Regulation of protein function by S-glutathiolation in response to oxidative and nitrosative stress.

Protein S-glutathiolation, the reversible covalent addition of glutathione to cysteine residues on target proteins, is emerging as a candidate mechanism by which both changes in the intracellular redox state and the generation of reactive oxygen and nitrogen species may be transduced into a functional response. This review will provide an introduction to the concepts of oxidative and nitrosative stress and outline the molecular mechanisms of protein regulation by oxidative and nitrosative thiol-group modifications. Special attention will be paid to recently published work supporting a role for S-glutathiolation in stress signalling pathways and in the adaptive cellular response to oxidative and nitrosative stress. Finally, novel insights into the molecular mechanisms of S-glutathiolation as well as methodological problems related to the interpretation of the biological relevance of this post-translational protein modification will be discussed.     

Regulation of plant cytosolic glyceraldehyde 3-phosphate dehydrogenase isoforms by thiol modifications

Cytosolic NAD-dependent glyceraldehyde 3-P dehydrogenase (GAPDH; GapC; EC 1.2.1.12) catalyzes the oxidation of triose phosphates during glycolysis in all organisms, but additional functions of the protein has been put forward. Because of its reactive cysteine residue in the active site, it is susceptible to protein modification and oxidation. The addition of GSSG, and much more efficiently of S-nitrosoglutathione, was shown to inactivate the enzymes from Arabidopsis thaliana (isoforms GapC1 and 2), spinach, yeast and rabbit muscle. Inactivation was fully or at least partially reversible upon addition of DTT. The incorporation of glutathione upon formation of a mixed disulfide could be shown using biotinylated glutathione ethyl ester. Furthermore, using the biotin-switch assay, nitrosylated thiol groups could be shown to occur after treatment with nitric oxide donors. Using mass spectrometry and mutant proteins with one cysteine lacking, both cysteines (Cys-155 and Cys-159) were found to occur as glutathionylated and as nitrosylated forms. In preliminary experiments, it was shown that both GapC1 and GapC2 can bind to a partial gene sequence of the NADP-dependent malate dehydrogenase (EC 1.2.1.37; At5g58330). Transiently expressed GapC-green fluorescent protein fusion proteins were localized to the nucleus in A. thaliana protoplasts. As nuclear localization and DNA binding of GAPDH had been shown in numerous systems to occur upon stress, we assume that such mechanism might be part of the signaling pathway to induce increased malate-valve capacity and possibly other protective systems upon overreduction and initial formation of reactive oxygen and nitrogen species as well as to decrease and protect metabolism at the same time by modification of essential cysteine residues.     

Irreversible thiol oxidation in carbonic anhydrase III: protection by S-glutathiolation and detection in aging rats

Proteins with reactive sulfhydryls are central to many important metabolic reactions and also contribute to a variety of signal transduction systems. In this report, we examine the mechanisms of oxidative damage to the two reactive sulfhydryls of carbonic anhydrase III. Hydrogen peroxide (H2O2), peroxy radicals, or hypochlorous acid (HOCl) produced irreversibly oxidized forms, primarily cysteine sulfinic acid or cysteic acid, of carbonic anhydrase III if glutathione (GSH) was not present. When GSH was approximately equimolar to protein thiols, irreversible oxidation was prevented. H202 and peroxyl radicals both generated S-glutathiolated carbonic anhydrase III via partially oxidized protein sulfhydryl intermediates, while HOCl did not cause S-glutathiolation. Thus, oxidative damage from H202 or AAPH was prevented by protein S-glutathiolation, while a direct reaction between GSH and oxidant likely prevents HOCl-mediated protein damage. In cultured rat hepatocytes, carbonic anhydrase III was rapidly S-glutathiolated by menadione. When hepatocyte glutathione was depleted, menadione instead caused irreversible oxidation. We hypothesized that normal depletion of glutathione in aged animals might also lead to an increase in irreversible oxidation. Indeed, both total protein extracts and carbonic anhydrase III contained significantly more cysteine sulfinic acid in older rats compared to young animals. These experiments show that, in the absence of sufficient GSH, oxidation reactions lead to irreversible protein sulfhydryl damage in purified proteins, cellular systems, and whole animals.     

