S-glutathionylation in protein redox regulation

Free Radical Biology & Medicine 43 (2007) 883 – 898 www.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

b

Department of Biology, University of Milan, I-20133 Milan, Italy Department of Neuroscience, University of Siena, I-53100 Siena, Italy Received 8 May 2007; revised 6 June 2007; accepted 6 June 2007 Available 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 cellular response to mild oxidative/nitrosative stress, but also occurs under basal (physiological) conditions. S-glutathionylation has now emerged as a potential mechanism for dynamic, posttranslational regulation of a variety of regulatory, structural, and metabolic proteins. Moreover, substantial recent studies have implicated S-glutathionylation in the regulation of signaling and metabolic pathways in intact cellular systems. The growing list of S-glutathionylated proteins, in both animal and plant cells, attests to the occurrence of S-glutathionylation in cellular response pathways. The existence 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 for disulfide analysis will help us better understand the full extent of the roles these modifications play in the regulation of cell function. In this review, we describe 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; Free radicals

Contents Oxidative modifications of protein thiols . Mechanisms of S-glutathionylation . . . . Roles of protein S-glutathionylation . . . Specificity of S-glutathionylation . . . . . Reversibility of S-glutathionylation . . . . Proteins regulated by S-glutathionylation . Enzymes with active-site thiols . . . . Signaling proteins. . . . . . . . . . . Transcription factors . . . . . . . . . Ras proteins. . . . . . . . . . . . . . Heat shock proteins . . . . . . . . . . Ion channels and Ca2+ pumps . . . .

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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, reactive oxygen species; RyR, ryanodine receptor channel; SERCA, sarcoplasmic/endoplasmic reticulum Ca2+ ATPase; Srx1, human sulfiredoxin; TNF-α, tumor necrosis factor α; TRX, thioredoxin(s). ⁎ Corresponding author. Fax: +39 02 50314781. E-mail address: [email protected] (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. . Cytoskeletal proteins . . Conclusions and perspectives Acknowledgments . . . . . . References . . . . . . . . . .

I. Dalle-Donne et al. / Free Radical Biology & Medicine 43 (2007) 883–898

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Oxidative stress is a situation in which cellular homeostasis is altered because of excessive production of reactive oxygen/ nitrogen species (ROS/RNS) and/or impairment of cellular antioxidant defenses, “leading to a disruption of redox signaling and control and/or molecular damage” [1]. ROS/RNS can cause specific, reversible and/or irreversible oxidative modifications on sensitive proteins that may lead to a change in the activity or function of the oxidized protein [2]. Most protein oxidation products are commonly considered as biomarkers of oxidative/ nitrosative stress/damage [3,4]. Under conditions of oxidative/nitrosative stress, the thiols in cysteine residues within proteins are among the most susceptible oxidant-sensitive targets and can undergo various reversible and irreversible redox alterations in response to ROS and/or RNS increase/exposure. All the modifications to protein thiols can potentially affect protein activity, with the degree depending on the importance of the cysteine residue in carrying out protein function. Actually, the oxidative modifications of the cysteine sulfhydryl group have recently attracted renewed interest, because Cys is present in the active site of many proteins and in protein motifs that function in protein regulation and trafficking, cellular signaling, and control of gene expression [5,6]. Some proteins may not contain cysteine residues important in protein function; however, modification of thiols may cause a conformational change that alters protein activity. Thus, because many redox alterations to protein thiols are readily reversible through mechanisms involving glutathione (GSH), thiol redox alteration, like phosphorylation, has been suggested to be an important mechanism of turning on and off proteins, i.e., protein redox regulation, particularly in response to oxidative and nitrosative stress. The exposure of cysteines on the protein surface is a functional necessity to prevent redox changes from spreading through the entire protein molecule. The surface-oriented Cys residues 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 to cellular redox balance. Changes in the cellular redox status (mainly due to a decrease in the GSH/GSSG ratio and/or depletion of GSH by the metabolism of drugs or other xenobiotic substances) 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 between protein sulfhydryl groups (PSH) and glutathione (S-glutathionylation) on multiple proteins, which makes of cellular glutathione a crucial modulating factor for an ever increasing number of proteins.

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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 physiologically relevant situations (i.e., basal conditions), in which it can produce discrete modulatory effects on protein function. This has rapidly increased the interest in S-glutathionylated proteins (PSSG) in the past few years and the list of proteins demonstrated to be S-glutathionylated increases continually [for reviews, see 5,7–10]. The fact that PSSG are involved in numerous physiological processes such as growth, differentiation, cell cycle progression, transcriptional activity, cytoskeletal functions, and metabolism, suggests that S-glutathionylation is a general mechanism of redox regulation. In fact, whereas posttranslational modifications such as phosphorylation, acetylation, and ubiquitinylation have been well established and understood for many years, the concept of protein S-glutathionylation as a posttranslational regulative modification, as opposed to a biomarker of oxidative damage, has gained acceptance only more recently [8–14]. The present review is meant to give just an overview of the potential role of protein sulfhydryl S-glutathionylation in the regulation of the structures/functions of a quite diverse range of cellular proteins. Oxidative modifications of protein thiols Cysteinyl thiols are particularly susceptible to oxidative modification and can undergo a diverse array of redox reactions, which are largely dependent on the species and concentration of oxidants they contact. The two major determinants of the susceptibility of thiols to redox regulation are the accessibility of the thiol within the three-dimensional structure of the protein and the reactivity of the cysteine, which is influenced by the surrounding amino acids. Most thiol modifications are unstable and can easily be reversed or replaced by other, more stable modifications. Although the thiol moiety on the side chain of cysteine is particularly sensitive to redox reactions, not all cysteinyl thiols are important as redox sensors, as most protein thiols do not react with oxidants under the conditions and at the concentrations they are found in cells. Nevertheless, some cysteine residues are susceptible to oxidation. The vast majority of cytoplasmic proteins contain cysteine sulfhydryls with a pKa value greater than 8.0 and, in the reducing environment of the cytoplasm, remain almost completely protonated at physiological pH. As a result, they are unlikely to be reactive with ROS/RNS. However, redox-sensitive proteins have specific Cys residues that exist as thiolate anions at neutral pH, due to a lowering of their pKa values as a result of charge interactions with neighboring


positively charged (i.e., basic) amino acid residues, becoming “active cysteines,” which are therefore more vulnerable to oxidation [15]. The thiolate anion of redox-sensitive Cys residues can readily be reversibly oxidized to sulfenic acid and protein or mixed disulfides or irreversibly oxidized to sulfinic and sulfonic acid (Fig. 1) [16,17], which are usually detrimental to protein function, although sulfiredoxin has recently been found to specifically reduce the sulfinic acid moiety in peroxiredoxins [18–21]. Reversible oxidation is believed to protect proteins from irreversible oxidation but may also modulate protein function.

