Free Radical Biology & Medicine 43 (2007) 883–898www.elsevier.com/locate/freeradbiomed

Review Article

S-glutathionylation in protein redox regulation

Isabella Dalle-Donne a,⁎, Ranieri Rossi b, Daniela Giustarini b, Roberto Colombo, Aldo Milzani a

a Department of Biology, University of Milan, I-20133 Milan, Italyb Department of Neuroscience, University of Siena, I-53100 Siena, Italy

Received 8 May 2007; revised 6 June 2007; accepted 6 June 2007Available online 15 June 2007

Abstract

Protein S-glutathionylation, the reversible formation of mixed disulfides between glutathione and low-pKa cysteinyl residues, not only is a cellularresponse to mild oxidative/nitrosative stress, but also occurs under basal (physiological) conditions. S-glutathionylation has now emerged as apotential mechanism for dynamic, posttranslational regulation of a variety of regulatory, structural, and metabolic proteins. Moreover, substantialrecent studies have implicated S-glutathionylation in the regulation of signaling andmetabolic pathways in intact cellular systems. The growing list ofS-glutathionylated proteins, in both animal and plant cells, attests to the occurrence of S-glutathionylation in cellular response pathways. Theexistence of antioxidant enzymes that specifically regulate S-glutathionylation would emphasize its importance in modulating protein function,suggesting that this protein modification too might have a role in cell signaling. The continued development of proteomic and analytical methods fordisulfide analysis will help us better understand the full extent of the roles thesemodifications play in the regulation of cell function. In this review, wedescribe recent breakthroughs in our understanding of the potential role of protein S-glutathionylation in the redox regulation of signal transduction.© 2007 Elsevier Inc. All rights reserved.

Keywords: Glutathione; Oxidative stress; Protein thiols; Redox regulation; Signal transduction; Mixed disulfides; GSH/GSSG ratio; Reactive thiolate anions; Freeradicals

Contents

Oxidative modifications of protein thiols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 884Mechanisms of S-glutathionylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 886Roles of protein S-glutathionylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 887Specificity of S-glutathionylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 888Reversibility of S-glutathionylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 889Proteins regulated by S-glutathionylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 890

Enzymes with active-site thiols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 890Signaling proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 890Transcription factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 891Ras proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 891Heat shock proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 891Ion channels and Ca2+ pumps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 891

Abbreviations: CFTR, cystic fibrosis transmembrane conductance regulator; EGF, epidermal growth factor; ERK, extracellular-signal-regulated kinase; GAPDH,glyceraldehyde-3-phosphate dehydrogenase; GRX, glutaredoxin(s); GS(O)SG, glutathione disulfide S-monoxide (also called glutathione thiosulfinate); GSH,glutathione; GSNO, S-nitrosoglutathione; GSOH, glutathione sulfenic acid; GSSG, glutathione disulfide; HDL, high-density lipoprotein; HSP, heat shock protein;LDL, low-density lipoprotein; MAPK, mitogen-activated protein kinase; MEKK1, MAPK/ERK kinase kinase 1; NF-κB, nuclear factor κB; PON1, paraoxonase 1;PSH, protein sulfhydryl group(s); PSSG, protein/glutathione mixed disulfide(s) (i.e., S-glutathionylated proteins); RNS, reactive nitrogen species; ROS, reactiveoxygen species; RyR, ryanodine receptor channel; SERCA, sarcoplasmic/endoplasmic reticulum Ca2+ ATPase; Srx1, human sulfiredoxin; TNF-α, tumor necrosisfactor α; TRX, thioredoxin(s).⁎ Corresponding author. Fax: +39 02 50314781.E-mail address: quack@unimi.it (I. Dalle-Donne).

0891-5849/$ - see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.freeradbiomed.2007.06.014

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Mitochondrial proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 892Cytoskeletal proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 892

Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 893Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 894References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 894

Oxidative stress is a situation in which cellular homeostasis isaltered because of excessive production of reactive oxygen/nitrogen species (ROS/RNS) and/or impairment of cellularantioxidant defenses, “leading to a disruption of redox signalingand control and/or molecular damage” [1]. ROS/RNS can causespecific, reversible and/or irreversible oxidative modificationson sensitive proteins that may lead to a change in the activity orfunction of the oxidized protein [2]. Most protein oxidationproducts are commonly considered as biomarkers of oxidative/nitrosative stress/damage [3,4].

Under conditions of oxidative/nitrosative stress, the thiols incysteine residues within proteins are among the most suscep-tible oxidant-sensitive targets and can undergo variousreversible and irreversible redox alterations in response toROS and/or RNS increase/exposure. All the modifications toprotein thiols can potentially affect protein activity, with thedegree depending on the importance of the cysteine residue incarrying out protein function. Actually, the oxidative modifica-tions of the cysteine sulfhydryl group have recently attractedrenewed interest, because Cys is present in the active site ofmany proteins and in protein motifs that function in proteinregulation and trafficking, cellular signaling, and control ofgene expression [5,6]. Some proteins may not contain cysteineresidues important in protein function; however, modification ofthiols may cause a conformational change that alters proteinactivity. Thus, because many redox alterations to protein thiolsare readily reversible through mechanisms involving glu-tathione (GSH), thiol redox alteration, like phosphorylation,has been suggested to be an important mechanism of turning onand off proteins, i.e., protein redox regulation, particularly inresponse to oxidative and nitrosative stress.

The exposure of cysteines on the protein surface is afunctional necessity to prevent redox changes from spreadingthrough the entire protein molecule. The surface-oriented Cysresidues are normally kept reduced and may therefore serve as“redox sensors” of the cells.

The tripeptide glutathione (L-γ-glutamyl-L-cysteinylglycine)is present in cells at millimolar concentrations (∼1–10 mM),and the ratio of GSH to glutathione disulfide (GSSG) is critical tocellular redox balance. Changes in the cellular redox status(mainly due to a decrease in the GSH/GSSG ratio and/or deple-tion of GSH by the metabolism of drugs or other xenobioticsubstances) as well as an increase in ROS and/or RNS generation(e.g., during inflammation), i.e., oxidative or nitrosative stress,may induce reversible formation of mixed disulfides betweenprotein sulfhydryl groups (PSH) and glutathione (S-glutathio-nylation) on multiple proteins, which makes of cellular gluta-thione a crucial modulating factor for an ever increasing numberof proteins.

S-glutathionylation, briefly the addition of GS− to low-pKa

Cys residues in proteins, occurs not only during oxidative/nitrosative stress, but also in a number of physiologicallyrelevant situations (i.e., basal conditions), in which it canproduce discrete modulatory effects on protein function. Thishas rapidly increased the interest in S-glutathionylated proteins(PSSG) in the past few years and the list of proteinsdemonstrated to be S-glutathionylated increases continually[for reviews, see 5,7–10]. The fact that PSSG are involved innumerous physiological processes such as growth, differentia-tion, cell cycle progression, transcriptional activity, cytoskeletalfunctions, and metabolism, suggests that S-glutathionylation isa general mechanism of redox regulation. In fact, whereasposttranslational modifications such as phosphorylation, acety-lation, and ubiquitinylation have been well established andunderstood for many years, the concept of protein S-gluta-thionylation as a posttranslational regulative modification, asopposed to a biomarker of oxidative damage, has gained accep-tance only more recently [8–14].

The present review is meant to give just an overview of thepotential role of protein sulfhydryl S-glutathionylation in theregulation of the structures/functions of a quite diverse range ofcellular proteins.