The utility of N,N-biotinyl glutathione disulfide in the study of protein S-glutathiolation

Glutathione disulfide (GSSG) accumulates in cells under an increased oxidant load, which occurs during neurohormonal or metabolic stimulation as well as in many disease states. Elevated GSSG promotes protein S-glutathiolation, a reversible post-translational modification, which can directly alter or regulate protein function. We developed novel strategies for the study of protein S-glutathiolation that involved the simple synthesis of N,N-biotinyl glutathione disulfide (biotin-GSSG). Biotin-GSSG treatment of cells mimics a defined component of oxidative stress, namely a shift in the glutathione redox couple to the oxidized disulfide state. This induces widespread protein S-glutathiolation, which was detected on non-reducing Western blots probed with streptavidin-horseradish peroxidase and imaged using confocal fluorescence microscopy and ExtrAvidin-FITC. S-Glutathiolated proteins were purified using streptavidin-agarose and identified using proteomic methods. We conclude that biotin-GSSG is a useful tool in the investigation of protein S-glutathiolation and offers significant advantages over conventional methods or antibody-based strategies. These novel approaches may find widespread utility in the study of disease or redox signaling models where GSSG accumulation occurs.     

Oxidant-induced S-glutathiolation inactivates protein kinase C-alpha (PKC-alpha): a potential mechanism of PKC isozyme regulation

Protein kinase C (PKC) isozymes are subject to inactivation by reactive oxygen species (ROS) through as yet undefined oxidative modifications of the isozyme structure. We previously reported that Cys-containing, Arg-rich peptide-substrate analogues spontaneously form disulfide-linked complexes with PKC isozymes, resulting in isozyme inactivation. This suggested that PKC might be inactivated by oxidant-induced S-glutathiolation, i.e., disulfide linkage of the endogenous molecule glutathione (GSH) to PKC. Protein S-glutathiolation is a reversible oxidative modification that has profound effects on the activity of certain enzymes and binding proteins. To directly examine whether PKC could be inactivated by S-glutathiolation, we used the thiol-specific oxidant diamide because its oxidant activity is restricted to induction of disulfide bridge formation. Diamide weakly inactivated purified recombinant cPKC-alpha, and this was markedly potentiated to nearly full inactivation by 100 microM GSH, which by itself was without effect on cPKC-alpha activity. Diamide inactivation of cPKC-alpha and its potentiation by GSH were both fully reversed by DTT. Likewise, GSH markedly potentiated diamide inactivation of a PKC isozyme mixture purified from rat brain (alpha, beta, gamma, epsilon, zeta) in a DTT-reversible manner. GSH potentiation of diamide-induced cPKC-alpha inactivation was associated with S-glutathiolation of the isozyme. cPKC-alpha S-glutathiolation was demonstrated by the DTT-reversible incorporation of [(35)S]GSH into the isozyme structure and by an associated change in the migration position of cPKC-alpha in nonreducing SDS-PAGE. Diamide treatment of NIH3T3 cells likewise induced potent, DTT-reversible inactivation of cPKC-alpha in association with [(35)S] S-thiolation of the isozyme. Taken together, the results indicate that PKC isozymes can be oxidatively inactivated by S-thiolation reactions involving endogenous thiols such as GSH.     