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Disulfides can form under oxidative conditions between two proteins (interprotein) or within a protein (intraprotein), causing changes in protein aggregation and conformation. As recently shown using proteomic techniques, intermolecular disulfide bonds are also formed in the cytoplasm upon exposure of cells to oxidative stress [16,22]. Interprotein disulfides can also have regulatory function, as recently shown, for instance, for the type I protein kinase A, which contains protein thiols that operate as redox sensors, forming an interprotein disulfide bond between its two regulatory RI subunits in response to cellular hydrogen peroxide [23]. This oxidative disulfide formation, which was

U

Fig. 1. Oxidative modifications of protein thiols. (A) The oxidation of a cysteine residue within a protein can result in the formation of a cysteinyl radical (Cys–S , not shown) or a sulfenic, sulfinic, or sulfonic acid derivative (the latter of which is always irreversible). (B) Alternatively, oxidation can result in a disulfide bridge (cystine). Disulfides can form under oxidative conditions between two adjacent proteins (intermolecular cystine or interprotein disulfide) or between two adjacent sulfhydryl groups within a protein (intramolecular cystine or intraprotein disulfide), causing changes in protein aggregation and conformation. (C) Reaction between protein cysteinyl residues and low-molecular-mass thiols such as glutathione and free cysteine can yield protein–glutathione or protein–cysteine mixed disulfides, respectively, i.e., S-glutathionylated or S-cysteinylated proteins. Each of these protein thiol modifications has the possibility of eliciting different cellular responses.

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already known but to date considered a constitutive structural bond, causes a subcellular translocation—from the cytosol to the nuclear and myofilament compartment and, to a lesser extent, a membrane fraction—and activation of the kinase, resulting in phosphorylation of multiple established substrate proteins. Protein sulfenic acids, which are principal products formed by protein thiols on contact with H2O2, are very unstable, being frequently an intermediate in sulfinic and/or sulfonic acid generation or readily reacting with other vicinal or accessible (lowmolecular-weight) thiols to form intra- or intermolecular disulfides, respectively [e.g., 24]. Despite their reactivity, it is now clear that some sulfenic acids are more stable, and these can be identified in proteins also under physiological conditions [24–26]. Protein sulfenate formation has important roles in redox signaling, particularly in the redox regulation of transcription factors and enzymes including tyrosine phosphatases, peroxiredoxins, and methionine sulfoxide reductases [27]. In isolated rat hearts treated with physiologically relevant concentrations of H2O2, Eaton and colleagues reported widespread protein sulfenic acid formation within cardiac tissue when H2O2 was elevated [24]. They concluded that protein sulfenic acids are widespread physiologically relevant posttranslational oxidative modifications that can be detected at basal levels in healthy tissue and are elevated in response to hydrogen peroxide. Oxidant exposure, in addition to resulting in irreversible oxidation of Cys residues to sulfonic acids, can lead to excessive disulfide bonding, protein misfolding, and aggregation [28,29]. Excessive disulfide bonding may lead to covalent aggregates that are difficult to reduce even when intracellular redox conditions are restored to normal. Mixed disulfides, i.e., S-thiolated proteins, form between protein sulfhydryls and low-molecular-mass thiols such as homocysteine, cysteinylglycine, free cysteine, and glutathione [30–33]. Because free Cys and GSH are the most abundant lowmolecular-mass thiols in vivo, S-cysteinylated and S-glutathionylated proteins will be the main mixed disulfides, which are not equally distributed between extracellular and intracellular settings. GSH shows high negative redox potential (high electrondonating capacity) combined with high (millimolar) intracellular concentration (∼ 1–10 mM), which generates great reducing power [34], and thus, GSH represents the major low-molecularmass antioxidant in cells. Major differences between cellular and extracellular compartments exist in terms of both the concentrations of sulfhydryl/disulfide systems and their relative redox states [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 represents the largest pool of low-molecular-mass thiols and disulfides in plasma and the extracellular compartment on the whole. The major low-molecular-mass sulfhydryl/disulfide system in cells, GSH/GSSG, is principally in the reduced form, whereas the major low-molecular-mass system in the extracellular compartment, Cys/cystine, is principally in the disulfide form, cystine. Thus, extracellular proteins may be prevalently S-cysteinylated,

whereas intracellular proteins may be prevalently S-glutathionylated. For example, whereas the fraction of S-thiolated hemoglobin in red blood cells is only S-glutathionylated [36,37], plasma proteins such as albumin are mainly Scysteinylated [33,38,39], possibly after formation of an intermediate sulfenic acid [25,26]. Because such a variety of protein cysteine oxidation states can be formed, this offers the potential for oxidant-specific functional effects in which particular ROS/RNS can differentially regulate the activity of redox-sensitive proteins on the basis that they form varied structural motifs. Mechanisms of S-glutathionylation Experimentally, S-glutathionylation of protein cysteinyl residues may be studied by using a number of different techniques [22,32,40–42], however, the mechanisms by which glutathione can react with protein thiols are not completely understood. The normal/physiological intracellular milieu is a reducing environment with a GSH/GSSG ratio around or even greater than 100, and thus the GSH/GSSG ratio constitutes a major redox buffer in cytosol [7,43]. Maintaining optimal GSH/GSSG ratios in the cell is critical to cell survival and is important in regulating the redox state of protein thiols [34]. Changes in GSH/GSSG ratios could potentially influence a number of target proteins by causing oxidation and disulfide exchange reactions at specific protein cysteinyl residues. As the GSH/GSSG ratio usually exceeds 100, the oxidation of a small amount of GSH to GSSG could also promote protein S-glutathionylation, shifting the 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 be formed by reaction with hydroxyl radical), sulfenic acid, or protein S-nitrosothiol (S-nitrosated protein) and GSH; (ii) thiol/ disulfide exchange reactions between protein thiols and GSSG or PSSG; (iii) reaction between protein thiols and intermediate S-nitrosothiols such as S-nitrosoglutathione (GSNO), which is able to modify PSH by both protein S-nitrosation and Sglutathionylation [44,45]; (iv) direct interaction between a free protein cysteinyl residue and GSH triggered by many oxidants. Furthermore, glutathione sulfenic acid (GSOH) and glutathione disulfide S-monoxide [GS(O)SG] are considered alternative mediators of S-glutathionylation [45–47]. The thiyl radical of U GSH, generated by reaction with hydroxyl radicals (HO ), can also form glutathionylated proteins. The reaction of glutathione thiyl radicals with proteins to generate PSSG is catalyzed by glutaredoxin [48], an enzyme normally acting as a reductant (see below). Indeed, the redox potential of most Cys residues is such that the ratio of GSSG versus GSH in cells would need to change ≈100-fold (i.e., from ∼ 100 to ∼1) to induce S-glutathionylation through a thiol exchange mechanism [43,49]. These considerations suggest that, although GSSG is capable of glutathionylating a number of proteins in vitro, such as the inhibitory κB kinase (IKK) β peptide [14], thiol exchange is an unlikely