Oxidative modifications of protein thiols

Cysteinyl thiols are particularly susceptible to oxidativemodification and can undergo a diverse array of redox reactions,which are largely dependent on the species and concentration ofoxidants they contact. The two major determinants of thesusceptibility of thiols to redox regulation are the accessibility ofthe thiol within the three-dimensional structure of the protein andthe reactivity of the cysteine, which is influenced by thesurrounding amino acids. Most thiol modifications are unstableand can easily be reversed or replaced by other, more stablemodifications.

Although the thiol moiety on the side chain of cysteine isparticularly sensitive to redox reactions, not all cysteinyl thiolsare important as redox sensors, as most protein thiols do not reactwith oxidants under the conditions and at the concentrations theyare found in cells. Nevertheless, some cysteine residues aresusceptible to oxidation. The vast majority of cytoplasmicproteins contain cysteine sulfhydryls with a pKa value greaterthan 8.0 and, in the reducing environment of the cytoplasm,remain almost completely protonated at physiological pH. As aresult, they are unlikely to be reactive with ROS/RNS. However,redox-sensitive proteins have specific Cys residues that exist asthiolate anions at neutral pH, due to a lowering of their pKa

values as a result of charge interactions with neighboring

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positively charged (i.e., basic) amino acid residues, becoming“active cysteines,” which are therefore more vulnerable tooxidation [15]. The thiolate anion of redox-sensitive Cys resi-dues can readily be reversibly oxidized to sulfenic acid andprotein or mixed disulfides or irreversibly oxidized to sulfinicand sulfonic acid (Fig. 1) [16,17], which are usually detrimentalto protein function, although sulfiredoxin has recently beenfound to specifically reduce the sulfinic acid moiety in peroxi-redoxins [18–21]. Reversible oxidation is believed to protectproteins from irreversible oxidation but may also modulateprotein function.

Fig. 1. Oxidative modifications of protein thiols. (A) The oxidation of a cysteine residshown) or a sulfenic, sulfinic, or sulfonic acid derivative (the latter of which is al(cystine). Disulfides can form under oxidative conditions between two adjacent prosulfhydryl groups within a protein (intramolecular cystine or intraprotein disulfide), cprotein cysteinyl residues and low-molecular-mass thiols such as glutathione and frrespectively, i.e., S-glutathionylated or S-cysteinylated proteins. Each of these protei

Disulfides can form under oxidative conditions between twoproteins (interprotein) or within a protein (intraprotein), causingchanges in protein aggregation and conformation. As recentlyshown using proteomic techniques, intermolecular disulfidebonds are also formed in the cytoplasm upon exposure of cells tooxidative stress [16,22]. Interprotein disulfides can also haveregulatory function, as recently shown, for instance, for the typeI protein kinase A, which contains protein thiols that operate asredox sensors, forming an interprotein disulfide bond betweenits two regulatory RI subunits in response to cellular hydrogenperoxide [23]. This oxidative disulfide formation, which was

ue within a protein can result in the formation of a cysteinyl radical (Cys–SU, notways irreversible). (B) Alternatively, oxidation can result in a disulfide bridgeteins (intermolecular cystine or interprotein disulfide) or between two adjacentausing changes in protein aggregation and conformation. (C) Reaction betweenee cysteine can yield protein–glutathione or protein–cysteine mixed disulfides,n thiol modifications has the possibility of eliciting different cellular responses.

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already known but to date considered a constitutive structuralbond, causes a subcellular translocation—from the cytosol to thenuclear and myofilament compartment and, to a lesser extent, amembrane fraction—and activation of the kinase, resulting inphosphorylation of multiple established substrate proteins.

Protein sulfenic acids, which are principal products formedby protein thiols on contact with H2O2, are very unstable, beingfrequently an intermediate in sulfinic and/or sulfonic acid gene-ration or readily reacting with other vicinal or accessible (low-molecular-weight) thiols to form intra- or intermoleculardisulfides, respectively [e.g., 24]. Despite their reactivity, it isnow clear that some sulfenic acids are more stable, and these canbe identified in proteins also under physiological conditions[24–26]. Protein sulfenate formation has important roles inredox signaling, particularly in the redox regulation oftranscription factors and enzymes including tyrosine phospha-tases, peroxiredoxins, and methionine sulfoxide reductases [27].

In isolated rat hearts treated with physiologically relevantconcentrations of H2O2, Eaton and colleagues reported wide-spread protein sulfenic acid formation within cardiac tissuewhen H2O2 was elevated [24]. They concluded that proteinsulfenic acids are widespread physiologically relevant post-translational oxidative modifications that can be detected atbasal levels in healthy tissue and are elevated in response tohydrogen peroxide.

Oxidant exposure, in addition to resulting in irreversibleoxidation of Cys residues to sulfonic acids, can lead to excessivedisulfide bonding, protein misfolding, and aggregation [28,29].Excessive disulfide bonding may lead to covalent aggregatesthat are difficult to reduce even when intracellular redox con-ditions are restored to normal.

Mixed disulfides, i.e., S-thiolated proteins, form betweenprotein sulfhydryls and low-molecular-mass thiols such ashomocysteine, cysteinylglycine, free cysteine, and glutathione[30–33]. Because free Cys and GSH are the most abundant low-molecular-mass thiols in vivo, S-cysteinylated and S-glutathio-nylated proteins will be the main mixed disulfides, which are notequally distributed between extracellular and intracellularsettings.

GSH shows high negative redox potential (high electron-donating capacity) combinedwith high (millimolar) intracellularconcentration (∼1–10 mM), which generates great reducingpower [34], and thus, GSH represents the major low-molecular-mass antioxidant in cells. Major differences between cellular andextracellular compartments exist in terms of both the concentra-tions of sulfhydryl/disulfide systems and their relative redoxstates [1,35]. The intracellular concentrations of free Cys,GSSG, and cystine are lower (micromolar) than those of GSH,whereas extracellular free Cys is more abundant than GSH:actually, the Cys/cystine redox couple quantitatively representsthe largest pool of low-molecular-mass thiols and disulfides inplasma and the extracellular compartment on the whole. Themajor low-molecular-mass sulfhydryl/disulfide system in cells,GSH/GSSG, is principally in the reduced form, whereas themajor low-molecular-mass system in the extracellular compart-ment, Cys/cystine, is principally in the disulfide form, cystine.Thus, extracellular proteins may be prevalently S-cysteinylated,

whereas intracellular proteins may be prevalently S-glutathio-nylated. For example, whereas the fraction of S-thiolatedhemoglobin in red blood cells is only S-glutathionylated[36,37], plasma proteins such as albumin are mainly S-cysteinylated [33,38,39], possibly after formation of an inter-mediate sulfenic acid [25,26].

Because such a variety of protein cysteine oxidation statescan be formed, this offers the potential for oxidant-specificfunctional effects in which particular ROS/RNS can differen-tially regulate the activity of redox-sensitive proteins on the basisthat they form varied structural motifs.

Mechanisms of S-glutathionylation

Experimentally, S-glutathionylation of protein cysteinylresidues may be studied by using a number of different tech-niques [22,32,40–42], however, the mechanisms by whichglutathione can react with protein thiols are not completelyunderstood.

The normal/physiological intracellular milieu is a reducingenvironment with a GSH/GSSG ratio around or even greaterthan 100, and thus the GSH/GSSG ratio constitutes a majorredox buffer in cytosol [7,43]. Maintaining optimal GSH/GSSGratios in the cell is critical to cell survival and is important inregulating the redox state of protein thiols [34]. Changes inGSH/GSSG ratios could potentially influence a number of targetproteins by causing oxidation and disulfide exchange reactionsat specific protein cysteinyl residues. As the GSH/GSSG ratiousually exceeds 100, the oxidation of a small amount of GSH toGSSG could also promote protein S-glutathionylation, shiftingthe equilibrium in the direction of mixed disulfide formation.