Site-specific S-glutathiolation of mitochondrial NADH ubiquinone reductase

The generation of reactive oxygen species in mitochondria acts as a redox signal in triggering cellular events such as apoptosis, proliferation, and senescence. Overproduction of superoxide (O2*-) and O2*--derived oxidants changes the redox status of the mitochondrial GSH pool. An electron transport protein, mitochondrial complex I, is the major host of reactive/regulatory protein thiols. An important response of protein thiols to oxidative stress is to reversibly form protein mixed disulfide via S-glutathiolation. Exposure of complex I to oxidized GSH, GSSG, resulted in specific S-glutathiolation at the 51 kDa and 75 kDa subunits (Beer et al. (2004) J. Biol. Chem. 279, 47939-47951). Here, to investigate the molecular mechanism of S-glutathiolation of complex I, we prepared isolated bovine complex I under nonreducing conditions and employed the techniques of mass spectrometry and EPR spin trapping for analysis. LC/MS/MS analysis of tryptic digests of the 51 kDa and 75 kDa polypeptides from glutathiolated complex I (GS-NQR) revealed that two specific cysteines (C206 and C187) of the 51 kDa subunit and one specific cysteine (C367) of the 75 kDa subunit were involved in redox modifications with GS binding. The electron transfer activity (ETA) of GS-NQR in catalyzing NADH oxidation by Q1 was significantly enhanced. However, O2*- generation activity (SGA) mediated by GS-NQR suffered a mild loss as measured by EPR spin trapping, suggesting the protective role of S-glutathiolation in the intact complex I. Exposure of NADH dehydrogenase (NDH), the flavin subcomplex of complex I, to GSSG resulted in specific S-glutathiolation on the 51 kDa subunit. Both ETA and SGA of S-glutathiolated NDH (GS-NDH) decreased in parallel as the dosage of GSSG increased. LC/MS/MS analysis of a tryptic digest of the 51 kDa subunit from GS-NDH revealed that C206, C187, and C425 were glutathiolated. C425 of the 51 kDa subunit is a ligand residue of the 4Fe-4S N3 center, suggesting that destruction of 4Fe-4S is the major mechanism involved in the inhibition of NDH. The result also implies that S-glutathiolation of the 75 kDa subunit may play a role in protecting the 4Fe-4S cluster of the 51 kDa subunit from redox modification when complex I is exposed to redox change in the GSH pool.     

Molecular basis and structural insight of vascular K(ATP) channel gating by S-glutathionylation

The vascular ATP-sensitive K(+) (K(ATP)) channel is targeted by a variety of vasoactive substances, playing an important role in vascular tone regulation. Our recent studies indicate that the vascular K(ATP) channel is inhibited in oxidative stress via S-glutathionylation. Here we show evidence for the molecular basis of the S-glutathionylation and its structural impact on channel gating. By comparing the oxidant responses of the Kir6.1/SUR2B channel with the Kir6.2/SUR2B channel, we found that the Kir6.1 subunit was responsible for oxidant sensitivity. Oxidant screening of Kir6.1-Kir6.2 chimeras demonstrated that the N terminus and transmembrane domains of Kir6.1 were crucial. Systematic mutational analysis revealed three cysteine residues in these domains: Cys(43), Cys(120), and Cys(176). Among them, Cys(176) was prominent, contributing to >80% of the oxidant sensitivity. The Kir6.1-C176A/SUR2B mutant channel, however, remained sensitive to both channel opener and inhibitor, which indicated that Cys(176) is not a general gating site in Kir6.1, in contrast to its counterpart (Cys(166)) in Kir6.2. A protein pull-down assay with biotinylated glutathione ethyl ester showed that mutation of Cys(176) impaired oxidant-induced incorporation of glutathione (GSH) into the Kir6.1 subunit. In contrast to Cys(176), Cys(43) had only a modest contribution to S-glutathionylation, and Cys(120) was modulated by extracellular oxidants but not intracellular GSSG. Simulation modeling of Kir6.1 S-glutathionylation suggested that after incorporation to residue 176, the GSH moiety occupied a space between the slide helix and two transmembrane helices. This prevented the inner transmembrane helix from undergoing conformational changes necessary for channel gating, retaining the channel in its closed state.     

S-cysteinylation is a general mechanism for thiol protection of Bacillus subtilis proteins after oxidative stress

S-Thiolation is crucial for protection and regulation of thiol-containing proteins during oxidative stress and is frequently achieved by the formation of mixed disulfides with glutathione. However, many Gram-positive bacteria including Bacillus subtilis lack the low molecular weight (LMW) thiol glutathione. Here we provide evidence that S-thiolation by the LMW thiol cysteine represents a general mechanism in B. subtilis. In vivo labeling of proteins with [(35)S]cysteine and nonreducing two-dimensional PAGE analyses revealed that a large subset of proteins previously identified as having redox-sensitive thiols are modified by cysteine in response to treatment with the thiol-specific oxidant diamide. By means of multidimensional shotgun proteomics, the sites of S-cysteinylation for six proteins could be identified, three of which are known to be S-glutathionylated in other organisms.     

