disul­ fide to form the dithiol in (a). Even in reactions catalyzed by glutaredoxin that has a bound GSH moiety, reduction of IPDs by GSH-glutaredoxin occurs only with few proteins where the geometry may be favorable (see see Fig. 2). On the other hand, reaction of (c) with dithiols either in a pro­ tein like thioredoxin or in reduced DTT, will result in a transient mixed disulfide in (b) where the second vicinal thiol of TXN or DTT will compete with that formed from the IPD. Scheme 2 shows an additional important aspect. Reaction of the protein dithiol in (a) with different reactive disulfides such as GSSG or DTNB, will result in the formation of the same intraprotein disulfide on expulsion of the activating mixed disulfide. For this reason, studies of S-thiolation in cells pre-loaded with radioactive GSH failed to identify VTPs converted to IPDs. However, reaction of GSSG or DTNB with the monothiol in (a) will form the different mixed disulfides protein-S-S-glutathione and protein-S-(5-thio-2-nitrobenzoic acid), respectively. Reaction of a cell extract with a reactive disulfide as shown in Scheme 2, is a mild procedure to oxidize all accessible thiols to the disulfides. L1210 lymphoblast cell extract thiol proteins were oxidized to disulfides with DTNB or glutathione-S-(5-thio-2-nitrobenzoic acid) (GSSNB). The oxidized cell extracts formed on reaction with DTNB or GSSNB are referred to as (oxCEDTNB) and (oxCEGSSNB), respectively. Because the 5-thio-2-nitrobenzoic acid (TNB) is a better leaving group than glutathione, GSSNB will react much faster than GSSG with protein monothiols to form exclusively protein-S-S-glutathione mixed disulfides and free thionitrobenzoate. The same protein disulfides formed in oxCEGSSNB can be obtained by incubation with GSSG. However, incubation has to be performed for longer periods and the efficiency of conversion is lower (see Fig. 9).

Mechanisms Controlling Redox Balance in Cells 265

3.4 NADPH-Dependent Protein Disulfide Reductase Activity Depends on the Mixed Disulfide Formed by Thioredoxin Both oxidized cell extracts contain the same intraprotein disulfides derived from the protein vicinal thiols. However, they differ in the protein monothiol mixed disulfides formed. We expected both extracts to show thioredoxin-catalyzed IPD reduction. However, results showed activity only in the oxCEGSSNB and complete inhibition in the TXN activity

3.4.1 Both Extracts Contain Active Thioredoxin and Glutaredoxin

Reductase

One of the assays for thioredoxin reductase activity 7 is based on the enzyme's capacity to catalyze the NADPH-dependent reduction of DTNB. The active site dithiol of thioredoxin reductase reduces the sub­ strate DTNB to form the active site disulfide and 2 molecules of TNB2". NADPH then reacts with the bound FAD of thioredoxin reductase to reform the active site dithiol. When oxCEDTNB and oxCEGSSNB were assayed for thioredoxin reductase by their capacity to use NADPH to reduce DTNB, both extracts had high activity (11.3-13.9 nmoles of DTNB reduced/min/mg protein). The activity was equivalent to that of a freshly prepared, untreated cell extract. Furthermore, the activity of glutaredoxin assayed by the removal of GSH from GSH-thiol protein mixed disulfides in both extracts was found to equivalent to that in fresh cell extracts. Thus, by incubating either extract with GSH or with NADPH, we determined which of the mixed disulfides or IPDs in the extracts could be reduced by GSH-glutaredoxin or by reduced thioredoxin.

3.4.2 Proteins in oxCEDTNB and oxCEGSSNB Reduced by GSH, NADPH or DTT Table 1 shows the quantitative results obtained on incubation of both extracts with the reductants GSH, NADPH or DTT. The IAIT incorpora­ tion was negligible in both extracts in the absence of reductants indicat­ ing that most thiol proteins were indeed in the disulfide form. Pre-treatment with DTT led to reduction of IPDs and mixed disulfides, markedly increasing IAIT incorporation in both extracts. Incubation with 1 mM GSH resulted in reduction of monothiol protein mixed disulfides

266 Cellular Implications ofRedox Signalling Table 1. Effect of NADPH on the reduction of IPDs to VTPs in oxCEDTNB and oxCEr [ 125 I]-IAIT Incorporation oxt_ii DTNB /o

No. 1 2 3 4

Treatment None 1 mM GSH 0.5 mM NADPH 5 mM DTT

of Total IAIT 0.6 3.60 0.7 28.9

OX(_r,GggNB

% of DTT

% of Total IAIT

2.2 12.5 2.5 100

0.5 3.5 11.5 24.6

% of D T T 1.91 14.3 46.7 100

0.175 ml of the oxidized cell extracts were incubated for 45 min at 37° with the reductants at the final concentrations shown. Then 0.1 ml was added to 10 uCi of IAIT and labeling was performed for 30 min at room temperature. Then 3 X concen­ trated sample buffer was added to stop the reaction. The radioactivity in the pro­ tein was determined by methanol precipitation.1

that was slightly higher in the oxCEGSSNB. We know reduction of IPDs to form VTPs was minor on incubation with GSH because labeling of the reduced proteins in the absence and presence of 25 uM PAO resulted in the same IAIT incorporation (see Fig. 2). The surprising finding was obtained on incubation of the extracts with NADPH. No increase in IAIT incorporation occurred in the oxCEDTNB. In marked contrast, effective reduction occurred on incubation with 0.5 mM NADPH in the oxCEGSSNB. In experiments where the concentration of the oxCEGSSNB was decrease in the incubation system, IAIT incorporation following incu­ bation with NADPH approached some 80% of that obtained with DTT reduction Under conditions where NADPH was incubated with oxCEDTNB, no evidence for protein disulfide reduction was observed. As mentioned above, the only difference between the two extracts resides in the nature of the monothiol protein mixed disulfides formed, namely protein-S-(5-thio-2nitrobenzoic acid) and protein-S-S-glutathione mixed disulfides. Figure 2 shows the pattern of the IAIT-labeled proteins formed on incubation of the extracts with GSH or NADPH. Labeling was negligible in oxCEDTNB and oxCEGSSNB in the absence of reductants (Fig. 2, lanes 3 and 5, respectively). Incubation with 1 mM GSH resulted in the reduction of similar proteins in both extracts (Fig. 2, lanes 1 and 4). However, the extent of reduction was higher in the oxCEGSSNB. Incubation with NADPH of the

Mechanisms Controlling Redox Balance in Cells 267

94674330-

20-

14.6-

^-TXN 1 2

3

4

5 6

Fig. 2. Disulfide proteins reduced in the L1210 lymphoblast extracts oxCEDTNB and oxCEGSSNB on incubation with GSH or NADPH. Lanes: (1). oxCEDTNB + 1 mM GSH; (2) oxCEDTNB + 0.5 mM NADPH; (3) oxCEDTNB + no addition; (4) oxCEGSSNB + 1 mM GSH; (5) oxCEGSSNB + no addition; (6) oxCEGSSNB + 0.5 mM NADPH. Conditions as in Table 1. oxCEDTNB resulted in only a few faint bands (Fig. 2, lane 2). In contrast, incubation of oxCEGSSNB with NADPH resulted in the reduction of many IPDs to form VTPs that were labeled with IAIT (Fig. 2, lane 6). The oxCEDTOB contains full thioredoxin reductase activity as mentioned above.

268

Cellular Implications ofRedox Signalling

12

3 4 5

Fig. 3. Protein disulfides reduced in the L1210 lymphoblast extract oxCEDTNB on incubation with 4 mM GSH or 4 mM GSH plus 0.5 mM NADPH. Lanes: (1) No addition; (2) 4 mM GSH + 20 pM PAO; (3) 4 mM GSH; (4) 4mM GSH, 0.5 mM NADPH; (5) 4mM GSH, 0.5 mM NADPH + 20 pM PAO. Conditions as in Table 1.

Nevertheless, it lacked activity of thioredoxin since n o IPD to VTP conversion could b e detected.

Mechanisms Controlling Redox Balance in Cells 269

3.4.3 Activation

of the Thioredoxin Activity

of OxCEDTNB by GSH

In contrast with bacterial thioredoxins, TXN in mammalian cells contains five thiols: two sets of vicinal thiols and a monothiol that form on oxidation, IPDs and a mixed disulfide, respectively. Since the vicinal thiols form the same IPDs in oxCEDTNB and oxCEGSSNB, the activity of thioredoxin in the oxCEDTNB appeared to be inhibited by formation of TXN-(5-thio-2-nitrobenzoic acid) mixed disulfide. These mixed disulfides are still highly reactive because of the 5-thio-2nitrobenzoic acid leaving group. Incubation with GSH results in the following reactions: Protein-S-(TNB2-) Protein-S-SG

+ +

GSH GSH

► ►

Protein-S-SG Protein-SH

+ +

TNB2" GSSG.

(1) (2)

Addition of GSH to oxCEDTNB resulted in the rapid appearance of a yellow color due to liberation of TNB2" as shown in Reaction (1) above. The protein-S-SG can further react with a second GSH to form the protein-SH [Reaction (1) catalyzed by glutaredoxin(s) present in the extracts]. If either reaction occurred on incubation with GSH, it should convert the inactive thioredoxin to the active form. That this was indeed the case is shown in Fig. 3. The oxCEDTNB alone showed no labeling by IAIT (Fig. 3, lane 1). On incubation with 4 mM GSH (Fig. 3, lane 3), labeled bands were found indicating that GSH reduced protein disulfides to protein-SH. However, the disulfides reduced were mainly monothiol protein mixed disulfides since labeling in the absence and presence of 20 uM PAO gave almost the same labeling pattern (compare Fig. 3, lane 3 and 2). On incubation with 4 mM GSH, active thioredoxin was present since a further incubation with 0.5 mM NADPH now resulted in the reduction of many IPDs to form VTPs including thioredoxin (Fig. 3, lane 4). The enhanced labeling was prevented by addition of 20 uM PAO prior to exposure to IAIT (Fig. 3, lane 5). Thioredoxin activity in the oxCEDTNB was also restored by pre-incubation for a few minutes with 0.4 mM GSH or with 25 uM DTT. These conditions released the total TNB2" present. 4 mM GSH was used to emphasize that GSH even at high concentrations cannot reduce the majority of the IPDs.

270 Cellular Implications ofRedox Signalling

946743-

30-

20-

14.51 2

3

Fig. 4. Incubation of oxCEDTNB with NADPH allows identification of the NADPH-dependent oxidoreductases in cell extracts. Conditions of incubation with 0.5 mM NADPH were as in Table 1. But labeling with I AIT was with 30 uCi to enhance the label incorporation into the active sites of the oxidoreductases. 3.4.4 NADPH Incubation of oxCEDTNB Allows Identification NADPH-Dependent Oxidoreductases

of

The finding that thioredoxin activity was blocked in the oxCEDTNB suggested that this extract could be used to directly identify pyridine nucleotide dependent disulfide oxidoreductases in cells. In these flavoprotein

Mechanisms Controlling Redox Balance in Cells 271

enzymes, incubation with NADPH reduces the active site disulfide to generate thiols that can be labeled with IAIT. Indeed, incubation of oxCEDTNB with NADPH resulted in the reduction of a few distinct proteins as shown in (Fig. 4, lane 2), while no labeling was found in the absence of NADPH (Fig. 4, lane 1). The same extract reduced with DTT (Fig. 4, lane 3) is shown to contrast with those reduced with NADPH alone. This again emphasizes the finding that DTNB pretreatment inhibits TXN. Incubation of oxCEDTNB with NADPH followed by addition of N-ethylmaleimide (NEM), resulted following removal of the NEM, in complete inhibition of the activities of both thioredoxin reductase and GSSG reductase and in the disappearance of the IAIT-labeled bands. However, NEM addition prior to incubation with NADPH, did not result in inhibition of enzyme activity since under these conditions the oxidoreductase active sites were in the disulfide form. Furthermore, incubation of the oxCEDTNB with NADPH, followed by affinity chromatography of the extract on As-Sepharose and elution with DTT resulted in a 100-fold enrichment in the specific activity of GSSGreductase in the eluate with a 22% yield. However, no thioredoxin reduc­ tase activity could be observed in the As-Sepharose DTT-eluates (Gitler et ah unpublished). The enzymatic activity was removed from the cell extract on incubation with As-Sepharose. However, no activity was eluted from the As-Sepharose. The selenol-thiol moiety has a stronger affinity for As-Sepharose. Elution requires incubation for long periods with BAL (Arner and Johanssen, personal communication). Some seven strong IAIT labeled bands are seen in Fig. 4. Thus, many pyridine nucleotide-disulfide oxidoreductases are present in the L1210 lymphoblast cell extract. Identification of different enzymes can be achieved either by specific active site labeling (8) or by their one-step purification by affinity chromatography.

3.4.5 Demonstration that in oxCEDTNB Thioredoxin Forms a 5-Thio-2-Nitrobenzoic Acid-Mixed Disulfide Reactions (1) and (2) above show that protein-S-(5-thio-2-nitrobenzoic) readily exchanges with GSH. Passage of the oxCEDTNB through a Sepharose-GSH (containing free GSH thiols) is expected to result in con­ version of protein-S-TNB 2 mixed disulfides to the Sepharose-GS-S-protein mixed disulfides with liberation of TNB2". On mixing of oxCEDTNB with the Sepharose-GSH, color due to free TNB2" was released immediately. When the absorbance at 412 nm

272 Cellular Implications ofRedox Signalling

TXN— Fig. 5. Thiol/disulfide exchange chromatography of the oxCEDTNB through a Sepharose-GSH column. SDS-Page of the DTT eluate of the selectively bound proteins labeled with IAIT.

indicated that all the mixed disulfide had reacted with the gel-thiols, the suspension was placed in a column and the non-adsorbed proteins were collected. The gel was washed until no protein was present in the effluent. Then, the proteins were eluted by incubating the gel with 20 mM DTT in buffer A (40 mM Hepes buffer, pH 7.4, 50 mM NaCl, 1.0 mM MgCl 2 , 1 mM EGTA, 10 ug/ml leupeptin, 10 ug/ml aprotinin and 1 mM PMSF). The proteins found in the eluate are shown in Fig. 5. The lower band of apparent mol. wt. of 15.6 kDa, which was strongly labeled by IAIT, was identified by N-terminal sequence to be thioredoxin.9

Mechanisms Controlling Redox Balance in Cells 273

It was also identified as thioredoxin by its capacity to catalyze reduction by DTT of oxidized glyceraldehyde-3-phosphate dehydrogenase or insulin. The oxCEDTNB proteins that did not bind to the Sepharose-GSH were dialyzed against buffer A to remove the TNB2" liberated. This nonbinding fraction contained all of the activity of thioredoxin reductase (assayed with DTNB as a substrate) of the original oxCEDTNB extract. Furthermore, it contained all the IPDs but lacked all the monothiol proteins, including TXN. The monothiol proteins eluted from the Sepharose-GSH were passed through a Sephadex G-25 spin column equilibrated in buffer A to remove the DTT. When combined with the non-binding fraction and incubated with NADPH, activity of NADPH-thioredoxin reductasethioredoxin system was readily detected. IAIT incorporation expressed as percentage of total (DTT reduced) increased from a value of 12.9% in the absence of NADPH to a value of 39% after incubation with 0.5 mM NADPH. IPDs were reduced to VTPs on incubation NADPH and the thioredoxin purified by its capacity to bind to Sephadex-GSH. Binding to Sephadex-GSH was due to the presence in oxCEDTNB of reactive protein-5-thio-2-nitrobenzoic acid mixed disulfides. Therefore, this results are definitive proof that thioredoxin formed this reactive mixed disulfide.

3.5 Implications of the Studies in vitro with Oxidized Cell Extracts The above results clearly indicate that incubation of cell extracts contain­ ing protein-thiols as disulfides with GSH at high concentrations, even in the presence of active glutaredoxin(s), results mainly in the reduction of monothiol protein mixed disulfides. Incubation of both oxCEDTNB and oxCEGSSNB with GSH formed monothiol proteins. Clearly, some IPDs can be reduced by GSH-glutaredoxin. Holmgren showed that in E. coli, muta­ tions that eliminated thioredoxin did not result in the inhibition of deoxyribonucleotide synthesis. The ribonucleoside-diphosphate reduc­ tase active site disulfide could be reduced by GSH-glutaredoxin. In addi­ tion, glutaredoxin(s) were shown to catalyze reduction of disulfides in the glucocorticoid and triiodotyronine hormone receptors and oxyR tran­ scription factor (see chapter by Carmer and Stoz). The results presented show that the main cellular IPD reductase is the NADPH-thioredoxin reductase-thioredoxin system. Surprisingly, many cell extract proteins contain their IPDs in a form accessible to proteinprotein reduction.

274

Cellular Implications ofRedox Signalling

In mammalian cells, thioredoxin contains additional thiols to those found in the same proteins in bacteria. This can be seen in the sequence of human thioredoxin: 1-MVKQIESKTA FQEALDAAGD KLVWDFSAT WCGPCKMINP FFHSLSEKYS NVIFLEVDVD 61-DCQDVASECE VKCTPTFQFF KKGQKVGEFS GANKEKLEAT INELW

In addition to the active site dithiol CGPC that is found in bacteria, there is a second dithiol CEVKC and a monothiol in position 62. Based on the cysteines present, mammalian reduced thioredoxin can be repre­ sented as TXN-(SH)2-(SH)2-SH, where the underlined dithiol represents the active site. TXN-S2-S2-S-(5-thio-2-nitrobenzoic acid) is the species formed on reaction with DTNB and TXN-S2-S2-S-S-glutathione is that formed on incubation with GSSNB (or GSSG). In the case of the extract oxCEGSSNB, no pre-incubation with GSH was required to obtain, on incubation with NADPH, high apparent TXN activity with effective IPD to VTP reduction (see Table 1 and Fig. 2). In contrast, even though oxCEDTNB contains equally active thioredoxin reductase, TXN-S2-S2-S(5-thio-2-nitrobenzoic acid) does not function as a substrate of the reduc­ tase and therefore, no protein disulfide reduction was observed. In vitro, both TXN mixed disulfides were poorly reduced by purified thioredoxin reductase (Gitler and Holmgren, unpublished). This latter finding sug­ gests that the difference in apparent thioredoxin reductase activity observed in the oxCEGSSNB versus oxCEDTNB resides in the capacity of the GSSNB-treated extract to form totally reduced thioredoxin. This capacity is absent in the oxCEDTNB. Irrespective of the explanation, the oxCEDTNB/ because of its apparent lack of thioredoxin activity, can be used to iden­ tify the cellular enzymes that utilize NADPH to reduce their active site disulfides. Furthermore, these extracts can be used to purify in one step all the cellular proteins containing monothiols.

4. Redox Regulation during Growth Initiation 9 Like phosphorylation, redox regulation is a covalent regulatory process. Systems are present in cells that modify the dithiol/intraprotein disulfide ratio (redox ratio) of different proteins. Dithiol/disulfide conversion alters the conformation of a protein. In the case of an enzyme, dithiol/disulfide conversion can function to alter the affinity for sub­ strates. In some proteins, notably TXN, a change in dithiol to disulfide can alter the capacity of the protein to bind to other proteins. As discussed above, the majority of the thiol proteins are found in cells in the reduced

Mechanisms Controlling Redox Balance in Cells 275

form. The NADPH-thioredoxin reductase-thioredoxin system continuously converts IPDs to VTPs. For redox regulation to be effective in modulating metabolic pathways, cells must possess enzymes that directly oxidize or inhibitors of reduction that transiently increase the disulfide forms of dif­ ferent proteins. Similar to phosphorylation where enzymes are present that remove (phosphatases) or attach the phosphate (kinases), so in redox regulation, mechanisms must be present that reduce the disulfides and others that inhibit reduction or selectively oxidize cellular dithiol proteins. Redox regulation has an addition facet that makes it important for cell function and survival. Redox ratios may also be altered by intrinsic or extrinsic oxidants. Hypoxia and hyperoxia clearly may affect redox ratios. Hydrogen peroxide and nitric oxide produced by cells involved in early responses to pathogens, or to local necrosis, would be expected to rapidly diffuse to cells in their vicinity to signal by altering redox ratios. Furthermore, many degenerative pathological conditions have been identified in which production of radicals a n d / o r oxidants are enhanced. The altered ratios of sensing thiol proteins may therefore also be critical to initiate signals that control regeneration, cell survival or apoptosis. The first step in identifying mechanisms that can modify redox ratios is to obtain the actual redox ratios of diverse proteins in cells undergoing a variety of metabolic processes. To determine the VTP/IPD ratios of proteins in intact cells we developed the post NEM-DTT procedure. See Refs. 2 and 9. The procedure involved rapid in situ blocking of protein thiols by treatment of cells with high concentrations of membrane-permeable N-ethylmaleimide (NEM). After detergent cell lysis, quenching of thiols with NEM was continued to block thiols in membrane proteins. NEM does not react with protein disulfides. After NEM removal, IPDs and protein-mixed disulfides were reduced by dithiothreitol (DTT). Then, the thiol proteins were either labeled by the thiol-specific reagent IAIT or enriched by selective binding of VTPs to As-Sepharose. Initial use of this procedure 2 showed that in exponentially growing L1210 lymphoblasts, the majority of the cellular VTPs were present in the reduced state. However, a minor but distinct set of proteins co-exists in these cells as IPDs. Oxidants such as diamide, shifted the redox ratios by increasing the formation of IPDs and mixed disulfides. Cells normally produce significant levels of H 2 0 2 as a byproduct of electron transport in mitochondria. In addition, phagocytic cells contain dispersed components of an NADPH-oxidase that can be speedily

276

Cellular Implications ofRedox Signalling

assembled during phagocytosis, to produce a burst of superoxide that rapidly dismutates to H 2 O z . A peroxide burst also occurs in nonphagocytic cells. Binding of a varied set of effectors (insulin, chemokines and mitogenic growth factors) to cell surface receptors initiates an immediate peroxide burst. Addition to cells of epidermal growth factor (EGF), platelet derived growth factor, or bradykinin induced a rapid burst of hydrogen peroxide formation.1011 The H 2 0 2 burst was transient, reaching a maximum in 1-2 min, decaying rapidly thereafter. It was mediated by a rise in intracellular calcium leading to the assembly of the NADPH-oxidase.11 It could be abrogated by electroporation of catalase,10 or transfection with high levels of peroxiredoxins I or II.12 This increased peroxide-destroying activity not only eliminated the transient peroxide burst but also the protein tyrosine phosphorylation initiated by binding of the growth factors to their tyrosine kinase receptors.10 Addition of low concentrations of H 2 O z to cells induces many of the cellular reaction normally elicited by binding of growth factors or insulin. Low levels of different peroxides induce activation of N F - K B that can be blocked by addition of relatively high levels of N-acetylcysteine.

4.1 Growth Factors Induce a Calcium-Dependent Transient Oxidation of TXN and Other Cell Proteins9 To assess the effect of mitogens on the redox state of cell proteins, immor­ talized human keratinocytes (HaCat) were exposed to bradykinin (or to epidermal growth factor or calcium ionophore A-23187). At different times thereafter, cellular oxidized disulfide-containing proteins were quantified by the post-NEM-DTT procedure. 2 ' 9 The [125I]-IAIT-labeled bands in the gels represented the proteins in the cells present as the oxidized protein disulfides. Radioautograms allowed quantification of the fraction oxidized when compared to cell extracts not treated with NEM, but reduced with DTT to show the total thiol proteins present in the cell extracts. In [Fig. 6(a)] are shown the kinetics of the changes in H 2 0 2 levels that occurred in HaCaT keratinocytes upon addition of bradykinin, EGF or the calcium ionophore A-23187. Quantitative estimates of oxidized thioredoxin measured under identical conditions are shown in [Fig. 6(b)]. That an increase in peroxide formation and oxidation of thioredoxin occur on addition of the calcium-ionophore A-23187, showed that cytosolic calcium mediates the generation of a cellular oxidizing environment. 911

Mechanisms Controlling Redox Balance in Cells 277

80 f

0 5 10 15 20 25 Time (min) (a)

0

5 10 15 20 Time (min) (b)

Fig. 6. Kinetics of peroxide formation (a) and thioredoxin oxidation (b) in HaCaT keratinocytes exposed to bradykinin, EGF or A23187. Hydrogen per­ oxide was measured as in Ref 11. Thioredoxin oxidized was measured as described in Ref. 9. Values shown are for bradykinin (-•-), EGF (-o-) or A23187 (-A-)

Both, the effect of A23187 and of the growth factors could be abrogated by pre-loading of the keratinocytes with a calcium-specific chelator 1,2-bis (o- amino phenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA).9 Furthermore, addition to the HaCaT cells of 0.1 mM of thapsigargin, an endoplasmic reticulum Ca-ATPase inhibitor, resulted within 10 min in the oxidation of 72% of cellular thioredoxin. Thus, calcium released from internal stores can also lead to oxidation of thioredoxin Figure 6 shows that the onset of H 2 0 2 formation and thioredoxin oxidation is rapid and parallel, as would be expected from the fact that both processes depend on a rise in cytosolic calcium. However, H 2 O z production decayed faster than the reduction of oxidized thioredoxin. For bradykinin, the peroxide peak fell to a plateau by 4 min, while ox-thioredoxin fell to a plateau only by 8 minutes. Similar persistence of oxidized thioredoxin beyond peroxide production was observed follow­ ing EGF or ionophore addition. Not only was oxidation of thioredoxin induced by growth factors or ionophore, but other proteins in the cell were oxidized above control levels. The dithiol proteins isolated following affinity chromatography on As-Sepharose of a HaCat keratinocyte extract prepared 2 min after addition

278 Cellular Implications ofRedox Signalling

-94 -67 -43 -30

-20 -14.6

1 2 Fig. 7. Affinity chromatography on As-Sepharose of the disulfide proteins formed 2 min following addition of bradykinin to HaCat keratinocytes. Polypeptides labeled with IAIT are shown after SDS-PAGE. Cell extract was pre­ pared by the NEM-DTT procedure. Lane 1: monothiol proteins that did not bind to the As-Sepharose. Lane 2: Dithiol proteins eluted with 20 mM DTT after selec­ tive binding to As-Sepharose. The strong band at -15 Kda was identified by sequence as thioredoxin.9 of bradykinin to the cells in culture are shown in Fig. 7 (Gitler et al. unpublished results). The protein bands labeled with IAIT in Fig. 7 were present in cells as protein disulfides. Several bands are seen in the non-adsorbed material (Fig. 7, lane 1). With a longer exposure, they are readily identified. These are monothiol proteins that were present in the stimulated cells as mixed disulfides. Easily, some 10 polypeptides are seen. The dithiol proteins bound to the As-Sepharose, and after removal of non-adsorbed proteins, they were eluted with 20 mM DTT and labeled with IAIT. At least 15

Mechanisms Controlling Redox Balance in Cells 279

polypeptides could be identified. These include the strongly labeled band at 15.6 KDa that corresponds to thioredoxin. These proteins were isolated by affinity purification and separated by SDS-PAGE. At this stage, they are of sufficient purity to be sequenced by mass spectrography. The procedure allows, therefore, not only kinetic analysis of the disulfide-containing proteins, but also direct identification of the proteins that are converted to disulfides during growth initiation.

4.2 Calcium Inhibits the in vitro Activity of Thioredoxin Reductase9 Normally, protein disulfides in cells are continuously converted to the reduced dithiols by reaction with reduced thioredoxin. The oxidized thioredoxin formed is normally effectively reduced by thioredoxin reduc­ tase. Figure 6 shows that a rise in cytosolic calcium leads to the rapid oxidation of thioredoxin. These findings suggested that calcium may be inhibiting the normal reduction of thioredoxin by thioredoxin reductase since an unlikely very high oxidative pressure is required to oxidize thioredoxin in the presence of an active thioredoxin reductase. Schallreuter et al.13 reported that thioredoxin reductase purified from human melanoma cells was inhibited by calcium. Lenartowicz 14 further showed that rat liver mitochondria contain a presumptive thioredoxin reductase that uses mitochondrial NADPH to reduce extra-mitochondrial DTNB. This activity was inhibited by < 20 uM calcium. In contrast, Oblong and Powis 15 reported that activity of a purified form of human placental thioredoxin reductase was not affected by mM concentrations of calcium. Addition of NADPH to a keratinocyte cell extract pre-oxidized by treatment with GSSG, (prepared as describe above in Scheme 2 and Fig. 2) resulted in the reduction of almost all cell protein disulfides.9 The effect of NADPH was slightly less than that of 2 mM DTT.9 However, no reduction was observed on addition of NADPH in the presence of 10 uM free calcium. Thus, either thioredoxin reductase is directly inhibited by calcium or proteins exist in the cell that modulate thioredoxin reductase activity in the presence of calcium.9 Preliminary results with purified thioredoxin reductase showed some inhibition by calcium but not equiva­ lent to that observed in the keratinocyte cell extracts. This suggests that proteins are present in the cytoplasm that may sensitize thioredoxin reductase to calcium. Calmidazolium, an inhibitor of calmodulin-dependent processes, when added to HaCat cells instead of inhibiting, actually

280

Cellular Implications of Redox Signalling

increased the fraction of ox-TXN to values of 60 to 80% of total TXN. The calcium-induced oxidation is not calmodulin-dependent but calmidazolium addition apparently increases intracellular calcium levels.

4.3 Implications of the Transient Oxidation of Thioredoxin and Other Cell Dithiol Proteins Thioredoxin modulates cellular reactions by two different mechanisms. The first involves its catalytic role in the conversion of protein disulfides to dithiols. The second consists of the selective regulatory binding of the dithiol form of thioredoxin to different functional proteins. Thus thio­ redoxin can function as a redox catalyst and as redox sensitive regulator through protein-protein interactions. Both functions are affected by the transient oxidation that is observed to occur in cells immediately after growth initiation. The absence of reduction, as would follow thioredoxin reductase inhibition, is not a very efficient means of generating intraprotein disulfides from vicinal thiol proteins. However, as discussed by Beckwith [see Ref. 16 and Beckwith chapter], oxidized thioredoxin is capable of func­ tioning as an effective dithiol oxidant when it accumulates on inactivation of thioredoxin reductase. It is unlikely that this oxidative capacity is selec­ tive. The majority of the cell thiol proteins are not affected by the inhibi­ tion of thioredoxin reductase. Our results 9 show that the transient oxidation of thioredoxin increases in parallel with the oxidized forms of several cellular proteins. But these are not representative of all the thiol proteins in the cell. What is the role of the transient rise in hydrogen peroxide that occurs in parallel with the inhibition of thioredoxin reductase? Hydrogen per­ oxide can oxidize protein thiols to sulfenic acid derivatives. These in turn react with vicinal thiols or with glutathione to form IPDs and protein-GSH mixed disulfides, respectively. But H 2 0 2 does not appear to be a very effective or selective oxidizing reagent in the reaction with thiols. However, the suggestion has been made that hydrogen peroxide does not exist free in cells.17 H 2 0 2 may bind to specific sites in proteins and DNA. The suggested binding to the histidyl side chains could, by means of the enhanced local concentration, selectively allow peroxide to oxidize thiols in the vicinity of histidines. This might be important for phosphotyrosine phosphatases that contain a His-Cys sequence in their active sites. In addition, H 2 0 2 reacts preferentially with the active site of

Mechanisms Controlling Redox Balance in Cells 281 Peroxiredoxin-SH Peroxiredoxin-SH

TXN-(

H 2 0,

Peroxircdoxin-S I Peroxiredoxin-S Scheme 3

peroxidases. Cells contain a set of thioredoxin peroxidases, also known as peroxiredoxins, that selectively react with H 2 0 2 by the reactions shown in Scheme (3) (see also chapter by Yodoi) Two homo- or hetero-subunits of the peroxiredoxin isoforms comprise the active peroxidase. These dimer proteins selectively transfer electrons from the active-site thiol of each subunit to the peroxide dioxygen to form water and the peroxiredoxin mfer-protein disulfide, 2[peroxiredoxin]-S2. Thioredoxin selectively reduces 2[peroxiredoxin]-S 2 to the peroxidasecatalytic dimer 2[peroxiredoxin-SH]. On inhibition of thioredoxin reductase observed during growth initia­ tion, the peroxide burst would lead to the selective and rapid accumula­ tion of the 2[peroxiredoxin]-S2. IPD<|

Peroxiredoxin-SH Peroxiredoxin-SH

VTP<

Scheme 4

The fact that peroxiredoxins are H 2 0 2 sinks presents two interesting alternatives: 2[peroxiredoxin]-S 2 rapidly oxidize thioredoxin (Scheme 3) and oxidized thioredoxin in turn is the agent that oxidizes other proteins (Scheme 4) or 2[peroxiredoxin]-S2 themselves are selective vicinal thiol protein oxidants (Scheme 4). This could provide for a selective mecha­ nism for protein dithiol oxidation since different isoforms or the perox­ iredoxins exist in cells. Note (Scheme 4) that both thioredoxin and the peroxiredoxins are reduced when they react with the different vicinal thiol proteins to con­ vert them to the intraprotein disulfide forms. This allows recycling of the peroxiredoxins as long as hydrogen peroxide is present.