intracellular mechanism for S-glutathionylation, because even the GSSG levels measured in oxidant-treated cells can be insufficient to trigger S-glutathionylation through a thiol/disulfide exchange reaction [e.g., 14]. Although the disulfide bond linking the protein and glutathione is readily reversible under reducing conditions, under oxidizing conditions the S-glutathionylation can be maintained indefinitely as a persistently glutathionylated protein [43,50]. However, in many situations protein S-glutathionylation is only transient, as an adjacent protein thiol displaces the glutathione moiety to form an intraprotein disulfide [43]. Thus, there are two main classes of S-glutathionylated proteins, those that are momentarily glutathionylated before protein disulfide formation and those that are persistently glutathionylated, of which the former seems to be more common [50]. The mechanistic reason for the tendency toward intraprotein disulfide formation is presumably that S-glutathionylation frequently occurs on protein thiols that are adjacent to a second thiol (vicinal thiols), in the same protein or in an adjacent protein, that rapidly displaces the glutathione moiety to form an intraprotein or an interprotein disulfide, respectively. Once the GSH/GSSG ratio has returned to its resting, high ratio, this will lead to reversal 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 the disulfide with the transient formation of a glutathionylated intermediate. Although a complete understanding of the formation pathways of S-glutathionylated proteins is still evolving and might provide scope for further mechanistic investigations, there is little doubt about the biochemical importance of protein Sglutathionylation. From a biochemical perspective, S-glutathionylation might fulfill several, often associated roles during oxidative stress, including reversible protein protection, regulation 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 PSSG under basal conditions can increase up to 20–50% under oxidative 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 as a storage mechanism for glutathione inside the cell, because GSH oxidized to GSSG would otherwise be rapidly extruded from the cell [52]. S-glutathionylation may also provide protection for PSH against irreversible modifications and protein damage in response to higher levels of oxidative stress. If a protein sulfhydryl group is glutathionylated, often at the expense of temporary loss in protein activity, it is not available for other oxidative reactions including irreversible oxidation to sulfinic and sulfonic acid (the latter is generally resistant to any type of cellular repair mechanism and leads to the proteasomal degradation of the protein) [7,34,53,54]. In this respect, S-glutathionylation is often

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considered a way to protect sensitive cysteinyl residues from other, possibly irreversible, forms of oxidation, thus allowing the cell to restore the cognate function of the protein when oxidative stress conditions are overcome. Supportive evidence for this arises, for instance, from the S-glutathionylation of the γglutamyl transpeptidase, which seems to protect this membranebound enzyme from the irreversible oxidative damage by hydrogen peroxide produced during γ-glutamyl transpeptidasemediated metabolism of GSH [55] and the S-glutathionylation, producing reversible inactivation, of α-ketoglutarate dehydrogenase in response to alterations in the mitochondrial GSH status [56], as well as the S-glutathionylation of HDL-associated paraoxonase 1 (PON1), which causes reversible inactivation of PON1 physiological activities, i.e., hydrolysis of specific oxidized lipids in LDL- and HDL-mediated cholesterol efflux from macrophages, under oxidative stress [57]. However, if the modified cysteine is functionally critical, S-glutathionylation not only will modify protein function, but could eventually compromise cellular activities [58,59]. In most cases this leads to an inhibition of protein activity, which is particularly evident in the case of enzymes. Also transcription factors, like c-Jun and NF-κB, are inhibited by S-glutathionylation [60–62]. Protein S-glutathionylation may even have a direct effect on ROS/RNS production and antioxidant defenses, because Cu/Znsuperoxide dismutase [47,63], thioredoxin [64,65], glutaredoxin [48], and 1-Cys peroxiredoxin [66–68] have been demonstrated to undergo S-glutathionylation of functionally sensitive cysteinyl residues. Furthermore, there is a reversible increase in the U production of O2 − by mitochondrial NADH-ubiquinone oxidoreductase in response to glutathionylation/deglutathionylation [69]. A correlation between protein S-glutathionylation and gene expression has also been proposed. A recent study reported several links between GSH levels and gene regulation in cultured HL60 cells exposed to H2O2 for various times [11]. This study showed that several genes, including those involved in NF-κB activation, transcription, and DNA methylation, in addition to several immune system-related genes, including cytokines and cytokine receptors, were regulated at different time points in response to H2O2 under conditions that resulted in increased protein S-glutathionylation [11]. In cultured human lung epithelial A549 and endothelial ECV304 cells, oxidative stress is linked to protein S-glutathionylation and multiple changes in specific mRNA levels [51]. The results indicate that the degree of S-glutathionylation of cellular proteins is closely correlated with the induction of a selective cluster of molecular chaperone genes common to both cell types, which includes not only a range of heat shock proteins but also DNA chaperones and transcriptional regulators [51]. Although it is difficult to derive a conclusive correlation between protein S-glutathionylation and gene expression using cells with an artificially disrupted GSH level, these studies point toward a significant role for PSSG formation in the cellular response to oxidative stress. This notion is supported by findings showing that S-glutathionylation and deglutathionylation are not random processes, but are partially under enzymatic control (see below).

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Remarkably, S-glutathionylation is a posttranslational modification that occurs not only during oxidative stress, but also under basal conditions [42,70,71]. Constitutive S-glutathionylation has been shown, for instance, in hemoglobin in red blood cells [36,72,73], γ-crystallin from human lens [74], and actin in human fibroblasts [75] and human epidermal A431 cells [76]. Protein S-glutathionylation under basal conditions suggests its possible involvement in cellular signaling and redox regulation of protein functions [8,10]. In signaling processes and redox regulation of proteins, especially for those of the “on–off switch” type exemplified by protein phosphorylation, two important requirements are specificity and reversibility. Specificity of S-glutathionylation At present it is unclear what features contribute to the sensitivity of a given cysteinyl residue to S-glutathionylation, although protein sulfhydryls exhibit a striking differential susceptibility to formation of mixed disulfides with glutathione, and the question about the factors that facilitate and confer site specificity on such modification is still debated. The identity of surrounding residues in the primary sequence or the tertiary structure may play a role in making a thiol more or less reactive. The reaction rate of most protein cysteines with ROS/RNS and/or GSH is too slow to be of physiological relevance under cellular conditions and concentrations. This situation changes drastically when cysteine is bound to a metal ion [17,77], such as Mg2+, Ca2+, or Zn2+, which can function as an allosteric effector to control PSH reactivity, or is in the thiolate anion (- S−) form [15]. As the pKa of cysteine is around 8.5, the same as for cysteine in GSH, dissociation to form a thiolate occurs only in unusual microenvironments in redox-sensitive proteins in which the nearby amino acid residues significantly lower the pKa through electrostatic interactions. Thus, formation of a cysteine thiolate anion (i.e., an “active cysteine”), which can then react to form a mixed disulfide with GSH or a protein disulfide [15,16], is favored by basic amino acids in its vicinity, whereas acidic vicinal amino acids will have the opposite effect. Therefore, a cationic environment renders the thiol group highly reactive and particularly susceptible to S-glutathionylation [60,78]. This provides a basis for specificity in S-glutathionylation. The concept of specificity of protein S-glutathionylation is supported, for instance, by rat hemoglobin, in which the low pKa value of fast-reacting thiols was explained, on the basis of threedimensional models, by charge stabilization of the thiolate form through 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 useful guide to its reactivity, this is not a generalizable rule. Little is known about the structural determinants favoring S-glutathionylation. Simple rules based on primary amino acid sequence, i.e., redox-active motifs equivalent to primary amino acid consensus sequences existing for protein kinase substrates, are likely to have some limitations on the susceptibility of a given cysteine, as the three-dimensional structure of the protein will influence sulfhydryl reactivity and its accessibility to ROS/RNS and/or glutathione. Of principal importance is whether the oxi-