Protein S-glutathionylation can occur by several reactions[5,7–9,44]: (i) direct interaction between partially oxidized(activated) protein sulfhydryls, i.e., thiyl radical (which can beformed by reaction with hydroxyl radical), sulfenic acid, orprotein S-nitrosothiol (S-nitrosated protein) and GSH; (ii) thiol/disulfide exchange reactions between protein thiols and GSSGor PSSG; (iii) reaction between protein thiols and intermediateS-nitrosothiols such as S-nitrosoglutathione (GSNO), which isable to modify PSH by both protein S-nitrosation and S-glutathionylation [44,45]; (iv) direct interaction between a freeprotein cysteinyl residue and GSH triggered by many oxidants.Furthermore, glutathione sulfenic acid (GSOH) and glutathionedisulfide S-monoxide [GS(O)SG] are considered alternativemediators of S-glutathionylation [45–47]. The thiyl radical ofGSH, generated by reaction with hydroxyl radicals (HOU), canalso form glutathionylated proteins. The reaction of glutathionethiyl radicals with proteins to generate PSSG is catalyzed byglutaredoxin [48], an enzyme normally acting as a reductant (seebelow).

Indeed, the redox potential of most Cys residues is such thatthe ratio of GSSG versus GSH in cells would need to change≈100-fold (i.e., from∼100 to∼1) to induce S-glutathionylationthrough a thiol exchange mechanism [43,49]. These considera-tions suggest that, although GSSG is capable of glutathiony-lating a number of proteins in vitro, such as the inhibitory κBkinase (IKK) β peptide [14], thiol exchange is an unlikely

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intracellular mechanism for S-glutathionylation, because eventhe GSSG levels measured in oxidant-treated cells can beinsufficient to trigger S-glutathionylation through a thiol/di-sulfide exchange reaction [e.g., 14].

Although the disulfide bond linking the protein andglutathione is readily reversible under reducing conditions,under oxidizing conditions the S-glutathionylation can be main-tained indefinitely as a persistently glutathionylated protein[43,50]. However, in many situations protein S-glutathionyla-tion is only transient, as an adjacent protein thiol displaces theglutathione moiety to form an intraprotein disulfide [43]. Thus,there are two main classes of S-glutathionylated proteins, thosethat are momentarily glutathionylated before protein disulfideformation and those that are persistently glutathionylated, ofwhich the former seems to be more common [50]. The mech-anistic reason for the tendency toward intraprotein disulfideformation is presumably that S-glutathionylation frequentlyoccurs on protein thiols that are adjacent to a second thiol(vicinal thiols), in the same protein or in an adjacent protein, thatrapidly displaces the glutathione moiety to form an intraproteinor an interprotein disulfide, respectively. Once the GSH/GSSGratio has returned to its resting, high ratio, this will lead toreversal of the S-glutathionylation by thiol/disulfide exchange.If the protein has formed an internal disulfide, then thiol/disulfide exchange with GSH could lead to the reduction of thedisulfide with the transient formation of a glutathionylatedintermediate.

Although a complete understanding of the formation path-ways of S-glutathionylated proteins is still evolving and mightprovide scope for further mechanistic investigations, there islittle doubt about the biochemical importance of protein S-glutathionylation. From a biochemical perspective, S-glutathio-nylation might fulfill several, often associated roles duringoxidative stress, including reversible protein protection, regula-tion of protein function, a temporary cutback in enzyme activity,and intracellular redox signaling [5,6,8,9,11,13,51].

Roles of protein S-glutathionylation

The small percentage of total cellular glutathione (GSH+GSSG+PSSG) that is present in the form of constitutive PSSGunder basal conditions can increase up to 20–50% underoxidative stress (overproduction or underscavenging of ROS/RNS), which is associated with a decrease in GSH levels [43].Under pro-oxidant conditions, S-glutathionylation may serve asa storage mechanism for glutathione inside the cell, becauseGSH oxidized to GSSG would otherwise be rapidly extrudedfrom the cell [52].

S-glutathionylation may also provide protection for PSHagainst irreversible modifications and protein damage inresponse to higher levels of oxidative stress. If a protein sulfhy-dryl group is glutathionylated, often at the expense of temporaryloss in protein activity, it is not available for other oxidativereactions including irreversible oxidation to sulfinic and sulfonicacid (the latter is generally resistant to any type of cellular repairmechanism and leads to the proteasomal degradation of theprotein) [7,34,53,54]. In this respect, S-glutathionylation is often

considered a way to protect sensitive cysteinyl residues fromother, possibly irreversible, forms of oxidation, thus allowing thecell to restore the cognate function of the protein when oxidativestress conditions are overcome. Supportive evidence for thisarises, for instance, from the S-glutathionylation of the γ-glutamyl transpeptidase, which seems to protect this membrane-bound enzyme from the irreversible oxidative damage byhydrogen peroxide produced during γ-glutamyl transpeptidase-mediated metabolism of GSH [55] and the S-glutathionylation,producing reversible inactivation, of α-ketoglutarate dehydro-genase in response to alterations in the mitochondrial GSH status[56], as well as the S-glutathionylation of HDL-associatedparaoxonase 1 (PON1), which causes reversible inactivation ofPON1 physiological activities, i.e., hydrolysis of specificoxidized lipids in LDL- and HDL-mediated cholesterol effluxfrom macrophages, under oxidative stress [57].

However, if the modified cysteine is functionally critical,S-glutathionylation not only will modify protein function, butcould eventually compromise cellular activities [58,59]. In mostcases this leads to an inhibition of protein activity, which isparticularly evident in the case of enzymes. Also transcriptionfactors, like c-Jun and NF-κB, are inhibited by S-glutathionyla-tion [60–62].

Protein S-glutathionylation may even have a direct effect onROS/RNS production and antioxidant defenses, because Cu/Zn-superoxide dismutase [47,63], thioredoxin [64,65], glutaredoxin[48], and 1-Cys peroxiredoxin [66–68] have been demonstratedto undergo S-glutathionylation of functionally sensitive cystei-nyl residues. Furthermore, there is a reversible increase in theproduction of O2

U− by mitochondrial NADH-ubiquinone oxido-reductase in response to glutathionylation/deglutathionylation[69].

A correlation between protein S-glutathionylation and geneexpression has also been proposed. A recent study reportedseveral links between GSH levels and gene regulation incultured HL60 cells exposed to H2O2 for various times [11]. Thisstudy showed that several genes, including those involved inNF-κB activation, transcription, and DNA methylation, inaddition to several immune system-related genes, includingcytokines and cytokine receptors, were regulated at differenttime points in response to H2O2 under conditions that resulted inincreased protein S-glutathionylation [11]. In cultured humanlung epithelial A549 and endothelial ECV304 cells, oxidativestress is linked to protein S-glutathionylation and multiplechanges in specific mRNA levels [51]. The results indicate thatthe degree of S-glutathionylation of cellular proteins is closelycorrelated with the induction of a selective cluster of molecularchaperone genes common to both cell types, which includes notonly a range of heat shock proteins but also DNA chaperonesand transcriptional regulators [51]. Although it is difficult toderive a conclusive correlation between protein S-glutathionyla-tion and gene expression using cells with an artificially disruptedGSH level, these studies point toward a significant role for PSSGformation in the cellular response to oxidative stress. This notionis supported by findings showing that S-glutathionylation anddeglutathionylation are not random processes, but are partiallyunder enzymatic control (see below).