282

Cellular Implications ofRedox Signalling

The nature of the target proteins that are oxidized to disulfides during growth initiation are not known. However, phosphotyrosine phosphateses (PTPases), are likely candidates because they behave as vicinal thiol proteins. PTPases are strongly inhibited by low concentrations (< 20 uM) of the dithiol-specific blocker phenylarsine oxide (PAO)18 and [Fig. 1(b)]. PAO does not inhibit monothiol proteins. In adipocytes, it has been known for some 50 years, that H 2 0 2 is insulinomimetic. When added in low concentrations to cells it mimics many of the effects induced by insulin. In turn, insulin stimulation has been shown to generate a burst of intracellular H 2 0 2 in insulin-sensitive hepatoma and adipose cells that is associated with reversible oxidative inhibition of up to 62% of overall cellular PTPase activity. The specific activity of immunoprecipitated PTP1B, a PTPase homologue implicated in the regulation of insulin sig­ naling, was also strongly inhibited by up to 88% following insulin stimu­ lation. Catalase pre-treatment abolished the insulin-stimulated production of H 2 0 2 as well as the inhibition of cellular PTPases.19 As mentioned above, thioredoxin functions also as a regulatory subunit in different protein complexes. E. coli thioredoxin binding increases processivity of T7 bacteriophage DNA polymerase. 20 A heterodimer of thioredoxin and I(B)2 cooperates with Sec 18p (NSF) to promote yeast vacuole inheritance.21,22 Recently, Ichijo and collaborators23"25 have reported in an intriguing function of thioredoxin. Apoptosis signal-regulating kinase (ASK-1) was identified as a mitogen-activated protein (MAP) kinase which activates the c-Jun N-terminal kinase (JNK) and p38 MAP kinase, kinase kinase pathways and is required for tumor necrosis factor (TNF)-alphainduced apoptosis. The so called stress pathway of MAP kinases is there­ fore dependant on activation of ASK-1. Thioredoxin was identified as an interacting regulatory subunit of ASK-1. Thioredoxin associated with the N-terminal portion of ASK-1 in vitro and in vivo. The interaction between thioredoxin and ASK-1 was found to be highly dependent on the redox status of thioredoxin. Moreover, oxidation of thioredoxin resulted in acti­ vation of endogenous ASK-1 activity, suggesting that thioredoxin is a physiological inhibitor of ASK-1. The oxidation of thioredoxin observed on binding of growth factors to receptors, should result in the transient activation of ASK-1 and therefore, of JNK and p38 MAP kinase pathways. Cytosolic calcium has a dual role in the cellular oxidative response (see Fig. 8). It activates the NADPH oxidase and inhibits thioredoxin reductase. Thus, cytosolic calcium, in addition to its many roles, is capable of initiating redox regulation by induction of protein disulfide oxidation. A transient Ca2+ rise may have a key regulatory role in growth initiation and

Mechanisms Controlling Redox Balance in Cells 283

Fig. 8. The role of calcium in redox regulation during growth initiation.

other receptor signaling processes. Persistence of calcium in the cytosol may lead to oxidative stress and apoptosis probably through the activa­ tion of ASK-1 Thioredoxin modulates cellular reactions by two different mecha­ nisms. The first involves its catalytic role in the conversion of protein disulfides to dithiols. The second consists of the selective regulatory bind­ ing of the dithiol form of thioredoxin to different functional proteins. Thus thioredoxin can function as a redox catalyst and as redox sensitive regulator through protein-protein interactions. Both functions are affected by the transient oxidation that is observed to occur in cells imme­ diately after growth initiation.

5. Concluding Remarks Two different mechanisms have been described that alter the functional status of thioredoxin. The first involves formation of mixed disulfides by

284

Cellular Implications ofRedox Signalling

the thioredoxin-monothiol. Mixed disulfide with 5-thio-2-nitrobenzoic acid results in complete inhibition of the disulfide reductase activity of thioredoxin, while the glutathione-mixed disulfide is as active as thioredoxin. No evidence is available that this type of regulation has physiological relevance. The second mechanism involves oxidation of thioredoxin that results from the calcium-dependent inhibition of thiore­ doxin reductase and the concomitant activation of the NADPH oxidase that increases cellular levels of hydrogen peroxide. This is the first description of a mechanism that can modulate the redox state of cellular proteins. It is critical to growth initiation since abrogating the peroxide burst inhibits receptor kinase phosphotyrosine phosphorylation. This suggests that an oxidizing cellular environment is critical for growth initiation. Specifically designed reductants might represent a pharmaco­ logical means to inhibit growth initiation.

Acknowledgements This work was supported by the Shvadsky-Gitler Fund created in memory of Gregorio and Sonia Shvadsky and Nathan and Pola Gitler. We thank Ana and Pablo Brener for sharing our meanings and for donations to this fund. Thanks to Dr. Avihai Danon for discussions that refined my views on the subject.

References 1.

2.

3.

4.

Gitler C, Mogyoros M, Kalef E. 1994. Specific labeling of protein vicinal dithiols to study the role of protein-S2 to protein-(SH)2 conversion in metabolic regulation and oxidative stress. Meth. Enzymol. 233: 403-415 Gitler C, Zarmi B, Kalef E. 1997. General method to identify and enrich vicinal thiol proteins present in intact cells in the oxidized, disulfide state. Anal. Biochem. 252: 48-55 Vincent JB, Averill BA. 1990. Sequence homology between purple acid phosphatases and phosphoprotein phosphatases. Are phosphoprotein phosphatases mctalloproteins containing oxide-bridged dinuclear metal centers? /. Trace Elem. Electrolytes Health Dis. 263: 265-268 Kalef E, Walfish PG, Gitler C. 1993. Arsenical-based affinity chromatography of vicinal dithiol-containing proteins: Purification of L1210 leukemia cytoplasmic proteins and the recombinant rat c-erb A (3-1 T3 receptor. Anal. Biochem. 212: 325-334

Mechanisms Controlling Redox Balance in Cells 285

5. Creighton TE. 1984. Disulfide bond formation in proteins. In Methods in Enzymology: Postranslational Modifications, eds. Wold F, Moldave K, Academic Press, Orlando 107 (Part B): 305-329 6. Gilbert HF. 1989. Molecular and cellular aspects of thiol/disulfide exchange. Adv. Enzymol. 63: 69-172 7. Luthman M, Holmgren A. 1982. Rat liver thioredoxin and thioredoxin reductase: Purification and characterization. Biochemistry 21: 6628-6633 8. Ye B, Gitler C, Gressel J. 1997. A high-sensitivity, single-gel, polyacrylamide gel electrophoresis method for the quantitative determi­ nation of glutathione reductases. Anal. Biochem. 246: 159-165 9. Gitler C, Zarmi B, Kalef E, Meller R, Zor U, Goldman R. 2002. Calcium-dependent oxidation of thioredoxin during cellular growth initiation. Biochem. Biophys. Res. Commun. 290: 624-628. 10. Bae YS, Kang SW, Seo MS, Baines IC, Tekle E, et ah 1997. Epidermal growth factor (EGF)-induced generation of hydrogen peroxide. Role in EGF receptor-mediated tyrosine phosphorylation. /. Biol. Chem. 272: 217-221 11. Goldman R, Moshonov S, Zor U. 1998. Generation of reactive oxygen species in a human keratinocyte cell line: Role of calcium. Arch. Biochem. Biophys. 350:10-18 12. Kang SW, Chae HZ, Seo MS, Kim K, Barnes IC, Rhee SG. 1998. Mammalian peroxiredoxin isoforms can reduce hydrogen peroxide generated in response to growth factors and tumor necrosis factoralpha. /. Biol. Chem. 273: 6297-6302 13. Schallreuter KU, Pittelkow MR, Wood JM. 1989. EF-hands calcium binding regulates the thioredoxin reductase/thioredoxin electron transfer in human keratinocytes. Biochem. Biophys. Res. Commun. 162: 1311-1316 14. Lenartowicz E. 1992. Ca(2+)-sensitive reduction of 5,5'-dithiobis(2-nitrobenzoic acid) by rat liver mitochondria. Biochem. Biophys. Res. Commun. 184: 1088-1093 15. Oblong JE, Powis G. 1993. A comment on the absence of calcium reg­ ulation of human thioredoxin reductase. FEES Lett. 334:1-2 16. Stewart EJ, Aslund F, Beckwith J. 1998. Disulfide bond formation in the Escherichia coli cytoplasm: An in vivo role reversal for the thioredoxins. EMBO. /. 17: 5543-5550 17. Schubert J, Wilmer JW. 1991. Does hydrogen peroxide exist "free" in biological systems? Free. Radio. Biol. Med. 11: 545-555 18. Garcia Morales P, Minami Y, Luong E, Klausner RD, Samelson LE. 1990. Tyrosine phosphorylation in T-cells is regulated by phosphatase activity: Studies with phenylarsine oxide. Proc. Natl. Acad. Sci. USA 87: 9255-9259

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19. Mahadev K, Zilbering A, Zhu L, Goldstein BJ. 2001. Insulinstimulated hydrogen peroxide reversibly inhibits protein-tyrosine phosphatase 1-B in vivo and enhances the early insulin action cas­ cade. /. Biol. Chem. 276: 48662^8669 20. Bedford E, Tabor S, Richardson CC. 1997. The thioredoxin binding domain of bacteriophage T-DNA polymerase confers processivity on Escherichia coli DNA polymerase I. Proc. Natl. Acad. Sci. USA 94: 479-484. 21. Xu Z, Wickner W. 1996. Thioredoxin is required for vacuole inheri­ tance in Saccharomyces cerevisiae. }. Cell. Biol. 132: 787-794. 22. Xu Z, Mayer A, Muller E, Wickner W. 1997. A heterodimer of thiore­ doxin and I(B)2 cooperates with Secl8p (NSF) to promote yeast vacuole inheritance. /. Cell. Biol. 136: 299-306. 23. Saitoh M, Nishitoh H, Fujii M, Takeda K, Tobiume K, et al. 1998. Mammalian thioredoxin is a direct inhibitor of apoptosis signalregulating kinase (ASK-1) inhibitors/biosynthesis/*metabolism. EMBO J. 17: 2596-2606 24. Ichijo H. 1999. From receptors to stress-activated MAP kinases. Oncogene 18: 6087-6093 25. Liu H, Nishitoh H, Ichijo H, Kyriakis JM. 2000. Activation of apoptosis signal-regulating kinase-1 (ASK-1) by tumor necrosis factor receptorassociated factor 2 requires prior dissociation of the ASK-1 inhibitor thioredoxin. Mol. Cell. Biol. 20: 2198-2208

Chapter 13 Regulatory Disulfides Controlling Transcription Factor Activity in the Bacterial and Yeast Responses to Oxidative Stress Orna Carmel-Harel, Matthew J. Wood, and Gisela Storz* Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 *[email protected]

Keywords: OxyR, RsrA, Yaplp, glutaredoxin, thioredoxin

1. Summary The ability of cells to modify their gene expression patterns in response to environmental changes is crucial to their survival and is largely dependent on the activities of specialized transcription factors. Understanding the events leading to cellular perception of the environmental changes as well as the mechanisms that allow transduction of the signal into a change in gene expression has been an important goal in studying the cellular effects of oxidative stress. In the following chapter we discuss two transcription factors, Escherichia coli OxyR and Saccharomyces cerevisiae Yaplp and a sigma factor/anti-sigma factor, Streptomyces coelicolor aR and RsrA, that are impor­ tant for survival under oxidizing conditions in bacteria and yeast. Oxidative stress leads to OxyR activation, o~R and RsrA dissociation, and Yaplp reten­ tion in the nucleus. Specific cysteine residues are critical for OxyR, RsrA and Yaplp function, and genetic and biochemical studies indicate that each of these proteins is regulated by a thiol-disulfide switch and may be a direct sensor of cellular-redox status. We suggest that other transcriptional regula­ tors also will be found to be modulated by oxidation and reduction and that the in vivo and in vitro approaches used to study OxyR, RsrA and Yaplp can be used to characterize disulfide bonds in other regulatory proteins. 287

288

Cellular Implications ofRedox Signalling

1.1 Introduction Oxidative stress has been defined as a disturbance in the prooxidantantioxidant balance in favor of prooxidants. 40 Thus oxidative stress can result from increased levels of reactive oxygen species such as superoxide anion (02»~), hydrogen peroxide (H 2 0 2 ), hydroxyl radical (HO») and alkyl hydroperoxides (ROOH). Reactive oxygen species are byproducts of nor­ mal metabolic processes but elevated levels of these prooxidants can be generated by environmental factors such as ionizing radiation, heavy met­ als or other redox-active chemicals such as the 0 2 » "generating compound menadione. If allowed to accumulate unchecked these molecules exceed the normal antioxidant buffering capacity of the cell leading to indiscrimi­ nate damage to cellular components including DNA, proteins and lipids. Cells also may experience oxidative stress resulting from a drop in the thiol/disulfide ratio within the cytosol or within an organelle. This drop in the thiol/disulfide ratio can occur under certain growth conditions or upon exposure to some oxidants such as the thiol-oxidant diamide. A distur­ bance in the thiol/disulfide ratio will lead to the formation of abnormal disulfide bonds and protein misfolding.32 To defend against oxidative stress, organisms have evolved redoxsensors whose activities are modulated by reactive oxygen species or by changes in the thiol/disulfide ratio. Upon oxidation, these regulators trig­ ger the expression of genes that restore the normal reducing environment of the cell. Here we discuss the Escherichia coli transcription factor OxyR, the Streptomyces coelicolor anti-sigma factor RsrA and the Saccharomyces cerevisiae transcription factor Yaplp whose activities all appear to be regu­ lated by disulfide bond formation. We describe evidence indicating that each of these regulators contains redox-sensitive cysteines and that oxidation of these cysteines results in changes in transcription factor activity and ultimately in the expression of protective activities.

2. OxyR 2.1 Identification of OxyR and Its Target Genes The locus encoding the OxyR transcription factor in E. coli was first identified via dominant point mutations that conferred increased resistance to H J O J and cumene hydroperoxide and led to elevated expression of H 2 0 2 -inducible proteins.8 Subsequently, recessive deletion mutations that conferred sensi­ tivity to H 2 0 2 and cumene hydroperoxide and prevented induction of a sub­ set of the H 2 0 2 -inducible proteins were constructed. A variety of different

Regulatory Disulfides Controlling Transcription Factor Activity

289

studies showed that OxyR activates oxyS encoding a small regulatory RNA,2 katG encoding hydroperoxidase 1/ ahpCF encoding an alkyl hydroperoxide reductase,7 gor encoding glutathione reductase, 7 grxA encoding glutaredoxin l,54 trxC encoding thioredoxin 237b, and dps encoding a nonspecific DNAbinding protein 1 and represses its own expression.8,47 Recent computational searches of the completed E. coli genome for additional OxyR binding sites revealed that fur encoding ferric uptake repressor,55 and dsbG encoding a disulfide bond isomerase,56 also are activated by OxyR, while fhuF encoding a ferric ion reductase,56 is repressed by OxyR. In addition, DNA microarrays analysis identified hemH encoding the last step in heme biosynthesis,57 and the sufABCDSF operon encoding activities likely to be involved in sulfur, selenium or Fe-S cluster metabolism,57 as OxyR-activated genes. The 34-kDa OxyR protein shares homology with the LysR family of bacterial regulators. 8 ' 46 OxyR levels are not changed after treatment with H 2 0 2 , 43 and in contrast to many other LysR-type transcriptional regulators, 1637 OxyR does not require a coinducer to activate its target genes. Instead, OxyR is activated directly by oxidation. Only the oxidized form of OxyR was able to activate transcription in experiments with puri­ fied components. 43 The oxidized and reduced forms of OxyR also have dif­ ferent DNA binding properties. Oxidized OxyR binds in four adjacent major grooves, while reduced OxyR contacts two pairs of adjacent major grooves separated by one helical turn.49 The reduced protein has diminished affinity for most of the OxyR-activated promoters indicating that only a subset of the OxyR-regulated promoters carry determinants for the bind­ ing of reduced OxyR. The two modes of OxyR binding allow the tran­ scription factor to repress some target genes during normal growth, while activating or repressing a different subset of genes upon oxidative stress.

2.2 OxyR Activation by Disulfide Bond Formation Between C199 and C208 The initial genetic and biochemical studies of OxyR led to the hypothesis that the transcription factor is a direct sensor of H 2 O z and that OxyR undergoes a conformational change upon oxidation. These findings also raised the question as to the nature of the redox-active center in OxyR. No transition metals were detected by inductively-coupled plasma metal ion analysis of two preparations of OxyR.54 The activity of OxyR also was unchanged after denaturation and renaturation in the presence of the metal chelator desferrioxamine, indicating that metal ions and other pros­ thetic groups were unlikely to constitute the redox-active center of OxyR.

290 Cellular Implications ofRedox Signalling

Fig.l. Sequence alignment of OxyR homologues. £. coli OxyR is represented schematically at the top of the figure. The helix-turn-helix (HTH) DNA binding domain is shaded in light grey. The protein fragment used to determine the crys­ tal structure extends from amino acid 80 to 305. The CLUSTAL W (1.8) program was used to align OxyR homologs from organisms in which the transcription factor has been shown to play a role in protecting against oxidative stress (reviewed in Ref. 44). Sequence alignment is shown for the redox-active site. Identical amino acids are marked with shaded boxes. Arrows indicate the posi­ tions of cysteine residues in E. coli OxyR. The E. coli OxyR protein does contain six cysteine residues. Of these, two cysteines (C199 and C208) are conserved among OxyR homologs in other bacteria (Fig. 1). To determine whether the cysteine residues in OxyR were important for sensing the oxidative stress signal, each of its six cysteines was individually changed to serine by site-directed mutagenesis. Each oxyR mutant strain then was assayed for its sensitivity to H 2 0 2 and cumene hydroperoxide. 29 The C25S, C143S, C180S, and C259S mutant strains showed the same sensitivity as a strain carrying the wild-type oxyR gene. In fact, a derivative in which C25, C143, C180 and C259 all were mutated to alanine (OxyR4C^A) also showed wild-type resistance and wild-type induction of the oxyS target gene.54 The C199S mutant strain was as hypersensitive to H 2 0 2 and cumene hydroperoxide as the control strain lacking oxyR, and the C208S strain was partially sensitive to H 2 0 2 . 29 oxyS expression also was not induced by H 2 O z in the C199S and C208S mutant strains. Under oxidizing conditions in vitro, the OxyRC25S, OxyRcl43S, OxyR^gQg, and OxyR,-^^ proteins showed the same DNase I footprints as the oxidized wild-type protein. In contrast, the OxyR cl99S and OxyR^gg proteins showed a reduced footprint under all conditions.

Regulatory Disulfides Controlling Transcription Factor Activity

291

To examine the oxidation states of the C199 and C208 residues in vitro, the reduced and oxidized OxyR4C^A proteins were digested with trypsin, and the molecular weights of the tryptic fragments were analyzed with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.54 Two peaks corresponding to fragments containing alkylated C199 and C208, were present in the trypsin digest of reduced OxyR. These two peaks were absent for the oxidized protein. Instead, a new peak corresponding to the sum of the C199- and C208-containing peptide frag­ ments joined by a disulfide bond, was detected. Quantitative thiol/ disulfide titrations also indicated that oxidized OxyR contains one intramolecular disulfide bond. Together these results show that the C199 and C208 residues form the redox center of OxyR. The redox potential of OxyR was determined to be -185 millivolts using in vitro transcription assays. This value is -100 millivolts above the intracellular redox potential, ensuring that OxyR is maintained in its reduced form in wild-type cells in the absence of oxidative stress. OxyR oxidation was characterized further using alkylation followed by altered mobility on reducing SDS-polyacrylamide gels.3,45 Complete oxidation of OxyR was achieved with 5 and 0.2 uM H 2 0 2 in vivo and in vitro, respec­ tively. These concentrations agree well with the submicromolar concentra­ tions required for the in vivo activation of an OxyR-regulated katG-lacZ fusion.14 Given the submicromolar levels of OxyR compared to the millimolar levels of GSH inside the cell, the reaction between OxyR and H 2 0 2 must be highly specific. It also was found that, in logarithmically growing wild-type cells treated with H 2 0 2 , OxyR is completely oxidized within 30 sec and remains oxidized for ~5 min, paralleling the increase and decrease of messenger RNAs transcribed from OxyR-regulated genes.3-45 Together these results show that OxyR is a very effective sensor of H 2 O z within the cell.

2.3 OxyR Reduction by Glutaredoxin The finding that OxyR was only oxidized for a defined period of time without a decrease in OxyR protein levels suggested the C199-C208 disul­ fide bond is specifically reduced within the cell.43 In vitro transcription assays with purified components showed that glutaredoxin-1 together with GSH and thioredoxin 1 (encoded by trxA) partnered with thioredoxin reductase (encoded by trxB) could catalyze the reduction of OxyR.54 In vivo, the OxyR-regulated response was prolonged in gor and grxA single mutants but not in trxA or trxB single mutants. 54 These results indicated

292

Cellular Implications ofRedox Signalling

H202 ►

<

glutaredoxin + GSH no transcriptional activation

transcriptional activation

Fig. 2. The ribbon structure of the reduced (left) and oxidized (right) forms of the OxyR regulatory domain. The two redox-active cysteines, C199 (C199S in the reduced form) and C208, are indicated by the ball-and-stick representations (adapted from Ref. 6). The individual monomers are shown in red and blue respectively. Full-length OxyR is a tetramer in solution while the regulatory domain is a dimer. The structural transition from reduced to oxidized OxyR occurs upon reaction with H 2 0 2 . Reduction of OxyR is accomplished by glutaredoxin-1 together with GSH.54 OxyR also can be activated through changes in the cellular thiol/disulfide status, at least in part through the reverse of the reaction required to reduce OxyR.3 Upon disulfide bond formation, a-helix C and /J-strand 8 in reduced OxyR are disrupted. Oxidized OxyR contains a newly formed /3-strand 8' and pseudo-helical loop (thick purple lines).6 The newly cre­ ated pseudo-helical loop plays an important role in the dimeric interaction. To highlight the dimeric structural transition between the reduced and oxidized forms, the red monomer in each form is displayed in the same orientation. The blue monomer rotates ~30° in the oxidized form relative to the reduced, bringing the N-termini closer together.

that, in the cell, the C199-C208 disulfide b o n d in OxyR is r e d u c e d p r e d o m i n a n t l y b y glutaredoxin-1 a n d that the OxyR r e s p o n s e is a u t o regulated, since the gor a n d grxA genes are themselves activated b y oxidized OxyR. The kinetics of OxyR oxidation b y low concentrations of H 2 0 2 are significantly faster than the kinetics of OxyR reduction, allowing for transient activation in an overall r e d u c i n g e n v i r o n m e n t . To gain further insight into OxyR oxidation a n d reduction in vivo, OxyR activity was examined in a katG~ahpCF~ strain lacking peroxidases a n d in trxA~gor~ a n d trxA~gshA~ strains carrying m u t a t i o n s affecting b o t h the GSHa n d t h i o r e d o x i n - d e p e n d e n t r e d u c i n g systems. 3 OxyR w a s constitutively

Regulatory Disulfides Controlling Transcription Factor Activity

293

active in the katG'ahpCF' mutant that has elevated levels of H 2 0 2 but a wild-type GSH/GSSG ratio. OxyR is partially active in the absence of oxidative stress in trxA~gor~ and trxA~gshA~ double mutant strains. The trxA'gor' and trxA'gshA' mutant strains do not have elevated levels of H 2 0 2 but do have a decreased GSH/GSSG ratio. 3 Other studies showed that OxyR also was activated in cells treated with the thiol-oxidant diamide, but at much higher concentrations (mM) than those required for activation by H 2 0 2 . M These results indicate that OxyR can be oxidized by two different mechanisms in vivo: by increases in the H 2 0 2 concentration and, less efficiently, by decreases in the thiol/disulfide ratio (Fig. 2).

2.4 Structure of the OxyR Regulatory Domain The recently-determined crystal structures 6 of the regulatory domain of OxyR (residues 80-305) in its reduced and oxidized forms have provided insight into the biochemical nature of the redox-active center and the conformation change caused by disulfide bond formation (Fig. 2). Understanding of the disulfide bond chemistry has come from looking at the structures of the monomers, each consisting of two domains. The C-terminal a/^-domain contains the two redox-active cysteines. In the reduced form, Cysl99 and Cys208 are separated by 17.3 A, and C199, which reacts directly with H2Oz, is mostly solvent excluded and sits in a hydrophobic pocket located between the N-terminal and C-terminal a//3domains. Directly contacting C199 is the positively charged side chain of R266. The C199-R266 interaction, together with the helix dipoles gener­ ated by the neighboring helices, should serve to stabilize the thiolate form of C199 thus lowering the pKa of the sulfide group and increasing its reactivity to H 2 0 2 . It is proposed that upon reaction with H 2 0 2 , C199 is oxidized to a sulfenic acid intermediate. This intermediate may not be sta­ ble in the pocket between N-terminal and C-terminal a//3-domains. Thus the oxidized cysteine will be forced out of the hydrophobic pocket allow­ ing C199 to react with C208. In the oxidized form of the regulatory domain, the environments surrounding Cysl99 and Cys208 are much less favorable for stabilizing an ionized sulfide group. This explains why the kinetics of OxyR reduction are significantly slower than the kinetics of OxyR oxidation. Intriguingly, examination of the structures of the dimers showed how localized "fold-editing" caused by the formation of the C199-C208 bond leads to global changes in the dimeric and tetrameric forms of OxyR. Conformational changes in the monomer caused by disulfide bond

294

Cellular Implications ofRedox Signalling

Fig. 3. Sequence alignment of RsrA and RsrA-related proteins. S. coelicolor RsrA is represented schematically at the top of the figure. The deduced amino acid sequence of RsrA was aligned with those of the products of uncharacterized ORFs lying immediately downstream of Mycobacterium tuberculosis sigH (RshA), sigL (RslA) and sigE (RseA); Myxococcus xanthus rpoEl (OrfE); Bacillus subtillis ylaC (YlaD) and sigW (YbbM); and S. coelicolor sigT (RstA) (adapted from Ref. 25). The sequence shown here is limited to the indicated region. Identical amino acids are marked with shaded boxes. Arrows indicate the positions of the cysteine residues in S. coelicolor RsrA. formation include the looping out of a-helix C, expulsion of C199 from its hydrophobic pocket, alteration of /3-strand 8 into a pseudo helical loop, and formation of a new /3-strand 8' from the flexible loop consisting of residues 212-217. These localized conformational changes in the monomers result in a dramatic reorganization of the dimer interface. An overlay of reduced and oxidized dimers showed that there is an approximately 30° difference in the rotation of the monomers relative to each other. Thus the N-termini of each monomer in oxidized-OxyR, where the DNA binding domains would be located, now are shifted closer together and are located on the front face of the dimer. This shift in the orientation of the DNA binding domains can explain the different binding properties of reduced and oxidized OxyR. However, full understanding of the effects of the quaternary structural changes on OxyR ability to bind DNA and regulate transcription awaits structural studies of OxyR-DNA and OxyR-RNA polymerase complexes.

3.
Regulatory Disulfides Controlling Transcription Factor Activity

295

a distinct set of promoters that are preferentially transcribed by the stationary phase RNA polymerase. 24 Deletion of the gene encoding o* (sigR) resulted in an increased sensitivity to both the thiol-specific oxidant diamide and to the redox-cycling compounds menadione and plumbagin. 36 sigR null mutants also demonstrated reduced levels of disulfide reductase activity and a failure to induce this activity upon exposure to diamide. This obser­ vation can be explained by the identification of the trxBA operon encoding thioredoxin reductase and thioredoxin, respectively, as a direct target for o* regulation. Additional o* targets include sigR itself and the recently dis­ covered trxC gene encoding a putative thioredoxin.35 More than 30 matches to a crR consensus motif [GGAAT(nl8)GTT] have been identified upstream of protein coding regions in the emerging S. coelicolor genome sequence; these sequences recently have been shown to represent additional promot­ ers recognized by crR.37a Transcription of the sigR gene initiates at two promoters, sigRpl and sigRp2, separated by 173 bp. The sigRpl promoter was transiently induced (70-fold) in cells treated with diamide, and the sigRp2 transcript was undetectable in a szgR-null mutant. 36 Purified (f~ also was shown to direct transcription from sigRp2 in vitro. Taken together these results indicate that transcription of sigR is induced by diamide in a c^-dependent man­ ner and that sigR is positively autoregulated. The trxBA operon also is transcribed from two promoters, trxBpl and trxBpl, separated by 5-6 bp. The trxBpl promoter is transiently induced at least 50-fold in response to diamide treatment in a cr R -dependent manner. 36 The transient o 8 dependent induction in cells treated with diamide suggested that o* is activated in response to cytoplasmic disulfide bond formation and its activity is switched off upon reestablishment of normal thiol levels.36 Since o~R itself contains no cysteines this model raised the question of what other components may be involved that would explain how a8- activity responds to disulfide stress. Moreover, the positive autoregulation of sigR transcription implied that there must be a negative regulatory factor within the o~R system. Analysis of the DNA sequence downstream of sigR revealed a gene (rsrA = Regulator of Sigma R) whose start codon overlaps the sigR stop codon (Fig. 3).25 A rsrA null mutant was constructed and found to have high constitutive levels of disulfide reductase activity and o^-dependent transcription. 35 The high levels of c^-dependent transcription observed in the mutant were no longer inducible by diamide. These results showed that RsrA acts as an anti-sigma factor of o~R in vivo. Interestingly, the rsrA null mutant also was blocked in sporulation and gave rise to white

296

Cellular Implications of Redox Signalling

H 2 02 or diamide ► < thioredoxin

no transcription

transcription of G R targets

Fig. 4. Model for the disruption of the c^-RsrA interaction upon RsrA oxida­ tion. Normally, RsrA interacts with o" inhibiting the cr^-dependent transcription of antioxidant genes. Upon oxidation with H 2 0 2 or diamide, 0* and RsrA disso­ ciate and 0* is free to transcribe its target genes.25 The RsrA residues displayed correspond to C l l , H37, C41 and C44. H37, C41 and C44 are all are conserved in RsrA-like proteins. C l l is present in four out of seven RsrA-like proteins, while a histidine residue is conserved in a similar position in the other homologs. Since stoichiometric amounts of zinc were found in purified RsrA, it is possible that the redox-sensitive cysteines and conserved H37 coordinate zinc. Oxidation by diamide could result in the formation of a disulfide bond(s) between these zinccoordinating cysteines leading to a conformational change in RsrA and sub­ sequent dissociation of o".35 The o^-RsrA interaction can be restored through reduction by thioredoxin. 25

colonies as o p p o s e d to gray colonies p r o d u c e d b y s p o r u l a t i o n proficient w i l d - t y p e cells. Purified RsrA w a s found to inhibit o^-directed transcrip­ tion in a reversible r e d o x - d e p e n d e n t manner. 2 5 Transcriptional inhibition only occurred in the presence of dithiothreitol (DTT) a n d w a s abolished in the p r e s e n c e of 1 m M H 2 0 2 or 1 m M d i a m i d e . In a d d i t i o n , a 1:1 o^-RsrA complex could b e detected in the presence of D T T b u t n o t w h e n d i a m i d e w a s i n c l u d e d in the b i n d i n g buffer. T h u s the r e d o x state of RsrA a p p e a r e d to affect its ability to b i n d a n d inhibit o* activity (Fig. 4).

3.2 Release of a1* U p o n Disulfide Bond Formation in RsrA Since the o^-RsrA interaction w a s DTT d e p e n d e n t a n d w a s abolished b y a d d i t i o n of H 2 0 2 or d i a m i d e , it w a s possible t h a t formation of disulfide

Regulatory Bisulfides Controlling Transcription Factor Activity

297

bonds might play a role in regulating this protein-protein interaction. RsrA contains seven cysteine residues. Of these, C41 and C44 are absolutely conserved among all other RsrA-related proteins (Fig. 3). C l l only is con­ served in four out of the seven RsrA-related proteins, but the other homologs contain a conserved histidine at an adjacent position (Fig. 3). To study the requirements for these residues in RsrA-dependent redox regu­ lation in vivo each of the seven cysteines was individually mutated to serine.35 The C3S, C31S, and C61S mutant strains sporulated normally. Thus these cysteines are not required for RsrA anti-sigma factor activity. In contrast, the C l l S , C41S and C44S mutants had a white colony phenotype, indicating these latter cysteines are critical for RsrA anti-sigma fac­ tor activity. Colonies carrying the remaining mutant allele, C62S, had a pale gray color and produced fewer spores than the wild-type, suggest­ ing that RsrAC62S was only partially active. To investigate the effect of the mutations on the ability of RsrA to sense disulfide stress, o^-dependent trxCpl activity was assessed following diamide treatment. Whereas the C3S, C31S, C61S and C62S mutant strains exhibited induction of the trxCpl promoter in response to diamide, trxCpl was expressed at high constitutive levels in the CllS, C41S and C44S mutants. The basal level of trxCpl activity was slightly higher in the C62A strain than in the wildtype, but was still strongly inducible by diamide. Each of the seven RsrA cysteine mutants also was overexpressed, puri­ fied and tested for its ability to inhibit c^-dependent transcription from the sigRp2 promoter in the presence of a range of DTT concentrations. 35 As demonstrated previously, 25 wild-type RsrA inhibited o^-dependent tran­ scription only when the DTT concentration was 0.25 mM or higher. RsrAC3S, RsrA^jjg, and RsrAC61s also inhibited o* activity in a DTT-dependent manner, but RsrA c n s , RsrAC41s, RsrAC44S, and RsrAC62S did not inhibit tran­ scription at any of the concentrations of DTT used. The in vitro activity of RsrAC3S, RsrA c n s , RsrAC31s, RsrAC41s, RsrAC44S and RsrAC61s agreed with their activity in vivo, but the finding that RsrAC62S was inactive in vitro con­ trasted with its partial activity in vivo. A RsrA mutant in which C3, C31, C61 and C62 were collectively replaced with either serine or alanine was still active as an anti-sigma factor and was able to sense disulfide stress. These experiments demonstrated that C l l , C41 and C44 in RsrA are crit­ ical for its anti-sigma factor activity both in vivo and in vitro and thus would appear to make up the redox-active center of RsrA. The ability of RsrA to form disulfide bonds in vitro was examined on non-reducing SDS-polyacrylamide gels.25 Compared to the reduced protein, an oxidized protein with intramolecular disulfide bonds usually

298

Cellular Implications ofRedox Signalling

migrates faster due to a decrease in chain flexibility and hydrodynamic volume while an oxidized protein linked to another protein with intermolecular disulfide bonds usually migrates slower due to the increased molecular weight. Oxidized RsrA had an apparent molecular weight of 14 kDa, close to the 11.96 kDa calculated mass. Treatment with increasing concentrations of DTT led to multiple bands corresponding to a range of size from 14 to 20 kDa apparent molecular weight. These results sug­ gested that intramolecular, rather than intermolecular disulfide bonds were formed under oxidizing conditions. To determine the number of free thiols in RsrA incubated with DTT or dialyzed against a buffer lack­ ing DTT, the protein samples were treated with iodoacetamide and then examined directly using electrospray mass spectrometry. 25 For DTTtreated RsrA, the major species corresponded to RsrA with seven thiols. For RsrA dialyzed against DTT-free buffer for 8 hrs, two major species, containing one and two disulfide bonds, were present. After 36 hrs of dialysis, the major species had two disulfide bonds, but there was also a minor peak representing a form containing three disulfide bonds. The specific disulfide bonds present in the different forms of RsrA were not determined, but the results of the mass spectrometric experiments con­ firmed that oxidation of RsrA leads to a change in the thiol-disulfide sta­ tus of this anti-sigma factor. Inductively coupled plasma atomic emission spectroscopy showed that stoichiometric amounts of zinc are present in RsrA.35 This finding and the fact that RsrA has three conserved and essential cysteines and one conserved histidine all within 30-residues, led to the suggestion that the C l l (or the corresponding histidine), H37, C41 and C44 may be involved in zinc coordi­ nation. These residues would constitute a novel zinc-binding motif in the form of C(/H)X25_26HX3CXXC, but further characterization of the role of zinc in RsrA anti-sigma factor activity needs to be investigated. Interestingly, the activity of the E. coli chaperone Hsp33 is regulated by disulfide bond forma­ tion, and Hsp33 was found to contain a novel zinc binding motif, in which the zinc is coordinated by the redox-sensitive cysteines.22,23 Thus, a switch between zinc coordination and disulfide bond formation may be a general mechanism for redox-regulation of protein activity (Fig. 4).