dant and/or glutathione can actually make contact with a potentially reactive protein thiol and then whether they will react under specific cellular conditions. Thus, Cys residue accessibility in the three-dimensional structure provides a basis for S-glutathionylation specificity, as illustrated, for instance, in the S-glutathionylation of thioredoxin [64] as well as of elongation factor 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 readily accessible regions of the respective protein molecules, i.e., in a loop region between two domains for elongation factor 1-α-1 and on an α-helix adjacent to three conserved glycines in the ATP-binding site for heat shock protein 60 [80]. Actin and carbonic anhydrase III are other examples of how S-glutathionylation may depend on the solvent accessibility of the Cys residues. Actin contains five cysteinyl residues existing in the reduced form. Native actin exposes one fast-reacting sulfhydryl group, that of Cys374 , next to the C-terminus and easily accessible to sulfhydryl-reactive agents. The exposed Cys374 residue is the most likely glutathionylation site both in vivo and in 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 a plausible candidate site for regulation of actin polymerization [80]. The three-dimensional structure of S-glutathionylated mammalian carbonic anhydrase III reveals that glutathione binds to Cys181 and Cys186 , the two highly surface-exposed of its five cysteinyl residues [78,82]. Cys181 and Cys186 are located in a rather neutral environment; nonetheless S-glutathionylation can be achieved by specific interactions between the glutathione moiety and the solvent-accessible Cys residues. Although both surface-exposed Cys181 and Cys186 are susceptible to S-glutathionylation, Cys186 is more readily modified both in vitro and in vivo [83]. Lys211 seems to be primarily responsible for the lowering of the pKa of Cys186, 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 to S-glutathionylation by GSSG, albeit to different extents, as recently shown by Cohen's group using isotope-coded affinity tag and mass spectrometry: the extent of S-glutathionylation of Cys118 by GSSG was 53%, and that of the terminal cysteines was 85% [84]. In cells, because the terminal cysteines are largely modified by lipid, Cys118, which is part of the guanine nucleotide binding site, is the prime target for S-glutathionylation and regulates p21ras activity, as indicated by the fact that both Sglutathionylation and stimulation of activity by oxidants are mostly 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 be involved in S-glutathionylation [e.g., 61,63]. An elegant study has recently analyzed the susceptibility to S-glutathionylation of the four Cys residues of cyclophilin A (Cys52, Cys62, Cys115, and Cys161 )—a ubiquitous intracellular protein, target of the immunosuppressive drug cyclosporin A, with multiple actions, including protein folding and chaperone activity—considering both the solvent exposure through molecular dynamics simulation and the influence of structural neighboring amino acids


through electrostatic calculations [13]. Using MALDI-MS Ghezzi and colleagues identified Cys52 and Cys62 as targets of glutathionylation in T lymphocytes, although molecular dynamic simulation showed that Cys52 and Cys161 exposed a larger surface of their side chains than Cys62 and Cys115. Therefore, a correlation between the solvent accessibility of Cys residues of cyclophilin A and glutathionylation cannot be made [13]. Electrostatic energy computations made to further define the nucleophilic reactivity of the various Cys residues in cyclophilin A showed that the changes in electrostatic energy involved in the conversion of a sulfhydryl to the corresponding thiolate anion follow the order Cys52 b Cys62 b Cys115 b Cys161, meaning that Cys52 and Cys62 form the thiolate anion with greater ease than Cys115 and Cys161 and can therefore be expected to be more reactive. Hence, the experimental observations, together with the theoretical calculations on the susceptibility of cyclophilin A Cys residues to glutathionylation, suggest that, even when a Cys residue has a small surface exposure, as in the case of cyclophilin A Cys62, electrostatic interactions can lead to susceptibility to S-glutathionylation [13]. The local pH and hydrophobic compartmentalization could be other important factors that render certain Cys residues highly reactive and particularly susceptible to S-glutathionylation [60,61]. Reversibility of S-glutathionylation The main feature that makes S-glutathionylation a possible regulatory mechanism is its reversibility. In fact, reversibility of posttranslational modifications is a requisite for cellular signaling. Deglutathionylation is the removal of the GSH moiety from

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protein mixed disulfides and can occur when the environment becomes more reducing in an enzyme-dependent or -independent manner (Fig. 2). To date, a limited number of proteins have been identified that are involved in deglutathionylation. S-glutathionylation of protein thiols can be reversed via direct thiol/disulfide exchange reactions with GSH once the reducing intracellular redox balance (mainly, an appropriate GSH/GSSG ratio) has been restored [34], by means of an enzymatically mediated reaction. Enzymes capable of reducing S-glutathionylated proteins include the glutaredoxins (GRX; also known as thioltransferases)/GRX reductase system [49,89], the thioredoxins (TRX)/TRX reductase system [90–92], and sulfiredoxin [19,20,93]. The TRX and GRX families contain a conserved -Cys-X-XCys-active site (Cys32 and Cys35 within thioredoxin-1), which is essential for their redox regulatory functions [49,89,90,92]. They catalyze the reduction of disulfide bonds and become concomitantly oxidized by forming an intramolecular disulfide in the -Cys-X-X-Cys- active site. The oxidized enzyme is then reduced by TRX reductase, in the case of TRX, or by GSH, in the case of GRX. In particular, TRX/TRX reductase can reduce protein disulfides and protein sulfenic acid intermediates. Thioredoxin-1 itself has been shown to be subject to Sglutathionylation, in addition to S-nitrosation and intramolecular disulfide formation, occurring not at cysteines spanning the redox-regulatory domain, but at three additional nonactive cysteine residues at positions 61, 68, and 72 [64]. The glutathionylation site was identified as Cys72 and this residue could be modified either by GSSG or by GSNO. Modification of the site abolished enzymatic activity of TRX. However, activity spontaneously recovered, suggesting that TRX was able to deglutathionylate itself. Such results support the concept of a