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Remarkably, S-glutathionylation is a posttranslational mod-ification that occurs not only during oxidative stress, but alsounder basal conditions [42,70,71]. Constitutive S-glutathiony-lation has been shown, for instance, in hemoglobin in red bloodcells [36,72,73], γ-crystallin from human lens [74], and actin inhuman fibroblasts [75] and human epidermal A431 cells [76].Protein S-glutathionylation under basal conditions suggests itspossible involvement in cellular signaling and redox regulationof protein functions [8,10]. In signaling processes and redoxregulation of proteins, especially for those of the “on–offswitch” type exemplified by protein phosphorylation, two im-portant requirements are specificity and reversibility.

Specificity of S-glutathionylation

At present it is unclear what features contribute to thesensitivity of a given cysteinyl residue to S-glutathionylation,although protein sulfhydryls exhibit a striking differentialsusceptibility to formation of mixed disulfides with glutathione,and the question about the factors that facilitate and confer sitespecificity on such modification is still debated. The identity ofsurrounding residues in the primary sequence or the tertiarystructure may play a role in making a thiol more or less reactive.

The reaction rate of most protein cysteines with ROS/RNSand/or GSH is too slow to be of physiological relevance undercellular conditions and concentrations. This situation changesdrastically when cysteine is bound to a metal ion [17,77], such asMg2+, Ca2+, or Zn2+, which can function as an allosteric effectorto control PSH reactivity, or is in the thiolate anion (-S−) form[15]. As the pKa of cysteine is around 8.5, the same as forcysteine in GSH, dissociation to form a thiolate occurs only inunusual microenvironments in redox-sensitive proteins in whichthe nearby amino acid residues significantly lower the pKa

through electrostatic interactions. Thus, formation of a cysteinethiolate anion (i.e., an “active cysteine”), which can then react toform a mixed disulfide with GSH or a protein disulfide [15,16],is favored by basic amino acids in its vicinity, whereas acidicvicinal amino acids will have the opposite effect. Therefore, acationic environment renders the thiol group highly reactive andparticularly susceptible to S-glutathionylation [60,78]. Thisprovides a basis for specificity in S-glutathionylation. Theconcept of specificity of protein S-glutathionylation is sup-ported, for instance, by rat hemoglobin, in which the low pKa

value of fast-reacting thiols was explained, on the basis of three-dimensional models, by charge stabilization of the thiolate formthrough a hydrogen bond between the anion of Cys-125β(H3)and Ser-123β(H1) [79].

Although the low pKa of a protein thiol provides a usefulguide to its reactivity, this is not a generalizable rule. Little isknown about the structural determinants favoring S-glutathio-nylation. Simple rules based on primary amino acid sequence,i.e., redox-active motifs equivalent to primary amino acidconsensus sequences existing for protein kinase substrates, arelikely to have some limitations on the susceptibility of a givencysteine, as the three-dimensional structure of the protein willinfluence sulfhydryl reactivity and its accessibility to ROS/RNSand/or glutathione. Of principal importance is whether the oxi-

dant and/or glutathione can actually make contact with apotentially reactive protein thiol and then whether they will reactunder specific cellular conditions. Thus, Cys residue accessi-bility in the three-dimensional structure provides a basis forS-glutathionylation specificity, as illustrated, for instance, in theS-glutathionylation of thioredoxin [64] as well as of elongationfactor 1-α-1 and heat shock protein 60, of which both the Cys411

of the former and the Cys447 of the latter are situated in readilyaccessible regions of the respective protein molecules, i.e., in aloop region between two domains for elongation factor 1-α-1and on an α-helix adjacent to three conserved glycines in theATP-binding site for heat shock protein 60 [80]. Actin andcarbonic anhydrase III are other examples of how S-glutathio-nylation may depend on the solvent accessibility of the Cysresidues. Actin contains five cysteinyl residues existing in thereduced form. Native actin exposes one fast-reacting sulfhydrylgroup, that of Cys374, next to the C-terminus and easilyaccessible to sulfhydryl-reactive agents. The exposed Cys374

residue is the most likely glutathionylation site both in vivo andin vitro [76,81]. However, in diamide-treated ECV304 cells,Cys217 was identified as a glutathionylation site of γ-actin,suggesting that this S-glutathionylation site could also be aplausible candidate site for regulation of actin polymerization[80]. The three-dimensional structure of S-glutathionylatedmammalian carbonic anhydrase III reveals that glutathione bindsto Cys181 and Cys186, the two highly surface-exposed of its fivecysteinyl residues [78,82]. Cys181 and Cys186 are located in arather neutral environment; nonetheless S-glutathionylation canbe achieved by specific interactions between the glutathionemoiety and the solvent-accessible Cys residues. Although bothsurface-exposed Cys181 and Cys186 are susceptible to S-gluta-thionylation, Cys186 is more readily modified both in vitro and invivo [83]. Lys211 seems to be primarily responsible for thelowering of the pKa of Cys

186, making its thiol more reactive[83]. Of the six cysteine residues of p21ras, four (i.e., 118, 181,184, and 186) are surface-exposed and are susceptible toS-glutathionylation by GSSG, albeit to different extents, asrecently shown by Cohen's group using isotope-coded affinitytag and mass spectrometry: the extent of S-glutathionylation ofCys118 byGSSGwas 53%, and that of the terminal cysteines was85% [84]. In cells, because the terminal cysteines are largelymodified by lipid, Cys118, which is part of the guanine nucleo-tide binding site, is the prime target for S-glutathionylation andregulates p21ras activity, as indicated by the fact that both S-glutathionylation and stimulation of activity by oxidants aremostly prevented in cells transfected with a Cys118 mutant[85–87], and is implicated in downstream signaling to Raf-1/Mek/Erk [see below and [85–88].

Electrostatic interactions too were hypothesized to beinvolved in S-glutathionylation [e.g., 61,63]. An elegant studyhas recently analyzed the susceptibility to S-glutathionylation ofthe four Cys residues of cyclophilin A (Cys52, Cys62, Cys115,and Cys161)—a ubiquitous intracellular protein, target of theimmunosuppressive drug cyclosporin A, with multiple actions,including protein folding and chaperone activity—consideringboth the solvent exposure through molecular dynamics simula-tion and the influence of structural neighboring amino acids

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through electrostatic calculations [13]. Using MALDI-MSGhezzi and colleagues identified Cys52 and Cys62 as targets ofglutathionylation in T lymphocytes, although moleculardynamic simulation showed that Cys52 and Cys161 exposed alarger surface of their side chains than Cys62 and Cys115.Therefore, a correlation between the solvent accessibility of Cysresidues of cyclophilin A and glutathionylation cannot be made[13]. Electrostatic energy computations made to further definethe nucleophilic reactivity of the various Cys residues in cyclo-philin A showed that the changes in electrostatic energy in-volved in the conversion of a sulfhydryl to the correspondingthiolate anion follow the order Cys52bCys62bCys115bCys161,meaning that Cys52 and Cys62 form the thiolate anion withgreater ease than Cys115 and Cys161 and can therefore beexpected to be more reactive. Hence, the experimental ob-servations, together with the theoretical calculations on thesusceptibility of cyclophilin A Cys residues to glutathionylation,suggest that, even when a Cys residue has a small surfaceexposure, as in the case of cyclophilin A Cys62, electrostaticinteractions can lead to susceptibility to S-glutathionylation[13].

The local pH and hydrophobic compartmentalization couldbe other important factors that render certain Cys residueshighly reactive and particularly susceptible to S-glutathionyla-tion [60,61].