3.3 RsrA Reduction by Thioredoxin The observation that o" target genes were induced only for a defined period of time suggested RsrA might be reduced, leading to the anti-sigma

Regulatory Bisulfides Controlling Transcription Factor Activity

299

Fig. 5. Sequence alignment of Yaplp homologues. S. cerevisiae Yaplp is repre­ sented schematically at the top of the figure. The bZip DNA binding domain is shaded in light grey and the amino- and carboxy-terminal cysteine-rich domains (w-CRD and c-CRD, respectively) are shaded in dark grey. A thick line marks the location of the nuclear export signal (NES). The CLUSTAL W (1.8) program was used to align Yaplp homologs from the indicated organisms. Sequence align­ ments are shown for the n-CRD and the c-CRD. Identical amino acids are marked with shaded boxes. Arrows indicate the positions of the S. cerevisiae Yaplp cysteine residues. It should be noted that the regulation of S. pombe Paplp has been found to be very similar to the regulation of S. cerevisiae Yaplp.48,50

factor binding to (F. Consistent with this model, in vitro o^-RsrA complex formation was observed in the presence of reduced S. coelicolor thioredoxin. 25 Complex formation was not seen upon treatment with glutathione. This result indicates that RsrA is reduced by thioredoxin and that a*, RsrA and thioredoxin comprise a homeostatic feedback loop that senses and responds to changes in the intracellular thiol/disulfide status. The role of £rx<4-encoded thioredoxin in RsrA reduction in vivo has not been examined. In addition, the discovery that trxC is a a* target, raises the possibility the putative thioredoxin encoded by trxC also may play a role in RsrA redox control.

4. Yapl 4.1 Identification of Yaplp and its Target Genes The S. cerevisiae Yaplp transcription factor was discovered and purified based on its binding to a site recognized by the mammalian AP-1 transcrip­ tion factor15 (Fig. 5). The corresponding gene then was cloned using anti­ bodies directed against purified Yaplp. 34 The YAP1 gene also was isolated independently in multicopy screens for resistance to sulfomeutron methyl and cycloheximide (PDR4, Ref. 18), resistance to 4-nitroquinoline-N-oxide,

300

Cellular Implications ofRedox Signalling

trenimon and nitrosoguanidine (SNQ3, Ref. 17), and resistance to the iron chelators 1,10-phenanthroline and l-nitroso-2-napthol (PAR1, Ref. 38). Subsequently, yapl null mutants were found to be hyper­ sensitive to oxidative stress. 39 Expression analysis of individual genes showed that in response to H 2 0 2 and diamide, Y a p l p regulates the expression of several genes whose products play roles in the oxidative stress tolerance. These targets include GSH1 encoding y-glutamylcysteine synthetase, 42,52 GLR1 encoding glutathione reductase, 14 GPX2 encoding glutathione peroxidase 19 , TRX2 encoding thioredoxin 2,26,33 TRR1 encoding thioredoxin reductase, 5,30 and the TSA1- and AHP1encoded thioredoxin peroxidases. 31 Whole genome expression analysis by two-dimensional protein gels 30 and DNA microarrays 12 showed that Yaplp regulates the expression of as many as 70 genes in response to treatment with H 2 0 2 as well as in response to treatment with menadione and diamide. A Yaplp recognition element was defined by studies of Yaplp binding to the TRX2 [5'-TTAG/CTAA] 26 and GSH1 [5'-TTAGTCA] 52 promoters and is present at most of the pro­ moters of genes whose expression is induced in a Yaplp-dependent fashion. 411 Because there are only modest increases in Yaplp-DNA binding activity in response to oxidative stress, it was suggested that Yaplp activation might be due to post-translational changes. 27 Confocal microscopy studies of Yaplp fused to the green fluorescent protein (GFP)27,53 revealed that the transcription factor localization changes dramatically in response to oxidative stress. While the Yaplp-GFP fusion is present throughout the cell during normal growth, the fusion is clearly concentrated in the nucleus upon treatment with H 2 O z or diamide. 10,27,53 Nuclear import of Yaplp is mediated by the Pselp recep­ tor but is not affected by oxidative stress. 20 Nuclear export is mediated by the C r m l p (Xpol) receptor. Yaplp is localized constitutively to the nucleus in a crml mutant defective in nuclear export. 28,53 Two-hybrid studies also showed an interaction between Yaplp and C r m l p in normally-growing cells.10,28,53 This binding requires a non-canonical, leucine-rich nuclear export signal (NES) located in the carboxy-terminus of the protein 28,53 (Fig. 6). Interestingly, the Y a p l p - C r m l p inter­ action is not detected in cells exposed to H 2 0 2 1 0 or diamide, 53 and in vitro, the interaction is strengthened in the presence of DTT.53 These results indicate that the interaction between Yaplp and C r m l p is redox-dependent.

Regulatory Disulfides Controlling Transcription Factor Activity

nuclear export

301

no nuclear export

Fig. 6. Model for the redox-sensitive Yaplp-Crmlp protein-protein interaction. Yaplp (blue) is displayed as a monomer with its N-terminal bZIP DNA binding domain, n-CRD and c-CRD. The cysteine residues correspond to C303, C310, C315, C598, C620 and C629. Crmlp interacts with the NES (yellow) located within the c-CRD of Yaplp, under normal growth conditions. It is proposed that H 2 0 2 leads to disulfide bond formation between cysteine residues in the n-CRD and c-CRD resulting in a conformational change that blocks Crmlp from export­ ing Yaplp.10 The oxidation of cysteines in the c-CRD also could block Crmlp from binding to the Yaplp NES.26a In this model, Yaplp would be reduced by thioredoxin.4'10'21

4.2 Block of Yaplp Nuclear Export by Disulfide Bond Formation The possibility that Yaplp might serve as the oxidative stress sensor was raised by the fact that Yaplp contains six cysteine residues, five of which are conserved among Yaplp homologs found in other yeast species (Fig. 5). The cysteines are located in two conserved regions that are sepa­ rated by ~280 amino acids and have been designated cysteine-rich domains (CRD). Three cysteines are present in the more amino-terminal domain (n-CRD) required for H 2 0 2 but not diamide resistance. 9 The more carboxy-terminal domain (c-CRD), that contains three Cys-Ser-Glu repeats and overlaps the NES-like sequence, was found to be important for Yaplp-dependent resistance to both H 2 0 2 and diamide. 27,51 One attrac­ tive scenario is that oxidation of the cysteine residues masks the NES, thus preventing export of Yaplp and allowing it to accumulate in the

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Cellular Implications ofRedox Signalling

nucleus.28,53 However, the results of mutational studies of the six cysteine residues in Yaplp have been more complicated than the findings for OxyR and RsrA. C315A and C620A mutant strains had wild-type resis­ tance to H 2 O z , and a GFP-Yaplp C620A fusions showed wild-type subcellular distribution.10'28,53 The C620A strain also had wild-type resistance to diamide, but a strain carrying a C620T allele was hyperresistant to diamide and the corresponding fusion protein showed constitutive nuclear localization.27,53 While the C303A, C310A, C598A, and C629A mutant strains all were hypersensitive to H202,9,10,53 C598T and C629T strains were reported to have wild-type resistance to H 2 0 2 . 27,53 The C303A and C598A strains had wild-type resistance to diamide, but the C629A strain was hyperresistant to the thiol oxidant.9,10,53 GFP-Yaplp C303A fusions were reported to be constitutively nuclear in one study 9 and constitutively cytoplasmic in another. 10 A GFP-Yaplp C310A fusion had delayed nuclear localization, a GFP-Yaplp C598A fusion was constitutively cytoplas­ mic, and a GFP-Yaplp C629A fusion was constitutively nuclear.10,53 Twohybrid experiments showed that while the interaction between wild-type Yaplp and C r m l p was abolished upon treatment with H 2 0 2 , the Yaplp C303A -Crmlp and Yaplp C598A -Crmlp interactions were maintained indicating that these two proteins are no longer sensitive to H 2 0 2 . 10 The lack of a strict correlation between H 2 0 2 resistance, diamide resistance and subcellular localization most likely reflects complexities in how H 2 0 2 and diamide lead to Yaplp nuclear localization. The effects of some sub­ stitutions of cysteines in the c-CRD also may solely be due to a disruption of the NES. In addition, some of the discrepancies probably can be attri­ buted to the different amino acid substitutions, constructs and assay con­ ditions used in the different studies. Nevertheless, two conclusions can be drawn from these mutational studies. First, at the mM concentrations tested, H 2 0 2 and diamide have different effects on Yaplp. Second, C303A and C598A mutants are hypersensitive to H 2 O z , implicating these cysteines in perceiving H 2 0 2 . The in vivo oxidation state of a Myc-YAPl fusion protein in response to H 2 0 2 and diamide treatment was examined using alkylation followed by altered mobility on reducing SDS-polyacrylamide gels.10 Compared to Myc-Yaplp from untreated cells, the protein from H 2 O z -treated cells ran with a faster mobility, possibly corresponding to an oxidized form of this Yaplp derivative. No change in mobility was observed for Myc-Yaplp from diamide-treated cells, consistent with the idea that H 2 0 2 and diamide modify Yaplp by different mechanisms. The faster mobility form of Myc-Yaplp was observed as early as 2.5 min after treatment with

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400 uM H 2 0 2 and disappeared at 60 min thus correlating with the kinetics of TRX2 expression. 10 The minimum concentration of H2G*2 required to detect this form of Yaplp was 100 uM. Examination of the effect of the cysteine substitutions on Yaplp mobility shift demonstrated that, similar to the wild-type fusion, Myc-YaplpC315A and Myc-YaplpC620A were oxidized in response to H 2 0 2 . Derivatives carrying the C303A and C598A substitu­ tions failed to be oxidized and Myc-Yaplp C310A and Myc-Yaplp c629A appeared to be partially oxidized. Delaunay et ah concluded that H 2 O z treatment causes a disulfide bond to be formed between C303 and C598 leading to a conformational change that prevents an interaction between Yaplp and Crmlp. As of yet, the presence of this disulfide bond has not been verified biochemically. The oxidation state of the three cysteines in the c-CRD of Yaplp was studied in vitro by mass spectrometry analysis.26"1 A GFP-c-CRD fusion was expressed in E. coli, purified and treated with either H 2 0 2 or diamide. Three different peaks corresponding to peptidase digestion fragments carrying single cysteine residues were detected in the absence of oxidant treatment. Following exposure to H 2 0 2 , all three peaks disappeared and instead a specific new peak appeared, indicating the formation of a disul­ fide bond between C598 and C620. Diamide treatment induced each possible pair of disulfide bonds between the three cysteines. Experiments in which the in vivo oxidation state of the c-CRD was examined by alkylation followed by altered mobility on reducing SDS-polyacrylamide gels were consistent with the results of the mass spectrometry analysis.263 Thus Kuge et al.26a concluded that H 2 0 2 and diamide can lead to disulfide bond formation among cysteines in the c-CRD, although the authors acknowl­ edged that additional disulfide bonds are likely to form in the full-length protein exposed to H 2 0 2 . More studies are needed to unify all of the find­ ings regarding the Yaplp cysteines. The model that H 2 O z and diamide both oxidize cysteine residues, such that the NES is blocked and Yaplp can no longer be bound by Crmlp and exported from the nucleus, remains attrac­ tive. However, the possibilities that Yaplp contains a redox-active metal such as zinc, that Yaplp activity is modulated through an as yet unidentified redox-sensitive protein, or that Yaplp regulation involves multiple steps, also still need to be considered. 4.3 Regulation of Yaplp Activity by Thioredoxin Genetic studies have shown that Yaplp is concentrated in the nucleus and is constitutively active in trxl trx2 mutants lacking both thioredoxins 1 and

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2,21 and in trrl mutants lacking thioredoxin reductase. 4 ' 10 These observa­ tions suggested that the thioredoxins might be required to reduce oxi­ dized Yaplp analogous to how glutaredoxin-1 reduces oxidized OxyR. When extracts from S. cerevisiae cells treated with H 2 0 2 were incubated with high concentrations of Spirulina thioredoxin, E. coli thioredoxin reductase and NADPH, the oxidation-dependent mobility shift of Yaplp was eliminated within 30 minutes. 10 Incubation of the purified GFP-o CRD protein with reduced Trx2p also converted this Yaplp derivative from an oxidized to reduced form.26a These findings are consistent with the model that thioredoxins directly reduce oxidized Yaplp. In contrast, the GSH-based reducing system does not appear to have a role in regu­ lating Yaplp in response to H 2 O z since gshl mutants show a normal adap­ tive response 41 and gshl and grxl grx2 mutants show wild-type induction of Yaplp target genes.10,21

5. Concluding Remarks A combination of genetic and biochemical studies have suggested that disulfide bond formation is a key step in regulating the activities of OxyR, RsrA and Yaplp. There are some interesting similarities between these three systems. The activities of all three of the regulators are modulated by H 2 0 2 . Since the oxidation of thiols is a major consequence of increased levels of H 2 0 2 , it makes physiological sense for regulators of defenses against H 2 0 2 to perceive oxidative stress via redox-active thiols. In addi­ tion, OxyR, RsrA and Yaplp all appear to be reduced by disulfide bond reducing systems whose expression is induced by these factors. Thus each system is autoregulated and only activated for a defined period of time. Some interesting differences between OxyR, RsrA and Yaplp also can be noted. OxyR only has one pair of critical cysteine residues and oxidation leads to the formation of only one disulfide bond. In contrast, several cysteine residues appear to be critical for RsrA and Yaplp activity, and it appears that several different disulfides might be formed. The possibility of forming multiple disulfide bonds should allow for more varied regula­ tion of the latter proteins. Regulation by a thiol/disulfide switch has several prominent advan­ tages. The formation of a disulfide bond does not require new protein synthesis and can occur extremely fast. Complete oxidation of OxyR was observed within 30 sec after the E. coli cells were treated with H 2 O z . The examples described here indicate that specific cysteines can be exquisitely

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sensitive to oxidants. The formation of a disulfide bond also is reversible allowing a regulator to be reduced and deactivated. In addition, redox regulation can support large conformational changes as was demon­ strated for OxyR. We anticipate that other transcription factors whose activities are regulated by a thiol/disulfide switch will be discovered. Transcription factors that modulate processes involving electron transfer, such as respiration and photosynthesis, or responses to microbial invasion, that involve reactive oxygen species, are good candidates for proteins whose activities might be responsive to the cellular redox environment. In addi­ tion, numerous transcription factors contain zinc-finger domains, and it is possible that the activities of some of these proteins will be modulated by oxidation of the cysteines required for zinc binding. Although much has been learned about how OxyR, RsrA and Yaplp activities are regulated in response to oxidative stress, many critical ques­ tions remain to be answered about the chemistry of the disulfide bond formation and reduction. What is the nature of the intermediates formed upon oxidation? It has been proposed that the C199 residue of OxyR is first oxidized to a sulfenic acid, but definitive proof for this intermediate is still lacking. Why are some cysteines particularly sensitive to oxidation? What is the basis for oxidant specificity? What are the contributions of surrounding residues? Further applications of the biochemical assays described above together with new assays should help to provide answers to these questions.

Acknowledgments We thank S. Kuge, J.-H. Roe, M. Paget and M. Toledano for sharing data prior to publication. We also appreciate the editorial comments of S. Kuge, S. Moye-Rowley, J.-H. Roe and M. Toledano. This work was sup­ ported by the intramural program of the National Institute of Child Health and Human Development (O. Carmel-Harel. and G. Storz) and a fellowship from the National Research Council (M.J. Wood.).

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2. Altuvia S, Weinstein-Fischer D, Zhang A, Postow L, Storz G. 1997. A small, stable RNA induced by oxidative stress: Role as a pleiotropic regulator and antimutator. Cell. 90: 43-53 3. Aslund F, Zheng M, Beckwith J, Storz G. 1999. Regulation of the OxyR transcription factor by hydrogen peroxide and the cellular thiol/disulfide status. Proc. Natl. Acad. Sci. USA. 96: 6161-6165 4. Carmel-Harel O, Stearman R, Gasch AP, Botstein D, Brown PO, Storz G. 2001. Role of thioredoxin reductase in the Yaplp-dependent response to oxidative stress in Saccharomyces cerevisiae. Mol. Microbiol. 39: 595-605 5. Charizanis C, Juhnke H, Krems B, Entian KD. 1999. The oxidative stress response mediated via Pos9/Skn7 is negatively regulated by the Ras/PKA pathway in Saccharomyces cerevisiae. Mol. Gen. Genet. 261: 740-752 6. Choi H-J, Kim S-J, Mukhopadhyay P, Cho S, Woo J-R, et al. 2001. Structural basis of the redox switch in the OxyR transcription factor. Cell. 105: 103-113 7. Christman MF, Morgan RW, Jacobson FS, Ames BN. 1985. Positive control of a regulon for defenses against oxidative stress and some heat-shock proteins in Salmonella typhimurium. Cell 41: 753-762 8. Christman MF, Storz G, Ames BN. 1989. OxyR, a positive regulator of hydrogen peroxide-inducible genes in Escherichia coli and Salmonella typhimurium, is homologous to a family of bacterial regulatory proteins. Proc. Natl. Acad. Sci. USA 86: 3484-3488 9. Coleman ST, Epping EA, Steggerda SM, Moye-Rowley WS. 1999. Yaplp activates gene transcription in an oxidant-specific fashion. Mol. Cell. Biol. 19: 8302-8313 10. Delaunay A, Isnard AD, Toledano MB. 2000. H 2 O z sensing through oxidation of the Yapl transcription factor. EMBO. }. 19: 5157-5166 11. DeRisi JL, Iyer VR, Brown PO. 1997. Exploring the metabolic and genetic control of gene expression on a genomic scale. Science 278: 680-686 12. Gasch AP, Spellman PT, Kao CM, Carmel-Harel O, Eisen MB, et al. 2000. Genomic expression programs in the response of yeast cells to environmental changes. Mol. Biol. Cell. 11: 4241-4257 13. Gonzalez-Flecha B, Demple B. 1997. Homeostatic regulation of intracellular hydrogen peroxide concentration in aerobically growing Escherichia coli. J. Bacteriol. 179: 382-388

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14. Grant CM, Maciver FH, Dawes IW. 1996. Stationary-phase induction of GLR1 expression is mediated by the yAP-1 transcriptional regula­ tory protein in the yeast Saccharomyces cerevisiae. Mol. Microbiol. 22: 739-746 15. Harshman KD, Moye-Rowley WS, Parker CS. 1988. Transcriptional activation by the SV40 AP-1 recognition element in yeast is mediated by a factor similar to AP-1 that is distinct from GCN4. Cell 53: 321-330 16. Henikoff S, Haughn GW, Calvo JM, Wallace JC. 1988. A large family of bacterial activator proteins. Proc. Natl. Acad. Sci. USA 85:6602-6606 17. Hertle K, Haase E, Brendel M. 1991. The SNQ3 gene of Saccharomyces cerevisiae confers hyper-resistance to several functionally unrelated chemicals. Curr. Genet. 19: 429-433 18. Hussain M, Lenard J. 1991. Characterization of PDR4, a Saccharomyces cerevisiae gene that confers pleiotropic drug resistance in high-copy number. Gene 101: 149-152 19. Inoue Y, Matsuda T, Sugiyama K, Izawa S, Kimura A. 1999. Genetic analysis of glutathione peroxidase in oxidative stress response of Saccharomyces cerevisiae. J. Biol. Chem. 274: 27002-27009 20. Isoyama T, Murayama A, Nonioto A, Kuge S. 2001. Nuclear import of the yeast AP-1-like transcription factor Yaplp is mediated by trans­ port receptor Pselp, and this import step is not affected by oxidative stress. /. Biol. Chem. 276: 21863-21869 21. Izawa S, Maeda K, Sugiyama K, Mano J, Inoue Y, Kimura A. 1999. Thioredoxin deficiency causes the constitutive activation of Yapl, an AP-1-like transcription factor in Saccharomyces cerevisiae. }. Biol. Chem. 274: 28459-28465 22. Jakob U, Eser M, Bardwell JC. 2000. Redox switch of Hsp33 has a novel zinc-binding motif. /. Biol. Chem. 275: 38302-38310 23. Jakob U, Muse W, Eser M, Bardwell JC. 1999. Chaperone activity with a redox switch. Cell 96: 341-352 24. Kang JG, Hahn MY, Ishihama A, Roe JH. 1997. Identification of sigma factors for growth phase-related promoter selectivity of RNA polymerases from Streptomyces coelicolor A3(2). Nucleic. Acids. Res. 25: 2566-2573 25. Kang JG, Paget MS, Seok YJ, Hahn MY, Bae JB, et al. 1999. RsrA, an anti-sigma factor regulated by redox change. EMBO. ]. 18: 4292-4298 26. Kuge S, Jones N. 1994. YAP1 dependent activation of Trx2 is essential for the response of Saccharomyces cerevisiae to oxidative stress by hydroperoxides. EMBO. }. 13: 655-664

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26a. Kuge S, Arita M, Murayama A, Maeta K, Izawa S, Inoue Y, Nomoto A. 2001. Regulation of the yeast Yap l p nuclear export signal is mediated by redox signal-induced reversible bond formation. Mol. Cell. Biol. 21: 6139-6150 27. Kuge S, Jones N, Nomoto A. 1997. Regulation of yAP-1 nuclear local­ ization in response to oxidative stress. EMBO. /. 16:1710-1720 28. Kuge S, Toda T, lizuka N, Nomoto A. 1998. Crml (Xpol) dependent nuclear export of the budding yeast transcription factor yAP-1 is sensitive to oxidative stress. Genes Cells. 3: 521-532 29. KuUik I, Toledano MB, Tartaglia LA, Storz G. 1995. Mutational analysis of the redox-sensitive transcriptional regulator OxyR: Regions important for oxidation and transcriptional activation. /. Bacteriol. 177: 1275-1284 30. Lee J, Godon C, Lagniel G, Spector D, Garin J, et al. 1999. Yapl and Skn7 control two specialized oxidative stress response regulons in yeast. /. Biol. Chem. 274:16040-16046 31. Lee J, Spector D, Godon C, Labarre J, Toledano MB. 1999. A new antioxidant with alkyl hydroperoxide defense properties in yeast. /. Biol. Chem. 274: 4537-4544 32. McDuffee AT, Senisterra G, Huntley S, Lepock JR, Sekhar KR, et al. 1997. Proteins containing non-native disulfide bonds generated by oxidative stress can act as signals for the induction of the heat shock response. /. Cell. Physiol. 171: 143-151 33. Morgan BA, Banks GR, Toone WM, Raitt D, Kuge S, Johnston LH. 1997. The Skn7 response regulator controls gene expression in the oxidative stress response of the budding yeast Saccharomyces cerevisiae. EMBO. J. 16: 1035-1044 34. Moye-Rowley WS, Harshman KD, Parker CS. 1989. Yeast YAP1 encodes a novel form of the Jun family of transcriptional activator proteins. Genes Dev. 3: 283-292 35. Paget MS, Bae JB, Hahn MY, Li W, Kleanthous C, et al. 2001. Mutational analysis of RsrA, a zinc-binding anti-sigma factor with a thiol/disulfide redox switch. Mol. Microbiol. 39: 1036-1047 36. Paget MS, Kang JG, Roe JH, Buttner MJ. 1998. oR, an RNA polymerase sigma factor that modulates expression of the thioredoxin system in response to oxidative stress in Streptomyces coelicolor A3(2). EMBO.}. 17: 5776-5782 37. Schell MA. 1993. Molecular biology of the LysR family of transcrip­ tional regulators. Ann. Rev. Microbiol. 47: 597-626

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Chapter 14 Redox Signaling During Light-Regulated Translation in Chloroplasts

Tal Alergand, Tova Trebitsh a n d Avihai D a n o n Department of Plant Sciences, Weizmann Institute of Science, Rehovot, Israel [email protected]

Keywords: mRNA-binding proteins, translational regulation, reductive signal, regulatory protein oxidation, protein disulfide isomerase-like protein, redox signal transduction, regulatory disulfide.

1. Summary Signaling by redox has been shown to regulate both transcriptional1,30,51 and posttranscriptional events controlling gene expression.12,24 However, the molecular mechanisms by which redox signals regulate the expression of specific genes are not well understood. The growing list of newly identified redox active regulators of gene expression1'2'7'9,13'15'18"20,23'29,36"38'50 suggests that redox signaling is mediated by specific proteins that are thought to use intrinsic redox changes to control their biological activity. While conceiv­ ably, the redox state of these proteins must change uniquely according to their regulatory function, the exact nature of these redox changes and the mechanism of their regulation is yet to be fully understood. To figure out regulation by redox signals in vivo, a better understand­ ing of the intracellular redox environment and the effects that it has on regulatory redox events is required. The intracellular milieu is highly reductive and includes antioxidative systems that function to counteract the effects of free radicals.8,16,17'21'35 Therefore, it imposes several restric­ tions on intracellular events of redox signaling. For example, because redox controlled gene expression requires changes in the redox state of regulatory proteins, from reduced to oxidized state or vice versa, the 311

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highly reductive state of the tntracellular milieu prescribes that regulatory proteins must, at least temporarily, oxidize for intracellular redox signal­ ing to occur. However, at the same time, the high tendency of the intra­ cellular milieu to oppose oxidation raises questions as to whether and how oxidation of specific proteins may occur. These questions are in par­ ticularly intriguing under conditions where the biological control requires continuous and dynamic modulation of gene expression by redox signaling as under this type of regulation the oxidized state of regulatory proteins must persist over time. Redox signaling of light-regulated translation in the chloroplast is a dynamic control that continuously adjusts the level of synthesis of several photosynthetic proteins to fluctuating light intensities. Hence, studying the properties of light-regulated redox signaling in the chloroplast should help clarify how redox signals can be transduced via specific pathways in the reductive intracellular milieu. From these studies we learn that the reduction and oxidation reactions of redox active regulators of gene expression can be specific, and that they are probably mediated by protein-protein interactions with unique oxidoreductases and not by global intracellular redox changes.

2. Light Regulated Translation in Chloroplasts: A Case Study of Dynamic Control by Redox Signaling Photosynthesis is a chloroplast unique set of membrane localized reac­ tions that converts light energy into the biological usable forms, ATP and NADPH. Light absorbance and conversion into beneficial chemical energy in the process of photosynthesis is accompanied by deleterious side effects to the proteins that are intimately involved in the primary reactions. This leads to photosynthetic proteins over turn that parallels light intensity.3,31'4S Light intensity and availability are not controlled by plants and fluctuate continuously during the course of the day. Therefore dynamic adjustment of gene expression to maintain photosynthetic capacity is required. Translational regulation provides the capacity to rapidly induce or reduce massive levels of protein synthesis from an existing pool of transcripts, and thereby can facilitate rapid adjustment of gene expression to fluctuating light intensities. In plants, a 50- to 100-fold increase in translation of psbA mRNA (encoding the Dl protein of photosystem II reaction center) has been observed upon illumination.14'25'26,28 Hence, plants have adopted a primary role to light-regulated translation

Redox Signaling During Light-Regulated Translation in Chloroplasts 313

Fig. 1. Translation of several chloroplast mRNAs is regulated in response to light via at least two major pathways. In the first, reduction of the plastoquinone (PQ) pool by photosystem II (PS II) activates a signal-transduction pathway which leads to the priming of the translational factors required for thioredoxinmediated modulation of translation. In the second, a thiol-mediated signal, pro­ portional to the reducing potential generated by photosystem I (PS I), is transduced by ferredoxin (Fd), ferredoxin-thioredoxin reductase (FTR) and thioredoxin (Trx) to stimulate translation.

in the homeostatic response that maintains fully functional photosynthesis under fluctuating light intensities.11 This leads to the conclusion, that the signaling pathways that link the perception of light intensity and the translational control of photosynthetic proteins in the chloroplast must have a dynamic capacity, as well. How does the fluctuating light-intensity control translation? It was found that perception of the light-signal is mediated by the light-capturing reactions of photosynthesis that are localized in the thylakoid membranes. 41 Light perception by the membranal photosynthetic reactions generates two signals (Fig. 1). The first light-signal (priming signal) turns on the translational regulatory pathways, that are inactive in the dark, and confers them receptive to the second light-signal. 41 Whereas, the second light-signal dynamically links light intensity with translational regulation, thereby modulating the rate of translation proportionally to the photosynthetically perceived light intensity. 41

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Fig. 2. Following illumination, the reducing power generated by photosynthesis is used to reduce thioredoxin through a series of oxidation-reduction reactions involving ferredoxin (Fd), ferredoxin-thioredoxin reductase (FTR), and thioredoxin (Trx). Reduced thioredoxin drives reduction of the psbA mRNA-binding proteins, increasing their capacity to bind the 5'-UTR of psbA mRNA. Binding of this protein complex to the psbA mRNA allows for recruitment of psbA mRNAs onto polysomes and subsequent translation of the Dl protein. The second light-signal w a s s h o w n to b e t r a n s d u c e d b y the ferredoxint h i o r e d o x i n s y s t e m t h a t w a s first i d e n t i f i e d b y B u c h a n a n a n d Colleagues. 5 1 0 In the light, electrons e m a n a t i n g from P h o t o s y s t e m I r e d u c e t h i o r e d o x i n t h r o u g h a series of oxidation-reduction reactions

Redox Signaling During Light-Regulated Translation in Chloroplasts 315

involving ferredoxin (Fd), ferredoxin-thioredoxin reductase (FTR), and thioredoxin (Trx) (Fig. 2). Reduced thioredoxin then drives the reduction of a regulatory disulfide in a protein complex implicated as translational regulator of psbA mRNA.12'13 Reduction of the regulatory disulfide stimu­ lates the binding of the regulatory protein complex to the 5'-untranslated region of psbA mRNA. 13 Binding of this protein complex to the psbA mRNA parallels the recruitment of psbA mRNAs onto polysomes and the subsequent synthesis of the Dl protein (encoded by psbA mRNA).13,48,49 This direct link between light and the synthesis of the Dl protein, a core protein of PS II reaction center, has the capacity to respond to fluctuating light levels and regulate the exchange of photooxidized reaction center proteins31,45 with de novo synthesized proteins at a rate proportional to the rate of photosynthesis.

3. The Properties of Light Regulated Redox Signal Transduction and Their Implications The three most striking features of redox signaling of light-regulated translation of chloroplast mRNAs are: (i) The stimulatory signal trans­ duced by the ferredoxin-thioredoxin system is reductive, therefore its per­ ception requires an oxidized translational regulator; (ii) the transduction of the stimulatory reductive signal takes place under "normal growth" conditions under which the intrachloroplast milieu is highly reductive; (iii) the redox signaling is a dynamic control continuously adjusting the rate of synthesis of specific proteins to fluctuating light intensities. Therefore, at least, partial oxidation of the pool of the translational regu­ lator proteins must persist over time. These properties of redox signaling of light-regulated translation of chloroplast mRNAs raise critical questions about the redox reactions of the translational regulator. Namely, how does the redox state of transla­ tional regulator change uniquely according to its regulatory function in the reductive environment of the chloroplast? To perceive the stimulatory reductive signal the redox sensor subunit of the translational regulator must be in a receptive oxidized state, and on reception of the translation stimulatory signal it should undergo reduction, thereby activating specific gene expression. Thus, how does the sensor protein oxidize inside the reductive environment of the chloroplast, and how does it get reduced specifically by the reductive stimulatory signal, carried by thio­ redoxin, and not by the global reductive state of the chloroplast?

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Cellular Implications of Redox Signalling

Furthermore, the dynamic nature of redox signaling controlling translation of chloroplast mRNAs suggests that the redox signals are not transient and that they must persist over long periods of time. Hence, at least partial oxidation of the pool of the translational regulators must be longlasting. This also suggests that signaling through changes in the global redox state of the organelle is not likely, as it would disrupt over extended periods of time the optimal redox state required for chloroplast metabo­ lism. Hence, how may redox regulation of chloroplast mRNAs translation be mediated through specific signaling pathways?

4. The Coupling of Regulatory Vicinal Dithiol Sites and Thioredoxins The binding of the regulatory protein complex to the leader sequence of psbA mRNA is under redox control and is thought to be required for stimu­ lating the translation of the mRNA.1213-32,33,48 It was found that oxidation inactivates and reduction activates the RNA-binding activity of the regu­ latory protein complex. 13 Interestingly, the RNA-binding of the oxidized regulatory protein complex could be restored only by dithiol reductants, thioredoxin and dithiothreitol, and not by monothiol reductants, such as ^?-mercaptoethanol.13 This high preference for reduction by dithiol reduc­ tants over monothiol reductants is reminiscent of a similar preference dis­ played by key chloroplast enzymes shown to be regulated by thioredoxin.4,6,34'44'46'47 This phenomenon is not limited to the chloroplast as similar preference of reduction by dithiols was identified in cytoplasmic enzymes, as well.22 Collectively, these data predicted that the psbA mRNA-binding protein complex contains a regulatory vicinal dithiol site (VDS) is regulated in vivo by thioredoxin rather than by glutathione, 13 and suggested a general mechanism by which proteins containing regulatory VDS perceive redox signals specifically through thioredoxin and not through monothiol reductants, such as glutathione. A vicinal dithiol site is comprised of two proximal cysteines (Fig. 3). The VDS is formed by two cysteines that are vicinal in protein sequence, or alternatively from distant cysteines that become vicinal by protein structure. In its oxidized state, the VDS forms a disulfide (Fig. 3). The vicinal configuration of the two thiols suggests a mode of action that explains the high preference of the VDS to reduction by dithiol reductants (Fig. 4).