Fig. 2. Induction and reversal of protein S-glutathionylation. Formation of protein–glutathione mixed disulfides, i.e., S-glutathionylated proteins (PSSG), in biological systems can occur after a decrease in the intracellular/cytoplasmic GSH/GSSG ratio that can induce the partial oxidation of protein sulfhydryls, yielding an “activated” protein thiol (thiyl radical or sulfenic acid). GSH can then interact with this activated thiol to give an S-glutathionylated protein. Alternatively, S-glutathionylation can be the result of thiol/disulfide exchange in the presence of increased cellular levels of GSSG or by other mediators (e.g., GS(O)SG or GSOH). Furthermore, PSSG formation can be promoted by nitric oxide, through various mechanisms including the formation of S-nitrosoglutathione (GSNO) and thiyl radicals. Finally, glutaredoxin can also catalyze the S-glutathionylation of certain proteins in the presence of a GS -generating system via a monothiol mechanism of thiol/disulfide exchange. Reversal of S-glutathionylation (deglutathionylation) can be achieved by changes in the intracellular redox status (increases in the GSH/GSSG ratio), by reduced thiols, or via enzymatic reduction by glutaredoxin (also known as thioltransferase) and sulfiredoxin, which selectively deglutathionylate PSSG by a GSHdependent or -independent mechanism, respectively, and, in a limited number of proteins, by the thioredoxin system.

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coordination of regulatory pathways through a possible cross talk between the glutathione and the TRX systems under conditions of oxidative stress [64]. Interestingly, chloroplast thioredoxin f, a key factor in the redox regulation of carbon-fixation enzymes, has recently been shown to undergo S-glutathionylation at a conserved, additional (other than its two active-site cysteines) cysteine that is not required for its activity. S-glutathionylation modulates thioredoxin f efficiency: the activation of its target enzymes is strongly decreased when thioredoxin f is S-glutathionylated [65]. Glutaredoxin was the first protein identified as a specific glutathionyl-mixed disulfide oxidoreductase [94]. Interestingly, GRX not only enzymatically deglutathionylates specific proteins [49,89] but has also been shown to catalyze the S-glutathionylation of certain proteins in the presence of a GS-radicalgenerating system [48,95] via distinct mechanisms [96]. Only one of the two Cys residues contained in the conserved -Cys-XX-Cys-motif of GRX is required for its oxidative function of S-glutathionylation via a monothiol mechanism of disulfide exchange involving the high reactivity of a GRX–SG mixed disulfide, whereas the presence of both Cys residues within the conserved -Cys-X-X-Cys-motif is required for its reductive function of deglutathionylation via a dithiol mechanism of disulfide exchange [49,89,96]. The formation of protein–SG mixed disulfides by GRX through a monothiol mechanism may play an important role in protecting against more drastic, irreversible modifications of protein thiols, particularly when the redox state of the cytoplasm becomes more oxidizing, as under conditions of oxidative stress. Human sulfiredoxin (Srx1) is the first protein identified that has been implicated specifically in the reductive deglutathionylation of proteins [93]. The specificity of Srx1 may be due to the presence of only one cysteinyl residue within its sequence. Although the conserved cysteinyl residue in Srx1 is essential for the deglutathionylation reaction, current data suggest that it is not a direct acceptor for the GSH moiety, because Srx1, in contrast to GRX, does not form a mixed disulfide with GSH during the deglutathionylation reaction, i.e., it is not itself glutathionylated during the removal of GSH moieties [93]. Proteins regulated by S-glutathionylation The existence of antioxidant enzymes that serve the unique role of specifically reducing protein–SG mixed disulfides emphasizes the importance of S-glutathionylation in modulating protein function. Actually, S-glutathionylation has been shown to regulate in a complex manner, either positively or negatively, the structure/function of a quite diverse range, and increasing 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 anhydrase 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 to regulation by S-glutathionylation, in most cases resulting in inhibition of enzyme activity. In the case of carbonic anhydrase III, S-glutathionylation of Cys186 is required for its phosphatase activity, whereas S-glutathionylation of Cys181 blocks activity [97]. Caspase-3 cleavage and activation are known to play central roles in apoptosis. However, the mechanisms that regulate caspase-3 cleavage remain elusive. S-glutathionylation has recently been shown to regulate TNF-α-induced caspase-3 cleavage and activation, and thus the resultant apoptosis, in endothelial cells [102]. In particular, an inverse correlation has been shown between caspase-3 S-glutathionylation and cleavage, and GRX plays an essential role in caspase-3 cleavage via regulation of caspase-3 S-glutathionylation. These findings demonstrate a novel mechanism of caspase-3 regulation in TNF-α-induced apoptosis. The HIV-1 protease is another enzyme that is regulated by S-glutathionylation. It contains conserved and critical Cys residues that, when S-glutathionylated, activate or deactivate the protease depending on the thiol group involved. In particular, S-glutathionylation of Cys95 inhibits the activity but S-glutathionylation of Cys67 stabilizes the HIV-1 protease [103]. Signaling proteins Redox status has dual effects on upstream signaling systems and downstream transcription factors. Oxidants can stimulate many upstream kinases in signaling pathway cascades and yet inhibit transcription factors AP-1 and NF-κB. Reductants can have opposite effects producing AP-1 and NF-κB activation [104]. Many signaling molecules and transcription factors fundamental for cell growth, differentiation, and apoptosis seem to be regulated by S-glutathionylation [7,105]. The S-glutathionylation of signaling molecules such as protein kinase A, protein kinase C, mitogen-activated protein kinase (MAPK)/extracellular-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 of kinases, modulates their activity. The catalytic subunit of cAMPdependent protein kinase (protein kinase A) is susceptible to inactivation through S-glutathionylation of Cys199 , which is located near the active site [107]. S-glutathionylation plays a key role in the regulation of the kinase activity of MEKK1 (MAPK/ERK kinase kinase 1; MAP3K), an upstream activator of the SAPK/JNK (stressactivated protein kinase/c-Jun N-terminal kinase) pathway, in response to oxidative stress [111]. MEKK1 was shown to be inhibited by site-specific S-glutathionylation in a single unique cysteinyl residue in the ATP-binding domain in vitro and MEKK1 isolated from menadione-treated cells was shown by mass spectrometry to be modified by glutathione on the same Cys residue. As MEKK1 provides a protein kinase with a demonstrated survival signaling function, its S-glutathionylation might participate in shifting the balance from cell survival to cell death by apoptosis in response to oxidative stress.