Reversibility of S-glutathionylation

The main feature that makes S-glutathionylation a possibleregulatory mechanism is its reversibility. In fact, reversibility ofposttranslational modifications is a requisite for cellular signal-ing. Deglutathionylation is the removal of the GSH moiety from

Fig. 2. Induction and reversal of protein S-glutathionylation. Formation of protein–glusystems can occur after a decrease in the intracellular/cytoplasmic GSH/GSSG ratio thprotein thiol (thiyl radical or sulfenic acid). GSH can then interact with this activatedbe the result of thiol/disulfide exchange in the presence of increased cellular levels oformation can be promoted by nitric oxide, through various mechanisms includiglutaredoxin can also catalyze the S-glutathionylation of certain proteins in the presexchange. Reversal of S-glutathionylation (deglutathionylation) can be achieved byreduced thiols, or via enzymatic reduction by glutaredoxin (also known as thioltrandependent or -independent mechanism, respectively, and, in a limited number of pro

protein mixed disulfides and can occur when the environmentbecomesmore reducing in an enzyme-dependent or -independentmanner (Fig. 2). To date, a limited number of proteins havebeen identified that are involved in deglutathionylation.

S-glutathionylation of protein thiols can be reversed via directthiol/disulfide exchange reactions with GSH once the reducingintracellular redox balance (mainly, an appropriate GSH/GSSGratio) has been restored [34], by means of an enzymaticallymediated reaction. Enzymes capable of reducing S-glutathiony-lated proteins include the glutaredoxins (GRX; also known asthioltransferases)/GRX reductase system [49,89], the thioredox-ins (TRX)/TRX reductase system [90–92], and sulfiredoxin[19,20,93].

The TRX and GRX families contain a conserved -Cys-X-X-Cys-active site (Cys32 and Cys35 within thioredoxin-1), which isessential for their redox regulatory functions [49,89,90,92].They catalyze the reduction of disulfide bonds and becomeconcomitantly oxidized by forming an intramolecular disulfidein the -Cys-X-X-Cys- active site. The oxidized enzyme is thenreduced by TRX reductase, in the case of TRX, or byGSH, in thecase of GRX. In particular, TRX/TRX reductase can reduceprotein disulfides and protein sulfenic acid intermediates.Thioredoxin-1 itself has been shown to be subject to S-glutathionylation, in addition to S-nitrosation and intramoleculardisulfide formation, occurring not at cysteines spanning theredox-regulatory domain, but at three additional nonactive cys-teine residues at positions 61, 68, and 72 [64]. The glutathio-nylation site was identified as Cys72 and this residue could bemodified either by GSSG or by GSNO. Modification of the siteabolished enzymatic activity of TRX. However, activityspontaneously recovered, suggesting that TRX was able to de-glutathionylate itself. Such results support the concept of a

tathione mixed disulfides, i.e., S-glutathionylated proteins (PSSG), in biologicalat can induce the partial oxidation of protein sulfhydryls, yielding an “activated”thiol to give an S-glutathionylated protein. Alternatively, S-glutathionylation canf GSSG or by other mediators (e.g., GS(O)SG or GSOH). Furthermore, PSSGng the formation of S-nitrosoglutathione (GSNO) and thiyl radicals. Finally,ence of a GS

U-generating system via a monothiol mechanism of thiol/disulfide

changes in the intracellular redox status (increases in the GSH/GSSG ratio), bysferase) and sulfiredoxin, which selectively deglutathionylate PSSG by a GSH-teins, by the thioredoxin system.

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coordination of regulatory pathways through a possible crosstalk between the glutathione and the TRX systems under con-ditions of oxidative stress [64]. Interestingly, chloroplast thiore-doxin f, a key factor in the redox regulation of carbon-fixationenzymes, has recently been shown to undergo S-glutathionyla-tion at a conserved, additional (other than its two active-sitecysteines) cysteine that is not required for its activity. S-gluta-thionylationmodulates thioredoxin f efficiency: the activation ofits target enzymes is strongly decreased when thioredoxin f isS-glutathionylated [65].

Glutaredoxin was the first protein identified as a specificglutathionyl-mixed disulfide oxidoreductase [94]. Interestingly,GRX not only enzymatically deglutathionylates specific pro-teins [49,89] but has also been shown to catalyze the S-gluta-thionylation of certain proteins in the presence of a GS-radical-generating system [48,95] via distinct mechanisms [96]. Onlyone of the two Cys residues contained in the conserved -Cys-X-X-Cys-motif of GRX is required for its oxidative function ofS-glutathionylation via a monothiol mechanism of disulfideexchange involving the high reactivity of a GRX–SG mixeddisulfide, whereas the presence of both Cys residues within theconserved -Cys-X-X-Cys-motif is required for its reductivefunction of deglutathionylation via a dithiol mechanism ofdisulfide exchange [49,89,96]. The formation of protein–SGmixed disulfides by GRX through a monothiol mechanism mayplay an important role in protecting against more drastic,irreversible modifications of protein thiols, particularly when theredox state of the cytoplasm becomes more oxidizing, as underconditions of oxidative stress.

Human sulfiredoxin (Srx1) is the first protein identified thathas been implicated specifically in the reductive deglutathiony-lation of proteins [93]. The specificity of Srx1 may be due to thepresence of only one cysteinyl residue within its sequence.Although the conserved cysteinyl residue in Srx1 is essential forthe deglutathionylation reaction, current data suggest that it isnot a direct acceptor for the GSH moiety, because Srx1, incontrast to GRX, does not form a mixed disulfide with GSHduring the deglutathionylation reaction, i.e., it is not itselfglutathionylated during the removal of GSH moieties [93].

Proteins regulated by S-glutathionylation

The existence of antioxidant enzymes that serve the uniquerole of specifically reducing protein–SG mixed disulfidesemphasizes the importance of S-glutathionylation in modulat-ing protein function. Actually, S-glutathionylation has beenshown to regulate in a complex manner, either positively ornegatively, the structure/function of a quite diverse range, andincreasing number, of cellular proteins, including enzymes,signaling molecules, transcription factors, heat shock proteins,ion channels, mitochondrial proteins, and cytoskeletal proteins[10].

Enzymes with active-site thiols

Activities of metabolic enzymes, including carbonic anhy-drase III [54,97], tyrosine hydroxylase [58], α-ketoglutarate

dehydrogenase [56], aldose reductase [98], creatine kinase[46,99], and GAPDH [59,100,101], among others, are subject toregulation by S-glutathionylation, in most cases resulting ininhibition of enzyme activity. In the case of carbonic anhydraseIII, S-glutathionylation of Cys186 is required for its phosphataseactivity, whereas S-glutathionylation of Cys181 blocks activity[97].

Caspase-3 cleavage and activation are known to play centralroles in apoptosis. However, the mechanisms that regulatecaspase-3 cleavage remain elusive. S-glutathionylation hasrecently been shown to regulate TNF-α-induced caspase-3 clea-vage and activation, and thus the resultant apoptosis, in endo-thelial cells [102]. In particular, an inverse correlation has beenshown between caspase-3 S-glutathionylation and cleavage, andGRX plays an essential role in caspase-3 cleavage via regulationof caspase-3 S-glutathionylation. These findings demonstrate anovel mechanism of caspase-3 regulation in TNF-α-inducedapoptosis.

The HIV-1 protease is another enzyme that is regulated byS-glutathionylation. It contains conserved and critical Cysresidues that, when S-glutathionylated, activate or deactivatethe protease depending on the thiol group involved. In particular,S-glutathionylation of Cys95 inhibits the activity but S-gluta-thionylation of Cys67 stabilizes the HIV-1 protease [103].