Effect of S-nitrosothiols on cellular glutathione and reactive protein sulfhydryls

S-Nitrosothiols may cause many of the biological effects of NO and cellular effects have been attributed to S-nitrosylation of reactive protein sulfhydryls. This report examines the effect of S-nitrosothiols on the low-molecular-weight thiols and protein thiols in NIH/3T3 cells. A low concentration of S-nitrosocysteine increased the cysteine content of the cells, with no evidence of either low-molecular-weight thiol or protein S-nitrosylation. Millimolar amounts of S-nitrosocysteine produced S-nitrosoglutathione (GSNO), cysteinyl glutathione, cysteine, and glutathione disulfide. Large amounts of protein S-nitrosylation and lesser amounts of protein S-glutathiolation and S-cysteylation were also observed. GSNO and S-nitroso-N-acetylpenicillamine (SNAP) were much less effective than S-nitrosocysteine, but a combination of cysteine and GSNO produced S-nitrosocysteine-like effects. In cultured hepatocytes, millimolar S-nitrosocysteine was significantly less effective since the cells contained three times more glutathione than NIH/3T3 cells. Results suggest that S-nitrosocysteine enters cells intact, and low concentrations do not significantly increase cellular pools of S-nitrosothiol or S-nitrosylated protein. Millimolar concentrations of S-nitrosocysteine generate S-nitrosylated, S-glutathiolated, and S-cysteylated proteins, as well as a variety of low-molecular-weight disulfides and S-nitrosothiols.     

Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis

Differential protein expression analysis based on modification of selected amino acids with labelling reagents has become the major method of choice for quantitative proteomics. One such methodology, two-dimensional difference gel electrophoresis (2-D DIGE), uses a matched set of fluorescent N-hydroxysuccinimidyl (NHS) ester cyanine dyes to label lysine residues in different samples which can be run simultaneously on the same gels. Here we report the use of iodoacetylated cyanine (ICy) dyes (for labelling of cysteine thiols, for 2-D DIGE-based redox proteomics. Characterisation of ICy dye labelling in relation to its stoichiometry, sensitivity and specificity is described, as well as comparison of ICy dye with NHS-Cy dye labelling and several protein staining methods. We have optimised conditions for labelling of nonreduced, denatured samples and report increased sensitivity for a subset of thiol-containing proteins, allowing accurate monitoring of redox-dependent thiol modifications and expression changes. Cysteine labelling was then combined with lysine labelling in a multiplex 2-D DIGE proteomic study of redox-dependent and ErbB2-dependent changes in epithelial cells exposed to oxidative stress. This study identifies differentially modified proteins involved in cellular redox regulation, protein folding, proliferative suppression, glycolysis and cytoskeletal organisation, revealing the complexity of the response to oxidative stress and the impact that overexpression of ErbB2 has on this response.     

Differential expression of annexins I and II in normal and malignant human mammary epithelial cells

Abstract. Collagen-binding proteins ( CBPs ) of rat mammary tumors are identical to Ca²⁺-binding annexins [49]. We have now isolated a protein of 38 kDa from the human mammary tumor cell line ALAB by collagen type I affinity chromatography as well as by extraction of calcium-binding proteins. The 38-kDa band of both preparations was identified as annexin II (calpactin I) by its reaction with an annexin II-specific monoclonal antibody in Western blot analysis. Annexin I (lipocortin I) was not detectable in these cells. Two other human cell lines, the SV40-transformed cell line SV3 and cell line HBL-100, both established from normal mammary glands, were also positive for annexin II and negative for annexin I.
In vivo expression of annexins was investigated by immunohistological staining of normal and malignant human mammary tissue. The annexin II-specific mAb reacted with normal and tumor parenchyme whereas the annexin I-specific mAb reacted with acini and ductal myoepithelium of the normal mammary gland but showed no reaction with tumor tissue. Immunolocalization studies also showed annexin II expression in both normal and tumor stroma while only tumor stromal cells were found to be reactive with the antibody against annexin I. The differential expression of annexins in normal and malignant human mammary tissue suggests special functions of these proteins in the mammary gland.     