Transcription factors By introducing the negative charges of GSH within their DNA binding sites, S-glutathionylation inhibits the DNA binding activity of the redox-sensitive transcription factors cJun and NF-κB [60–62]. It has been well established that NF-κB, a central regulator of immunity, is subject to multiple regulation by redox changes. S-glutathionylation of Cys62 of the p50 subunit is known to prevent binding of the transcription factor to κB sites in the promoter regions of genes [61]. Remarkably, Sglutathionylation of the p65 subunit of NF-κB, which is mainly responsible for transcriptional activation, has been shown to inhibit p65–NF-κB binding to DNA, resulting in decreased resistance to hypoxia in pancreatic cancer cells supplemented with GSH via N-acetylcysteine [62]. It has now been reported that Cys179 of the IKK β subunit of the IKK signalosome is a central target for oxidative inactivation by means of Sglutathionylation, which is responsible for the repression of kinase activity by H2O2. S-glutathionylation of IKK-β Cys179 is reversed by GRX, which restores kinase activity [14]. Reynaert and colleagues propose that GRX1-dependent reversal of Sglutathionylation of IKK-β constitutes a protective mechanism that modulates the extent and timing of activation of NF-κB in response to redox changes by protecting IKK-β from irreversible inactivation and allowing for rapid regeneration of enzymatic activity [14]. Collectively, these data suggest that S-glutathionylation could be a physiologically relevant mechanism for controlling the magnitude of activation of the NF-κB pathway. Pax-8 belongs to a family of transcription factors that contain a paired domain as a DNA-binding domain. Pax-8 stimulates expression of a set of thyroid-enriched proteins, thyroglobulin, thyroperoxidase, and sodium/iodide symporter. Recent evidence proves that a decrease in the GSH/GSSG ratio induces Sglutathionylation of the first two cysteine residues, Cys45 and Cys57, of three present in the paired domain of Pax-8, resulting in a loss of DNA binding [112]. The authors suggest that the GSH/ GSSG-dependent oxidative inactivation of Pax-8 could account for the decreased thyroglobulin expression in the cells with a low level of GSH. Therefore, S-glutathionylation of these cysteines has a regulatory role in paired domain binding and provides a new insight into the molecular basis for the modulation of Pax function [112]. Furthermore, S-glutathionylation of OxyR, a bacterial DNA binding protein, stimulates transcription of multiple redox response genes [113]. Ras proteins The Ras proteins are small guanine nucleotide exchange proteins that play a critical role as signal transducers in the regulation of a number of cellular processes, including cell growth, differentiation, and apoptosis. Reactions of ROS/RNS with Cys118, which resides near the guanine nucleotide binding site, has been implicated in activation of p21ras leading to downstream signaling to Raf-1/MEK/ERK to mediate cellular responses [88]. Cohen's group has recently demonstrated that increased levels of oxidants in both endothelial and smooth muscle cells can directly activate p21ras by S-glutathionylation

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of a reactive thiol on Cys118 and trigger downstream signaling through phosphorylation of ERK and AKT [85,87]. They could likewise show that S-glutathionylation of Cys118 on Ras was the critical step mediating angiotensin II-induced hypertrophy of vascular smooth muscle cells, thus identifying Ras as the upstream molecular target of ROS/RNS in this model [85]. More recently, Cohen and colleagues have investigated if oxidized LDL could regulate insulin signaling as a direct result of increasing oxidant-mediated p21ras activation. In this elegant study, the authors demonstrate that S-glutathionylation and activation of p21ras caused by peroxynitrite generated in endothelial cells by oxidized LDL promotes MEK-dependent ERK activation, resulting in ERK-dependent phosphorylation of insulin receptor substrate 1 and inhibition of insulin-mediated AKT phosphorylation [86]. These findings suggest a novel molecular mechanism by which oxidants could induce endothelial insulin resistance via S-glutathionylation of p21ras and ERK-dependent inhibition of insulin signaling [86]. Ras also plays a crucial role in the regulation of hypertrophic growth in cardiac myocytes in response to stimuli such as αadrenergic receptor agonists and mechanical strain. The role of S-glutathionylation in the regulation of Ras has also been studied in cardiomyocytes, in models of ROS-mediated cardiac hypertrophy stimulated by mechanical strain or α-adrenergic receptor [114,115]. Results support the hypothesis that Sglutathionylation of Ras is functionally relevant for ROSmediated signal transduction leading to cardiomyocyte hypertrophy. In particular, these findings indicate that mechanical strain causes ROS-dependent S-glutathionylation of Ras at Cys118, leading to myocyte hypertrophy via activation of the Raf/MEK/ERK growth pathway [115]. Heat shock proteins Correct protein folding is an essential cellular function controlled by heat shock proteins (HSPs), which stabilize exposed hydrophobic surfaces of nonnative proteins to facilitate correct folding into functional structures. Members of the HSP60 and HSP70 group have been found to be modified by Sglutathionylation in T lymphocytes stressed with hydrogen peroxide or diamide [116] as well as in ECV304 endothelial cells during constitutive metabolism or diamide-induced oxidative stress [70,80]. S-glutathionylation of heat shock cognate protein 70, a constitutively expressed homologue of HSP70, in retinal pigment epithelium increases its chaperone function, increasing fivefold its ability (compared to the reduced HSP) to prevent heat-induced protein aggregation [117]. Maintaining active chaperones is important for regulation of oxidative misfolding of proteins, an event linked to cellular damage and disease. Thus, redox regulation of HSP chaperone activity may be a physiologic response to oxidative stress relevant to major disease processes. Ion channels and Ca2+ pumps Other targets for S-glutathionylation, affecting ion transport activity, include ion channels, such as the Ca2+ -release/