Signaling proteins

Redox status has dual effects on upstream signaling systemsand downstream transcription factors. Oxidants can stimulatemany upstream kinases in signaling pathway cascades and yetinhibit transcription factors AP-1 and NF-κB. Reductants canhave opposite effects producing AP-1 and NF-κB activation[104]. Many signaling molecules and transcription factors fun-damental for cell growth, differentiation, and apoptosis seem tobe regulated by S-glutathionylation [7,105]. The S-glutathiony-lation of signaling molecules such as protein kinase A, proteinkinase C, mitogen-activated protein kinase (MAPK)/extracel-lular-signal-regulated kinase (ERK) [106,107], T cell p59fyn

kinase [108], and protein tyrosine phosphatase-1B [109,110],one phosphatase that can participate in the deactivation ofkinases, modulates their activity. The catalytic subunit of cAMP-dependent protein kinase (protein kinase A) is susceptible toinactivation through S-glutathionylation of Cys199, which islocated near the active site [107].

S-glutathionylation plays a key role in the regulation ofthe kinase activity of MEKK1 (MAPK/ERK kinase kinase 1;MAP3K), an upstream activator of the SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase) pathway, inresponse to oxidative stress [111]. MEKK1 was shown to beinhibited by site-specific S-glutathionylation in a single uniquecysteinyl residue in the ATP-binding domain in vitro andMEKK1 isolated from menadione-treated cells was shown bymass spectrometry to be modified by glutathione on the sameCys residue. As MEKK1 provides a protein kinase with ademonstrated survival signaling function, its S-glutathionylationmight participate in shifting the balance from cell survival to celldeath by apoptosis in response to oxidative stress.

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Transcription factors

By introducing the negative charges of GSH within theirDNA binding sites, S-glutathionylation inhibits the DNAbinding activity of the redox-sensitive transcription factors c-Jun andNF-κB [60–62]. It has beenwell established that NF-κB,a central regulator of immunity, is subject to multiple regulationby redox changes. S-glutathionylation of Cys62 of the p50subunit is known to prevent binding of the transcription factor toκB sites in the promoter regions of genes [61]. Remarkably, S-glutathionylation of the p65 subunit of NF-κB, which is mainlyresponsible for transcriptional activation, has been shown toinhibit p65–NF-κB binding to DNA, resulting in decreasedresistance to hypoxia in pancreatic cancer cells supplementedwith GSH via N-acetylcysteine [62]. It has now been reportedthat Cys179 of the IKK β subunit of the IKK signalosome is acentral target for oxidative inactivation by means of S-glutathionylation, which is responsible for the repression ofkinase activity by H2O2. S-glutathionylation of IKK-β Cys179 isreversed by GRX, which restores kinase activity [14]. Reynaertand colleagues propose that GRX1-dependent reversal of S-glutathionylation of IKK-β constitutes a protective mechanismthat modulates the extent and timing of activation of NF-κB inresponse to redox changes by protecting IKK-β from irreversibleinactivation and allowing for rapid regeneration of enzymaticactivity [14]. Collectively, these data suggest that S-glutathio-nylation could be a physiologically relevant mechanism forcontrolling the magnitude of activation of the NF-κB pathway.

Pax-8 belongs to a family of transcription factors that containa paired domain as a DNA-binding domain. Pax-8 stimulatesexpression of a set of thyroid-enriched proteins, thyroglobulin,thyroperoxidase, and sodium/iodide symporter. Recent evidenceproves that a decrease in the GSH/GSSG ratio induces S-glutathionylation of the first two cysteine residues, Cys45 andCys57, of three present in the paired domain of Pax-8, resulting ina loss of DNA binding [112]. The authors suggest that the GSH/GSSG-dependent oxidative inactivation of Pax-8 could accountfor the decreased thyroglobulin expression in the cells with a lowlevel of GSH. Therefore, S-glutathionylation of these cysteineshas a regulatory role in paired domain binding and provides anew insight into the molecular basis for the modulation of Paxfunction [112]. Furthermore, S-glutathionylation of OxyR, abacterial DNA binding protein, stimulates transcription ofmultiple redox response genes [113].

Ras proteins

The Ras proteins are small guanine nucleotide exchangeproteins that play a critical role as signal transducers in theregulation of a number of cellular processes, including cellgrowth, differentiation, and apoptosis. Reactions of ROS/RNSwith Cys118, which resides near the guanine nucleotide bindingsite, has been implicated in activation of p21ras leading todownstream signaling to Raf-1/MEK/ERK to mediate cellularresponses [88]. Cohen's group has recently demonstrated thatincreased levels of oxidants in both endothelial and smoothmuscle cells can directly activate p21ras by S-glutathionylation

of a reactive thiol on Cys118 and trigger downstream signalingthrough phosphorylation of ERK and AKT [85,87]. They couldlikewise show that S-glutathionylation of Cys118 on Ras was thecritical step mediating angiotensin II-induced hypertrophy ofvascular smooth muscle cells, thus identifying Ras as theupstreammolecular target of ROS/RNS in this model [85]. Morerecently, Cohen and colleagues have investigated if oxidizedLDL could regulate insulin signaling as a direct result ofincreasing oxidant-mediated p21ras activation. In this elegantstudy, the authors demonstrate that S-glutathionylation andactivation of p21ras caused by peroxynitrite generated inendothelial cells by oxidized LDL promotes MEK-dependentERK activation, resulting in ERK-dependent phosphorylation ofinsulin receptor substrate 1 and inhibition of insulin-mediatedAKT phosphorylation [86]. These findings suggest a novelmolecular mechanism by which oxidants could induce endothe-lial insulin resistance via S-glutathionylation of p21ras andERK-dependent inhibition of insulin signaling [86].

Ras also plays a crucial role in the regulation of hypertrophicgrowth in cardiac myocytes in response to stimuli such as α-adrenergic receptor agonists and mechanical strain. The role ofS-glutathionylation in the regulation of Ras has also beenstudied in cardiomyocytes, in models of ROS-mediated cardiachypertrophy stimulated by mechanical strain or α-adrenergicreceptor [114,115]. Results support the hypothesis that S-glutathionylation of Ras is functionally relevant for ROS-mediated signal transduction leading to cardiomyocyte hyper-trophy. In particular, these findings indicate that mechanicalstrain causes ROS-dependent S-glutathionylation of Ras atCys118, leading to myocyte hypertrophy via activation of theRaf/MEK/ERK growth pathway [115].

Heat shock proteins

Correct protein folding is an essential cellular functioncontrolled by heat shock proteins (HSPs), which stabilizeexposed hydrophobic surfaces of nonnative proteins to facilitatecorrect folding into functional structures. Members of theHSP60 and HSP70 group have been found to be modified by S-glutathionylation in T lymphocytes stressed with hydrogen per-oxide or diamide [116] as well as in ECV304 endothelial cellsduring constitutive metabolism or diamide-induced oxidativestress [70,80]. S-glutathionylation of heat shock cognate protein70, a constitutively expressed homologue of HSP70, in retinalpigment epithelium increases its chaperone function, increasingfivefold its ability (compared to the reduced HSP) to preventheat-induced protein aggregation [117]. Maintaining activechaperones is important for regulation of oxidative misfolding ofproteins, an event linked to cellular damage and disease. Thus,redox regulation of HSP chaperone activity may be a physio-logic response to oxidative stress relevant to major diseaseprocesses.