Similarity and dissimilarity of thiols as anti-nitrosative agents in the nitric oxide-superoxide system

Concomitant production of nitric oxide and superoxide in biological systems has been proposed to generate numerous reactive oxygen and nitrogen species that cause oxidative and nitrosative stress. Thiols, especially glutathione, play an important role in cellular defense against radical species. In the present study, we investigated and compared the anti-nitrosative activity of a wide range of thiols in a simplified chemical system of co-generated nitric oxide and superoxide. Of the 13 thiols studied, three groups of thiols are distinguishable: (i) Group I includes cysteine and its four congeners (cysteine methyl ester, cysteine ethyl ester, homocysteine, cysteamine); they are subject to rapid oxidative decomposition and have the least anti-nitrosative activity. (ii) Group II consists of glutathione, penicillamine, tiopronin and mesna; they have the greatest effect on delaying the nitrosation reaction. (iii) Group III comprises N-acetylcysteine, N-acetylpenicillamine, captopril, and thioglycolate; they all have high pK_a for the mercapto group and show the strongest inhibitory effect on the rate and extent of nitrosation in the system studied.     

Quantification of oxidative posttranslational modifications of cysteine thiols of p21ras associated with redox modulation of activity using isotope-coded affinity tags and mass spectrometry

p21ras GTPase is the protein product of the most commonly mutated human oncogene and has been identified as a target for reactive oxygen and nitrogen species. Posttranslational modification of reactive thiols, by reversible S-glutathiolation and S-nitrosation, and potentially also by irreversible oxidation, may have significant effects on p21ras activity. Here we used an isotope-coded affinity tag (ICAT) and mass spectrometry to quantitate the reversible and irreversible oxidative posttranslational thiol modifications of p21ras caused by peroxynitrite (ONOO(-)) or glutathione disulfide (GSSG). The activity of p21ras was significantly increased after exposure to GSSG, but not to ONOO(-). The results of LC-MS/MS analysis of tryptic peptides of p21ras treated with ONOO(-) showed that ICAT labeling of Cys(118) was decreased by 47%, whereas Cys(80) was not significantly affected and was thereby shown to be less reactive. The extent of S-glutathiolation of Cys(118) by GSSG was 53%, and that of the terminal cysteines was 85%, as estimated by the decrease in ICAT labeling. The changes in ICAT labeling caused by GSSG were reversible by chemical reduction, but those caused by peroxynitrite were irreversible. The quantitative changes in thiol modification caused by GSSG associated with increased activity demonstrate the potential importance of redox modulation of p21ras.     

Acetaminophen hepatotoxicity in vivo is not accompanied by oxidant stress

Hepatotoxic doses of acetaminophen in Fischer 344 rats did not increase biliary efflux of oxidized glutathione. Pretreatment of the animals with bis(2-chloroethyl)-N-nitrosourea inhibited hepatic glutathione reductase by 73 percent but did not potentiate the hepatotoxicity of acetaminophen and did not produce an increase in biliary efflux of oxidized glutathione in response to acetaminophen. Hepatic protein thiol content was not depleted by acetaminophen. A proposed role for oxidant stress mechanisms mediated either by reactive oxygen species or by the direct oxidant action of a reactive metabolite in acetaminophen-induced hepatotoxicity is unsubstantiated and unlikely.     

The crystal structure of annexin Gh1 from Gossypium hirsutum reveals an unusual S3 cluster.