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ryanodine receptor channels (RyR) and the cystic fibrosis transmembrane conductance regulator (CFTR), a phosphorylation/ATP-dependent chloride channel that modulates salt and water transport across lung and gut epithelia [118,119]. NADPH addition to microsomes isolated from heart muscle significantly enhances, via NADPH oxidase activation, both ryanodine receptor type 2 S-glutathionylation and Ca2+-induced Ca2+ release [120]. The highly redox-sensitive ryanodine receptor type 1 (RyR1), which is essential for skeletal muscle excitation– contraction coupling, is S-glutathionylated by H2O2 in vitro in the presence of GSH, which enhances channel activity [118,121]. Additionally, the RyR1 protein is susceptible to S-nitrosation [118]. A successive work by the same authors shows that the skeletal muscle T-tubules possess a functional nonphagocytic NADPH oxidase activity. This enzyme generates H2O2, which, in triads isolated from mammalian skeletal muscle composed of junctional T-tubule and sarcoplasmic reticulum membranes, decreases the total sulfhydryl content and activates RyR1 through S-glutathionylation, enhancing calcium release [122]. Increased S-glutathionylation of RyR, eliciting calcium release and sequentially enhancing CREB (the transcription factor cAMP/Ca2+ response element binding protein) phosphorylation via ERK activation, has been confirmed in mouse N2a neuroblastoma cells and in primary rat hippocampal neurons [123]. A restricted number of cysteines, of the 100 cysteines in each RyR1 subunit, that are likely to be involved in the functional response of the skeletal muscle Ca2+ release channel (RyR1) have recently been identified as specific substrates of S-nitrosation, S-glutathionylation, and oxidation to disulfides [124]. Human CFTR channels are reversibly inhibited by reactive glutathione species, i.e., GSSG, GSNO, and glutathione treated with diamide. Three lines of evidence indicate that the likely mechanism for this inhibitory effect is S-glutathionylation of a CFTR cysteine [119]. Regulatory effects of S-glutathionylation have also been described for the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) [125,126] and the small GTPase transforming protein, p21ras (Ras) [53,85–87,114]. Cohen and colleagues recently found that SERCA is S-glutathionylated by NO (in the form of its active “effector” peroxynitrite) in intact cells or arteries, as well as in vitro, resulting in accelerated Ca2+ uptake activity of the protein, explaining cGMP-independent arterial relaxation due to NO, thereby relaxing vascular smooth muscle by lowering intracellular free Ca2+ [125,126]. Whereas several other cysteines can be S-glutathionylated, the site where most of the GSH was bound is the most reactive thiol on SERCA (Cys674), which regulates enzyme function. In atherosclerotic aorta, NO does not increase S-glutathionylation of SERCA and, as a result, fails to stimulate its activity because of the irreversible oxidation of the Cys674 thiol (more than 50%) to sulfonic acid [125,126]. Mitochondrial proteins Mitochondria represent one of the most oxidative environments inside cells, and the S-glutathionylation of mitochondrial proteins 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 production by mitochondrial complex I increases in response to oxidation of the mitochondrial GSH pool to GSSG. This correlates with S-glutathionylation of thiols on the 51- and 75kDa subunits of complex I in the isolated complex, bovine heart mitochondrial membranes, and intact mitochondria. S-glutathionylation of complex I increases superoxide production by the complex and, when the mixed disulfides are reduced, superoxide production returns to basal levels. Within intact mitochondria, most of this superoxide produced after S-glutathionylation of complex I is converted to hydrogen peroxide, which can then diffuse into the cytoplasm. This mechanism of reversible mitochondrial ROS production suggests how mitochondria might regulate redox signaling and shows how oxidation of the mitochondrial glutathione pool could contribute to the pathological changes to mitochondria that occur during oxidative stress [69]. A wide range of mitochondrial membrane proteins contain exposed, reactive thiols that reacted with GSSG by thiol–disulfide exchange to form a mixed disulfide. However, only a few thiol proteins remained glutathionylated, with most displacing the GSH to form an intraprotein disulfide [50]. Consequently, the proportion of protein thiols that was persistently glutathionylated was far smaller than that which formed intraprotein disulfides. Complex I was the most prominent mitochondrial membrane protein that was persistently S-glutathionylated by GSSG in the presence of mitochondrial thiol transferase glutaredoxin 2 (GRX2) [50]. The mechanistic reason for greater intraprotein disulfide formation is likely that most S-glutathionylated protein thiols are formed adjacent to a second thiol that rapidly displaces GSH to form an intraprotein disulfide. The activity of α-ketoglutarate dehydrogenase too seems to be modulated through enzymatic glutathionylation and deglutathionylation catalyzed by GRX [56]. Mitochondrial NADP+dependent isocitrate dehydrogenase was shown to be susceptible to inactivation by S-glutathionylation of Cys269 [128]. The inactivated NADP+-dependent isocitrate dehydrogenase was reactivated enzymatically by GRX2 in the presence of GSH [128]. Cytochrome oxidase subunit Va within human T lymphocyte cells was S-glutathionylated in response to diamide [116], and cytochrome oxidase subunit Vb in rat hepatocytes was S-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 redox regulation of a variety of cytoskeletal proteins, including tubulin and actin. S-glutathionylation of β-tubulin has been reported in endothelial-like cells [48,70], whereas a direct thiol-disulfide exchange between GSSG and native tubulin was demonstrated in vitro [130]. Actin is perhaps one of the best examples of physiologically relevant regulation by S-glutathionylation. Actin has been shown to polymerize into filaments, translocate from a uniform cytosolic distribution to the cellular periphery, and rearrange into


membrane ruffles in response to epidermal growth factor (EGF) in A431 cells [76] and in response to fibroblast growth factor in NIH3T3 cells [131]. Treatment of A431 cells with EGF leads to deglutathionylation of actin despite an increase in intracellular ROS [76]. In fact, under normal (basal) cellular conditions, a portion of G-actin is S-glutathionylated at Cys374. This likely inhibits polymerization into F-actin, as deglutathionylation leads to an increased rate of polymerization, and inhibition of actin polymerization by Cys374 glutathionylation has been well demonstrated in vitro [81,132]. The deglutathionylation is catalyzed by GRX in cells [76]. Furthermore, specific knockout of GRX1 in NIH3T3 cells by tetracycline-inducible RNAi abolished growth factor-mediated actin polymerization, translocalization to the cell periphery, and membrane ruffling [131]. These studies suggest that reversible S-glutathionylation of actin by GRX contributes to the regulation of the cellular functions of actin. Regulation of actin function by S-glutathionylation of Cys374 decreases the rate of actin polymerization most likely due to a change in protein conformation [81]. Another evidence of in vivo actin redox regulation by a physiological source of ROS, specifically those generated by integrin receptors during integrin-mediated cell adhesion, has recently been shown in murine NIH3T3 fibroblasts [12]. Actin oxidation takes place via S-glutathionylation of Cys374; this modification is essential for cell spreading and for cytoskeleton organization. Impairment of actin S-glutathionylation, through either GSH depletion or expression of the C374A redox-insensitive mutant, greatly affects cell spreading and the formation of stress fibers, leading to inhibition of the disassembly of the actinomyosin complex. The control of cytoskeleton organization is mainly due to the action of the members of the family of the small GTPases Rho. Nevertheless, it was previously shown that ROS can act as second messengers in the organization of cytoskeleton in response to integrin-mediated cell adhesion [133]. These recent data suggest that actin S-glutathionylation is essential for cell spreading and cytoskeleton organization and that it plays a key role in disassembly of the actinomyosin complex during cell adhesion [12]. Furthermore, other recent results suggest that S-glutathionylation of actin in the thin filament during ischemia–reperfusion injury may alter the contractile performance of the myocardium [134]. Finally, actin has been identified as a specific target of Srx1-dependent deglutathionylation both in vitro and in vivo [96]. Moreover, many proteins interact with actin to enhance its organization, such as actin-cross-linking proteins that connect actin filaments to each other and actin-binding proteins that connect the actin cytoskeleton to the cell membrane, such as annexins, one group of multifunctional, Ca 2+ -dependent heterotetrameric proteins. Among these, S-glutathionylation of two (i.e., Cys8 and Cys132) of four cysteines of tetrameric annexin II causes an inhibition of annexin binding activity that is reversible by GRX [135]. At the cellular level, S-glutathionylation of annexin II has been identified in HeLa cells after stimulation with TNF-α [136]. Collectively, these data provide a basis for proposing that S-glutathionylation could regulate cellular actin both directly and indirectly through annexin II or perhaps other accessory actin proteins. Indeed, other cytoske-