Ion channels and Ca2+ pumps

Other targets for S-glutathionylation, affecting ion transportactivity, include ion channels, such as the Ca2+-release/

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ryanodine receptor channels (RyR) and the cystic fibrosistransmembrane conductance regulator (CFTR), a phosphoryla-tion/ATP-dependent chloride channel that modulates salt andwater transport across lung and gut epithelia [118,119]. NADPHaddition to microsomes isolated from heart muscle significantlyenhances, via NADPH oxidase activation, both ryanodinereceptor type 2 S-glutathionylation and Ca2+-induced Ca2+

release [120]. The highly redox-sensitive ryanodine receptortype 1 (RyR1), which is essential for skeletal muscle excitation–contraction coupling, is S-glutathionylated by H2O2 in vitro inthe presence of GSH, which enhances channel activity[118,121]. Additionally, the RyR1 protein is susceptible toS-nitrosation [118]. A successive work by the same authorsshows that the skeletal muscle T-tubules possess a functionalnonphagocytic NADPH oxidase activity. This enzyme generatesH2O2, which, in triads isolated from mammalian skeletal musclecomposed of junctional T-tubule and sarcoplasmic reticulummembranes, decreases the total sulfhydryl content and activatesRyR1 through S-glutathionylation, enhancing calcium release[122]. Increased S-glutathionylation of RyR, eliciting calciumrelease and sequentially enhancing CREB (the transcriptionfactor cAMP/Ca2+ response element binding protein) phosphor-ylation via ERK activation, has been confirmed in mouse N2aneuroblastoma cells and in primary rat hippocampal neurons[123]. A restricted number of cysteines, of the 100 cysteines ineach RyR1 subunit, that are likely to be involved in thefunctional response of the skeletal muscle Ca2+ release channel(RyR1) have recently been identified as specific substrates ofS-nitrosation, S-glutathionylation, and oxidation to disulfides[124]. Human CFTR channels are reversibly inhibited by re-active glutathione species, i.e., GSSG, GSNO, and glutathionetreated with diamide. Three lines of evidence indicate that thelikely mechanism for this inhibitory effect is S-glutathionylationof a CFTR cysteine [119].

Regulatory effects of S-glutathionylation have also beendescribed for the sarcoplasmic/endoplasmic reticulum Ca2+

ATPase (SERCA) [125,126] and the small GTPase transformingprotein, p21ras (Ras) [53,85–87,114]. Cohen and colleaguesrecently found that SERCA is S-glutathionylated by NO (in theform of its active “effector” peroxynitrite) in intact cells orarteries, as well as in vitro, resulting in accelerated Ca2+ uptakeactivity of the protein, explaining cGMP-independent arterialrelaxation due to NO, thereby relaxing vascular smooth muscleby lowering intracellular free Ca2+ [125,126]. Whereas severalother cysteines can be S-glutathionylated, the site where most ofthe GSH was bound is the most reactive thiol on SERCA(Cys674), which regulates enzyme function. In atheroscleroticaorta, NO does not increase S-glutathionylation of SERCA and,as a result, fails to stimulate its activity because of the ir-reversible oxidation of the Cys674 thiol (more than 50%) tosulfonic acid [125,126].

Mitochondrial proteins

Mitochondria represent one of the most oxidative environ-ments inside cells, and the S-glutathionylation of mitochondrialproteins such as complex I (NADH-ubiquinone oxidoreductase)

[50,69] and aconitase, during heart ischemia–reperfusion injury[30] and after incubation with GSSG [127], can regulate ROS/RNS generation and/or metabolism. In particular, ROS produc-tion by mitochondrial complex I increases in response tooxidation of the mitochondrial GSH pool to GSSG. Thiscorrelates with S-glutathionylation of thiols on the 51- and 75-kDa subunits of complex I in the isolated complex, bovine heartmitochondrial membranes, and intact mitochondria. S-glutathio-nylation of complex I increases superoxide production by thecomplex and, when the mixed disulfides are reduced, superoxideproduction returns to basal levels. Within intact mitochondria,most of this superoxide produced after S-glutathionylation ofcomplex I is converted to hydrogen peroxide, which can thendiffuse into the cytoplasm. This mechanism of reversible mito-chondrial ROS production suggests how mitochondria mightregulate redox signaling and shows how oxidation of the mito-chondrial glutathione pool could contribute to the pathologicalchanges to mitochondria that occur during oxidative stress [69].

A wide range of mitochondrial membrane proteins containexposed, reactive thiols that reacted with GSSG by thiol–di-sulfide exchange to form amixed disulfide. However, only a fewthiol proteins remained glutathionylated, with most displacingthe GSH to form an intraprotein disulfide [50]. Consequently,the proportion of protein thiols that was persistently glutathio-nylated was far smaller than that which formed intraproteindisulfides. Complex I was the most prominent mitochondrialmembrane protein that was persistently S-glutathionylated byGSSG in the presence of mitochondrial thiol transferase gluta-redoxin 2 (GRX2) [50]. The mechanistic reason for greaterintraprotein disulfide formation is likely that most S-glutathio-nylated protein thiols are formed adjacent to a second thiol thatrapidly displaces GSH to form an intraprotein disulfide. Theactivity of α-ketoglutarate dehydrogenase too seems to bemodulated through enzymatic glutathionylation and deglu-tathionylation catalyzed by GRX [56]. Mitochondrial NADP+-dependent isocitrate dehydrogenase was shown to be susceptibleto inactivation by S-glutathionylation of Cys269 [128]. Theinactivated NADP+-dependent isocitrate dehydrogenase wasreactivated enzymatically by GRX2 in the presence of GSH[128]. Cytochrome oxidase subunit Va within human T lympho-cyte cells was S-glutathionylated in response to diamide [116],and cytochrome oxidase subunit Vb in rat hepatocytes wasS-glutathionylated after exposure to menadione [129]. However,the physiological significance of this alteration is unclear.

Cytoskeletal proteins

S-glutathionylation has recently been implicated in the redoxregulation of a variety of cytoskeletal proteins, including tubulinand actin. S-glutathionylation of β-tubulin has been reported inendothelial-like cells [48,70], whereas a direct thiol-disulfideexchange between GSSG and native tubulin was demonstratedin vitro [130].

Actin is perhaps one of the best examples of physiologicallyrelevant regulation by S-glutathionylation. Actin has beenshown to polymerize into filaments, translocate from a uniformcytosolic distribution to the cellular periphery, and rearrange into

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membrane ruffles in response to epidermal growth factor (EGF)in A431 cells [76] and in response to fibroblast growth factor inNIH3T3 cells [131]. Treatment of A431 cells with EGF leads todeglutathionylation of actin despite an increase in intracellularROS [76]. In fact, under normal (basal) cellular conditions, aportion of G-actin is S-glutathionylated at Cys374. This likelyinhibits polymerization into F-actin, as deglutathionylation leadsto an increased rate of polymerization, and inhibition of actinpolymerization by Cys374 glutathionylation has been welldemonstrated in vitro [81,132]. The deglutathionylation iscatalyzed by GRX in cells [76]. Furthermore, specific knockoutof GRX1 in NIH3T3 cells by tetracycline-inducible RNAiabolished growth factor-mediated actin polymerization, translo-calization to the cell periphery, and membrane ruffling [131].These studies suggest that reversible S-glutathionylation of actinby GRX contributes to the regulation of the cellular functions ofactin. Regulation of actin function by S-glutathionylation ofCys374 decreases the rate of actin polymerization most likely dueto a change in protein conformation [81].