Redox Signaling During Light-Regulated Translation in Chloroplasts 317

Fig. 3.1. A vicinal dithiol site (VDS) is comprised by two cysteines (C) which are in close proximity of each other. X designate any amino acid. II. In its oxidized form the VDS forms a disulfide. Reduction by two electrons is required to convert an oxidized VDS (II) to the reduced form (I). III. The two cysteines can be become vicinal either by sequence (II), or alternatively by protein structure (III).

J\

Monothiol reductant

PS Dithiol reductant

Fig. 4. Reduction of the disulfide to the dithiol form is a two-step reaction requir­ ing two electrons. Reduction by a monothiol reductant (A) such as glutathione produces an intermediate form of a mixed disulfide of glutathione and one of the vicinal thiols, and the second thiol reduced. Because of its near location reduction by the second vicinal thiol competes very efficiently with reduction by a second glutathione molecule, thereby driving the reaction back. In contrast, the two-step reduction (B) by a vicinal dithiol reductant, such as thioredoxin, is able to drive the reaction to completion.

318

Cellular Implications ofRedox Signalling

Reduction of the disulfide to the dithiol form is a two-step reaction requiring two electrons. Therefore, the reduction of a VDS requires two molecules of a monothiol reductant, such as glutathione, or one molecule of a dithiol reductant, such as thioredoxin or glutaredoxin. A monothiol reductant can reduce one cysteine and thus produce an unstable mixed disulfide intermediate comprised of a glutathione and one cysteine disulfide, and a second reduced vicinal thiol [Fig. 4(a)]. Because of its proximity, the second vicinal thiol competes very efficiently with the second reduction step by a second glutathione molecule, thereby favoring the back reaction and reoxidation of the VDS and resulting in an apparent resistance of the VDS to reduction by monothiols. In contrast, the two-step reduction by a vicinal dithiol reductant, such as thioredoxin, is able to effectively drive the reaction to completion (Fig. 4). Once oxidized, a regulatory VDS-containing protein may be reduced in vivo specifically by a thioredoxin and not by glutathione. Hence, this property of an oxidized regulatory VDS can perceivably confer it responsive specifically to reductive signals carried by thioredoxin, and to resist reduction by the highly reduced intracellular state of glutathione. In light-regulated translation, the high preference of the reg­ ulatory VDS to reduction by dithiols entrains the translational regulatory complex receptive to the light-signal that is transduced by thioredoxin.

5. Oxidation of the Regulatory VDS in Chloroplasts To perceive the reductive signal carried by thioredoxin the regulatory VDS-containing protein must be in a receptive oxidized state. To deter­ mine whether the regulatory VDS of the protein complex implicated as a translational regulator of the chloroplast psbA mRNA is oxidized in organello, and to test whether its redox state is affected specifically by the light-generated reductive signal carried by thioredoxin, we characterized the changes of its redox state in chloroplasts in response to light.42 First, we showed that the regulatory VDS is carried by RB60, a member of the regulatory protein complex showing high similarity to protein disulfide isomerases.42'43 Characterization of the redox state of RB60 in chloroplasts showed that within minutes after illumination the pool of RB60 under­ goes oxidation by a yet unknown mechanism. 42 The oxidation of RB60 was not shared by the majority of chloroplast proteins, indicating that it was specific and not a result of global oxidation of the intrachloroplast milieu. This was the first demonstration of specific oxidation of a regulatory protein under normal growth conditions. Thus, the redox state of the

Redox Signaling During Light-Regulated Translation in Chloroplasts 319

regulatory VDS of RB60 seems to be independent of the global redox state of the chloroplast. High light intensities stimulate translation of specific chloroplast mRNAs, such as psbA mRNA, by increasing the flow of reductive equi­ valents through the ferredoxin-thioredoxin system (Fig. 2). To test whether the redox state of RB60 is affected specifically by the light generated reductive signal carried by thioredoxin, we characterized the changes of its redox state in response to increasing light intensities in translation active-chloroplasts. We found that under higher light intensities chloro­ plasts contained higher pool of reduced RB60 and exhibited stimulated rate of translation of psbA mRNA.42 Thus, the redox state of the pool of RB60 in the chloroplast appears to be controlled by a counter-balanced action of reductive and oxidizing activities that act specifically on RB60 (Fig. 5). The oxidation of RB60 renders it receptive to the reductive signal, and the properties of the regulatory VDS of RB60 entrains it respon­ sive specifically to reduction by thioredoxin carrying a reductive signal which is proportional to light intensity (Fig. 5).

6. Intracellular Oxidation of Proteins Evidently, oxidation of regulatory proteins, as shown for RB60, is essen­ tial for intracellular redox signaling. However, is oxidation of RB60 in the chloroplast a special case? Is it observed also for other proteins in other cell compartments, and how is it mediated? Recent findings suggest that oxidation of proteins in other cell compartments, including the cytosol, is possible, and imply that oxidation of intracellular proteins is mediated by specific protein-protein interactions. For example, using genetic approach to study disulfide formation of alkaline phosphatase in Escherichia coli cytoplasm, Beckwith and his colleagues showed that in the absence of thioredoxin reductase activity a cytoplasmic alkaline phosphatase is maintained oxidized in an otherwise reductive cytosol.40 The cytosolic alkaline phosphatase remained oxidized in the presence of reduced glutathione and glutaredoxin. Interestingly, both the reduction and the formation of the disulfides of alkaline phosphatase were catalyzed in the cytosol specifically by thioredoxin. 40 In a second example, in the cyto­ plasmic folding of vaccinia virus proteins, an ERVl/ALR-like protein oxidizes unique glutaredoxins.27-39 Thus, stable disulfides may form in the cytoplasm, formation of the disulfide is probably mediated through protein-protein transfer of oxidizing equivalents.

320

Cellular Implications ofRedox Signalling

Fig. 5. Light activates specific oxidation of RB60 by a yet unknown factor. Oxidation of RB60 inactivates the translational regulator complex and confers it receptive to the reductive signal. This form of the translational regulator enters a cycle of activation by reduction, mediated by the ferredoxin-thioredoxin system in proportion to photosynthetic light intensity, and inactivation by oxidation by the RB60-specific activity. Higher light intensity increases the portion of reduced translational activator pool, and thereby translation of psbA mRNA. Whereas under lower light intensity, oxidization of RB60 increases the portion of inactive pool of the translational regulator complex and diminishes translation.

Interestingly, t h e r e g u l a t i o n of activity of t h e e n z y m e alkaline p h o s p h a t a s e in E. coli cytosol 40 is a n a l o g o u s to the p h o t o - r e g u l a t i o n of chloroplast e n z y m e s 5 (Fig. 2). In b o t h p l a n t chloroplasts a n d in E. coli, the activity of certain proteins containing disulfides is specifically controlled b y thioredoxin according to the extent of its r e d u c t i o n b y thioredoxin reductase. In E. coli cytosol, the reduction of thioredoxin is b y N A D P H d e p e n d e n t thioredoxin reductase. In chloroplasts, thioredoxin is reduced b y thioredoxin reductase that accepts electrons from ferredoxin. A s ferredoxin

Redox Signaling During Light-Regulated Translation in Chloroplasts 321

is the electron acceptor of photosystem I, it confers light-responsiveness to the activity of the thioredoxin-regulated enzymes.

7. Concluding Remarks In conclusion, data presented here show that oxidation of specific proteins is possible in the reductive intracellular milieu and indicate that the trans­ fer of oxidizing equivalents is probably mediated through specific proteinprotein interactions. In addition, once oxidized specialized proteins, such as VDS-containing proteins, can resist reduction by the global milieu while being reduced by specific proteins, such as thioredoxins. These attributes are essential for redox signaling regulating specific gene expression, as shown here for light-regulated translation of chloroplast mRNAs. Further studies are required to determine how widespread is this type of redox regulation, and to identify the yet unknown key factors such as those that may drive the oxidation of the regulatory proteins in vivo.

Acknowledgments Avihai Danon holds The Judith and Martin Freedman Career Develop­ mental Chair and is supported by grants from the Israel Science Foundation, the Minerva Foundation, BARD, and from Levy R. & R. Foundation.

References 1. 2.

3. 4.

Abate C, Patel L, Rauscher F, Curran T. 1990. Redox regulation of fas and jun DNA-binding activity in vitro. Science 249: 1157-1161 Aono S, Ohkubo K, Matsuo T, Nakajima H, Ashton A, Johnson M. 1998. Redox-controlled ligand exchange of the heme in the COsensing transcriptional activator CooA. /. Biol. Chem. 273: 25757-25764 Barber J, Anderson B. 1992. Too much of a good thing: Light can be bad for photosynthesis. Trends Biochem. Sci. 17: 61-66 Breazeale V, Buchanan B, Wolosiuk R. 1978. Chloroplast sedoheptulose 1,7-bisphosphatase: Evidence for regulation by the ferredoxin/ thioredoxin system. Z. Naturfarsch 33c: 521-528

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5. Buchanan BB. 1991. Regulation of C 0 2 assimilation in oxygenic photosynthesis: The ferredoxin/thioredoxin system. Arch. Biochem. Biophys. 288: 1-9 6. Buchanan BB, Schurmann P, Kalberer PP. 1971. Ferredoxin-activated fructose diphosphatase of spinach chloroplasts. Resolution of the sys­ tem, properties of the alkaline fructose diphosphatase component, and physiological significance of the ferredoxin-linked activation. /. Biol. Chem. 246: 5952-5959 7. Cabrillac D, Cock JM, Dumas C, Gaude T. 2001. The S-locus receptor kinase is inhibited by thioredoxins and activated by pollen coat pro­ teins. Nature 410: 220-223 8. Carmel-Harel O, Storz G. 2000. Roles of the glutathione- and thioredoxin-dependent reduction systems in the Escherichia coli and Saccharomyces cerevisiae responses to oxidative stress. Ann. Rev. Microbiol. 54: 439-461 9. Coleman ST, Epping EA, Steggerda SM, Moye-Rowley WS. 1999. Yaplp activates gene transcription in an oxidant-specific fashion. Mol. Cell. Biol. 19: 8302-8313 10. Dai S, Schwendtmayer C, Schurmann P, Ramaswamy S, Eklund H. 2000. Redox signaling in chloroplasts: Cleavage of disulfides by an iron-sulfur cluster. Science 287: 655-658 11. Danon A. 1997. Translational regulation in the chloroplast. Plant Physiol. 115: 1293-1298 12. Danon A, Mayfield SP. 1991. Light-regulated translational activators: Identification of chloroplast gene specific mRNA binding proteins. EMBO. }. 10: 3993^001 13. Danon A, Mayfield SP. 1994. Light-regulated translation of chloroplast messenger RNAs through redox potential. Science 266:1717-1719 14. Fromm H, Devic M, Fluhr R, Edelman M. 1985. Control of psbA gene expression: In mature Spirodela chloroplasts light regulation of 32-kd protein synthesis is independent of transcript level. EMBO. J. 4: 291-295 15. Georgellis D, Kwon O, Lin EC. 2001. Quinones as the redox signal for the arc two-component system of bacteria. Science 292: 2314-2316 16. Gilbert HF. 1990. Molecular and cellular aspects of thiol/disulfide exchange. Adv. Enzymol. Rel. Areas Mol. Biol. 63: 69-172 17. Halliwell B. 1999. Antioxidant defence mechanisms: From the begin­ ning to the end (of the beginning). Free Radic. Res. 31: 261-272 18. Hentze MW, Kuhn LC. 1996. Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress. Proc. Natl. Acad. Sci. USA 93: 8175-8182

Redox Signaling During Light-Regulated Translation in Chloroplasts 323

19. Hidalgo E, Ding H, Demple B. 1997. Redox signal transduction via iron-sulfur clusters in the SoxR transcription activator. Trends Biochem. Sci. 22: 207-210 20. Hill S, Austin S, Eydmann T, Jones T, Dixon R. 1996. Azotobacter vinelandii NIFL is a flavoprotein that modulates transcriptional acti­ vation of nitrogen-fixation genes via a redox-sensitive switch. Proc. Natl. Acad. Sci. USA 93: 2143-2148 21. Hwang C, Sinskey AJ, Lodish HF. 1992. Oxidized redox state of glutathione in the endoplasmic reticulum. Science 257: 1496-1502 22. Kiss F, Wu MX, Wong JH, Balogh A, Buchanan BB. 1991. Redox active sulfhydryls are required for fructose 2,6-bisphosphate activation of plant pyrophosphate fructose-6-phosphate 1-phosphotransferase. Arch. Biochem. Biophys. 287: 337-340 23. Klatt P, Lamas S. 2000. Regulation of protein function by S-glutathiolation in response to oxidative and nitrosative stress. Eur. }. Biochem. 267: 4928-4944 24. Klausner R, Rouault T, Harford J. 1993. Regulating the fate of mRNA: The control of cellular iron metabolism. Cell 72: 19-28 25. Klein RR, Mason HS, Mullet JE. 1988. Light-regulated translation of chloroplast proteins: I. Transcripts of psaA-psaB, psbA and rbcL are associated with polysomes in dark-grown and illuminated barley seedlings. /. Cell Biol. 106: 289-301 26. Krupinska K, Apel K. 1989. Light-induced transformation of etioplasts to chloroplasts of barley without transcriptional control of plastid gene expression. Mol. Gen. Genet. 219: 467-473 27. Locker JK, Griffiths G. 1999. An unconventional role for cytoplasmic disulfide bonds in vaccinia virus proteins. /. Cell. Biol. 144: 267-279 28. Malnoe P, Mayfield SP, Rochaix J-D. 1988. Comparative analysis of the biogenesis of photosystem II in the wild-type and Y-l mutant of Chlamydomonas reinhardtii. J. Cell. Biol. 106: 609-616 29. Markus M, Benezra R. 1999. Two isoforms of protein disulfide isomerase alter the dimerization status of E2A proteins by a redox mechanism. /. Biol. Chem. 274: 1040-1049, in process citation 30. Matthews JR, Wakasugi N, Virelizier JL, Yodoi J, Hay RT. 1992. Thioredoxin regulates the DNA binding activity of NF-kappaB by reduction of a disulphide bond involving cysteine 62. Nucleic Acids Res. 20: 3821-3830 31. Mattoo AK, Pick U, Hoffman-Falk H, Edelman M. 1981. The rapidly metabolized 32,000-dalton polypeptide of the chloroplast is the "proteinaceous Shield" regulating photosystem II electron transport

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and mediating diuron herbicide sensitivity. Proc. Natl. Acad. Sci. USA 78:1572-1576 Mayfield SP, Cohen A, Danon A, Yohn CB. 1994. Translation of the psbA mRNA of Chlamydomonas reinhardtii requires a structured RNA element contained within the 5' untranslated region. /. Cell. Biol. 127: 1537-1545 Mayfield SP, Yohn CB, Cohen A, Danon A. 1995. Regulation of chloroplast gene expression. Ann. Rev. Plant Physiol. Plant Mol. Biol. 46:147-166 McKinney D, Buchanan B, Wolosiuk R. 1978. Activation of chloro­ plast ATPase by reduced thioredoxin. Phytochemistry 17: 794-795 Noctor G, Foyer CH. 1998. Ascorbate and glutathione: Keeping active oxygen under control. Ann. Rev. Plant Physiol. Plant Mol. Biol. 49: 249-279 Pellicena-Palle A, Stitzinger SM, Salz HK. 1997. The function of the Drosophila thioredoxin homologue encoded by the deadhead gene is redox-dependent and blocks the initiation of development but not DNA synthesis. Mech. Dev. 62: 61-65 Rebbapragada A, Johnson MS, Harding GP, Zuccarelli AJ, Fletcher HM, et al. 1997. The Aer protein and the serine chemoreceptor Tsr inde­ pendently sense intracellular energy levels and transduce oxygen, redox, and energy signals for Escherichia coli behavior. Proc. Natl. Acad. Sci. USA 94: 10541-10546 Saitoh M, Nishitoh H, Fujii M, Takeda K, Tobiume K, et al. 1998. Mammalian thioredoxin is a direct inhibitor of apoptosis signalregulating kinase (ASK-1. EMBO }. 17: 2596-2606 Senkevich TG, White CL, Koonin EV, Moss B. 2000. A viral member of the ERV1/ALR protein family participates in a cytoplasmic path­ way of disulfide bond formation. Proc. Natl. Acad. Sci. USA 97: 12068-12073 Stewart EJ, Aslund F, Beckwith J. 1998. Disulfide bond formation in the Escherichia coli cytoplasm: An in vivo role reversal for the thioredoxins. EMBO. }. 17: 5543-5550 Trebitsh T, Danon A. 2001. Translation of chloroplast psbA mRNA is regulated by signals initiated by both photosystems I and II. Proc. Natl. Acad. Sci. USA 98: 12289-12295 Trebitsh T, Levitan A, Sofer A, Danon A. 2000. Translation of chloro­ plast psbA mRNA is modulated in the light by counteracting oxidi­ zing and reducing activities. Mol. Cell. Biol. 20: 1116-1123

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43. Trebitsh T, Meiri E, Ostersetzer O, Adam Z, Danon A. 2001. The pro­ tein disulfide isomerase-like RB60 is partitioned between stroma and thylakoids in Chlamydomonas reinhardtii chloroplasts. /. Biol. Chem. 276: 4564-4569 44. Wendt UK, Wenderoth I, Tegeler A, Von Schaewen A. 2000. Molecular characterization of a novel glucose-6-phosphate dehydrogenase from potato {Solarium tuberosum L.). Plant/. 23: 723-733 45. Wettern AK, Ohad I. 1984. Light-induced turnover of thylakoids polypeptides in Chlamydomonas reinhardtii. Isr. /. Bot. 33: 253-263 46. Wolosiuk R, Buchanan B. 1978. Activation of chloroplast NADPlinked glyceraldehyde 3-phosphate dehydrogenase by the ferredoxin/ thioredoxin system. Plant Physiol. 61: 669-671 47. Wolosiuk RA, Buchanan BB. 1978. Regulation of chloroplast phosphoribulokinase by the ferredoxin/thioredoxin system. Arch. Biochem. Biophys. 189: 97-101 48. Yohn CB, Cohen A, Danon A, Mayfield SP. 1996. Altered mRNA binding activity and decreased translational initiation in a nuclear mutant lacking translation of the chloroplast psbA mRNA. Mol. Cell Biol. 16: 3560-3566 49. Yohn CB, Cohen A, Rosch C, Kuchka MR, Mayfield SP. 1998. Translation of the chloroplast psbA mRNA requires the nuclearencoded Poly(A)-binding protein, RB47. /. Cell. Biol. 142: 435-442 50. Zheng M, Aslund F, Storz G. 1998. Activation of the OxyR transcrip­ tion factor by reversible disulfide bond formation. Science 279: 1718-1721 51. Zheng M, Storz G. 2000. Redox sensing by prokaryotic transcription factors. Biochem. Pharmacol. 59: 1-6

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Chapter 15 Regulation of mRNA Translation and Stability in Iron Metabolism: Is there a Redox Switch?

Lukas C. Kuhn Chemin des Boveresses 155, Swiss Institute for Experimental Cancer Research (ISREC), CH-1066 Epalinges, Switzerland Lukas. [email protected].

1. Summary Iron is both essential and potentially toxic for living organisms. Cellular iron homeostasis needs, therefore, to be maintained within an optimal concentration range by coordinate regulation of iron absorption, storage and utilization. In animals, this is achieved by post-transcriptional regu­ latory mechanisms that involve mRNA-protein interactions. Particular RNA hairpin structures, so called iron responsive elements, are present in the untranslated regions of mRNAs coding mainly for proteins that play a central role in cellular iron-metabolism. Iron regulatory proteins bind to these structures and thereby influence the rate of mRNA translation or degradation. These iron regulatory proteins are themselves regulated by iron. This chapter will discuss the activity of iron regulatory proteins, their targets and to which extent these control mechanisms of iron homeostasis are subject to effects of redox.

2. Iron Homeostasis Due to its capacity to donate or accept electrons, iron was selected since the earliest times of natural evolution for redox reactions in living organisms. Thus, iron in heme, in iron-sulfur clusters or directly bound to proteins is a constituent of many essential proteins with functions in oxygen transport, energy metabolism, electron transfer, deoxynucleotide synthesis or detoxi­ fication. Its presence in the active sites of numerous enzymes implies that cells must have a sufficient pool of "free" iron available for protein 327

328

Cellular Implications ofRedox Signalling

biosynthesis. While non-proliferating cells may reutilize the iron liberated from local protein degradation, cells that proliferate or have particular needs for iron, like those of the erythroid lineage, accumulate iron from their environment. For this, vertebrate cells bind iron-loaded serum transferrin to their cell surface transferrin receptor, endocytose the complexes and release iron in the acidic endosomal compartment (for reviews, see Refs. 1^1). Iron gets then reduced and transported to the cytoplasm across the endosomal membrane by the recently identified proton-coupled metal transporter DMT1 (also known as nramp2 or DCT1).5""7 Once transported, iron enters a cytoplasmic chelatable iron pool prior to its utilization or stor­ age.8,9 The exact nature of this pool remains unknown, but iron is thought to interact with counter-ions such as citrate, nucleotides or amino acids. Unless incorporated into proteins, divalent cytoplasmic iron on transit is potentially toxic as it catalyzes in the presence of H 2 0 2 the formation of hydroxyl radicals that damage DNA, proteins and membrane lipids. In order to diminish excess cytoplasmic iron, cells synthesize H and L chain ferritin subunits that assemble to hollow protein shells of 24 subunits into which up to 3500 ions of iron get deposited (reviewed by Harrison.10) Ferritin can, therefore, be considered as a detoxifying as well as a storage site for iron. Thus, a delicate balance between the uptake, storage and utilization of iron maintains the "free" iron pool at an appropriate steady state level. Interestingly, as I will discuss in this chapter, the principle proteins that influ­ ence directly the transit iron pool are themselves regulated in their expression by cytoplasmic iron. An increase in intracellular free iron results in the induc­ tion ferritin mRNA translation and reduction of transferrin receptor mRNA stability. In contrast, deprivation of iron has opposite effects. An unbalance of iron in the entire organism can profoundly affect cellu­ lar iron availability and provoke disease. Iron deprivation often due to poor absorption of nutritional iron, important bleeding or pregnancy can induce anemia. Excessive iron accumulation due to whole blood transfusion or the genetic predisposition known as idiopathic hemochromatosis may lead to iron overload if untreated. This can induce irreversible tissue damage, pre­ sumably because the binding capacity of transferrin and storage into ferritin are exceeded and no longer capable of neutralizing the toxicity of free iron.

3. Iron Regulatory Proteins and Feedback Regulation of Iron Within the limits of normal physiological conditions, the homeostasis of cytoplasmic iron is sensed and regulated by the iron regulatory proteins-1

Regulation of mRNA Translation and Stability in Iron Metabolism

G A

G U

CG„ C GC AU AU GC GC UA AU UA UA AU (a)

329

A

U

CG^ AU c AU CG UG UA C GC U CG CG UA UA

Loop

Proximal helix

Bulge

Distal helix

(b)

Fig. 1. Typical examples of iron responsive elements of human transferrin receptor (a) and human H ferritin (b) mRNA. They consist of a paired RNA stemloop structure that extends usually over 30 to 35 bases. The three-dimensional structure of the IRE was predicted and since solved by NMR spectroscopy.13"15 The loop consists of a highly conserved 6-base sequence CAGUGN (where N is all but G) at the tip of a proximal helix of 5 paired bases. There exist only few exceptions to this loop sequence in natural mRNAs. The loop bases 1 and 5 interact.13-17 The helix sequence is also conserved in evolution, but varies in IREs of different genes. Its length is critical for optimal IRP binding. In most IREs, the proximal stem is flanked 5-by a small asymmetrical bulge with an unpaired C (a). However, all fer­ ritin IREs (b) contain an alternative bulge with the 5' sequence UGC and an oppo­ site C that pairs with the G.17 A distal helix region is required for sufficient stability of the stem-loop structure. The bulge is important to give the IRE a slight bend. a n d -2 (IRP-1 a n d IRP-2). These p r o t e i n s act on the post-transcriptional fate of m R N A s b y interacting directly w i t h iron r e s p o n s i v e elements (IREs; Fig.l) in u n t r a n s l a t e d regions (UTRs) in certain m R N A s . D e p e n d ­ ing on the position of these elements, 5' or 3 ' of the c o d i n g region, IRP b i n d i n g blocks either m R N A translation efficiency or m R N A d e g r a d a t i o n (reviewed in Refs 11 a n d 12). IRP-1 a n d IRP-2 are soluble cytoplasmic proteins that are themselves post-translationally regulated b y iron levels: they exhibit RNA-binding activity only u n d e r conditions of iron deprivation, b u t are mostly inactive w h e n iron-supply is sufficient. In the case of IRP-1, this change is posttranslational a n d does not affect the protein level. The isolation and subse­ quent sequencing of IRP-1 c D N A s b r o u g h t an explanation for these properties. 1 8 1 9 IRP-1 is ubiquitously expressed w i t h 889 a m i n o acids a n d a

330

Cellular Implications of Redox Signalling

Fig. 2. Regulation of IRP-1 and IRP-2 conformations and binding of the IRE. A hinge region between domains 1-3 and 4 gives IRPs flexibility. While IRP-1 is reg­ ulated between an "open" and a "closed" (aconitase) conformation by iron or NO, IRP-2 is regulated through its stability. IRP-1 is in addition rapidly activated by extracellular H 2 0 2 . The mechanism of activation remains unknown but may involve a phosphorylation event of either IRP-1 or a cofactor and the disassembly of the [4Fe-4S]-cluster. IREs interact with several regions on the IRP-1 surface (darker areas), which are probably to a large extent conserved in IRP-2. These regions determine the affinity and specificity of the IRE-IRP interaction. They are only accessible in the "open" conformation. molecular mass of 98 kDa.19,20 Strikingly, IRP-1 shares sequence homology with a family of proteins that comprises aconitases and isopropylmalate isomerases.21~23 Based on the conservation of all active site residues, IRP-1 was predicted to resemble mitochondria! aconitase,21 an extensively stud­ ied enzyme of the citric acid cycle that inserts a [4Fe-4S]-cluster as part of its active site.24"26 This protein has three ammo-terminal domains linked by a flexible hinge-region to a fourth larger carboxy-terminal domain (Fig. 2). The arrangement is such that it creates an extensive cleft that gives access for citrate or isocitrate to the iron-sulfur cluster. The [4Fe-4S]-cluster is attached to three cysteine residues. Consequently, IRP-1 was also found to

Regulation ofmRNA Translation and Stability in Iron Metabolism

331

insert a [4Fe-4S]-cluster under normal cellular iron supply.27""29 This holo-protein form of IRP-1 is nothing else than a cytoplasmic aconitase.30 It converts citrate to isocitrate, with aconitate as an intermediate, at a similar Km and Vmax as the mitochondrial enzyme. However, the enzymatic confor­ mation of IRP-1 precludes its binding to mRNA.28'29 For RNA-binding, IRP1 has to remain in the apo-protein form without the iron-sulfur cluster, and this is mainly observed under the condition of cellular iron deprivation.29-31 This conclusion was also directly confirmed by site-directed mutagenesis of the 3 cysteines that bind the iron-sulfur cluster. Changing them to serines produces constitutively active IRP-1.32"34 We will see later in this chapter that certain conditions other than iron-deprivation also prevent the ironsulfur cluster formation or promote its disassembly. IRP-2 was subsequently identified, isolated and sequenced from cDNA clones.35"37 The protein is less abundant than IRP-1 in most cells, and its tis­ sue distribution appears to be more restricted with strongest expression in intestine and brain.35-38 IRP-2 shows extensive homology with IRP-1.18-38-39 However, IRP-2 was not observed to insert an iron-sulfur cluster and to convert to an aconitase. Instead, the IRE-binding activity of IRP-2 increases in iron deprived cells due to a change in protein stability.36 The protein is rapidly degraded by the proteasome when iron is plentiful in cells .4tW3 This requires a unique 73 amino acid domain. 40 Active IRPs bind with high affinity to mRNA hairpin structures known as iron-responsive elements (IREs)44,45 (Fig. 1). The first and best-studied cases of IREs are those of ferritin H and L chain mRNA.46,47 Their IRE is located in the 5' UTR about 45 nucleotides downstream of mRNA cap struc­ ture and about 150 nucleotides from the initiation AUG codon. The IRE is both sufficient and necessary to make ferritin mRNA translation irondependent. 48 Ferritin H and L chain mRNAs are only fully translated when IRPs are inactivated. However, in iron-deprived cells, a large fraction of fer­ ritin mRNA remains untranslated because IRE-bound IRP prevents bind­ ing of ribosomal subunits to the mRNA. 49 This effect is more efficient when the IRE is at a short distance from the cap structure, but fades when the IRE is placed further away.50 IREs located more than 65 nucleotides away in the 5' UTR exert no effect on translation initiation, but may attenuate translation elongation in a position remote of the cap structure.51 Subsequently, 5 IREs were discovered in the 3' UTR of transferrin receptor mRNA. Each of these IREs can bind one copy of active IRP. As a result of this interaction, transferrin receptor mRNA becomes 10- to 20-fold more stable in iron deprived cells.52"55 This stabilization is rapidly reversed by culture conditions in high iron. Destabilized transferrin receptor mRNA decays with a half-life of about 30 to 40 minutes. This destabilization

332 Cellular Implications ofRedox Signalling

Fig. 3. Post-transcriptional regulation of ferritin and transferrin receptor expression and the feedback control of the cytoplasmic "free" iron pool. As explained in the text, IRP-1 and IRP-2, whose RNA-binding activity is iron-dependent, correct any deviation from the optimal concentration of cytoplasmic iron. According to this con­ cept, cytoplasmic iron controls its own steady state. It tends to establish itself at the concentration needed to assemble an iron-sulfur cluster for cytoplasmic aconitase. depends on 3' UTR sequences adjacent to the IREs. Deletion of these sequences produces a stable mRNA. According to the prevalent hypothe­ sis, bound IRP protects the mRNA against attack by a hypothetical ribonuclease. The decay appears to be initiated by endoribonucleolytic cleavage at the instability elements, since transferrin receptor mRNA, unlike other short-lived mRNAs, is not deadenylated prior to its degradation. 56 How­ ever, this pathway and enzymes involved awaits further investigation. To put these two major mechanisms into perspective, one may conclude that ferritins and transferrin receptor are regulated in opposite ways by IRP (Fig. 3). Yet, the regulation of ferritins and transferrin receptor tends to com­ pensate coordinately any deviation from optimal cellular iron levels. Thus, in iron deprived cells, translation inhibition of ferritin prevents iron storage and this increases the free iron pool. Similarly, increased transferrin recep­ tor expression and, hence, cellular iron uptake in deprived cells contributes

Regulation of mRNA Translation and Stability in Iron Metabolism

333

to replenish cells with iron. Ferritin and transferrin receptor regulations have, therefore, cumulative effects that make sense in terms of cell physiol­ ogy. Once iron levels exceed a certain threshold concentration, IRPs are inac­ tivated and the regulation gets inverted such that ferritins increase and transferrin receptors diminish. Both effects lower the free iron pool. Free iron levels should thus reach a steady equilibrium. Since iron is essentially sensed by IRPs it would seem that the optimal cellular iron level reached at equilibrium is situated close to the concentration required for the inactivation of IRPs. For IRP-1, this should represent the free cytoplasmic iron con­ centration needed to form the iron-sulfur cluster. Based on direct measurements of the chelatable iron-pool, this concentration can be esti­ mated as about 1 (iM.9'57,58 In normal growing cell cultures about 85% of all IRP-1 molecules have a Fe-S cluster and function as an aconitase. However, we still know little about how iron-sulfur clusters of cytoplasmic proteins are formed. The cytoplasmic aconitase cluster is easily reconstituted in vitro suggesting a spontaneous process also in cells, but recent data in yeast indi­ cate the involvement of mitochondrial proteins.59 It is also not entirely clear whether IRP-2 is inactivated by precisely the same concentration of iron as IRP-1, even if IRP-1 and IRP-2 seem to be regulated in parallel. Since iron concentrations determine whether or not IRPs are active, and IRPs influence iron levels indirectly by the regulatory mechanisms described, one may also conclude that iron, through IRP, is actually con­ trolling its own cytoplasmic concentration. This opens the perspective that additional factors interfering with iron-sulfur formation like the sulfide pool, the redox state of free iron or molecules interacting with iron may also influence IRP activity and the iron steady state level. As dis­ cussed below, it was actually found that NO, oxidative compounds and oxygen-pressure modify the RNA-binding activity of IRPs.