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letal proteins than actin, such as vimentin, myosin, tropomyosin, cofilin, profilin, and α-actinin, have been shown to be S-glutathionylated in human T cell blasts and human platelets in response to oxidants [116,137]. Increased S-glutathionylation of actin and vimentin has been detected in isolated rat aortic ring stimulated with acetylcholine [138]. In contrast, our data demonstrate that NO donors do not elicit actin S-glutathionylation [45]; therefore it is puzzling that the in vivo delivery of NO elicited by acetylcholine may result in actin S-glutathionylation. Furthermore, S-glutathionylation of membrane skeletal proteins has been detected in red blood cells exposed to diamide to model the effects of oxidative stress [139]. Thus, understanding the regulation via S-glutathionylation of actin and other cytoskeletal components could reveal another example of regulation of translocation and signal transduction widespread throughout the cell. Conclusions and perspectives An increasing body of evidence has highlighted an important role for a variety of redox signaling mechanisms in the control of a plethora of cellular processes. One mechanism by which GSH can regulate cellular functions is through S-glutathionylation of protein sulfhydryls, a protein modification that has been detected also under basal/physiological conditions and not only after oxidative/nitrosative stress. S-glutathionylation accounts for the reversible regulation of structure and function of a wide variety of proteins that are integral to cell structure, signaling, and metabolism. Despite the abundance of cellular protein thiols, Sglutathionylation occurs with a certain specificity, although not yet completely established. Where phosphorylation depends on kinases for specificity, S-glutathionylation depends on protein structure and thiolate anion reactivity for specificity. Some additional specificity may be provided by the action of GRXs, and possibly Srx, which enzymatically add or remove GSH from proteins. Thus, like phosphorylation, the reversible S-glutathionylation of protein thiols may be a mechanism to turn signaling pathways on or off through modification of important cysteinyl residues or through the induction of conformational changes in the protein. If S-glutathionylation may be considered a redox-dependent posttranslational modification with potential relevance to signal transduction, then glutathione is not only an essential antioxidant, but also an essential signaling molecule, also considering that, in the context of redox regulation, the ratio between GSH and GSSG determines the redox state of redoxsensitive cysteines in some proteins [9,11]. Redox signaling may contribute to the control of cell development, differentiation, growth, death, and adaptation and has been implicated in diverse physiological and pathological processes [140,141]. If GSH is also an essential signaling molecule, one should wonder whether the deleterious consequences that glutathione depletion has under many oxidative stress conditions are not only due to the lack of an antioxidant, thiol-sparing molecule, but also due to the blockade of S-glutathionylation-dependent signaling mechanisms, similar to the findings that ATP depletion can affect phosphorylation-mediated signaling. Overproduction or

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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 from excessive generation of ROS/RNS and/or impaired antioxidant defenses. After moderate elevation of ROS/RNS levels and/or moderate impairment of antioxidant defenses (moderate oxidative stress), reversible S-glutathionylation of protein thiols maintains glutathione inside the cells (often at the expense of temporary loss of protein function) and protects protein sulfhydryl groups from irreversible oxidation. At high ROS/RNS levels and/or strong impairment of antioxidant defenses (severe oxidative stress), irreversible oxidation of protein sulfhydryl groups can change protein function permanently, thus impairing the physiological redox regulation of proteins with redox-sensitive cysteines. Irreversible modifications are usually associated with permanent loss of function and may lead to the elimination of the damaged proteins by the proteasome system or to their accumulation as insoluble aggregates.

underscavenging of ROS/RNS (oxidative/nitrosative stress) can irreversibly oxidize protein thiols. This irreversible oxidation could cause aberrant cellular signaling, eliminating GSHdependent signaling mediated by protein sulfhydryl glutathionylation (Fig. 3). The application of redox proteomics techniques [22,32,40– 42,142] will enable an increasing number of the target protein thiols, representing pivotal regulatory control points, to be uncovered. These data will provide an important platform to probe the molecular mechanisms underpinning S-glutathionylation and other reversible oxidative modifications of protein thiols. It is anticipated that future advances in redox proteomics may provide novel opportunities for both basic research and human disease research [142]. In particular, the continued development of proteomic and analytical methods for disulfide analysis will help us better understand the full extent that these modifications play in the regulation of cell function [142]. However, in exploring all these processes, the first challenge is to identify those proteins whose thiol redox state is affected by oxidative/nitrosative stress and/or redox signaling and those proteins that undergo reversible oxidative modifications under physiological conditions, determine whether the changes are due to the formation of a protein disulfide or to persistent Sglutathionylation or other reversible oxidation of cysteinyl thiols, find out which cysteinyl residues are modified, and explain how the redox changes affect protein function. At present, little is known about the details of these proteins and, especially, the physiological consequences of oxidation of their thiols. The identification of S-glutathionylated proteins and exploration of the cellular significance of this modification are really still in their infancy. Nevertheless, advances in structural biology and redox proteomics are now providing the molecular

tools to address these issues. This is a rich and promising area for future research. Acknowledgments Our apologies for any relevant reports we were unable to cite, due to either the need to selectively choose examples or our oversight. The authors' research was supported by FIRST 2006 (Fondo Interno Ricerca Scientifica e Tecnologica) University of Milan. References [1] Jones, D. P. Redefining oxidative stress. Antioxid. Redox Signal. 8:1865–1879; 2006. [2] Finkel, T.; Holbrook, N. J. Oxidants, oxidative stress and the biology of ageing. Nature 408:239–247; 2000. [3] Dalle-Donne, I.; Scaloni, A.; Giustarini, D.; Cavarra, E.; Tell, G.; Lungarella, G.; Colombo, R.; Rossi, R.; Milzani, A. Proteins as biomarkers of oxidative/nitrosative stress in diseases: the contribution of redox proteomics. Mass. Spectrom. Rev. 24:55–99; 2005. [4] Dalle-Donne, I.; Rossi, R.; Colombo, R.; Giustarini, D.; Milzani, A. Biomarkers of oxidative damage in human disease. Clin. Chem. 52:601–623; 2006. [5] Jacob, C.; Knight, I.; Winyard, P. G. Aspects of the biological redox chemistry of cysteine: from simple redox responses to sophisticated signalling pathways. Biol. Chem. 387:1385–1397; 2006. [6] Biswas, S.; Chida, A. S.; Rahman, I. Redox modifications of protein-thiols: emerging roles in cell signaling. Biochem. Pharmacol. 71:551–564; 2006. [7] Klatt, P.; Lamas, S. Regulation of protein function by S-glutathiolation in response to oxidative and nitrosative stress. Eur. J. Biochem. 267:4928–4944; 2000. [8] Giustarini, D.; Rossi, R.; Milzani, A.; Colombo, R.; Dalle-Donne, I. S-glutathionylation: from redox regulation of protein functions to human diseases. J. Cell. Mol. Med. 8:201–212; 2004.

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