The three-dimensional crystal structure of recombinant annexin Gh1 from Gossypium hirsutum (cotton fibre) has been determined and refined to the final R-factor of 0.219 at the resolution of 2.1 A. This plant annexin consists of the typical 'annexin fold' and is similar to the previously solved bell pepper annexin Anx24(Ca32), but significant differences are seen when compared to the structure of nonplant annexins. A comparison with the structure of the mammalian annexin AnxA5 indicates that canonical calcium binding is geometrically possible within the membrane loops in domains I and II of Anx(Gh1) in their present conformation. All plant annexins possess a conserved tryptophan residue in the AB loop of the first domain; this residue was found to adopt both a loop-in and a loop-out conformation in the bell pepper annexin Anx24(Ca32). In Anx(Gh1), the conserved tryptophan residue is in a surface-exposed position, half way between both conformations observed in Anx24(Ca32). The present structure reveals an unusual sulfur cluster formed by two cysteines and a methionine in domains II and III, respectively. While both cysteines adopt the reduced thiolate forms and are separated by a distance of about 5.5 A, the sulfur atom of the methionine residue is in their close vicinity and apparently interacts with both cysteine sulfur atoms. While the cysteine residues are conserved in at least five plant annexins and in several mammalian members of the annexin family of proteins, the methionine residue is conserved only in three plant proteins. Several of these annexins carrying the conserved residues have been implicated in oxidative stress response. We therefore hypothesize that the cysteine motif found in the present structure, or possibly even the entire sulfur cluster, forms the molecular basis for annexin function in oxidative stress response.     

Stress-induced protein S-glutathionylation in Arabidopsis

S-Glutathionylation (thiolation) is a ubiquitous redox-sensitive and reversible modification of protein cysteinyl residues that can directly regulate their activity. While well established in animals, little is known about the formation and function of these mixed disulfides in plants. After labeling the intracellular glutathione pool with [35S]cysteine, suspension cultures of Arabidopsis (Arabidopsis thaliana ecotype Columbia) were shown to undergo a large increase in protein thiolation following treatment with the oxidant tert-butylhydroperoxide. To identify proteins undergoing thiolation, a combination of in vivo and in vitro labeling methods utilizing biotinylated, oxidized glutathione (GSSG-biotin) was developed to isolate Arabidopsis proteins/protein complexes that can be reversibly glutathionylated. Following two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry proteomics, a total of 79 polypeptides were identified, representing a mixture of proteins that underwent direct thiolation as well as proteins complexed with thiolated polypeptides. The mechanism of thiolation of five proteins, dehydroascorbate reductase (AtDHAR1), zeta-class glutathione transferase (AtGSTZ1), nitrilase (AtNit1), alcohol dehydrogenase (AtADH1), and methionine synthase (AtMetS), was studied using the respective purified recombinant proteins. AtDHAR1, AtGSTZ1, and to a lesser degree AtNit1 underwent spontaneous thiolation with GSSG-biotin through modification of active-site cysteines. The thiolation of AtADH1 and AtMetS required the presence of unidentified Arabidopsis proteins, with this activity being inhibited by S-modifying agents. The potential role of thiolation in regulating metabolism in Arabidopsis is discussed and compared with other known redox regulatory systems operating in plants.     

Detection and mapping of widespread intermolecular protein disulfide formation during cardiac oxidative stress using proteomics with diagonal electrophoresis

Regulation of protein function by reversible cysteine-targeted oxidation can be achieved by multiple mechanisms, such as S-glutathiolation, S-nitrosylation, sulfenic acid, sulfinic acid, and sulfenyl amide formation, as well as intramolecular disulfide bonding of vicinal thiols. Another cysteine oxidation state with regulatory potential involves the formation of intermolecular protein disulfides. We utilized two-dimensional sequential non-reducing/reducing SDS-PAGE (diagonal electrophoresis) to investigate intermolecular protein disulfide formation in adult cardiac myocytes subjected to a series of interventions (hydrogen peroxide, S-nitroso-N-acetylpenicillamine, doxorubicin, simulated ischemia, or metabolic inhibition) that alter the redox status of the cell. More detailed experiments were undertaken with the thiol-specific oxidant diamide (5 mm), a concentration that induces a mild non-injurious oxidative stress. This increase in cellular oxidation potential caused global intermolecular protein disulfide formation in cytosolic, membrane, and myofilament/cytoskeletal compartments. A large number of proteins that undergo these associations were identified using liquid chromatography-mass spectrometry/mass spectrometry. These associations, which involve metabolic and antioxidant enzymes, structural proteins, signaling molecules, and molecular chaperones, were confirmed by assessing "shifts" on non-reducing immunoblots. The observation of widespread protein-protein disulfides indicates that these oxidative associations are likely to be fundamental in how cells respond to redox changes.