4. Other Targets of Iron Regulatory Proteins The network of mRNAs regulated by IRPs is broader than initially sus­ pected. Several other mRNAs bear an IRE in their 5'UTR and are translationally regulated. One of them is the erythroid amino-levulinate synthase (eALAS) an enzyme in the biosynthesis of proto-porphyrin IX, the precursor of heme required in hemoglobin synthesis.60-62 Hemoglobin incorporates about 70% of all body iron in mammals. Yet under conditions of anemia, iron availability is clearly limiting to its biosynthesis. Under such conditions IRPs are activated and reduce eALAS translation.63-64 It is thought that this

334

Cellular Implications ofRedox Signalling

will prevent proto-porphyrin IX synthesis when there is insufficient iron for heme completion. This may represent a way to limit porphyria. Yet, physio­ logical investigations to prove this concept are still missing. Two other mRNAs contain an IRE in their 5' UTR and show translational control: the mitochondrial aconitase of vertebrates62,65,66 and succi­ nate dehydrogenase subunit B of insects.66,67 Both enzymes are part of the mitochondrial citric acid cycle and involved in energy metabolism. Both are translated as precursors in the cytoplasm and then imported to the mitochondrial matrix and both have an iron-sulfur cluster. While translational inhibition in iron-deprived cells was experimentally confirmed, it remains unknown why this regulatory loop was selected in evolution. Several hypotheses were put forward, but need to be further investigated: (a) Citrate could represent an important component to keep free iron as a soluble complex in the cytoplasm. It was also postulated to function as a carrier for its import into mitochondria. Thus, when iron gets scarce it would seem favorable to lower mitochondrial aconitase in order to pre­ serve citrate. This idea is attractive in view of recent evidence that all ironsulfur clusters, including those for cytoplasmic proteins, might be assembled in mitochondria. 59 (b) Taking into consideration that insect cells control succinate dehydrogenase rather than aconitase, one might also invoke a direct connection to energy metabolism. Scarcity of iron would not permit cell growth and duplication. Thus, diminished energy supply under low iron conditions might provide a signal for the cell to avoid cell proliferation, (c) Alternatively, it may be deleterious to pro­ duce mitochondrial precursors for enzyme protein complexes, when these precursors do not mature due to lack of iron. If this hypothesis were correct one would have to explain why other iron-sulfur containing enzymes are not translationally controlled by IRPs. Several candidate sequences for IRE-like structures were put forward. Transferrin mRNA comprises a sequence that resembles vaguely an IRE and forms an RNA-protein complex resembling that of IRP-1.68,69 However, the proposed RNA-IRP-1 complex could not be confirmed with recombinant IRP-1.16 Glycolate oxidase mRNA was picked up in an enriched cDNA library of mRNA that bound to a recombinant IRP-1 column followed by a screen for the IRE-IRP-1 interaction.70 This mRNA comprised a single puta­ tive IRE in the 3' UTR. However, this IRE-like structure had a mismatch in the stem close to the loop, and subsequent analysis showed that it was unstable and non-functional at physiological temperature. 70 Recently, two iron transporters have been isolated and cloned from intestinal tissue and are thought to play an important role in iron absorption

Regulation of mRNA Translation and Stability in Iron Metabolism

335

Fig. 4. Possible role of IRPs in regulating intestinal iron absorption. The recently cloned iron transporters DMTl and IREG1 are strongly induced by iron depriva­ tion and contain IRE-like elements in their mRNA. However, direct evidence for post-transcriptional regulation is still missing. from nutrition. These are the D M T l (also n a m e d n r a m p 2 or DCT1), 5,6 a n d IREG1 (also k n o w n as MTP1 or ferroportin-1). 71 " 73 D M T l besides its

336

Cellular Implications ofRedox Signalling

expression in endosomes was mainly localized to the brush-border apical membrane and is thought to mediate iron absorption, whereas IREGl is located at the basolateral membrane and probable responsible for iron export from the epithelium (Fig. 4). Since iron absorption is itself a highly regulated event that contributes to maintain the overall balance of iron in the organism, it is of interest that both DMT1 and IREGl mRNAs are induced in iron-deprived mice and in idiopathic hemochromatosis, the inherited disease of iron overload.6'71,74 This induction might be connected to activation of IRPs (see hypotheses in Refs. 75 and 76). An IRE-like structure was identified in the 3' UTR of the DMT1 mRNA sequence6 suggesting that this transporter might be regulated by a posttranscriptional stabilization of its mRNA similar to transferrin receptor mRNA. The idea received support by studies showing that only the mRNA with this IRE-like element was increased upon iron deprivation, whereas an alternatively spliced mRNA without the IRE was not.75 Direct assays of the IRE-IRP-1 complex formation confirmed an RNA-protein interaction, which was somewhat weaker for the DMT1 IRE than the ferritin IRE.77 Yet these authors did not detect evidence for a regulatory role of this IRE in mouse fibroblast and erythroleukemia cell lines. In conclusion DMT1 mRNA induction seems to be regulated by a different mechanism. In the case of IREGl mRNA a perfect IRE is present in the 5' UTR.71-72 This IRE showed a good affinity for IRP-1 in binding assays. Yet, the ele­ ment is somewhat too remote of the cap structure to inhibit translation ini­ tiation, but might function as a translational attenuating element. 51 Attenuation of translation would, however, seem opposite to what might be expected in an iron-deprived animal where IREGl mRNA is induced and iron transport should be increased rather than diminished. It has not been excluded that this IRE may influence mRNA stability. Alternatively, IREGl mRNA might be induced by a transcriptional mechanism.

5. Regulation of IRE Binding Activity of IRP-1 by Iron The mutually exclusive function of IRE-binding and aconitase activity in IRP-1 indicates that the RNA-binding determinants in the protein become hidden by the insertion of the iron-sulfur cluster. Since the mitochondrial aconitase structure25 can perfectly serve as a model for the cytoplasmic aconitase/IRP-1 structure, folding predictions for IRP-1 indicate a cleft between domains 1-3 and 4 with a flexible hinge region connecting the two parts. The active site with the 4Fe-4S-cluster is buried at the center of the inner surface of this cleft in domain 2. Three cysteines at

Regulation ofmRNA Translation and Stability in Iron Metabolism 337 A. c-Aconitase form

437Cys-SVv/|

/No—\

B. inactive form \ / ^ C y s -S«

13 Cys-S

C, IRE-binding form

\

s

J\ 1/

>

/

437 C V S - SH ?

8-

■/l

t^^O5

1/

S-Cys 506 Thr y \

S-Cys 6 0 6 Thr V \

D. IRP-1: IRE-binding form

E. inactive forms (two)

dlamlde

<

>

10mM 2-me, red. ferredoxin HS-Cys 606

Thry

\

S-Cys5M Thr y \

Fig. 5. Different molecular forms of the cytoplasmic aconitase/IRP-1, its iron-sulfur cluster and factors influencing conversion. IRP-1 is synthesized as an apo-protein (D) and matures to the holo-protein with a [4Fe-4S]-cluster, the aconitase form (A) via intermediates (B). Of these only the IRP-1 form has IRE-binding properties. Cys437 needs to be reduced for the binding, which is the case in the environment of a normal cellular cytoplasm. However, upon incubation of cells with the oxidizing diamide, IRP-1 gets reversibly inactivated (E), due to intramolecular disulfide bridges between Cys437 and Cys503 or Cys506. A disulfide bridge between Cys503 and Cys506 has no inactivating effect.33 Disassembly of the [4Fe-4S]- cluster is slow, but can be induced in cells by iron chelation or NO. Formation and disassembly of the cluster can also be obtained in vitro. Furthermore, all forms with an iron-sulfur cluster are converted in vitro to an IRE-binding form (C) by extreme reducing conditions.

338

Cellular Implications ofRedox Signalling

positions 437,503 and 506 bind three iron atoms of the cluster [Fig. 5(A.)] The substrate interacts both with the fourth iron and with several amino acids of the active site, including Arg580 and Arg644 in domain 4 (for details, see Ref. 78). Therefore, the aconitase form of IRP-1 has a closed conformation when substrate is bound. Conversely it was postulated that the apo-protein IRP-1 is unable to bind substrate and, thus, may have a more open conformation in which domain 4 moves apart to open the cleft such that IRE binding sites get exposed. The predictions are twofold: (a) in order to expose such sites, the substrate and possibly part of the cluster must be removed and (b) IRE inter­ action sites are possibly located on the inner surface of the cleft. Studies to determine IRP activity rely on gel-retardation assays in which RNA is transcribed and radiolabeled in vitro and then incubated with a cytoplasmic protein extract.44 The IRE-IRP complexes are separated in non-denaturing polyacrylamide gels. Radioactivity observed at the position of the RNA-protein complex is proportional to the active IRP. In this way IRPs were first discovered in rat liver,44 and then mainly studied in cell cultures. It was observed that, independently of cell culture condi­ tions or iron loading, all IRP-1 molecules including those which had incor­ porated the iron-sulfur cluster could be fully activated by the in vitro addition of 2% 2-mercaptoethanol to the extract just prior to the assay.79 This incited the authors to advertise their discovery as an indication for redox regulation of IRP-1. The in vitro activation within seconds seemed to mimic the in vivo activation of IRP-1 by iron deprivation, where the maxi­ mal activity was quantitatively similar after culturing cells for 15 hours with an iron chelator. Consistent with this idea, IRP-1 of iron-deprived cells [Fig. 5(D)] was already activated prior to cell fractionation, since it did not respond further to in vitro treatment with 2-mercaptoethanol. However, the exposure to a 300-mM solution of a strong reducing agent in vitro is of course incomparable to any physiologically relevant redox component in the cell. The precise molecular modification that occurs on IRP-1 under these conditions is still not fully elucidated. Exposure of the aconitase form to 2-mercaptoethanol does not irreversibly destroy the ironsulfur cluster. RNA binding is only activated for the time of its exposure to the reducing agent. When 2-mercaptoethanol is removed by a gel-filtration column, the protein regains most of its enzyme activity, suggesting that the cluster remained associated with the protein. 33 In view of results pre­ sented below, the strongly reducing environment may modify the aconi­ tase protein conformation by disrupting the substrate-cluster interaction and maybe compete in the most accessible cysteine-iron-sulfur cluster interaction, for example, at Cys437 [Fig. 5(C)].

Regulation ofmRNA Translation and Stability in Iron Metabolism

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The similarity in the extent of activation by iron-deprivation and 2-mercaptoethanol has given rise to the most popular control used in IRE-IRP-binding assays. In order to know what fraction of IRP is active in an extract, RNA-protein complexes are formed once in the absence of 2-mercaptoethanol and once in its presence. The first condition mea­ sures spontaneously active IRP, while the second indicates the total amount of IRP present. Using such assays it was shown for cells in cul­ ture that the total amount of IRP-1 does not significantly change in ironrich and iron-deprived medium. 54,79 Yet, the spontaneously active fraction is entirely different: in iron-rich medium only 10 to 15% of total IRP-1 is active, whereas after culture in medium with the iron chelator desferrioxamine up to 100% of IRP-1 becomes active. Full activation of IRP-1 after addition of the iron chelator requires about 15 hours. When the chelator is removed and iron salt or hemin added back to the medium, the IRP-1 activity returns to the original value within less than 1 hour. The slow response to chelator may have several reasons: (a) the limited access of certain chelators to the cytoplasm, (b) the time required to exhaust the intracellular iron pool, notably of ferritin, or (c) the sta­ bility of the iron-sulfur cluster, or a combination of all three. Initially, the loss of a single iron atom of the cluster was supposed to be sufficient to activate IRP-1 [Fig. 5(B)]. This was based on the similarity with mitochondrial aconitase where the fourth Fe-atom is labile.80 However, the cluster of cytoplasmic aconitase appears to be much more stable.30 Moreover, IRP-1 expressed as a recombinant protein in bacteria is also isolated to a large extent with a [4Fe-4S]-cluster.81 It can be converted in presence of P^Oj to a [3Fe-4S] cluster form with a typical EPR-signal81 [Fig. 5(B)]. This form is unable to bind the IRE. In contrast, recombinant IRP-1 made in baculovirus-infected cells in the absence of iron supply lacks the iron-sulfur cluster and is active in IRE binding.29,31 The active IRP-1, similar to the mito­ chondrial apo-protein,24 can be converted to the aconitase form in vitro in a solution with divalent iron, sulfide and reductant like cysteine or dithiothreitol.29,82 While gaining enzyme-activity the protein looses IRE bind­ ing activity, similar to recombinant IRP-1 made in the presence of iron. As for mitochondrial aconitase,24 in vitro conversion of the cytoplasmic aconitase form back to the RNA-binding form requires rather drastic oxidative disas­ sembly of the iron-sulfur cluster with ferricyanide followed by reduction of intra-molecular disulfide bridges33,83 [Fig. 5(E)]. These data suggest that the complete absence of the cluster is needed to activate IRE-binding properties. To confirm that absence of the [4Fe-4S]-cluster is required to activate IRE binding of IRP-1, the three cysteine residues holding this cluster were

340

Cellular Implications of Redox Signalling

mutated, separately or together, to serine by site-directed mutagenesis. As predicted, any of these cysteine mutants of IRP-1 shows constitutive RNA-binding activity in mammalian cells.32"34 Interestingly recombinant bacterial IRP-1 with mutations in Cys503 or Cys506 are not active when freshly isolated or incubated with the disulfide-oxidizing agent diamide. For activation they require the presence of 10 mM 2-mercaptoethanol. In contrast, the mutant in Cys437 and the double mutant in Cys503/Cys506 are spontaneously active. It indicates that formation of intra-molecular disulfide bridges between Cys437 and Cys503 or 506 prevent IRE binding [Fig. 5(E)]. Cys437 has to be reduced for binding, indicating that the pro­ tein conformation at or near this cysteine is critical for optimal affinity. Moreover, binding is lost when Cys437 is alkylated by the bulky N-ethylmaleimide, but not by iodoacetamide. 33 It suggests that Cys437 is not directly involved in the contact with the RNA. The evidence for the formation of intra-molecular disulfide bridges in vitro suggests that such bridges might also form in cells and their for­ mation controlled by redox. Indeed, experiments with diamide on cell cultures show a rapid inactivation of IRP-1 activity within 1 hour.84 This inactivation is, however, as rapidly reversed when diamide is removed. It shows that disulfide bridges can form in the cytoplasm under artificial oxidizing conditions, but that the natural redox potential of the cell is suf­ ficient to reduce them rapidly. Indeed, generally in fresh cytoplasmic extracts the RNA-binding form of IRP-1 is found reduced. It will, how­ ever, oxidize upon storage and loose its RNA-binding activity. This can then be recovered by mild reduction. The rapid re-activation of IRP-1 after washing away diamide from cells is different from the much slower activation after iron-chelation. It shows that activation of IRP-1 in iron deprived cells is not simply a phe­ nomenon of a redox reaction on an intra-molecular disulfide bridge. The slow response to iron chelators of cultured cells may have several reasons: (a) the limited access of certain chelators to the cytoplasm, (b) the time required to exhaust the intracellular iron pool, notably of ferritin, or (c) the stability of the iron-sulfur cluster. Following the finding that full cluster disassembly is required a fourth hypothesis is attractive as well. Active IRP-1 may represent apo-aconitase prior to its maturation to the enzyme and iron chelation just prevents the formation of iron-sulfur clus­ ters. The slow response to iron chelation may then reflect the protein turnover and de novo synthesis of IRP-1. Indeed, the presence of the trans­ lation inhibitor cycloheximide during iron chelation retards the appearance of active IRP-1 for 4 to 8 hours in cultured fibroblast cell lines, but not beyond.42,54 It suggests that both de novo synthesis as well as slow

Regulation ofmRNA Translation and Stability in Iron Metabolism

341

disassembly of the 4Fe-4S-cluster of cytoplasmic aconitase contribute to the active IRP-1 pool in iron deprivation.

6. Regulation of IRP-2 Activity by Iron The discovery and subsequent analysis of IRP-2 were first made with rodent tissue and cells in culture.35"37,39'44 The protein is slightly larger than IRP-1, but its sequence shows 57% identical and 79% similar amino acids. IRP-1 and IRP-2 from rodent cells give rise to distinct complexes with a different migration. However, the complexes of human IRP-1 and IRP-2 co-migrate, such that it is difficult to distinguish them. For human cell extracts, specific reagents such as antibodies or specific IREs are needed to make this distinction. With respect to its regulation by iron, IRP-2 behaves quite differently from IRP-1. As already mentioned, IRP-2 changes in protein levels at dif­ ferent iron concentrations. It is virtually undetectable in cells cultured in iron-rich medium, but is clearly induced after addition of an iron chelator. As for IRP-1, the IRE-binding activity of IRP-2 reaches maximal levels within about 15 hours. However, addition of 2% 2-mercaptoethanol in vitro does not produce a strong enhancement of IRP-2 activity. IRP-2 protein decays very rapidly after addition of iron salts or hemin to cells, unless a proteasome inhibitor is present.37,41-42,85 From this, it was con­ cluded that IRP-2 is probably targeted to the proteasome for degradation. The rapid decay depends on a 73 amino-acid sequence, which is unique to IRP-2.40 Grafting this sequence into IRP-1 is sufficient to render the IRP-1 protein unstable in high iron.40 This rapid protein decay does not require insertion of an iron-sulfur cluster. The 73 amino acid protein sequence comprises several cysteines that are important to initiate IRP-2 degradation. They may directly be involved in binding iron and initiating oxidative damage. 40 In vitro studies suggest that oxidative modification of this region might be at the origin of subsequent recognition by the ubiquitination machinery. 43 These data have led to the concept that IRP-2 might sense high iron levels by the increase of oxidative damage.

7. Do IRP-1 and IRP-2 have Similar or Different Functions? This important question is still under investigation and cannot be fully answered yet. However, there exists increasing evidence that pathways activating IRP-1 and IRP-2 show major differences that may lead to a

342

Cellular Implications ofRedox Signalling

refined response in specific tissues. This aspect will be further discussed below. At least theoretically, there exists the possibility that IRP-1 and IRP-2 may bind preferentially to certain downstream targets as the result of either differential activation or differences in affinity.86 It is clear that IRP-1 and IRP-2 can independently of one another regulate IRE-containing mRNAs in cells.34,87-89 There is also space for the discovery of specific, yet unknown targets for either IRP. The question whether IRP-1 and IRP-2 have different functions was recently approached through the analysis of animals lacking these genes. Thus far, only results of mice lacking IRP-2 were reported. 89 These mice grow and develop normally suggesting that IRP-1 is sufficient for most of the basic regulatory functions. However with 6 months of age the mice develop a neurodegenerative disease. They show increased ferritin expression, most prominently in the central nervous system and intesti­ nal mucosa. This age-dependent iron overload goes together with a decrease of transferrin receptor expression in affected tissues. The authors noticed that the tissues that are most affected are those in which IRP-2 is expressed at a relatively high proportion compared to IRP-1.35 In order to know more about the possible differential recognition of tar­ get mRNAs, the detailed analysis of the RNA protein interaction may be instructive. The crystal structures of IRP-1 or IRP-2 are still not solved, and information about the interaction with IREs relies largely on cross-linking and mutagenesis experiments. IRP-1 and IRP-2 do not contain any classical RNA-binding motifs. Moreover, phylogenetic comparison shows that IREbinding properties were acquired late in the evolution of aconitases and are only found in animals, but are absent from prokaryotes or other eukaryotes such as yeast and plants.38,90 It is remarkable that IRE binding properties seem to be highly conserved between IRP-1 and IRP-2 as both proteins show a similar affinity for the ferritin IRE.35"37 It suggests a strong structural conservation and probably recent gene duplication in evolution. Yet for cer­ tain target IREs, differential binding parameters were reported.86,91,92 It is too early to know whether this has any physiological impact. Presently available data for the IRE/IRP-1 complex indicate multiple interaction sites on the protein in domains 1-3 and 4 as well as a require­ ment of the entire protein for binding (Fig. 2). UV-cross-linking experi­ ments of a radio-active IRE to recombinant IRP-1 identified a cross-link between amino acid 120 and 131, more precisely at Serl27.93,94 This site is in the area of the iron-sulfur cluster at the inner surface of the cleft in domain 1 (Fig. 2). Access to this site by the IRE would require the open­ ing of the cleft. One other study identified instead amino acids 480-623 as

Regulation ofmRNA Translation and Stability in Iron Metabolism 343

a cross-linked region in rabbit IRP-1 95 suggesting that extensive contact sites may exist between the protein and the RNA. By a quite different approach surface residues that get protected against proteolysis by the interaction with the IRE were mapped. 96 This foot-printing assay indicated that two regions on IRP-1 are probably in direct contact with the RNA: a region between amino acids 80 and 187, as well as a region in domain 4 between amino acids 721 and 735 encom­ passing the two Arg721 and Arg728 residues. Moreover, these regions are more protected in the aconitase than the IRP form. A third approach used site directed mutagenesis of surface areas on IRP-1. Such regions were predicted from the mitochondrial aconitase structure and sequence alignments. They were then mutated to the corre­ sponding sequences of bacterial aconitase, which does not bind to IREs. Two mutants in domain 4 at amino acids 685-689 and 732-737 diminished strongly the affinity, but did not affect the folding of IRP-1.97 In another study active site residues of the aconitase were mutated and shown to abolish IRE binding. 98 Since these residues are located in central coordi­ nates of the IRP-1 structure, it is not possible verify that all these mutants are folded normally. Thus, the conclusion that active site residues contact the RNA directly should be considered with caution. Selection of IRE variants by selex procedures showed that several mutations in the conserved loop and bulge sequences of the IRE are tol­ erated, although binding affinities are usually lower with mutated IREs than the wild-type IRE.16,91,92 Interestingly, certain mutated IREs show binding only for IRP-1 or IRP-2, suggesting differences at the interacting protein surface. The specificity of these reagents was used to identify interacting IRP-1 surface regions grafted into IRP-2. The results indicate that Arg728 and Arg732 are part of a region that contacts the IRE bulge, whereas a region between 685-689 is necessary for recognition of the IRE loop. 97 The distance between the two regions corresponds perfectly to the distance between the loop and the bulge of the IRE. Both binding sites are located near the edge of the cleft. They form surface loops that are miss­ ing in bacterial or mitochondrial aconitase, suggesting their acquisition during evolution. Taken together the different approaches show complementary results that indicate at least three IRE interaction sites, one on the inner cleft sur­ face of domains 1-3 and two regions in domain 4 (Fig. 2). The data sug­ gest that IRPs lacking the iron-sulfur-cluster open their structure to accommodate the IRE. More direct evidence for a conformational change was recently presented by studies using differential sensitivity to protease

344

Cellular Implications ofRedox Signalling

cleavage. 96 " Moreover, the identification of IRP-1 and IRP-2 specific IREs suggests the possible existence of mutated IREs in natural mRNAs and their specific recognition as targets by IRP-1 or IRP-2. However, extensive computer-assisted searches of databases have to date not revealed the exis­ tence of such IRE mutants at relevant positions in cDNAs.

8. Modulation of IRP-1 and IRP-2 by Nitric Oxide Nitric oxide (NO) is a gas and radical synthesized from L-arginine in most cell types in response to physiological stimuli. There exist three different nitric oxide synthases, of which one is induced by cytokines and prominent in macrophages. N O serves as a second messenger and modulates physio­ logical processes such as vascular tone, neuronal signaling or antibacterial defense mechanisms. It reacts readily with transition metals or sulfhydryl groups of proteins and interferes with the function of various iron-containing proteins (reviewed in Refs. 100 and 101). Notably, enzymes with iron-sulfur clusters including the mitochondrial aconitase are fully inhibited after induc­ tion of nitric oxide synthase in macrophages.102 Diffusion of NO can also inhibit enzymes in neighboring cells and is thought to contribute to its bacte­ ricidal role. The similarity of IRP-1 with mitochondrial aconitase suggested that NO might also modulate IRP-1 and IRP-2. Indeed, stimulation of pri­ mary macrophages or macrophage cell lines with interferon-y and lipopolysaccharide induces nitric oxide synthase, and the RNA-binding form of IRP-1 accumulates within about 12 hours. 103104 The specific nitric oxide synthase inhibitor N G -monomethyl-arginine inhibits this process. A similar effect was observed upon NO-synthesis or in the presence of NOreleasing drugs in rat brain slices,105 the erythroid cell line K562,104106 transfected murine fibroblast cell lines107 or hepatoma cells.108 Activation of IRP-1 by NO modulates ferritin and transferrin receptor expression essentially as does iron chelation.104,106'107 However, in certain cases ferritin109110 or transferrin receptor108 were not regulated, or even opposite to what was expected. This is possibly caused by additional overriding effects of cytokines or N O on cell proliferation a n d / o r gene transcription. It was also postulated that regulation by IRP-2 might dominate under cer­ tain condition.111 Whereas IRP-2 was to some extent activated by NO in transfected mouse fibroblasts107 and liver during inflammation,111 it was much less induced by N O than IRP-1 in macrophages and rat hepatoma cells.103,108 In at least two studies with a macrophage cell lines, cytokines down-regulated IRP-2 levels109112 and ferritin expression was increased and

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transferrin receptor diminished.109 It was concluded that IRP-1 and IRP-2 are differentially regulated in these cells, and that IRP-2 may play a major role in the control of ferritin levels in macrophages during inflammation.109 The mechanism of downregulation of IRP-2 remains to be studied. Recently, it was also observed that IRP-1 mRNA and protein diminished after stimulation of nitric oxide synthase, while RNA-binding activity was induced.113 Oxidative destabilization of IRPs and/or transcription factors by NO or peroxynitrite might explain such parallel phenomena. The mechanism of IRP-1 activation by NO could either be similar to the one by iron chelators and either prevent the iron-sulfur cluster formation or directly provoke its disassembly. NO was reported to have a chelating effect on the free iron pool.114115 Yet, NO also interacts directly with the [4Fe-4S]cluster of aconitases in vitro and inhibits the enzyme activity.116 Initial stud­ ies in vitro could not demonstrate full activation of c-aconitase to its RNA-binding apo-protein form by N O in vitro.103 However, recent data show not only disassembly of the cluster but, in addition, that NO oxidizes intra-molecular disulfides117 previously observed as inhibitory to IREbinding.33 The addition of 10 mM 2-mercaptoethanol or the reconstitution of thioredoxin-thioredoxin reductase is capable of activating the IRP-binding of NO-treated c-aconitase.117 Rapid activation of IRP-1 in co-cultured cells by NO-secreting cells118 speaks also in favor of an immediate disassembly of the iron-sulfur cluster in vivo as soon as a certain NO-concentration is reached. As mentioned before, the reductive environment in the cell, notably thioredoxin-thioredoxin reductase system appears sufficient to avoid the intramolecular disulfide bridge formation. The previously observed slow response to N O in cells might be due to the delay required to induce the synthesis of nitric oxide synthase and to reach a critical NO concentration. With regard to the IRP-1 activation, it was also debated whether the NO radical itself or a derivative was responsible. Notably peroxynitrite (ONOO~) which is generated from NO and superoxide (0 2 ~) under pathophysiological conditions was in certain studies a more effective inactivator of aconitases than NO. 119120 This view has been challenged recently by a careful in vitro study of EPR signals observed with purified mitochondrial and cytoplasmic aconitases.116 According to these authors, NO is sufficient to promote enzyme inhibition and iron-sulfur cluster disas­ sembly. Experiments carried out with peroxynitrite in vitro confirmed inactivation of aconitase, but showed no concomitant activation of the mRNA-binding activity of IRP-1, unless a small amount of 2-mercap­ toethanol was present.121 It suggests again oxidation of critical SH-groups in the iron-sulfur cluster-free protein [Fig. 5(E)]. This result is reminiscent of

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other strong oxidants, which disassemble the iron-sulfur cluster in vitro, such as ferricyanide33,83 or exposure to an Oj generating system.122 The physiological meaning of a regulation of IRP-1 and IRP-2 and ulti­ mately iron metabolism by NO is not entirely clear. Besides the mentioned effect of NO as a bacteriostatic agent acting possibly through iron-chelation or other cytotoxic effects, one may search for a connection to inflammatory situations. It is known that iron is retained in ferritin by reticuloendothelial macrophages under these conditions. The results observed in certain stud­ ies would fit a picture according to which inflammatory cytokines induce NO-synthesis and reduce IRP-2, which increases then ferritin synthesis.109 However, it needs to be confirmed that IRP-2 really plays a predominant role over IRP-1 in reticuloendothelial cells. Inflammatory cytokines may exert direct effects on ferritin transcription.123124 Most other studies show in fact activation of IRP-1 by NO and induction of ferritin, which does not fit to explain the inflammatory response.

9. Activation of IRP-1 by Hydrogen Peroxide Another interesting link exists between iron metabolism and oxidative stress (for recent review, see Ref. 125). As mentioned above, free Fe2+ in conjunction with H 2 O z is responsible for the formation by Fenton chem­ istry of highly reactive hydroxyl radicals. Cellular damage by reactive oxy­ gen species is often referred to as an important cause of degenerative disorders and hypoxia-reperfusion injury.126 Since IRP-1 and IRP-2 directly regulate free iron, it is of considerable interest that both proteins are influ­ enced in their IRE-binding activity by oxidative stress. As already men­ tioned, IRP-2 is rapidly degraded in response to increased iron levels, and this correlates with oxidative modifications in a 73-amino acid region of the protein that predispose for ubiquitination and proteasome degradation.40,43 Moreover, it was observed that H 2 0 2 strongly activates IRP-1, but not IRP-2.127128 This activation only takes place when H 2 0 2 is added extracellularly. The effect is almost immediate and already measur­ able after 15 minutes. 128 No such effect could be observed upon drugmediated induction of intracellular H 2 0 2 and 0 2 by quinones,129 nor by catalase inhibition,130 nor when H 2 0 2 was added in vitro to recombinant cytoplasmic aconitase.81 The rapid response to H 2 O z involves the inactivation of the aconitase activity and provokes the regulation of ferritin transla­ tion and transferrin receptor synthesis expected for activated IRE-binding.128

Regulation ofmRNA Translation and Stability in Iron Metabolism 347

The mechanism by which H 2 0 2 activates IRP-1 is clearly different from the one proposed for iron deprivation or NO, already by the fact of its rapidity and the requirement of extracellular initiation. Several models were put forward to explain this phenomenon 131 among which two seem most pertinent: (a) the possibility of a direct modification of IRP-1 which rapidly converts the aconitase to an IRE-binding form (Fig. 2) or (b) the activation of a protein that modifies IRP-1 and facilitates the conversion to an IRE-binding conformation. In favor of these mechanisms, activation by H 2 0 2 is diminished by the type I/IIa phosphatase inhibitor okadaic acid, whereas activation by iron deficiency and N O are insensitive to the inhibitor.128132 In a recent study these authors have reconstituted the effect with permeabilized fibroblast cells in vitro.133 The activation requires both the cytoplasm and an insoluble component, possibly of the membrane, and is sensitive to alkaline phosphatase. The activation is also completely blocked by non-hydrolyzable nucleotide analogs. These studies are com­ patible with a signaling pathway and a phosphorylation or dephosphorylation event that modifies the cytoplasmic aconitase and possibly triggers iron-sulfur cluster disassembly. An entirely different line of evidence shows that IRP-1 and IRP-2 are phosphorylated both in vitro and in vivo by protein kinase C and that these modifications induce the a 2-fold increase in the affinity of IRE-IRP inter­ actions.134135 This regulation is thought to involve Serl38, a site that is much more accessible in the apo-protein form of IRP-1." This phosphorylation site was mutated to Glu or Asp, amino acids that exhibit a charge similar to the phosphate. 136 The [4Fe-4S]-cluster of these mutants was less stable and disassembled readily when exposed to oxygen. The data are interpreted as evidence that phosphorylation of IRP-1 may modulate the stability of the [4Fe-4S]-cluster independent of the cellular iron status. At present it is tempting to speculate that phosphorylation may provide a frame to under­ stand the activation of IRP-1 by extracellular H2Oz. The physiological meaning of the activation of IRP-1 by extracellular H 2 O z and of the phosphorylation events by protein kinase C remains, however, largely obscure.

10. Regulation of IRP-1 by Hypoxia Recently, it was discovered that culturing rat hepatoma cells in low oxygen concentration (1-3%) lowers the IRE-binding activity of IRP-1 and

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increases that of IRP-2.137'138 This effect required the presence of normal iron concentrations and was reversible after reoxygenation.137 The inactivation of IRP-1 was essentially post-translational and did not affect the protein level, nor did protein synthesis inhibitors influence it. These results suggest a direct effect of hypoxia on the stability or formation of the [4Fe-4S]-cluster in IRP-1. The authors argue that low oxygen concentration may lower the cytoplasmic 0 2 concentration and thus preserve the cluster from disassembly.125 In contrast to IRP-1, IRP-2 was increased in protein levels, which might be due to protein stabilization.138 It suggests that the mechanism of IRP-2 oxidation and subsequent proteasomal degradation depend on both the iron and oxygen concentration. The oxygen sensing mechanism and pathways leading to IRP-2 stabilization in hypoxia need to be defined further. The physiological relevance of these findings will also require further exploration.

11. Conclusions The regulation of IRP-1 and IRP-2 appears in the first place designed to maintain the steady state level of iron in all tissues and thereby ensure an appropriate balance in the entire organism. However, we have learnt in recent years that IRP-1 and IRP-2 have complementary functions and may be differentially regulated by the additional presence of NO, H 2 0 2 or dif­ ferent 0 2 derivatives and concentrations. Thus, there exists the possibility of local and probably tissue-specific responses that may influence the iron balance. It seems clear that the necessity of iron for cell survival leaves only a limited margin for these post-transcriptional mechanisms. However, they are perfectly adapted to sense and correct very subtle changes in iron metabolism. The iron regulatory proteins seem to have a better capacity to react to iron deprivation or activating conditions than to iron overload since most of the IRP is inactivated under normal physiological conditions. With respect to the role of redox in the control of IRP-1 and IRP-2, it seems clear that an efficient cytoplasmic reducing capacity is essential to obtain IREbinding activity. This seems to be provided by the thioredoxin-thioredoxin reductase system. To my knowledge there exists no good evidence for any physiological situation in vivo, in which oxidizing conditions are suffi­ ciently strong to overcome the reduced state of the intramolecular sulfhydryls in IRPs. However, NO, 0 2 or derivatives thereof act directly or indirectly on the redox state of iron, as well as the formation and/or disas­ sembly of the iron-sulfur cluster in IRP-1 and the stability of IRP-2. In turn,

Regulation ofmRNA Translation and Stability in Iron Metabolism 349

changes in free iron levels and its redox state can influence the formation reactive oxygen species. It seems rather in this context that redox may exert its effect on iron-sulfur cluster assembly and IRP activities. Unfortunately, we are still quite ignorant about the precise conditions, which govern the iron-sulfur cluster assembly in vivo, and we do not know in detail the mech­ anism by which IRP-2 is modified by oxidative conditions in vivo.