Another evidence of in vivo actin redox regulation by aphysiological source of ROS, specifically those generated byintegrin receptors during integrin-mediated cell adhesion, hasrecently been shown in murine NIH3T3 fibroblasts [12]. Actinoxidation takes place via S-glutathionylation of Cys374; thismodification is essential for cell spreading and for cytoskeletonorganization. Impairment of actin S-glutathionylation, througheither GSH depletion or expression of the C374A redox-in-sensitive mutant, greatly affects cell spreading and the formationof stress fibers, leading to inhibition of the disassembly of theactinomyosin complex. The control of cytoskeleton organizationis mainly due to the action of the members of the family of thesmall GTPases Rho. Nevertheless, it was previously shown thatROS can act as second messengers in the organization of cyto-skeleton in response to integrin-mediated cell adhesion [133].These recent data suggest that actin S-glutathionylation isessential for cell spreading and cytoskeleton organization andthat it plays a key role in disassembly of the actinomyosincomplex during cell adhesion [12]. Furthermore, other recentresults suggest that S-glutathionylation of actin in the thinfilament during ischemia–reperfusion injury may alter the con-tractile performance of the myocardium [134]. Finally, actin hasbeen identified as a specific target of Srx1-dependent deglu-tathionylation both in vitro and in vivo [96].

Moreover, many proteins interact with actin to enhance itsorganization, such as actin-cross-linking proteins that connectactin filaments to each other and actin-binding proteins thatconnect the actin cytoskeleton to the cell membrane, such asannexins, one group of multifunctional, Ca2+-dependentheterotetrameric proteins. Among these, S-glutathionylation oftwo (i.e., Cys8 and Cys132) of four cysteines of tetramericannexin II causes an inhibition of annexin binding activity that isreversible by GRX [135]. At the cellular level, S-glutathionyla-tion of annexin II has been identified in HeLa cells afterstimulation with TNF-α [136]. Collectively, these data provide abasis for proposing that S-glutathionylation could regulatecellular actin both directly and indirectly through annexin II orperhaps other accessory actin proteins. Indeed, other cytoske-

letal proteins than actin, such as vimentin, myosin, tropomyosin,cofilin, profilin, and α-actinin, have been shown to be S-gluta-thionylated in human T cell blasts and human platelets inresponse to oxidants [116,137]. Increased S-glutathionylation ofactin and vimentin has been detected in isolated rat aortic ringstimulated with acetylcholine [138]. In contrast, our datademonstrate that NO donors do not elicit actin S-glutathionyla-tion [45]; therefore it is puzzling that the in vivo delivery of NOelicited by acetylcholine may result in actin S-glutathionylation.Furthermore, S-glutathionylation of membrane skeletal proteinshas been detected in red blood cells exposed to diamide to modelthe effects of oxidative stress [139]. Thus, understanding theregulation via S-glutathionylation of actin and other cytoskeletalcomponents could reveal another example of regulation oftranslocation and signal transduction widespread throughout thecell.

Conclusions and perspectives

An increasing body of evidence has highlighted an importantrole for a variety of redox signaling mechanisms in the control ofa plethora of cellular processes. One mechanism by which GSHcan regulate cellular functions is through S-glutathionylation ofprotein sulfhydryls, a proteinmodification that has been detectedalso under basal/physiological conditions and not only afteroxidative/nitrosative stress. S-glutathionylation accounts for thereversible regulation of structure and function of a wide varietyof proteins that are integral to cell structure, signaling, andmetabolism. Despite the abundance of cellular protein thiols, S-glutathionylation occurs with a certain specificity, although notyet completely established. Where phosphorylation depends onkinases for specificity, S-glutathionylation depends on proteinstructure and thiolate anion reactivity for specificity. Someadditional specificity may be provided by the action of GRXs,and possibly Srx, which enzymatically add or remove GSH fromproteins. Thus, like phosphorylation, the reversible S-glutathio-nylation of protein thiols may be a mechanism to turn signalingpathways on or off through modification of important cysteinylresidues or through the induction of conformational changes inthe protein.

If S-glutathionylation may be considered a redox-dependentposttranslational modification with potential relevance to signaltransduction, then glutathione is not only an essentialantioxidant, but also an essential signaling molecule, alsoconsidering that, in the context of redox regulation, the ratiobetween GSH and GSSG determines the redox state of redox-sensitive cysteines in some proteins [9,11]. Redox signalingmay contribute to the control of cell development, differentia-tion, growth, death, and adaptation and has been implicated indiverse physiological and pathological processes [140,141]. IfGSH is also an essential signaling molecule, one should wonderwhether the deleterious consequences that glutathione depletionhas under many oxidative stress conditions are not only due tothe lack of an antioxidant, thiol-sparing molecule, but also dueto the blockade of S-glutathionylation-dependent signalingmechanisms, similar to the findings that ATP depletion canaffect phosphorylation-mediated signaling. Overproduction or

Fig. 3. Role of protein cysteine S-glutathionylation in redox signaling and oxidative stress responses. At low ROS/RNS levels (basal, or physiological, conditions),reversible S-glutathionylation of protein cysteine thiols regulates protein activity/function and can subserve cell signaling responses. Oxidative stress results fromexcessive generation of ROS/RNS and/or impaired antioxidant defenses. After moderate elevation of ROS/RNS levels and/or moderate impairment of antioxidantdefenses (moderate oxidative stress), reversible S-glutathionylation of protein thiols maintains glutathione inside the cells (often at the expense of temporary loss ofprotein function) and protects protein sulfhydryl groups from irreversible oxidation. At high ROS/RNS levels and/or strong impairment of antioxidant defenses (severeoxidative stress), irreversible oxidation of protein sulfhydryl groups can change protein function permanently, thus impairing the physiological redox regulation ofproteins with redox-sensitive cysteines. Irreversible modifications are usually associated with permanent loss of function and may lead to the elimination of thedamaged proteins by the proteasome system or to their accumulation as insoluble aggregates.

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underscavenging of ROS/RNS (oxidative/nitrosative stress) canirreversibly oxidize protein thiols. This irreversible oxidationcould cause aberrant cellular signaling, eliminating GSH-dependent signaling mediated by protein sulfhydryl glutathio-nylation (Fig. 3).

The application of redox proteomics techniques [22,32,40–42,142] will enable an increasing number of the target proteinthiols, representing pivotal regulatory control points, to beuncovered. These data will provide an important platform toprobe the molecular mechanisms underpinning S-glutathionyla-tion and other reversible oxidative modifications of proteinthiols. It is anticipated that future advances in redox proteomicsmay provide novel opportunities for both basic research andhuman disease research [142]. In particular, the continueddevelopment of proteomic and analytical methods for disulfideanalysis will help us better understand the full extent that thesemodifications play in the regulation of cell function [142].However, in exploring all these processes, the first challenge isto identify those proteins whose thiol redox state is affected byoxidative/nitrosative stress and/or redox signaling and thoseproteins that undergo reversible oxidative modifications underphysiological conditions, determine whether the changes are dueto the formation of a protein disulfide or to persistent S-glutathionylation or other reversible oxidation of cysteinylthiols, find out which cysteinyl residues are modified, andexplain how the redox changes affect protein function. Atpresent, little is known about the details of these proteins and,especially, the physiological consequences of oxidation of theirthiols. The identification of S-glutathionylated proteins andexploration of the cellular significance of this modification arereally still in their infancy. Nevertheless, advances in structuralbiology and redox proteomics are now providing the molecular

tools to address these issues. This is a rich and promising area forfuture research.

Acknowledgments

Our apologies for any relevant reports we were unable tocite, due to either the need to selectively choose examples or ouroversight. The authors' research was supported by FIRST 2006(Fondo Interno Ricerca Scientifica e Tecnologica) University ofMilan.

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