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Love P, Tresser N, Rouault TA. 2001. Targeted deletion of the gene encoding iron regulatory protein-2 causes misregulation of iron metabolism and neurodegenerative disease in mice. Nat. Genet. 27: 209-214 Rothenberger S, Mullner EW, Kiihn LC. 1990. The mRNA-binding protein which controls ferritin and transferrin receptor expression is conserved during evolution. Nucleic Acids Res. 18:1175-1179 Henderson BR, Menotti E, Kiihn LC. 1996. Iron regulatory proteins-1 and-2 bind distinct sets of RNA target sequences. /. Biol. Chem. 271: 4900-4908 Butt J, Kim HY, Basilion JP, Cohen S, Iwai K, Philpott CC, Altschul S, Klausner RD, Rouault TA. 1996. Differences in the RNA binding sites of iron regulatory proteins and potential target diversity. Proc. Natl. Acad. Sci. USA. 93: 4345-4349 Basilion JP, Rouault TA, Massinople CM, Klausner RD, Burgess WH. 1994. The iron-responsive element-binding protein: Localization of the RNA-binding site to the aconitase active-site cleft. Proc. Natl. Acad. Sci. USA. 91: 574-578 Neupert B, Menotti E, Kuhn LC. 1995. A novel method to identify nucleic acid binding sites in proteins by scanning mutagenesis — Application to iron regulatory protein. Nucleic Acids Res. 23: 2579-2583 Swenson GR, Walden WE. 1994. Localization of an RNA binding element of the iron responsive element binding protein within a proteolytic fragment containing iron coordination ligands. Nucleic Acids Res. 22: 2627-2633 Gegout V, Schlegl J, Schlager B, Hentze MW, Reinbolt J, Ehresmann B, Ehresmann C, Romby P. 1999. Ligand-induced structural alterations in human iron regulatory protein-1 revealed by protein footprinting. /. Biol. Chem. 274: 15052-15058 Kaldy P, Menotti E, Moret R, Kiihn LC. 1999. Identification of RNA-binding surfaces in iron regulatory protein-1. EMBO /. 18: 6073-6083 Philpott CC, Klausner RD, Rouault TA. 1994. The bifunctional ironresponsive element binding protein/cytosolic aconitase — The role of active-site residues in ligand binding and regulation. Proc. Natl. Acad. Sci. USA. 91: 7321-7325 Schalinske KL, Anderson SA, Tuazon PT, Chen OS, Kennedy MC, Eisenstein RS. 1997. The iron-sulfur cluster of iron regulatory protein1 modulates the accessibility of RNA binding and phosphorylation sites. Biochemistry 36: 3950-3958

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100. Henry Y, Lepoivre M, Drapier J-C, Ducrocq C, Boucher J-C, Guissani A. 1993. EPR characterization of molecular targets for N O in mammalian cells and organelles. FASEB }. 7: 1124-1134 101. Drapier J-C, Bouton C. 1996. Modulation by nitric oxide of metalloprotein regulatory activities. Bioessays 18: 549-556 102. Drapier J-C, Hibbs JB, Jr. 1986. Murine cytotoxic activated macrophages inhibit aconitase in tumor cells. Inhibition involves the iron-sulfur prostethic group and is reversible. /. Clin. Invest. 78:790-797 103. Drapier J-C, Hirling H, Wietzerbin J, Kaldy P, Kuhn LC. 1993. Biosynthesis of nitric oxide activates iron regulatory factor in macrophages. EMBO J. 12: 3643-3649 104. Weiss G, Goossen B, Doppler W, Fuchs D, Pantopoulos K, Werner-Felmayer G, Wachter H, Hentze MW. 1993. Translational regulation via iron-responsive elements by the nitric oxide/NOsynthase pathway. EMBO }. 12: 3651-3657 105. Jaffrey SR, Cohen NA, Rouault TA, Klausner RD, Snyder SH. 1994. The iron-responsive element binding protein — A target for synaptic actions of nitric oxide. Proc. Natl. Acad. Sci. USA. 91:12994-12998 106. Richardson DR, Neumannova V, Nagy E, Ponka P. 1995. The effect of redox-related species of nitrogen monoxide on transferrin and iron uptake and cellular proliferation of erythroleukemia (K562) cells. Blood 86: 3211-3219 107. Pantopoulos K, Hentze MW. 1995. Nitric oxide signaling to ironregulatory protein: Direct control of ferritin mRNA translation and transferrin receptor mRNA stability in transfected fibroblasts. Proc. Natl. Acad. Sci. USA. 92: 1267-1271 108. Phillips JD, Kinikini DV, Yu Y, Guo B, Leibold EA. 1996. Differen­ tial regulation of IRP-1 and IRP-2 by nitric oxide in rat hepatoma cells. Blood 87: 2983-2992 109. Recalcati S, Taramelli D, Conte D, Cairo G. 1998. Nitric oxidemediated induction of ferritin synthesis in J774 macrophages by inflammatory cytokines: Role of selective iron regulatory protein-2 downregulation. Blood 91:1059-1066 110. Oria R, Sanchez L, Houston T, Hentze MW, Liew FY, Brock JH. 1995. Effect of nitric oxide on expression of transferrin receptor and ferritin and on cellular iron metabolism in K562 human erythroleukemia cells. Blood 85: 2962-2966 111. Cairo G, Pietrangelo A. 1995. Nitric-oxide-mediated activation of iron-regulatory protein controls hepatic iron metabolism during acute inflammation. Eur. }. Biochem. 232: 358-363

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112. Bouton C, Oliveira L, Drapier J-C. 1998. Converse modulation of IRP-1 and IRP-2 by immunological stimuli in murine RAW 264.7 macrophages. /. Biol. Chan. 273: 9403-9408 113. Oliveira L, Drapier J-C. 2000. Down-regulation of iron regulatory protein-1 gene expression by nitric oxide. Proc. Natl. Acad. Sci. USA. 97: 6550-6555 114. Hibbs JB, Jr, Taintor RR, Vavrin Z. 1984. Iron depletion: Possible cause of tumor cell cytotoxicity induced by activated macrophages. Biochem. Biophys. Res. Commun. 123: 716-723 115. Hibbs JB, Jr, Taintor RR, Vavrin Z. 1987. Macrophage cytotoxicity: Role for L-arginine deiminase and imino nitrogen oxidation to nitrite. Science 235: 473-476 116. Kennedy MC, Antholine WE, Beinert H. 1997. An EPR investigation of the products of the reaction of cytosolic and mitochondrial aconitases with nitric oxide. /. Biol. Chem. 272: 20340-20347 117. Oliveira L, Bouton C, Drapier J-C. 1999. Thioredoxin activation of iron regulatory proteins. Redox regulation of RNA binding after exposure to nitric oxide. /. Biol. Chem. 274: 516-521 118. Bouton C, Raveau M, Drapier J-C. 1996. Modulation of iron regula­ tory protein functions. Further insights into the role of nitrogenand oxygen-derived reactive species. /. Biol. Chem. 271: 2300-2306 119. Hausladen A, Fridovich I. 1994. Superoxide and peroxynitrite inactivate aconitases, but nitric oxide does not. /. Biol. Chem. 269: 29405-29408 120. Castro L, Rodriguez M, Radi R. 1994. Aconitase is readily inactivated by peroxynitrite, but not by its precursor, nitric oxide. /. Biol. Chem. 269: 29409-29415 121. Bouton C, Hirling H, Drapier J-C. 1997. Redox modulation of iron regulatory proteins by peroxynitrite. /. Biol. Chem. 272: 19969-19975 122. Cairo G, Castrusini E, Minotti G, Bernelli-Zazzera A. 1996. Superoxide and hydrogen peroxide-dependent inhibition of iron regula­ tory protein activity: A protective stratagem against oxidative injury. FASEB } 10: 1326-1335 123. Kwak EL, Larochelle DA, Beaumont C, Torti SV, Torti FM. 1995. Role for NF-kappaB in the regulation of ferritin H by tumor necrosis factor-alpha. /. Biol. Chem. 270: 15285-15293 124. Tran TN, Eubanks SK, Schaffer KJ, Zhou CY, Linder MC. 1997. Secretion of ferritin by rat hepatoma cells and its regulation by inflammatory cytokines and iron. Blood 90: 4979-4986

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125. Hanson ES, Leibold EA. 1999. Regulation of the iron regulatory proteins by reactive nitrogen and oxygen species. Gene Exp. 7: 367-376 126. Hershko C. 1994. Control of disease by selective iron depletion: A novel therapeutic strategy utilizing iron chelators. Bailliere's Clin. Haematol. 7: 965-1000 127. Martins EAL, Robalinho RL, Meneghini R. 1995. Oxidative stress induces activation of a cytosolic protein responsible for control of iron uptake. Arch. Biochem. Biophys. 316:128-134 128. Pantopoulos K, Hentze MW. 1995. Rapid responses to oxidative stress by iron regulatory protein. EMBO }. 14: 2917-2924 129. Gehring NH, Hentze MW, Pantopoulos K. 1999. Inactivation of both RNA binding and aconitase activities of iron regulatory protein-1 by quinone-induced oxidative stress. /. Biol. Chem. 274: 6219-6225 130. Pantopoulos K, Mueller S, Atzberger A, Ansorge W, Stremmel W, Hentze MW. 1997. Differences in the regulation of iron regulatory protein-1 (IRP-1) by extra- and intra-cellular oxidative stress. /. Biol. Chem. 272: 9802-9808 131. Hentze MW. 1996. Iron-sulfur clusters and oxidant stress responses. Trends Biochem. Sci. 21: 282-283 132. Pantopoulos K, Weiss G, Hentze MW. 1996. Nitric oxide and oxidtive stress (H 2 0 2 ) control mammalian iron metabolism by different pathways. Mol. Cell. Biol. 16: 3781-3788 133. Pantopoulos K, Hentze MW. 1998. Activation of iron regulatory protein-1 by oxidative stress in vitro. Proc. Natl. Acad. Sci. USA. 95: 10559-10563 134. Eisenstein RS, Tuazon PT, Schalinske KL, Anderson SA, Traugh JA. 1993. Iron-responsive element-binding protein. Phosphorylation by protein kinase C. /. Biol. Chem. 268: 27363-27370 135. Schalinske KL, Eisenstein RS. 1996. Phosphorylation and activation of both iron regulatory proteins-1 and-2 in HL-60 cells. /. Biol. Chem. 271: 7168-7176 136. Brown NM, Anderson SA, Steffen DW, Carpenter TB, Kennedy MC, Walden WE, Eisenstein RS. 1998. Novel role of phosphorylation in Fe-S cluster stability revealed by phosphomimetic mutations at Ser-138 of iron regulatory protein-1. Proc. Natl. Acad. Sci. USA. 95: 15235-15240 137. Hanson ES, Leibold EA. 1998. Regulation of iron regulatory protein-1 during hypoxia and hypoxia/reoxygenation. /. Biol. Chem. 273: 7588-7593 138. Hanson ES, Foot LM, Leibold EA. 1999. Hypoxia post-translationally activates iron-regulatory protein-2. /. Biol. Chem. 274: 5047-5052

Chapter 16 Redox Flow as an Instrument for Gene Regulation Samuel Kaplan, Jesus M. Eraso, Jeong-Il Oh, Jung Hyeob Roh Department of Microbiology and Molecular Genetics, The University of Texas Medical School, 6431 Fennin Street, Houston, USA [email protected]

Keywords: Rhodobacter sphaeroid.es, redox control, gene expression, photosynthetic apparatus, electron flow, signal transduction

1. Summary Rhodobacter sphaeroides 2.4.1 is a facultative, photoheterotrophic bacterium classified to the subgroup ct-proteobacteria. It enjoys a diverse lifestyle being able to grow as a chemoheterotroph, chemolithotroph, photoheterotroph or photolithotroph. It can fix both carbon dioxide and nitrogen. This physiologic versatility is a product of the very wide array of electron donors and acceptors capable of being used under these diverse growth conditions. A central question accompanying the study of this organism is how is it able to regulate and adapt to these diverse growth modes? When oxygen tensions fall below < 3% (semiaerobic conditions) the photosynthetic apparatus or intracytoplasmic membrane (ICM) of the facultative photoheterotroph, Rhodobacter sphaeroides 2.4.1, is induced and arises as invaginations from the cytoplasmic membrane. 38 The ICM con­ tains a reaction center (RC) encoded by the puf and puh genes and two light harvesting (LH) pigment-protein complexes, the B800-850 and the B875 encoded by the puc and puf opeions respectively. Thus oxygen is the primary regulator of ICM formation. Once photosynthesis (PS) gene expression is turned on in the absence of 0 2 , then light intensity incident to the culture is inversely related to the levels of PS gene expression and ICM content. 361

362 Cellular Implications ofRedox Signalling

Fig. 1. Prr transcripts. Schematic representation of the prr region. PrrB, prrC, prrA and spb are depicted as boxes. The arrows indicate the direction of transcription, and the different transcripts. Stem-loops refer to putative transcription termina­ tors. Numbers represent lengths of genes and intergenic regions. In the accompanying discussions we have described the four major regulatory systems controlling PS gene expression and how the flow of cellular redox can manifest itself as an instrument of gene regulation.

2. The Prr System Three major redox-responsive regulatory systems regulate PS gene expression in Rhodobacter sphaeroides 2.4.1: the Prr system, the PpsR-AppA repressor/antirepressor system and FnrL.93 A fourth regulator, TspO works through the PpsR/AppA system. The Prr (Photosynthetic response regulator) system is a signal transduction regulatory pathway involved in the positive regulation of PS gene expression in response to the redox state and / o r electron flow through the electron transport chain (ETC).15"1719,54"56,68 Prr is comprised of the prrA gene product, which func­ tions as a response regulator, the prrB gene product, which functions as a sensor histidine kinase/phosphatase, and prrC, which encodes a transmembrane associated protein (Fig. 1). These three genes form a cluster which maps outside the PS gene cluster on Chromosome I of R. sphaeroides 2.4.1. prrC and prrA form an operon, although prrA also has its own promoter, 19 and prrB is diver­ gently transcribed from prrCA (Fig. 1). Prr is similar to regBA in Rhodobacter capsulatus,48,72 Rhodovulum sulfidophilum45'*7 and Roseobacter denitrificans,46,47 the Sinorhizobium meliloti actRS system, 78 and the Bradyrhizobium japonicum RegSR system. 1 The response regulators of these systems are involved in regulation of different downstream processes. RegA in R. capsulatus, R. sulfidophilum and R. denitrificans is involved in regulation of photosystem formation, and it has been recently

Redox Flow as an Instrument for Gene Regulation

363

found that in R. capsulatus RegBA also regulates the molybdenumcontaining nitrogenase and the uptake hydrogenase. 11 ActR in S. meliloti is involved in acid tolerance, and it controls synthesis of formate, formaldehyde and methanol dehydrogenases. 21 RegR in B. japonicum activates many genes involved in symbiotic nitrogen fixation.22 The Prr system regulates transcription of both photosynthesis and respiratory genes,1516-27, as well as the expression of genes involved in CO z and N 2 fixation34,64 and more recently genes involved in chemotaxis (J. Armitage, personal communication). It also regulates its own transcrip­ tion. These and other data reveal that PrrA is a new form of global tran­ scription regulator. 2.1 PrrA PrrA is a 184 amino acid, cytoplasmically localized response regulator, with a conserved amino-terminal phosphorylation receiving domain. Similar to other response regulators, PrrA is believed to become active upon phosphorylation (Fig. 2). When the oxygen concentration is low or absent, PrrA is activated, and it positively regulates PS gene expression, see Table 1 and Fig. 2. Aerobically growing wild-type cells aberrantly produce spectral com­ plexes and have increased PS gene activity when prrA is present in multi­ copy.15 PrrA can function both as a transcriptional activator and a repressor of specific gene expression Ref. 18 e.g. the ccoNOQP operon which encodes the cbb3 terminal oxidase (Fig. 2). Inactivation of prr A causes a PS" phenotype, due to the complete inability of cells to produce photochemical complexes. Transcriptional activities of all PS genes measured, are severely reduced under these conditions. RegA, RegR, ActR and PrrA contain virtually identical carboxy-terminal regions, containing a putative helix-turn-helix (H-T-H), involved in DNA binding,1,40,47 although the crystal structure of the protein has not been solved, and predictions as to the conformation of this region differ, according to the algorithm used. A constitutively active mutant of RegA, RegA*, has been found to bind to the promoter regions of the R. capsula­ tus, puc, puf and hupSLC operons, as well as the regBA regulatory region, the nifA2 gene, and the R. sphaeroides cycA gene.1011'27,76 RegR binds to the fixR-nifA operon13 of B. japonicum. A consensus DNA binding motif has been proposed for RegR.14 RegA is phosphorylated by RegSc, a truncated form of RegS12 suggesting the interchangeability of the components comprising these systems. In vivo, RegR, RegA and ActR have been shown to be func­ tionally similar.12 In addition, RegA has been postulated to bind to DNA

364

Cellular Implications of Redox Signalling

Redox signal

ADP

Activation I Repression

HM W M

*s

*

+ pucBAC LHII + puhA RC + pufKBALMX LHI and RC + hemA -, tetrapyrrole + hemN I biosynthesis + hemZ J + crtA \ Crt biosynthesis + crtIB J + bchEJGP Bchl biosynthesis + cycA Cytc2 - dorS DMSO reductase - ccoNOQP, rdxBH cbbz oxidase and Rdx + ctaD aaz oxidase + cbb RubisCo + nif Nitrogenase Fig. 2. PrrBA, two-component activation. Abbreviations are as follows: PrrA-P, phosphorylated PrrA; LHI, light-harvesting complex I; LHII, light-harvesting complex II; RC, reaction center; Bchl, bacteriochlorophyll; Crt, carotenoids; DMSO, dimethyl sulfoxide; (-), negative redox signal on PrrB. + and - signs represent transcriptional activation and repression of gene expression, respectively.

Redox Flow as an Instrument for Gene Regulation

365

targets in both the phosphorylated (active) and non-phosphorylated (inactive) state.40 2.2 PrrB PrrB is a 462-residue histidine kinase/phosphatase. PrrB is anchored in and crosses the membrane six times, with it carboxy-terminal end facing the cytoplasm. 60 PrrB is believed to act as a sensor which responds indi­ rectly to changes in oxygen tension by sensing a signal derived from the electron transport chain (ETC). Sensing this signal is believed to occur within the amino-terminal membrane-spanning region of the protein, since the carboxy-terminal region appears to contain the histidine kinase consensus activation site. Evidence suggests that PrrB interacts with PrrC, 19 in the signal transduction pathway, see below. PrrB putatively autophosphorylates at a conserved histidine residue, and it has been postulated to activate PrrA through phosphorylation, and deactivate PrrA through dephosphorylation. In R. sphaeroides a truncated form of PrrB, PrrBTMA, appears to rapidly dephosphorylate PrrA in vitro,20 whereas in R. capsulatus, a truncated form of the PrrB homologue, RegB, phosphorylates the PrrA homologue RegA.32 A point muta­ tion involving a Leu to Pro change at position 78 of the PrrB protein (PRRB78), confers an oxygen-insensitive phenotype to the cell,16 i.e. PS gene expression is "full-on" under both aerobic and anaerobic conditions. Mutants defective in prrB are PS" when grown at low to medium light inten­ sities, but do grow at high light intensities, albeit with severely reduced lev­ els of LH complexes. Therefore, PrrA can be phosphorylated in the absence of PrrB, suggesting the following: (1) it is the phosphatase activity of PrrB which is under oxygen control, (2) PrrA can either be phosphorylated by heterologous histidine kinases and/or PrrA is capable of autophosphorylation. We have evidence in R. sphaeroides of these possibilities. Therefore, although the centrality of PrrA and PrrB to the regulation of PS gene expres­ sion is without dispute, the precise alternative physiologic states which modulate the functions of these proteins is yet to be fully understood. 2.3 PrrC PrrC is a protein with 231 amino acid residues. It crosses the membrane once, with its carboxy-terminus facing the periplasm, and the amino terminus facing the cytoplasm.19 Deletion of prrC leads to substan­ tial increases in the amount of photosynthetic complexes and PS gene

366

Cellular Implications ofRedox Signalling

expression under aerobic growth conditions, when compared to the wild-type. 19 This is consistent with results obtained with deletions of the cbb3 cytochrome c oxidase, the Rdx proteins and the CcoQ subunit of the cbb3 cytochrome c oxidase,53,55 to be discussed below, and indicates that PrrC is probably in the same signal transduction pathway as the former described membrane proteins. PrrC is believed to be the link between CcoQ, and the PrrBA two-component activation system. In contrast to PrrA and PrrB, cells with an inactivated PrrC are photosynthetically competent under all conditions of growth and light intensities. PrrC is highly conserved in both prokaryotes and eukaryotes. 19 SenC is the PrrC homologue in R. capsulatus.5 The best studied homologues are the Saccharomyces cerevisiae Scol and Sco2 proteins. Both proteins are localized to the mitochondrial inner-membrane of this organism,25,71 and the carboxy-terminal end of Scol was found to be located in the innermitochondrial space. 3 Disruption of scol, unlike that of scol, results in the lack of a cytochrome c terminal oxidase, and the effect was found to be post-transcriptional. 25,70 Thus, Scol has been proposed to be involved in cytochrome c oxidase assembly.43,70 In addition, Scol has been proposed to bind copper using the highly conserved (-CxxxC-) domain, and thus make copper available for the assembly of the oxidase.25,66 Alternatively, the (-CxxxC-) domain in SenC has been proposed to be an iron binding "half-site", similar to the 4 Cys domain present in bacterial ferredoxins.4,5 In the case of PrrC, the -CxxxC- domain resides in the periplasm and both cys residues are essential for PrrC activity in the signal transduction pathway.

3. Electron Transport Since the ETC plays a crucial role in the energy metabolism of R. sphaeroides under diverse growth conditions (Fig. 3), it was suggested by Cohen-Bazire et al.7 that the redox state of the ETC might be the source of a signal that controls the ability of R. sphaeroides to switch between aer­ obic and anaerobic photosynthetic metabolism (Fig. 4). As described below, we recently demonstrated that regulation of photosynthesis gene expression in R. sphaeroides is closely coupled to the functional state of the cbb3 cytochrome c oxidase and the redox state of the quinone pool.56 The respiratory ETC of R. sphaeroides contains those basic components com­ monly found in the mitochondria (quinone pool, ubiquinone: cytochrome c oxidoreductase [bc^ complex], and a mobile periplasmic cytochrome c2) (Fig. 3). These basic electron transfer components are terminated with two

Redox Flow as an Instrument for Gene Regulation 367

Fig. 3. Aerobic respiratory transport pathways of R. sphaeroides. The thickness of the arrows is a measure of the relative contribution of cytochromes c2 and c to channel electrons from the bcx complex to the cbb3 cytochrome c oxidase. Under aerobic conditions 0 2 is the terminal electron acceptor for both cytochrome c oxi­ dases and quinol oxidase. Abbreviations: Q-pool, quinone pool; bcv bcx complex; c2 and c , cytochromes c2 and cy; aay aa3 cytochrome c oxidase; cbb3, cbb3 cytochrome c oxidase; Qxt, quinol oxidase.

Fig. 4. Photosynthetic electron transport. Under anaerobic conditions the cbb3 oxidase is the exclusive cytochrome c oxidase synthesized in R. sphaeroides and some extent of electron flow occurs through the cbb3 oxidase to an unknown elec­ tron acceptor. Abbreviations: Q-pool, quinone pool; bcv bcx complex; c2 and c , cytochromes c2 and c ; cbby cbb3 cytochrome c oxidase, hv, light

functional cytochrome c oxidases and at least one functional quinol oxidase (Qxt).24-31-51'73'79 In addition to cytochrome c2 the membrane bound cytochrome c also contributes to the respiratory electron transfer between the bCj complex and the two cytochrome c oxidases.9,52 The aa3- and cbb3type cytochrome c oxidases both belong to the heme-copper oxidase superfamily where a low spin heme and a high spin heme exist in the cat­ alytic subunit, the latter being associated with CuB in a bimetallic Fe-Cu center, where molecular oxygen is reduced to H 2 0. 23 In the high spin heme denoted by the subscript 3 (Fig. 5), only one of the axial coordination places is occupied by a histidine residue. The other free axial coordination

368

Cellular Implications ofRedox Signalling

ccoNOQP operon

ccoN

ccoO

ccoQ

ccoP

cbb3 cytochrome coxidase

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C-X-X-C-H-Xn-M-P CcoP: H126, H223 CcoO:H72 Heme c binding motif

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°2 1

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High spin heme

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Fig. 5. Genomic organization of the ccoNOQP operon encoding the cbb3 oxidase and schematic diagram of the cbb3 oxidase. The redox centers and intramolecular electron transfer within the cbb3 oxidase are depicted. The catalytic subunit of the oxidases belonging to the heme-copper superfamily contains a low spin heme (Low) and a binuclear center composed a high spin heme (High) and CuB. The axial histidine ligands for both hemes and three histidine residues ligating to CuB are numbered on the basis of the amino acid sequence of CcoN of jR. sphaeroides. CcoO and CcoP are mono-heme and di-heme cytochromes c, respectively. The histidine residue in the heme c binding motif is numbered using the amino acid sequence of the corresponding subunit of R. sphaeroides.

Redox Flow as an Instrument for Gene Regulation

369

position can be used to bind to other molecules such as 0 2 , CO, or cyanide. On the other hand, two conserved histidine residues coordinate to the low spin heme ligand. The aa3 cytochrome c oxidase is homologous to the mitochondrial aa3 oxidase. The bacterial aa3 cytochrome c oxidase consists of four subunits, which represents a simple form of the oxidase when compared to the eukaryotic counterpart consisting of 13 subunits.23'80 The cbb3 cytochrome c oxidase with a high affinity for 0 2 is found in rhizobia and free-living diazotrophs in the rhizosphere (Agrobacterium tumefaciens, Azorhizobium caulinodans, Azospirillum brasilense, Bradyrhizobium japonicum, Rhizobium etli, and Sinorhizobium meliloti), as well as pathogenic and non-pathogenic aerobes (Campylobacter jejuni, Helicobacter pylori, Pseudomonas aeruginosa, Pseudomonas stutzeri, Vibrio cholerae, and MagnetospiriUum magnetotacticum) where the oxidase is required for microaerophillic respiration. Particularly in symbiotic rhizobia, the aerobic respiration using the cbb3 cytochrome c oxidase during root nodule symbiosis meets the high energy demand for nitrogen fixation, an energy-consuming process requiring up to 40 ATP to reduce one molecule of N 2 to NH3.36,62 The cbb3 cytochrome c oxidase is also present in the purple non-sulfur photosynthetic bacteria R. capsulatus and R. sphaeroides (where it is not required for N2-fixation per se), as well as in their close non-photosynthetic relative Paracoccus denitrificans. In photosynthetic bacteria, we have shown it to play an additional role as an oxygen sensor, which will be described below.

3.1 cbb 3 Cytochrome Oxidase The cbb3 cytochrome c oxidase is encoded by the ccoNOQP (fixNOQP) operon (Fig. 5) and its genomic organization is well conserved among various bacteria 79 (Fig. 6). The ccoN (fixN) gene encodes the catalytic subunit of the oxidase which is homologous to the subunit I of the aa3 oxi­ dase. The ccoO ifixO) and ccoP (fixP) genes encode membrane-bound mono- and di-heme cytochrome c with the C-terminal domains facing the periplasm, respectively. On the basis of determined redox potentials, it was predicted that electrons are transferred from cytochrome c (cytochromes c2 and c in R. sphaeroides and R. capsulatus) to CcoN via CcoP and CcoO, Fig. 5,30. The CcoQ (FixQ) is the smallest subunit of the oxidase, consisting of 48 - 73 amino acids. Its primary structure shows a basal level of homology to the subunit IV (CtaH) of the aa3 oxidase in the membrane spanning helix region.83 As inferred from the known crystal

370 Cellular Implications ofRedox Signalling R. sphaeroides R. capmlatus

I hemZ }fnr0p»nr^ I M*AFE | — N ^ H

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Fig. 6. Diagrams illustrating the genetic organization of the cco(fix)NOQP and rdxBHIS (fixIccoGHIS) operon and their flanking regions from R. sphaeroides,66 R. capsulatus,11 B. japonicum,61 R. meliloti,35 P. denitrificans (Genebank acces­ sion number U34353), and A. caulinodans.u The fnrL and fnrP genes are anaerobiosis transcriptional regulators. The fixJ-fixL is a two-component regulation system. The hisAFE genes are involved in histidine biosynthesis. The hemZ and hemN genes encode isoenzymes of coproporphyrinogen III oxidase.

structure of the aa3 oxidase, 3 3 C c o Q a p p e a r s to b e b o u n d w i t h i n the m e m ­ b r a n e w i t h its C-terminus p r o t r u d i n g to the periplasmic space. R e m o v a l of C c o Q (FixQ) w a s s h o w n not to significantly affect the catalytic p r o p e r ­ ties of the oxidase. 55,94 H o w e v e r , recent u n p u b l i s h e d d a t a i n our labora­ tory indicate that C c o Q m i g h t b e i n v o l v e d in the stabilization of the cbb3 oxidase i n the presence of oxygen. T h e catalytic s u b u n i t (CcoN or FixN) of the cbb3 oxidase contains six conserved histidine residues w h i c h p r o v i d e ligands to the low spin h e m e a n d the binuclear center (Fig. 5). 95 All of these histidines are also conserved i n the catalytic s u b u n i t of m e m b e r s of the h e m e - c o p p e r oxidase superfamily. Additionally, t h e histidine r e s i d u e (H397 in C c o N of R. sphaeroides) involved in the ligation to a Mn 2 + or Mg 2 * is conserved in the catalytic s u b u n i t of the aa3 a n d cbb3 cytochrome c oxidases. The low spin h e m e a n d binuclear center a r e non-covalently b o u n d t o the apoprotein, CcoN subunit, w h e r e a s the h e m e c moieties found in CcoO a n d CcoP are covalently b o u n d via thioether b o n d s to the cysteines of the h e m e binding motif, CXXCH. The histidine i n this motif serves as an axial ligand of the h e m e iron. The second axial ligand is p r o ­ v i d e d b y m e t h i o n i n e located far from the h e m e b i n d i n g motif. Consistent w i t h the physiological role of the cbb3 oxidase in aerobic r e s p i r a t i o n u n d e r microaerobic c o n d i t i o n s , t h e ceoNOQP (fixNOQP)

Redox Flow as an Instrument for Gene Regulation

371

operon is induced in response to reduced 0 2 tensions.22-50,69,81'82 In most cases, the induction is exerted by regulatory proteins which are classified to the Fnr family of regulatory proteins on the basis of their involvement in redox-dependent regulation and their primary structure similarity to £. coli Fnr. The prototype of Fnr homologues contains four cysteine residues in the N-terminal region which have been shown to ligate the [4Fe-4S]2+ cluster, and a helix-turn-helix secondary structure in the C-terminal region.39 Since they have the [4Fe-4S]2+ center as a redox cofactor, the Fnr homologues in this group apparently sense 0 2 directly as sug­ gested for E. coli Fnr, and thereby regulate the expression of the ccoNOQP (fixNOQP) operon in response to 0 2 availability. FnrL is also involved in the regulation of a selective set of PS genes in R. sphaeroides (Table 1), but not R. capsulatus 58,92. However, it will not be discussed further here, since we have no evidence that FnrL "senses" the state of the ETC. Nonetheless we should note that FnrL regulation of selective PS gene expression increases the stringency of the overall regulatory network and it acts in consort with the above described regulatory proteins.

4. Redox Control The first indication that the cbb3 cytochrome c oxidase is involved in a redox-related signal transduction pathway came from the finding that inactivation of the cbb3 oxidase leads to spectral complex formation in R. sphaeroides, even under highly aerobic conditions.53,91 The spectrum of PS genes that are derepressed under aerobic conditions by the inactiva­ tion of the cbb3 oxidase (see Table 1) is coincident with those genes which are shown to be regulated by the PrrBA two-component system (Table 1, Fig. 2). Inactivation of the PrrBA system in a Ceo null mutant overrides the Cco-minus phenotype. 54 Both findings allowed us to suggest that the PrrBA two-component system resides downstream of, but within the same signal transduction pathway as the cbb3 oxidase and that the cbb3 oxidase generates a signal under aerobic conditions which shifts the rela­ tive equilibrium of PrrB activity from the kinase mode to the phosphatase mode, resulting in the repression of PS gene expression (Fig. 7). Inactivation of the major aa3 cytochrome c oxidase which shares the upstream ETC components with the cbb3 oxidase and is responsible for the bulk of the total cytochrome c oxidase activity under highly aerobic conditions (Fig. 3), leads to no change in PS gene expression (puc and puf),

372 Cellular Implications of Redox Signalling Table 1. Regulation of photosynthesis gene expression by three major regulatory systems in R. sphaeroides.

Gene or Operoii Gene Product or/and Function PuhA PufKBALMX PucBAC hemA hetriN hemZ cycA bchFNBHLM bchEJGP bchlDO bchCXYZ crtA crtlB crtDc crtEF C 0 2 & N 2 Fix Upstream redox sensor DNA-binding motif

Regulatory System PrrBA

Re RC and B875 B800-850 ALA-synthase Coproporphyrinogen III -oxidase cytochrome c2

+ + + + + + +

Bchl biosynthesis

+

Crt biosynthesis

+ +

PpsR

FnrL

+/+/+ + + -

+

-

+ cbb3 oxidase noconsensus

AppA

Internal

TGT-N12ACA

TTGATATCAA

The regulation of PS genes, which is either established experimentally or pre­ dicted on the basis of sequence analyses, is depicted using the "+" and " - " signs which indicate induction under 0 2 -limiting conditions (semi-aerobic or anaerobic) and repression under high 0 2 conditions, respectively. The active form of FnrL is thought to be a dimer containing two [4Fe-4S] centers as redox-sensing prosthetic groups and thereby it appears to sense 0 2 directly. The operon structure of bchl and crt genes is predicted on the basis of sequence information. Abbreviations: ALA, 5-aminolevulinic acid; Bchl, bacteriochlorophyll; Crt, carotenoid; RC, photochemical reaction center; B875 and B800-850, light harvesting complexes I and II, respectively.

r u l i n g out the possibility that d e r e p r e s s i o n of the PS genes o b s e r v e d in the Ceo n u l l m u t a n t s results from c h a n g e s in the r e d o x state of electron

Redox Flow as an Instrument for Gene Regulation 373

Fig. 7. Model for 0 2 signaling through the cbb3-PrrBA signal transduction pathway. In the presence of 0 2 electron flow through the cbb3 oxidase generates the signal which shifts the equilibrium of PrrB activity from the kinase mode to the phosphatase mode, resulting in dephosphorylation of PrrA. This signal is transduced to PrrB via the CcoQ subunit of the cbb3 oxidase and PrrC. The "?" mark indicates that imposition of PrrC between the cbb3 oxidase and PrrB in the signal transduction pathway is established only genetically and that the physical interaction of PrrC with other components remain to be studied. In the absence of 0 2 , electron flow through the cbb3 oxidase is greatly reduced (we have evidence that some extent of electron flow through the cbb3 oxidase occurs under anaerobic conditions). This abolishes the signal which keeps PrrB in the phosphatasedominant mode, which leaves PrrB in the default state, i.e. the kinase-dominant mode.

carriers upstream of the cbb3 oxidase.56 Furthermore, under aerobic condi­ tions the ccoQ in-frame deletion mutant retaining a catalytically func­ tional cbb3 oxidase produces spectral complexes accompanied by the aerobic derepression of PS genes, suggesting that the cbb3 oxidase itself is an O z /redox sensor.55 Inactivation of either the bc1 complex or both cytochrome c2 and c , through which electrons are transferred from the quinone pool to both cytochrome c oxidases (Fig. 3), also brings about derepression of PS genes under aerobic conditions, as observed for the Ceo mutants. 56 This finding indicates that it is the interruption of electron flow into the cbb3 oxidase that leads to the aerobic derepression of PS genes (Fig. 7). Using the bct

374

Cellular Implications ofRedox Signalling

complex-specific inhibitor myxothiazol, the correlation between the extent of electron flow through the bc^ complex and PS gene expression was exam­ ined under highly aerobic conditions.56 It was demonstrated that it is the rate or volume of electron flow through the cbb3 oxidase, rather than the binding per se of an oxygen molecule to the cbb3 oxidase that ultimately determines PS gene regulation by the PrrBA two-component system. Additional experiments using a protonophore in combination with myxo­ thiazol clearly revealed that it is not a decrease in Proton Motive Force and/or cellular levels of ATP, but the inhibition of electron flow to the cbb3 oxidase which leads to the aerobic derepression of the PS genes.56 The greater the volume of electron flow to the cbb3 oxidase, the stronger the sig­ nal generated by the oxidase to shift the equilibrium of PrrB activity toward the phosphatase mode, resulting in the repression of PS genes (Fig. 7). At this point we are confronted with the question: how is the extent of electron flow through the cbb3 oxidase translated into the regulation of the activity of the PrrB histidine kinase/phosphatase?

5. Signal Transduction Pathway Two broad mechanisms are conceivable. Firstly, electrons are transferred via the cbb3 oxidase to a redox protein(s) which in turn regulates PrrB activity in accordance with its redox state. In this case, an additional redox system which can reoxidize the protein is necessary to keep the redox signal relay recycling. The second possibility is that the conforma­ tion of the cbb3 oxidase determined by the volume of electron flow is the signal, which is transmitted to the PrrBA two-component system (Fig. 7). As mentioned previously, the removal of the CcoQ subunit affects neither cbb3 oxidase activity nor the assembly of the remaining three subunits in the membrane, implying that near normal electron flow occurs through the cbb3 oxidase lacking CcoQ. This is also true in a PrrC mutant strain. However, this form of cbb3 oxidase (lacking CcoQ) cannot generate the inhibitory signal which keeps PrrB in the phosphatase-dominant mode in the presence of 0 2 . A series of mutant forms of the cbb3 oxidase itself, were constructed by virtue of site-directed mutagenesis of the ccoN gene, which leads to variations in cytochrome c oxidase activity but which are assem­ bled properly in the membrane. 59 The results obtained from these mutants revealed that the cytochrome c oxidase activity of the altered cbb3 is not proportionally correlated with the strength of the inhibitory signal emanating from the cbb3 oxidase, as judged by the extent of spectral

Redox Flow as an Instrument for Gene Regulation

375

complex formation and pw/expression levels under aerobic conditions. Taken together, these results suggest that it is a conformational change of the cbb3 oxidase which is determined by the volume of electron flow through the oxidase and which is "sensed" and "transmitted" to control PrrBA activity. Our finding that removal of the low spin heme of the CcoNsubunit also turns on PS gene expression, 56 despite a functional cbb3 oxidase, can also be interpreted as resulting in a conformational change as a result of the loss of CcoQ binding (Fig. 5). This remains to be deter­ mined. Although the role of the CcoQ subunit in this signal transduction pathway is not unequivocally revealed, CcoQ is likely either to transpond the conformational change of the cbb3 oxidase to the membrane-localized PrrC as suggested previously19,55 or to be required for maintaining the correct conformation of the cbb3 oxidase. If the latter is true, the lack of the CcoQ subunit might lock the conformation of the oxidase in a "state" incapable of generating an inhibitory signal in the presence of 0 2 . The cbb3 oxidase has two excellent properties, which the aa3 oxidase does not share, as an oxygen sensor in order to provide for the orderly control of PS gene expression in R. sphaeroides: (1) it has a high affinity for Oj. 63 This property enables R. sphaeroides not to activate the PrrBA twocomponent system to induce the formation of the photosynthetic appara­ tus as well as N 2 - and C 0 2 fixation systems until 0 2 tensions in the environment fall to sufficiently low levels, (2) it is present in cells grown under anaerobic conditions,55,74 which allows R. sphaeroides both to main­ tain transcriptional control of those genes belonging to the PrrBA regulon under anaerobic conditions and to turn off the PrrBA system quickly as soon as it is re-exposed to 0 2 and high volume electron flow through the cbb3 oxidase resumes. Although the two-component systems (RegBA) homologous to the PrrBA two-component system were identified in the phylogenetically related photosynthetic bacteria such as R. capsulatus, R. sulfidophilum, and R. denitrificans, the involvement of the cbb3 oxidase in this signal trans­ duction pathway remains to be established in these organisms. Data sug­ gest a similar regulatory pathway may exist in a Ceo mutant of R. capsulatus.5 R. sulfidophilum and R. denitrificans are "aerobic photosyn­ thetic bacteria" in which the formation of the photosynthetic apparatus is only slightly repressed under aerobic conditions. 47 The fact that the regB and regA genes derived from R. sulfidophilum and R. denitrificans fully complement the corresponding mutant strains of R. capsulatus with regard to both aerobic repression and anaerobic induction of the PS genes suggests that an 0 2 sensor involved in the generation of an

376

Cellular Implications ofRedox Signalling

inhibitory signal to repress the RegBA system under aerobic conditions is missing in the aerobic photosynthetic bacteria.

6. PpsR/AppA, Repressor/Antirepressor Additional experiments conducted in our laboratory suggest that light regulation of PS gene expression and in part, elements of the oxygen reg­ ulatory pathway of photopigment gene expression in JR. sphaeroides, is mediated through the redox state of the quinone pool (Fig. 4). We have data which suggests that the antirepressor protein AppA (Table 1) is able to monitor the redox state of the quinone pool via a bound flavin.33 Other ligands believed to be associated with the antirepressor AppA, can inac­ tivate the repressor PpsR, which binds as an oligomer29 to the sequence GTG-N12-CAC. The presence of two PAS domains in the repressor, PpsR, could be involved in oligomerization. 29 Thus, when the quinone pool is relatively oxidized (aerobic growth or high light anaerobic growth; Figs. 3 and 4) the antirepressor is marginally functional (not reduced) and the repressor is active. As light intensity decreases, the quinone pool becomes more reduced and the antirepressor becomes active (reduced), thereby inactivating the repressor. The importance of the antirepressor, AppA, in this regulatory circuit cannot be overstated.26"28 We have estimated that the cellular levels of repressor, PpsR are virtually invariant under different growth conditions and PpsR itself is not activated nor inactivated by the presence or absence of oxygen, respectively.27 The genes under the control of the PpsR/AppA system are listed in Table 1. Thus, the antirepressor, AppA would appear to be the critical effector in this regulatory circuit by being able to control the functional state of the repressor. Preliminary characterization of AppA reveals that it contains a bound flavin at the amino end, a bound heme and an iron:sulfur center towards the central and carboxy terminal regions of the protein. 28 In an AppA null mutant, PS gene expression is diminished at all light intensities. 26 However, suppressor mutations can be isolated, all of which map to the ppsR gene. 27 This result suggests the importance of the inter­ action between these two proteins.

7. TspO TspO is an outer-membrane localized protein which is a homologue of the mammalian mitochondrial peripheral benzodiazepine receptor

Redox Flow as an Instrument for Gene Regulation

377

protein.84,87 The protein is approximately 17KDal and is rich in L-tryptophan and other aromatic residues (Tryptophan rich sensory protein). When this protein is absent, photopigment gene expression turns on at an acceler­ ated rate when cells are subject to semiaerobic conditions. 84 Interestingly, the genes which are affected by TspO are the same as those belonging to the PpsR/AppA regulon. 84 Recently, using a series of double mutant strains we have generated data supporting the idea that TspO in fact, works through this regulatory circuit. How? Although this is still a work in progress, the results suggest that TspO is involved in the efflux of intermediates in the porphyrin biosynthetic pathway and we believe that one of these intermediates, derived from coproporphyrinogen III, is a co-activator of AppA.88,89 Thus, the level of this co-activator is normally low in the wild-type, but in the TspO mutant it builds up, serving to activate AppA, which in turn inactivates PpsR. Supporting this hypothesis is the observation that extra copies of the hemN gene (Table 1), encoding an aerobically active coproporphyrinogen III oxidase, will produce in wild-type, a TspO" like phenotype. 88 This observation fits with the idea that AppA binds some form of tetrapyrrole. Thus, the TspO system merely modulates or dampens the turn on of the release from the repressor effect on PS gene expression. Very recently a paper by Davey and de Brujn8 revealed the presence of a TspO-like protein in S. meliloti which works through the FixL system to regulate downstream gene expression. The R. sphaeroides TspO can func­ tionally substitute for the S. meliloti protein. Interestingly, the other regulon involving the Prr activation system is also modulated by the action of FnrL which increases the expression of ccoNOQP as oxygen levels decrease (Table 1) thus, dampening the diminu­ tion of the cbb3 generated inhibitory signal as oxygen levels decline.

8. Rdx In our investigations of the signal transduction pathway described above we identified an additional member of this pathway, whose precise role is not yet understood. The RdxBHIS (Redox) protein complex was identi­ fied following the observation that inactivation of the rdxB gene also resulted in PS gene expression under aerobic conditions. 53 Accompanying this defect was the increased synthesis of the carotenoid (Crt) spheroidenone (SO) at the expense of the Crt, spheroidene (SE), also observed when electron transport through the cbb3 oxidase is interrupted.53""55,68

378

Cellular Implications ofRedox Signalling

All of RdxBHIS components of R. sphaeroides 2.4.1 (these are FixGHIS in Rhizobium and Ceo GHIS in R. capsulatus) appear to be membrane pro­ teins containing at least one membrane-spanning region.53'68 The rdxBHIS gene cluster is located immediately downstream of the ccoNOQP operon on Chromosome I (Fig. 6, Ref. 90). The first gene, rdxB, was initially iden­ tified as a homologue of the rdxA gene located on Chromosome II.49,53 The amino acid sequence of RdxA and RdxB reveal 65% identity and 80% similarity.49 In the proposed RdxA model which was shown to be membrane localized following an analysis using phoA fusions, all 12 cysteine residues containing the 2[4Fe-4S] binding motifs are located on the cytoplasmic side of the membrane. 49 The 2[4Fe-4S] binding motifs are also well conserved in other redox proteins such as bacterial ferredoxin, fumarate reductase, and succinate dehydrogenase. Thus, it was proposed that RdxB is likely to be involved in cellular redox processes.53,55,68 Rdxl contains all of the sequence motifs for a membrane localized CPx-type ATPase.65,77 Because CPx-type ATPases are important enzymes for metal (Cu2+) homeostasis, 2,57 Rdxl is assumed to function as a copper transport protein.35,42,44,61,68 Unlike the above two proteins, RdxH and RdxS have no recognizable protein motifs.35,42,44,61,68 The gene clusters ccoNOQP and rdxBHIS from several bacteria have been completely sequenced.35,41,44,53,61,68 In most cases, the structural genes are in the same order, ccoNOQP followed by rdxBHIS, although flanking regions are different. Several examples of this gene organization are shown in Fig. 6.

8.1 rdxBHIS Early studies using S. meliloti fixGHIS:Ti\5 insertion mutations suggested that fixGHIS constitutes a single transcription unit.35 More recently, it was reported using lacZ fusions that the ccoG, ccoH, ccol, and ccoS genes from R. capsulatus are expressed independently. 42 Expression of rdxBHIS from R. sphaeroides 2.4.1 is more complicated. Under aerobic conditions, the rdxBHIS cluster is expressed predominantly as two transcripts, rdxBH and rdxIS. Under anaerobic conditions, a ccoNOQPrdxBH co-transcript form exists in addition to the rdxIS transcript. 67 The growth of a ccoGHIS dele­ tion mutation of R. capsulatus was complemented with both ccoNOQP and ccoGHIS, but not with the ccoGHIS cluster alone.42 The co-transcription of ccoNOQP-rdxBH could provide the answer to this phenotype as observed for R. sphaeroides.

Redox Flow as an Instrument for Gene Regulation

379

The presence of an "anaerobox", a DNA-binding site for the Fnr/ FixK-like anaerobic transcriptional regulators (Table 1), upstream of the rdxBHIS/fixGHIS cluster suggests that oxygen regulation of gene expres­ sion is mediated, in part, through the anaerobic regulator. The Fnrbinding motif is present in the sequences upstream of rdxB/fixG from S. meliloti,35 B. japonicum,61 and R. sphaeroides,53'68-90 but is not found upstream of the sequences for A. caulinodans44 and R. capsulatus.41 Expression of the B. japonicum fixGHIS is highly induced in cells grown microaerobically or anaerobically, 61 while in R. sphaeroides rdxB is expressed to a greater extent under aerobic conditions. 67 The effect of mutation on rdxBHIS (fix/ccoGHIS) has been described for several species including S. meliloti,35 A. caulinodans,44 B. japonicum,61 R. capsulatus,41'61 and R. sphaeroides.53,68 In S. meliloti, mutations in ccoGHIS caused a defect in symbiotic nitrogen fixation (Fix") similar to the phenotype of a ccoNOQP mutant of S. meliloti.35 More detailed characterizations of fixGHIS mutations were performed in B. japonicum.61 The fixGHIS dele­ tion mutation of B. japonicum had less than one-third of the whole cell res­ piratory activity when compared to wild type, and showed 60-70% decreased levels of membrane-bound b- and c- type cytochromes. This phenotype is caused by a defect of the post-transcriptional modification of the cbb3 cytochrome oxidase. Thus, it was suggested that the fixGHIS gene products are involved in the assembly and/or stability of the cbb3 cytochrome oxidase necessary for nitrogen fixation. However, mutations in both gene clusters in A. caulinodans caused only a partial Fix" pheno­ type. This partial phenotype is explained by the functional compensation, for the loss of the cbb3 cytochrome oxidase, by a d-type cytochrome oxi­ dase that is not present in B. japonicum.*4 Specific in-frame, non-polar mutations were created to investigate the function of each gene of the rdxBHIS (ccoGHIS) cluster in both R. sphaeroides68 and R. capsulatus.42 The absence of the ccoG from R. capsulatus does not significantly affect cbb3 cytochrome oxidase activity or amount. However, ccoH and ccol in-frame mutations of R. capsulatus show no cbb3 cytochrome oxidase activity nor detectable proteins by Western blot analysis. Mutation of the metal bind­ ing domain of Ccol, also exhibited decreased amounts of the cbb3 cytochrome oxidase, suggesting that metal ions (Cu2+) transported by Ccol have an important role in cbb3 cytochrome oxidase maturation (Fig. 8). A CcoS mutant strain of R. capsulatus contains wild-type amounts of inactive cbb3 cytochrome oxidase protein. Reduced minus oxidized spectra and FTIR difference spectra analysis showed the absence of heme b, heme b„ and CuR cofactor in the membranes of the ccoS in-frame mutation while

380

Cellular Implications ofRedox Signalling

c-type heme was present. Thus, it was suggested that CcoH and Ccol are needed to maintain the normal steady-state amounts of the enzyme while the small protein, CcoS, is essential for the incorporation of the redox-active prosthetic group (heme b, heme b 3 , and CuB) into the cbb3 cytochrome oxidase.42 In R. sphaeroides, and in R. capsulatus the rdxB in-frame mutation exhibited a phenotype having normal cbb3 cytochrome oxidase activity. But only in R. sphaeroides was it shown that the mutant also revealed increased PS gene expression under aerobic conditions. Reduced minus oxidized spectra of the membrane fractions of RdxH, Rdxl, and RdxS mutants of R. sphaeroides revealed that both cytochromes b and c are decreased. The activity and protein levels of the cbb3 cytochrome oxidase was also impaired in RdxH, Rdxl, and RdxS mutant strains. The RdxBHIS gene products appear to have a high measure of specificity affecting the cbb3 cytochrome oxidase, but showing no effect on the closely related aa3 type cytochrome oxidase in both R. sphaeroides and B. japonicum.61'68

8.2 RdxB Although the rdxBHIS mutations from different species are shown to have different phenotypes, RdxH, Rdxl, and RdxS function to maintain an active cbb3 cytochrome oxidase by post-transcriptional modification (Fig. 8) in R. sphaeroides.68 Unlike the other components, RdxB does not show any effect on the cbb3 cytochrome oxidase, but does reveal increased PS gene expression under aerobic conditions as well as altered carotenoid levels under anaerobic photosynthetic conditions.53'55,68 Considering the close functional relationship of RdxH, Rdxl, and RdxS on the structure of the cbb3 cytochrome oxidase, it is speculated that RdxB interacts with the cbb3 oxidase by being able to receive electrons from the cbb3 cytochrome oxidase, and thus mutants of RdxB alter the flow of reductant through cbb3 cytochrome oxidase in the presence of 0 2 , by causing a negative "back pressure" to reductant flow to the cbb3 oxidase resulting in PS gene expression (Fig. 8). The fact that RdxB mutants give rise to an altered carotenoid compo­ sitions under anaerobic photosynthetic conditions, we believe, supports the idea of reductant flow to RdxB from the cbb3 oxidase under both aerobic and anaerobic conditions. The carotenoid content of R. sphaeroides is primarily composed of spheroidene (SE) and spheroidenone (SO) in the ratio of ~ 95:5 respectively

Redox Flow as an Instrument for Gene Regulation

381

x=o SE{B800-B850} Electron transport chain

CrtA SO(RC,B875) X-OH

Fig. 8. Hypothetical pathway of electron flow through the RdxBHIS for R. sphaeroides 2.4.1. X = O is a hypothetical 2-oxo donor to SE in the presence of CrtA, SE is converted to SO. X = O is believed to be converted to X-OH, by an electron. under anaerobic photosynthetic conditions. 85 However, the carotenoid composition in each of the CcoNOP and RdxBHIS mutants, except for the CcoQ mutant grown under photosynthetic conditions, 55 is altered so that high levels of SO to SE (90:10 respectively) are observed.53,55,68 Because the cbb3 cytochrome oxidase is unaffected in the CcoQ mutant, as is the carotenoid composition, it is assumed that alteration in the redox state of a critical redox donor (Fig. 8) is the reason for SO accumulation. 55 In the RdxB mutant, like the CcoQ mutant, the cbb3 cytochrome oxidase is not affected, but in the former the carotenoid composition is altered to favor SO accumulation. 55,68 Therefore, it is sug­ gested that RdxB accepts electrons originating from the cbb3 cytochrome oxidase under anaerobic photosynthetic conditions and that this reduc­ ing power is involved in maintaining the reduced Crt, SE.55,68,85 In the absence of these reducing equivalents, the 2-OXO containing Crt, SO accumulates.55,68,85 Since the 2-OXO of SO does not come from water under anaerobic conditions, 86 we hypothesize an organic donor, which can be kept in the reduced state via the flow of reductant through the cbby RdxB pathway, (X = O -» H-X-OH) (Fig. 8). When that flow of reductant is removed, then X = O accumulates enhancing the conver­ sion of SE to SO by the CrtA protein. 53 The importance of this pathway illustrates two concepts: (1) there is reductant flow through the terminal oxidase cbb3 under anaerobic condi­ tions, and (2) this is an important post-transcriptional regulatory pathway in maintaining the levels of the B800-850 and B875 spectral complexes. In the former, SE is the preferred Crt for assembly of the spectral complex, whereas for the latter, there is no preferred Crt.8S

382

Cellular Implications ofRedox Signalling

9. Conclusions We have described some of the key components involved in the regulation of PS gene expression in R. sphaeroides 2.4.1 and how these components respond to oxygen and light to effect the regulation of the PS genes. Central to this regulatory system is the electron transport chain whose state can be monitored upstream and downstream of the ubiquinol bc1 oxidoreductase complex. Upstream it is the redox state of the quinone pool and downstream it is the volume of electron flow through the cbb3 terminal oxidase which provide the signals to ultimately control PS gene expression. The signal transducing components comprising each of these regulatory circuits have been identified. Each circuit ends with a unique set of regulatory proteins, the PpsR/AppA, repressor/antirepressor and the PrrBA two component activation system. The latter represents a new and wide-spread global regulator and the former introduces a new redox active protein pair which connects gene control to electron transport.

10. Perspectives We have developed a model to explain PS gene expression in R. sphaeroides. This model is backed by considerable experimental evi­ dence, but much remains to be accomplished, e.g: (1) the nature of the inhibitory signal generated at the level of the cbb3 oxidase and the mecha­ nisms by which it is transmitted, (2) to prove the physical interactions of suspected members of the signal transduction pathway, (3) to determine precisely how the membrane-localized histidine kinase switches, in the dynamic sense, between a kinase dominant and phosphatase dominant mode, (4) to reveal the mechanisms of interaction between AppA and PpsR, (5) and to ascertain how AppA monitors redox state of the quinone pool. An additional critical question is to define the extent of the Prr acti­ vation system in R. sphaeroides and how widespread is it amongst differ­ ent bacterial systems? Thus, there is much to do. However, and most importantly is that it is unlikely that the ability of an electron transport chain to act as an instrument of gene regulation was invented once, in R. sphaeroides. There is every reason to believe that this form of gene control will be wide spread in both prokaryotes and eukaryotes and it will be incumbent upon others to make these revelations. There are numerous experimental systems in bacteria, plants and animals which point to this direction. It is also likely that such a mechanism of gene

Redox Flow as an Instrument for Gene Regulation

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regulation will be of fundamental importance because it possesses the inherent ability to coordinate the activities of diverse regulatory pathways.

Acknowledgments This work is supported by NIH grants GM15590 and GM55481.

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Chapter 17 The Permeability Transition Pore as Source and Target of Oxidative Stress Paolo Bernardi CNR Unit for the Study of Biomembranes, University ofPadova Medical School, Italy. [email protected]

Keywords: permeability transition, oxidative stress, cell injury, mitochondria

1. Summary This review summarizes our work on the regulation of the permeability transition pore, a cyclosporin A-sensitive mitochondrial channel that may play a role in a variety of forms of cell death. The basic bioenergetics aspects of pore modulation are discussed, with some emphasis on the links between oxidative stress and pore dysregulation as a potential cause of mitochondrial dysfunction that may be relevant to cell injury. The initial event of energy conservation is charge separation at the inner mitochondrial membrane. Electrons deriving from intermediary metabo­ lism are donated to complexes I or II, and funneled to oxygen through the respiratory chain in a process coupled to H + ejection on the redox H + pumps. Since the passive membrane permeability is low, H + ejection results in the establishment of a H+ electrochemical gradient, which can then be utilized as the energy source for ATP synthesis as well as for a variety of other processes.73 Mitochondrial respiration also generates the superoxide anion (02~) in a process that is estimated to account for 0.4 to 4% of the total 0 2 consumption. 12 Under physiological conditions 02~ is reduced to H 2 O z by superoxide dismutases (SOD), and is then converted to H 2 0 by catalase and glutathione peroxidases. Yet, excess 02~ and H 2 0 2 can participate in the Fenton reaction, which produces OH' and can start the generation of other highly damaging reactive oxygen species (ROS).40 The pathophysiological 393

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Cellular Implications ofRedox Signalling

relevance of these processes is illustrated most clearly by the consequences of genetic inactivation of SOD, whose phenotype is particularly severe when the mitochondrial form of the enzyme, SOD2, has been suppressed. 72 SOD2-deficient mice indeed die at about 8 days of age from dilated cardiomyopathy, a condition that can be eliminated by injection of antioxidants that convert 02~ to H 2 0 2 . 72 Mitochondrially-generated ROS are suspected to play a role in a variety of pathological conditions such as Alzheimer and Parkinson diseases, many mitochondrial diseases, amyotrophic lateral sclerosis, Friedreich ataxia, and ischemic diseases.105 The clinical heterogeneity of these conditions suggests that different cells may have different susceptibility to ROS-dependent damage. Identification of specific targets of ROS, and of the self-amplifying circuits that may mediate cell death, appear therefore to be of paramount importance for a better understanding and treatment of these high-prevalence conditions.105 An attractive link between oxidative damage and cell death is the mito­ chondrion itself.63 ROS-induced damage can affect energy conservation through (1) inactivation of aconitase,39 with inhibition of the Krebs cycle (lack of supply of reducing equivalents); (2) inhibition of respiratory com­ plexes (interruption of electron flow); and (3) increased permeability of the inner membrane via opening of the mitochondrial permeability transition pore (FTP), an inner membrane voltage-dependent channel that displays a striking modulation by redox events. 5 PTP opening in vivo may be followed by mitochondrial swelling and release of apoptogenic proteins such as cytochrome c,66 apoptosis inducing factor98 and Smac-Diablo27,29 [see Ref. 7 for a recent review], and it is currently one of the most studied targets for therapeutic intervention in diseases where ROS are suspected to play a key role. Of particular relevance to the present discussion are the findings that an increased probability of pore opening is caused by membrane depolar­ ization;4 that this effect is potentiated by oxidation of vicinal thiols and of matrix pyridine nucleotides;20,21 and that the pore open-closed transitions display a striking modulation by electron flow through complex I.34 The importance of the PTP as a target of oxidative damage is stressed by the recent finding that mitochondria from SOD2 (+/-) mice were sensitized to PTP opening, an event that correlated with early onset of apoptosis. 58

2. The Permeability Transition The mitochondrial permeability transition (PT) is a generalized perme­ ability increase of the inner membrane which may result from the in vitro

The Permeability Transition Pore as Source and Target ofOxidative Stress

395

accumulation of Ca2+, or simply as a consequence of in vitro aging.46,48,49 The permeability pathway has an exclusion limit of approximately 1500 Za, and can be induced by a wide variety of compounds that have no structural or functional similarity [see Ref. 5 for review]. In the 1970s, Pfeiffer and coworkers proposed the "membrane" theory of the PT. The defect was traced to the membrane itself, which would undergo major changes of permeability as a result of the accumulation of acyllysophospholipids following activation of Ca 2+ -dependent phospholipase A2.3,87 This theory accounted for the effects of many inducers and inhibitors and could readily explain the lack of selectivity of the permeability pathway. An alternative theory, which had already been proposed in the early 1970s71 considered the PT as linked to reversible opening of a pore regu­ lated by a variety of effectors. The pore theory was fully developed in the late 1970s by Hunter and Haworth,46,48,49 but it only gained consensus with the later demonstration that the PT could be inhibited by nanomolar concentrations of cyclosporin A (CsA).13,22,37 The electrophysiological demonstration that the mitochondrial megachannel, a high-conductance inner membrane channel,55,86 is inhibited by CsA101 and responds to most inducers and inhibitors as does the PT9,69,100102 left little doubt that the PT is mediated by opening of this high conductance channel. The PTP is regulated by a variety of factors. While modulation by oxidation-reduc­ tion events is covered in the following paragraphs, I briefly list here the major regulatory factors of the PTP open-closed transitions (Fig. 1). A more detailed account can be found in Ref. 5.

2.1 Divalent Cations The PT is greatly favoured by accumulation of Ca2+ ions in the matrix, while it is counteracted by other Me2* ions like Mg2+, Si2"1" and Mn2+. Pore opening in vitro can be easily achieved at micromolar Ca2+ concentrations and the Ca2+ requirement should therefore not be intended in the sense of a massive overload. The general effects of Me2+ ions on the pore can be rationalized with the existence of two separate binding sites: an external site whose occupancy by Me2+ ions (like Sr2*, Mn2+ and Ca2+ itself, apparent I50 = 0.2 mM) decreases the probability of pore opening; and an internal site whose occupancy by Ca2+ increases the probability of pore opening, all other Me2+ (Sr2+, Mn2+) being inhibitory and in apparent competition with Ca2+.10 The well-known inducing effects of Pi can be partly explained by its ability of decreasing the intramitochondrial free [Mg2+].

396 Cellular Implications ofRedox Signalling

The Permeability Transition Pore

Fig. 1. Regulation of the PTP. The scheme illustrates the factors favoring a closed or an open conformation of the PTP. Plus and minus signs, transmembrane elec­ trical potential; Q, quinones. For details, see text and references therein.

2.2 CsA and Mitochondrial Cyclophilin Most biological effects of the immunosuppressant peptide CsA are mediated by binding to intracellular receptors, the cyclophilins, which all possess a peptidyl-prolyl-cz's-trans-isomerase (rotamase) activity that is inhibited by CsA binding. Mitochondria possess a unique matrix cyclophilin, cyclophilin D,107 but apparently no other cyclosporin-binding proteins. 75 The most plausible hypothesis is that cyclophilin D binding from the matrix side of the inner membrane modulates the pore conduc­ tance, and that CsA may be inhibiting the pore indirectly by modifying its interactions with cyclophilin D.18-75 We have established that calcineurin inhibition is not involved in pore modulation by cyclophilin D since N-MethylVal-4-cyclosporin, a derivative that binds cyclophilin but not calcineurin, is as effective as CsA at pore inhibition. 75 It appears that the enzymatic activity of cyclophilin is not essential for its effects on the pore, since diethylpyrocarbonate promotes pore opening at concentrations that fully inhibit the rotamase activity.91

The Permeability Transition Pore as Source and Target of Oxidative Stress 397

Fig. 2. Mitochondrial production and scavenging of reactive oxygen species. Monovalent reduction of oxygen leads to the production of superoxide anion, which is scavenged to hydrogen peroxide by superoxide dismutase. Glutathione peroxidase (GP) reduces hydrogen peroxide at the expense of GSH, which is regenerated by glutathione reductase (GR) at the expense of NADPH. Note that NADPH can be regenerated by transhydrogenation from NADH by transhydrogenase (TH), a proton pump that utilizes the proton electrochemical gradient to shift the equilibrium towards NADPH.47 Through TH the proton electrochemical gradient is directly utilized to maintain antioxidant defenses, and depolarization mediated by a PT or other causes makes mitochondria more prone to damage deriving from production of hydrogen peroxide.

2.3 Matrix pH and Pi Since the early work of Hunter and Haworth it had been appreciated that the PT is inhibited at acidic p H values and stimulated by matrix Pi.48 Our demonstration that inhibition by acidic p H is exerted from the matrix side 9 through protonation of critical histidyl residue(s) 76 repre­ sented the first indication that the pore could be indirectly controlled by proton pumping, and that matrix alkalinization accompanying Ca2+ uptake could be a factor in PTP opening. In a recent study we have found, however, that inhibition of the PTP by acidic p H may be offset in respiring mitochondria by an increased Pi uptake, which instead favors PTP opening. 60

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Cellular Implications ofRedox Signalling

2.4 Membrane Potential The PTP open-closed transitions are affected by the transmembrane voltage, in the sense that physiological (more negative) potentials tend to stabilize the pore in the closed conformation while depolarization may cause PTP opening. 4 The voltage dependence can be observed by modulating the membrane potential with proton 4,80 or potassium currents, 93 as well as by modulating the applied voltage in patch-clamp experiments. 103 However, many pore effectors appear to modify the threshold voltage (the "gating potential") at which opening occurs rather than affecting the membrane potential as such. 81 Pore inducers shift the apparent gating potential to more negative (physiological) values, thereby favoring pore opening, while pore inhibitors have the opposite effect and favor its closure. 82 The PTP may also sense changes of the surface potential, a more positive surface potential favoring closure. 62 This could explain why atractylate and bongkrekate, which both inhibit the adenine nucleotide translocator (ANT), have opposite effects on the pore [atractylate favors while bongkrekate inhibits the PTj 46,48,49 i n d e e d atractylate and bongkrekate lock the ANT in two different conformations, i.e. the "c" and "m" conformation, respectively [Ref. 90 and references therein]. Given that the "c" conformation exposes a large number of negative charges of the abundant ANT to the cytosolic surface of the inner membrane, thus decreasing the surface potential, 68 the effects of the ANT conformation can be accomodated within the framework of the pore voltage dependence. We postulated the existence of a sensor decoding the voltage changes into variations of the probability of pore opening, 81 and showed that arginine residues may play an important role in voltage and Ca2+ sensing. 30 It appears plausible that the PTP-inducing effects of GD3 ganglioside 94 and of arachidonic adic85,92 may be mediated by surface potential effects at the voltage sensor.

2.5 Electron Flux at Complex I The PTP is regulated by electron flux through Complex I of the respira­ tory chain in the sense that an increased electron flux through Complex I is a favoring condition for PTP opening, an effect that could be clearly dis­ sociated from oxidation of pyridine nucleotides. 35

The Permeability Transition Pore as Source and Target ofOxidative Stress

399

2.6 Quinones We have discovered that quinones are potent PTP effectors,36 and our studies revealed the existence of three classes of quinones: (1) PTP inhibitors [ubiquinone 0; decylubiquinone; ubiquinone 10; 2,3-dimethyl6-decyl-l / 4-benzoquinone; 2,3,5-trimethyl-6-geranyl-l,4-benzoquinone]; (2) PTP inducers [2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-l,4benzoquinone; 2 / 5-dihydroxy-6-undecyl-l,4-benzoquinone]; and (3) PTPinactive quinones that counteract the effects of both inhibitors and inducers [ubiquinone 5; 2,3,5-trimethyl-6-(3-hydroxyisoamyl)-l,4-benzoquinone]. 106 The structure-function correlation indicates that minor modifications in the isoprenoid side chain can turn an inhibitor into an activator, and that the methoxy groups are not essential for the effects of quinones on the PTP. These quinone analogs have a similar midpoint potential and decrease mitochondrial production of reactive oxygen species to the same extent, supporting the hypothesis that quinones may modulate the PTP through a common binding site rather than through oxidation-reduction reactions.106 Occupancy of this site could modulate the PTP open-closed transitions through Ca 2+ -binding affinity.106

2.7 Oxidative Stress The relationships between oxidative stress and PTP are discussed in the following paragraphs.

3. The Pore as a Target of Oxidative Stress Oxidative stress has long been known to favor the PT.108 Pore opening is favored by oxidants of both pyridine nucleotides (like acetoacetate and oxaloacetate), glutathione (like fert-butylhydroperoxide), and of dithiols (like diamide), as well as by dithiol crosslinkers (like phenylarsine oxide and arsenite). Since mitochondrial levels of pyridine nucleotides and glutathione are connected through energy-linked transhydrogenase 1,47 and glutathione reductase (Fig. 2), it has long been debated whether NADH, NADPH or GSH levels were the relevant factor in pore modulation. 108 We have been able to resolve this long-standing issue by independently modulating the levels of reduced pyridine nucleotides and glutathione.

400 Cellular Implications ofRedox Signalling

S site "~*SH

Reduction

s-

—SH

Oxidation

I S"

\MBM(NEM} DTT

.S—MBM(NEM)

HO—As

•SH lower '"^•^jg—

Open probability

higfier -w*"5*'

Fig. 3. Modulation of the PTP open-closed transitions at the S-site. A lower open probability corresponds to the dithiol while a higher open probability accompanies the transition to the disulfide. Blocking the S-site with low concentrations (10-20 (J.M) of monobromobimane (MBM) or NEM prevents PTP opening by oxidants like diamide. The oxidized state of the S-site can be mimicked by trivalent arsenicals like arsenite (as shown above) or phenylarsine oxide, in a reaction that can be fully reversed by DTT. The scheme is based on the original work described in Ref. 82.

Our results indicate that three redox-sensitive sites can be experimentally distinguished, and that all can contribute to pore modulation by oxidat­ ing and reducing agents under specific conditions. (i) A first site (which we dubbed the "S-site") coincides with an oxida­ tion-reduction sensitive dithiol (Fig. 3). Cross-linking of the S-site by arsenite or phenylarsine oxide, or its oxidation by glutathione favors the PT under conditions where the pyridine nucleotides pool is demonstrably reduced. 20 Dithiothreitol can fully revert the effects of cross-linking or oxidation at this site, which is blocked by low concentrations (10-20 uM) of N-ethylmaleimide (NEM)82 or mono­ bromobimane. 19 Glutathione is in apparent equilibrium with the S-site, and it is possible that hydroperoxides affect the pore through changes in the levels of reduced glutathione rather than by direct oxidation.17

The Permeability Transition Pore as Source and Target ofOxidative Stress

401

(ii) A second site (which we dubbed the "P-site") is in apparent oxidation-reduction equilibrium with the pool of pyridine nucleotides. It was possible to demonstrate that pyridine nucleotides modulate the PT independent of the S-site because the regulation is observed even when the glutathione pool is kept in the fully reduced state or when the dithiol is reacted with arsenite. At variance from the S-site, the P-site cannot be blocked by monobromobimane, while it is sensi­ tive to NEM in the same concentration range as the S-site.17'20 (iii) A third site has been recently discovered. Treatment of mitochondria with low concentrations of the sulfhydryl oxidant copper-orf/tophenanthroline promotes pore opening. 21 At variance from all reagents acting at the S-site, with this inducer pore opening is pro­ moted rather than inhibited by micromolar concentrations of NEM, while pore closure is observed upon reduction with dithiothreitol or ^-mercaptoethanol. 21 It has long been known that the PT can also be induced by high (millimolar) concentrations of NEM.87 We unexpect­ edly found that pore opening by 1 mM NEM could be fully prevented by the subsequent addition of reductants, suggesting that pore open­ ing by substituted maleimides is a two step event. The first step would be binding to the primary reactive SH group(s). This, in turn, would cause a change in conformation unmasking a secondary, oxidizable site (probably a thiol group) mediating the second step, i.e. an oxidation leading to an increased probability of pore opening. This interpretation is supported by the finding that the PT is fully inhib­ ited by adding a reductant (dithiotreitol or fj-mercaptoethanol) after NEM, proving that oxidation occurs downstream of the NEM reac­ tion step in the pore-activating sequence. This site is distinct from the previously identified S-site since the former cannot be blocked by monobromobimane and, obviously, by NEM.21

4. The Pore as a Source of Oxidative Stress The consequences of PTP opening in isolated mitochondria are relatively well understood, and are summarized in Fig. 4: (1) Opening of the PTP causes membrane depolarization, due to proton backflow through the permeability defects, and this is followed by loss of ion homeostasis. (2) Because the proton electrochemical gradient has been dissipated, any available (glycolytic) ATP is hydrolyzed in a process that by itself can precipitate cell death by energy deprivation. 24 (3) Due to the PTP cutoff of

402

Cellular Implications ofRedox Signalling

Fig. 4. Consequences of a PT on mitochondrial energy balance, Ca2+ and volume homeostasis. Panel A: Respiring mitochondria pump protons at the energy-conserving sites (squiggle), and a proton electrochemical difference is established thanks to the low inner membrane permeability to ions and solutes. 73 ADP is transported inside the matrix in exchange with ATP (a process which is driven by the transmembrane electrical potential difference), while Pi is taken up in symport with protons (a process which is driven by the pH difference) (omit­ ted for clarity), and the proton gradient drives the synthesis of ATP.73 Owing to the high electrochemical gradient for Ca2+, this cation can be easily taken up through the Ca2+ channel (uniporter). The buildup of a large Ca2+ concentration gradient is normally prevented by Ca2+ efflux on two separate pathways [omitted for clarity, but see Ref. 5], but rises of cytosolic Ca2+ are accompanied by large rises of matrix Ca2+ that can reach the near-millimolar range. 89 Panel B: Opening of the PTP (twin barrels) causes membrane depolarization, due to proton backflow through the permeability defects (box 1), which is followed by loss of ion homeostasis, as depicted here for Ca2+. Because the proton electrochemical gradi­ ent has been dissipated, any available (e.g. glycolytic) ATP is hydrolyzed (box 2) in a process that by itself can precipitate cell death by energy deprivation [see Ref. 24 for review]. Due to the PTP cutoff of about 1500 Da solutes may be lost accord­ ing to their concentration gradient (box 3), which leads to depletion of substrates and pyridine nucleotides.25,104 As soon as this event takes place, the initial uncoupling (box 4) is followed by respiratory inhibition (box 5), which is also favored by the depletion of cytochrome c (not depicted here, see Fig. 5) that follows matrix swelling and outer membrane rupture. Note that only the inner membrane has been drawn here for the sake of clarity.

The Permeability Transition Pore as Source and Target ofOxidative Stress 403

o

SOD

H202

1= SH SH

GSSG

Fig. 5. Cytochrome c release, reactive oxygen species and FTP opening. A vicious circle. Release of cytochrome c interrupts electron flow at cytochromes b, creating a situation that is similar to that seen after mitochondrial poisoning with antimycin A. The resulting increase in the rate of generation of the superoxide anion15 may cause a surge in the production of hydrogen peroxide (box 1), lead­ ing in turn to glutathione and/or S-site oxidation which can cause PTP opening (box 2). The ensuing osmotic swelling may cause further cytochrome c release. Note that PTP opening could also be the initiating cause rather than the conse­ quence of increased production of hydrogen peroxide, although once the process is initiated it may be difficult to separate cause from effect. about 1500 Da solutes may be lost according to their concentration gradient, and this leads to depletion of substrates and pyridine nucleotides. 25104 (4) The initial uncoupling is thus followed by respiratory inhibition, which is also favored by the depletion of cytochrome c that follows matrix swelling and outer membrane rupture. 53 Release of cytochrome c interrupts electron flow at cytochromes b, and this creates a situation that is similar to that seen after mitochondrial poisoning with antimycin A, which indeed causes a very high rate of production of reactive oxygen species.59 The increased rate of generation of the superoxide anion15 may cause a surge in the production of hydro­ gen peroxide, leading in turn to glutathione a n d / o r S-site oxidation which can cause PTP opening (Fig. 5). It should be noted that PTP opening

404

Cellular Implications of Redox Signalling

can thus be both the cause or the consequence of cytochrome c release; and that in principle a PT can follow the release of cytochrome c even when the initiating event is outer membrane insertion of tBID following its caspase-8-dependerit cleavage, with BAX/BAK clustering in the outer mitochondrial membrane.31,43,64'67 It must be stressed that the consequences of PTP opening in situ are much more difficult to assess. This is partly a methodological issue, in the sense that mitochondrial function in situ is monitored with indirect tools whose limits are not always adequately appreciated [see Refs. 7, 8 for a specific discussion]; and partly due to the fact that the PTP flickers between closed and open states86 but the transitions in individual mito­ chondria may not be synchronous, a problem that becomes extremely relevant when the PTP open time is short. This behaviour is illustrated most dramatically by a study where mitochondrial membrane potential changes were resolved based on the fluorescence changes of individual mitochondria loaded with the fluorescent potentiometric probe tetramethylrhodamine ethyl ester. This study demonstrated the occurrence of transient, asynchronous cycles of depolarization-repolarization in indi­ vidual mitochondria, which became long-lasting only after a variable length of time, when they were also accompanied by permeabilization to calcein (MW = 620 Da).50 Pharmacological evidence indicates that both the reversible depolarizations (which were not accompanied by calcein diffusion) and the long-lasting depolarizations (which were accompanied by calcein diffusion) are due to PTP opening. 50 It appears permissible to conclude that the PTP can open for times that are too short for diffusion of calcein. It must be stressed that such transient opening would be missed in population studies (which includes studies on intact cells) unless individual mitochondria are resolved, and that evidence for PTP flickering in healthy cells in situ is now available.83

5. Oxidative Stress and Carcinogenesis by 2-Acetylaminofluorene The molecular definition of early alterations that might explain the selec­ tion of precancerous cells is a major goal in oncology. Since the establish­ ment of the model of the "resistant hepatocyte" by Solt and Farber, one of the key notions has been that precancerous cells are able to expand in the tissue due to their resistance against a toxic environment. 96 In a thorough search for early alterations induced in the rat liver by the carcinogen

The Permeability Transition Pore as Source and Target ofOxidative Stress 405

T

Myxothiazol

AAF =^> NO-F

-O-NH-F * •

■"

of"""""' o2 SOD V

Fig. 6. Redox cycling by 2-nitrosofluorene, a toxic metabolite of the hepatic carcinogen 2-acetylaminofluorene. The scheme illustrates the redox interactions of NOF, which is generated in the liver by the carcinogenic arylamine AAF, with the mitochondrial respiratory chain. NOF can accept electrons from the respira­ tory chain and bypass the block of myxothiazol, but the fluoronitrosyl radical can also reduce oxygen directly producting the superoxide anion.57

2-acetylaminofluorene (AAF),74 Neumann and coworkers showed that 2-nitrosofluorene (NOF), a toxic metabolite of AAF, undergoes redox cycling at the level of mitochondria 57 (Fig. 6). Interestingly, NOF impaired oxidative phosphorylation and caused PTP opening in isolated liver mitochondria at very low concentrations. 56 To elucidate whether these effects are of relevance for the tumor-promoting properties of AAF, liver mitochondria isolated from rats fed with a diet containing 0.04% AAF were studied. Strikingly, these mitochondria were highly resistant to induction of the permeability transition by NOF, indicating an adaptive response of liver mitochondria during metabolism of AAF in vivo.56 Since the mitochondrial PTP appears to play an important role in the control of apoptosis 7,61 (see also the next paragraph) the increased resistance of mitochondria against the PTP may indicate that the thres­ hold for apoptosis has been altered during feeding of AAF, which may in turn result in an increased persistance of mutated cells. Taken together, these results suggest that liver cells adapt to AAF toxicity by mechanisms

406

Cellular Implications ofRedox Signalling

that include a modified, higher threshold to PTP opening. As a result of this adaptation, increased cell survival may favor the persistance of precancerous lesions. We are currently studying the mitochondrial responses in primary cultures of hepatocytes isolated from AAF-fed rats.

6. Nature of the Permeability Transition The PT is affected by a large number of compounds with no obvious common functional or structural features.45 Among these, atractylate (which induces the PT) and bongkrekate (which inhibits it) have attracted considerable interest. Both are inhibitors of the adenine nucleotide translocase while they affect the PTP in opposite directions, atractylate favoring and bongkrekate inhibiting the PT. Direct electrophysiological evidence shows that the adenine nucleotide translocase reconstituted in giant liposomes exhibits a high-conductance channel activity stimulated by Ca2+ that exhibits a marked voltage dependence. 14 The channel displays prominent gating effects, with abrupt closures at high membrane potentials of either sign that are consistent with the voltage dependence of the pore in intact mitochondria81,82 and mitoplasts, 103 but it is insensitive to CsA.14 Complexes prepared by low detergent extraction of mitochondria and enriched in hexokinase, porin and the adenine nucleotide translocase exhibit Ca 2+ -dependent and CsA-sensitive high-conductance channel activity when reconstituted in planar lipid bilayers, and Ca 2+ -dependent and CsA-sensitive ATP and malate diffusion after incorporation in proteoliposomes.11 The most active fractions were not enriched in translocase or porin, however, but rather in a 67 kDa species which may be composed of heterodimers of these proteins. 11 In my views, subsequent studies with a similar approach have not resolved the problem, because the purity of the reconstituted fractions remains too low to assign the permeability changes to the ANT rather to any of the many other proteins present in the vesicles.70 It should be noted, however, that Crompton and coworkers have reported CsA-sensitive permeabilization of liposomes by Ca2+ plus Pi after incorporation of VDAC and the ANT purified by affinity chromatography on immobilized cyclophilin D, which was also included in the reconstituted liposomes. 23 What remains unclear is whether the permeabilizing activity can be univocally attributed to the abundant VDAC a n d / o r ANT or rather to less abundant proteins present in the fraction

The Permeability Transition Pore as Source and Target of Oxidative Stress

407

eluted from the affinity matrix. Furthermore, no evidence was presented that the CsA-treated proteoliposomes still retained fluorescein and could undergo a fluorescence increase upon the addition of melittin [see Fig. 5 in Ref. 23]. On balance, I think that a direct involvement of the ANT in pore for­ mation has not been demonstrated beyond doubt. Furthermore one should wonder why, besides atractylate and bongkrekate, no other pore inducers and inhibitors (including CsA) affect the activity and the con­ formation of the translocase. In my views, an indirect effect of the ANT on the pore still appears more likely (see Sec. 2). A univocal answer about the role of the ANT in PTP regulation should come from the analysis of mito­ chondria where the genes encoding for the ANT have been deleted by genetic methods. 41

7. A Role for the Permeability Transition in Ca+2 Homeostasis? Mitochondria participate in intracellular Ca2+ homeostasis, and they can rapidly accumulate Ca2+ after cell stimulation by a variety of signals increasing cytosolic Ca2+ in the physiological range.2,88 This is made possible by the existence of two Ca2+ transport systems: (1) the Ca2+ uniporter, a channel that allows fast (Vmax above 1200 nmol Ca2+ x mg pro­ tein^1 x min"1) Ca2+ transport along its electrochemical gradient;44 and (2) the rapid uptake mode, an efficient system for sequestration of submicromolar Ca2+ pulses which is sensitive to their frequency.97 At high mem­ brane potential, steady state Ca2+ efflux is only possible through the slow H+-Ca2+ and Na+-Ca2+ antiporters, whose combined Vmax cannot exceed about 20 nmol Ca2+ x mg protein"1 x min"1.44 While this may be adequate to compensate Ca2+ uptake when extramitochondrial free [Ca2+] is in the submicromolar range, a kinetic imbalance becomes apparent when the steady-state Ca2+ concentration is increased, posing the potential hazard of mitochondrial Ca2+ overload in vivo. We have proposed that fast mitochondrial Ca2+ release in vivo can be achieved through transient opening of the PTP operating as an inducible mitochondrial Ca2+ release channel [see Ref. 6 for a thorough discussion]. This provides an appealing link with a variety of pathological conditions characterized by Ca2+ over­ load. In this scenario, pore opening might contribute to cellular Ca2+ over­ load both because of mitochondrial release and of ATP depletion, causing

408 Cellular Implications ofRedox Signalling

Cvtosol

BCL-2

BAX/BAK

BCL-XL

Jl

Jl

V

V

f>

*'■■■'■■■■■

iiiijMifliiijjuiii

outer membrane

VDAC I

<3> CD

innmjiiiiiii

\smac>

pore

inner membrane

Matrix Fig. 7. Mitochondrial proteins involved in cell death. The scheme depicts the submitochondrial distribution of proteins involved in cell death: The inner membrane PTP; the intermembrane cytochrome c, Smac-Diablo, and apoptosis inducing factor; and the outer membrane VDAC and BCL-2 family members with antiapoptotic (e.g. BCL-2 and BCL-XL) or proapoptotic (BAX/BAK) activity.

in turn impaired Ca2+ clearance by both the endoplasmic reticulum and the plasma membrane. An intriguing link with oxidative stress is the stimulation of mitochondrial ROS production by Ca2+ uptake/ 6 which may represent a further amplifying loop that would potentiate the untowards effects of cytochrome c release on mitochondrial ROS production.

8. The Permeability Transition and Cell Death Occurrence of a long-lasting PT in vivo would represent a bioenergetic catastrophe. Mitochondria would cease to make ATP, and would rather start hydrolyzing it at the maximal attainable rates, thus contributing to ATP depletion;24 Ca2+ dysregulation would ensue both as a direct consequence of mitochondrial dysfunction and as an indirect consequence of ATP deple­ tion; respiration would slow down following release of mitochondrial NAD+,104 and its hydrolysis by outer membrane glicohydrolase24'95 could

The Permeability Transition Pore as Source and Target ofOxidative Stress

409

provide ADP-ribose with production of cyclic ADP-ribose and further release of Ca2+ from intracellular stores.26 In this light, it is not surprising that initial evidence for an involvement of the PT in cell death came from established models like ischemia-reperfusion or treatment with toxicants (oxidants in particular) or other conditions where Ca2+ overload is a common event.28,52'79 Besides ATP depletion and Ca2+ dysregulation, however, intriguing links between mitochondria and cell death have emerged in the context of apoptosis. Figure 7 summarizes a series of mitochondrial proteins that control apoptosis: the inner membrane PTP; the intermembrane cytochrome c,66 apoptosis inducing factor" and SmacDiablo;27,29 and the outer membrane members of the BCL-2 family, which can be antiapoptotic (like BCL-2 itself and BCL-XL) or proapoptotic (like BAX and BAK).42 Cytochrome c, Smac-Diablo and apoptosis inducing factor exert their proapoptotic effects only after they have been released in the cytosol, and BCL-2 family members affect the release process in a way that is consistent with their overall effects on cell survival (i.e. antiapoptotic members prevent and proapoptotic members promote the release process).42 The mechanistic aspects remain controversial, in particular the role of the outer membrane VDAC [see Ref. 7 for discussion]. While a coverage of this field is beyond the scope of this short review, it is interest­ ing to note that mitochondrial swelling following pore opening in saltcontaining media has long been known to cause release of several matrix proteins 51 and of cytochrome c.84 What needs to be stressed is that the link between PTP opening and release of proapoptotic proteins appears to be indirect, and that a major role in this process could be played by osmotic swelling of the matrix. Limited matrix swelling might increase the availability of cytochrome c for release through the tBID-dependent BAX-BAK path­ way, while extensive swelling could release all intermembrane proteins fol­ lowing outer membrane rupture [see Ref. 7 for discussion]. Although the evidence is not always as convincing as it is often assumed, 7 a causal role of the PT appears likely in many forms of cell death, and this makes the PTP an appealing target for pharmacological intervention, which appears very promising in several forms of experimental brain injiary. s2-33^38-54-65-77'78

9. Conclusions In summary, mitochondria are both sources and targets of reactive oxygen species. The exquisite sensitivity of the PTP to oxidative stress makes it an important target for ROS-dependent damage; and the consequences of

410

Cellular Implications ofRedox Signalling

PTP opening (increased ROS production, decreased ROS scavenging) may play a role in apoptotic signal transduction to the mitochondrion. These features make the PTP an attractive target for therapeutic interven­ tion in diseases where ROS play a key role.105

Acknowledgements I would like to thank Prof. Raffaele Colonna for critical reading of the manuscript. Research in my laboratory is supported by the Italian Ministry for the University and Scientific Research (Progetto "Bioenergetica e Trasporto di Membrana"), the National Research Council, Telethon-Italy (Grant No. 1147), the Armenise Harvard Foundation and the NIH-PHS (USA).

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Author Index

Soloyvov Anton, 233 Arne Holmgren, 1, 233 Avihai Danon, v, 311 Batia Zarmi, 257 Bob B. Buchanan, 99

Jesus M. Eraso, 361 Johanna Lundstrom-Ljung, 233 Jon Beckwith, 213 Jung Hyeob Roh, 361 Junji Yodoi, 115 Lukas C. Kiihn, 327

Carlos Gitler, v, 257 Christine C Winterbourn, 175 Christine H Foyer, 191 Edna Kalef, 257 Elias S.J. Arner, 27 Gisela Storz, 287

Matthew J. Wood, 287 Melissa Schwaller, 233 Orna Carmel-Harel, 287 Paolo Bernardi, 393 Peter Schurmann, 73

Hajime Nakamura, 115 Helmut Beinert, 47 Helmut Sies, 141 Hiroshi Masutani, 115

Robert Mallis, 141 Ruoyu Xiao, 233

Itaro Hattori, 115

Tal Alergand, 311 Tova Trebitsh, 311

James A Thomas, 141 Jeong-Il Oh, 361

Samuel Kaplan, 361

Yumiko Nishinaka, 115

421

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Subject Index

l-chloro-2,4-dinitrobenzene 46, 48,53 2-cys peroxiredoxin 204, 205, 208 2-mercaptoethanol 338-341, 345 Arabidopsis thaliana 198-204 aconitase 330, 331, 333, 334, 336, 338-341, 343-347, 350-352, 354, 356-358, 360 aconitase/IRP-1 336 active oxygen species 191 affinity chromatography 257-260, 262, 263, 271, 277 alkaline phosphatase 214, 215, 222, 223, 225, 226, 229, 230, 232 alkylhydroperoxidase 226 allergenicity 99,105, 111 allergens 105,106,108 antioxidant defenses 175,177 antioxidants 191,194, 204, 205 apoplast 191,194,199, 208, 211 aquaporins 208 arsenical 257, 259, 260, 262,284 arsines 257, 259 ascorbate 176,177,180,181,184-189, 191,192,194,196-199, 203-205, 207-212 ascorbate-glutathione cycle 191,192, 197,198, 200 AU-rich elements 40, 46, 54, 55 baking quality 99 Benson-Calvin cycle 196 C3 pathway of photosynthesis 195 C4 plant 195 catalase 191, 195, 204, 207-212 catalases 192 cell injury 393 cetiltrimethyammonium bromide (CTABr) 257, 258 chaperone 219, 229, 232, 233, 235

chloroplast 191,192,194-197,199, 203-205,208, 209, 211, 312, 313, 315, 316, 318-325 chloroplastic 196,199, 204, 205, 207 chloroplasts 196,197, 200, 204, 208-211 cluster interconversions 1, 8, 24 cluster stability 1 covalent regulatory process 257, 274 cytoplasm 213-216, 221-223, 225-227, 229, 231,232 cytoplasmic 328, 329, 331, 333, 334, 336, 338-341, 345-349, 352, 353, 356 cytoplasmic iron 328, 333 cytosol 197,199, 200, 203-205 defense 191-194,198, 205, 209, 212 defense responses 191,193, 205 dehydroascorbate reductase 197, 209-212 development 191, 200, 205, 211 DHAR's 204 diamide 340 dinitrohalobenzenes 40, 44, 45, 48, 50, 51, 53, 56 disulfide bond isomerization 215, 219, 231 disulfide bonds 213-216, 219, 220, 222, 223, 225-232 disulfide bridges 339-340 disulfide cascade 227 disulfide formation 319 disulfide isomerization 233-235, 238, 239, 242, 245, 252 disulfides 1, 5-7,16,18-20 DMT1 335, 336, 355 DNA synthesis 1, 4, 5 domains of PDI 233, 242, 251 drought 193, 211 dsbA 216-223, 227-229, 231 dsbB 216-218, 221, 222, 227, 230 DsbC 218-222, 226-232 423

424

Subject Index

DsbD 220-222, 229, 230, 232 DTNB5 DTT 257-260, 262-266, 269, 271-273, 275, 276, 278, 279

growth factors 257, 262, 276, 277, 282, 285 growth initiation 257, 279, 280-285 Grxl 233,234, 251, 252

ECS regulation 202 electron flow 361, 362, 373-375, 382 electron transfer 1,14,15,18 endoplasmic reticulum (ER) 233-235 erythroid amino-levulinate synthase 333 extra-chloroplastic 207 extremes of temperature 193

hydrogen peroxide (H 2 0 2 ) 328, 339, 346, 347, 348, 356, 359, 360 hydroxyl radicals 197,198, 211 hydroxyproline-rich glycoproteins 198 hypersensitive 193, 207, 210 hypoxia 346-348, 360

ferredoxin 85-87, 89, 90, 93, 95-97, 102-110,196, 204,211 ferredoxin /thioredoxin system 85, 86, 90, 95, 97,102-105,109 ferredoxin: thioredoxin reductase 105 ferredoxin-thioredoxin system 314, 315, 319 ferritin 328, 331, 333 food improvement 99 fructose 1, 6-bisphosphatase 85, 90, 100,104 fructose-1, 6-bisphosphatase 196 GDP-mannose pyrophosphorylase 198 gel-retardation assays 338 gene expression 311, 312, 315, 321-324, 361-363, 365, 366, 371, 374-377, 379, 380, 382-385, 387-390 g-glutamylcysteine synthetase 200 glutaredoxin 1-4, 6, 7 15-18, 21-23, 25, 26, 287, 289, 291, 304 glutathione 1-4, 6, 8-13,15,16,18-26, 176-178,180,185-192,194, 199-205, 207, 209, 211-213, 225, 226, 257, 258, 264, 266, 280, 285 glutathione oxidoreductase 225 glutathione peroxidases (GPX) 192 glutathione reductase 1, 2, 8,10-13, 15, 23, 24,196, 209 glutathione-S-(5-thio-2-nitrobenzoic acid GSSNB 257 glutathione-S-conjugates 200 glycollate oxidase 195

intra-molecular disulfide bridges 339, 340 IREG1 335, 336, 355 iron homeostasis 327, 352 iron metabolism 327, 346, 348, 350, 355, 357, 358, 360 iron regulatory proteins-1 328, 356, 357,360 iron responsive element (IRE) 350 iron-sulfur (Fe-S) clusters 1 IRP-1 329-331, 333, 334, 336, 338-348, 360 IRP-1 and IRP-2 329, 333, 341, 342, 344-348, 352, 358, 359 IRP-2 331, 341-346, 348, 349, 352 isomerase activity 233, 234, 237, 242, 243, 245, 247, 249, 250-252, 254, 256 L-galactono-g-lactone dehydrogenase 198 light-regulated redox signaling 312 light-signal 313, 314, 318 lymphoblast cell extract 257, 264, 271 MDHA 197-199, 204 Medicago truncatula 201 Mehler reaction 194, 196 Mehler-peroxidase 196,197 Mehler-peroxidase cycle 196,197 metal transporter DMT1 328 mitochondria 197,199, 208 mitochondrial 393-396, 399, 403-419 mitochondrial citric acid cycle 334

Subject Index

mitotic cycle 198 monodehydroascorbate reductase 196, 210 NADH 213, 226, 227 NADP-dependent malate dehydrogenase 90,101 NADPH 1, 2, 3, 5, 7-10,12,15,16, 22-24, 213,225-227 NADPH-dependent oxidoreductases 257, 270 NADPH-thioredoxin reductase-thioredoxin system 257, 260, 273, 275 N-ethylmaleimide NEM 257 NF-kappa B 207 N-iodoacetyl-3-iodotyrosine 257, 258 nitric oxide 344, 345, 350, 358-360 NO 333, 344-348, 358 non-specific peroxidases 192 oxalate 198 oxCEDTNB 257,264-267,269-271, 273, 274 oxCEGSSNB 257, 264-267, 269, 273, 274 oxidase 198 oxidase activity 233, 236, 237, 242, 250, 252 oxidation of regulatory proteins 319 oxidative burst 192,194, 210 oxidative load 195,196, 204, 205, 207, 208, 211 oxidative protein folding 233, 238, 253, 254, 256 oxidative stress 175, 179,181,185,188, 202, 206, 207, 393, 399, 401,404, 408, 409,412, 415 OxyR 287-294, 302, 304-306, 308-310 pathogen attack 195,198 pathogens 193, 207, 208 periplasm 213-216, 218, 220,222, 223, 226, 227, 229, 230, 232 permeability 393-395, 401, 405-408, 410-419 peroxiredoxin 199, 204, 205, 208

425

peroxiredoxins 1, 6,192 peroxisomal 194,195, 207 peroxisome 191,194,195 peroxisomes 197, 204 phenylarsine oxide PAO 257 phosphatases 257,260, 262, 263, 275, 280, 284 phosphotyrosine 257, 260, 262, 280, 282, 284 photorespiration 194, 195, 211 photosynthesis 191,192,194-196, 199, 208, 209, 211, 312, 313, 315, 321, 322 photosynthetic apparatus 361, 375 plasma lemma 199, 208 plasma membrane 199 plastoquinone 208 pool 327, 328, 332, 333, 339, 340, 341, 345, 349, 354 post NEM-DTT procedure 257,260, 275 prolyl hydroxylase 198 protein cysteine 141-145,154,161, 168,169 protein disulfide isomerase (PDI) 233, 235, 239 protein disulphide isomerases 204 protein folding 214, 228-230, 232 protein phosphorylation 202 protein thiol labeling 257, 258 protein-S-S-glutathione mixed disulfides 257, 264, 266 PSII196 pyridine nucleo-tide dependent disulfide oxidoreductases 257, 270 quinones 218 radical scavenging 175, 177,179-181, 183,184 receptive oxidized state 315, 318 redox buffer 233-235, 238, 242, 243, 247, 251, 252, 256 redox control 361, 371 redox potential 85, 86, 93, 98,105 redox potential of 7 redox potentials 1,15-18 redox ratio 257, 274, 275

426

Subject Index

redox regulation 5, 6,18, 20, 26 redox sensor 315 redox signaling 311, 312, 315, 316, 319, 321, 322 regulation of growth 191,193 regulatory disulfides 85, 86, 99,102, 103,106 Rhodobacter sphaeroides 2.4.1 361, 362, 384, 385, 387, 388, 390 ribonucleotide reductase 1, 3, 4, 5, 6,19, 21 ribulose-1, 5-bisphosphate carboxylase-oxygenase (Rubisco) 194 RNA-binding activity 316 RsrA 287, 288, 294-299, 302, 304,305, 307, 308 scanning and escape 233, 245, 256 SECIS 39, 42-44, 46, 53 secreted proteins 233, 235, 236, 249 secretion in yeast 233, 249, 254 sedohepulse-1, 7-bisphosphatase 196 selenazol drug ebselen 2,15 selenenylsulfide 44, 50, 53 selenite 8, 9, 22 selenocysteine 2, 8-10,12, 22-25, 39, 41-44,47, 48, 51-54, 57 selenodiglutathione 8, 9, 22 selenolate 41 selenoprotein 39-44, 52, 55 Sepharose-GSH 257, 271, 273 signal transduction 361, 362, 365, 366, 371, 374, 375, 377, 382, 385, 388 signal transfer 85, 95 singlet oxygen 197 site-directed mutagenesis 85, 99,101, 102,105 S-Nitrosylation 141,144,146-148,150, 153,155,156,158,161-163, 167-170 S-Thiolation 141, 144-149, 157, 158, 160-162,167,169 structural disulfides 233, 234, 238 substrate inhibition 233, 247-249 sulfhydryl 1, 4, 7,19, 25 sulfhydryl oxidation 141,160,162,170

sulfur nutrition 207 superoxide 176,179-184,186,187,194, 196,197, 209, 210, 211, 345, 359 superoxide dismutases 192 synthetase (GS) 200 systemic acquired resistance 195 target enzymes 85, 86, 90, 93, 97-100, 103,105,106 tartrate 198 T-cell 207 thiol redox control 1, 2, 3, 6, 7,18 thiolation 207 thiols 175,178,180,181,184,186,187 thioredoxin 1-9,11,12,14-25, 39-42, 44, 46,48, 50-57,115-118, 120-140,196, 218, 219,221-223, 225-227, 229-231, 233, 234,236, 237, 239-242, 249, 251,252, 254-256, 287, 289, 291, 295, 298-300, 303, 304, 306-309 thioredoxin f 85, 90-92, 93-95, 98,100, 103-108 thioredoxin m 85, 90-94, 98,103, 107,108 thioredoxin peroxidases 1, 6,18 thioredoxin, redox regulation 99 thioredoxin reductase 1-5, 7-15, 20, 22-25, 39, 41,42, 44, 46,48, 51-57, 223, 225, 227, 231 thioredoxin system 40, 47, 51 thioreoxin family 233, 239 three-dimensional structure 4, 7, 9,19, 25 thylakoid membranes 192,196 tocopherols 197 transferrin receptor 328, 331-333, 336, 342, 344-346, 349, 353, 354, 356, 357, 358 transition 393, 394, 405-408, 410-419 translational regulation 311-313, 322 translational regulator 315, 318 transport of hormones 200 triplet repeat 226, 231 vicinal dithiol site 316 vicinal thiols 181

Subject Index 427

vitamin C 191,194, 209,212 VTP/IPD 257, 275

xanthophyll cycle 197 xenobiotics 200

water cycle 196 water-water cycle 196 wounding 193

Yaplp 287, 288,299-307

Cellular Implications of Redox Signaling Redox regulation, like phosphorylation, is a covalent regulatory system that controls many of the normal cellular functions of all living cells and organisms. In addition, it controls how cells respond to stress involving oxidants and free radicals, which underlie many degenerative diseases. This area is undergoing a transition from general knowledge to specific description of the c o m p o n e n t s and mechanisms involved. This invaluable book provides a timely basic description of a field whose relevance to cell biology and degenerative diseases is of the utmost importance. It describes the state of the art, lays the foundations for understanding the reactions involved, and presents the prospects for future developments. It can serve as a basic text for any undergraduate or graduate course that deals with redox regulation, oxidative stress and free radicals under normal and pathological conditions in bacterial, plant and animal cells.

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