S-Glutathiolation by peroxynitrite activates SERCA during arterial relaxation by nitric oxide

Nitric oxide (NO) physiologically stimulates the sarco/endoplasmic reticulum calcium (Ca(2+)) ATPase (SERCA) to decrease intracellular Ca(2+) concentration and relax cardiac, skeletal and vascular smooth muscle. Here, we show that NO-derived peroxynitrite (ONOO(-)) directly increases SERCA activity by S-glutathiolation and that this modification of SERCA is blocked by irreversible oxidation of the relevant cysteine thiols during atherosclerosis. Purified SERCA was S-glutathiolated by ONOO(-) and the increase in Ca(2+)-uptake activity of SERCA reconstituted in phospholipid vesicles required the presence of glutathione. Mutation of the SERCA-reactive Cys674 to serine abolished these effects. Because superoxide scavengers decreased S-glutathiolation of SERCA and arterial relaxation by NO, ONOO(-) is implicated as the intracellular mediator. NO-dependent relaxation as well as S-glutathiolation and activation of SERCA were decreased by atherosclerosis and Cys674 was found to be oxidized to sulfonic acid. Thus, irreversible oxidation of key thiol(s) in disease impairs NO-induced relaxation by preventing reversible S-glutathiolation and activation of SERCA by NO/ONOO(-).     

Peroxynitrite modification of protein thiols: oxidation, nitrosylation, and S-glutathiolation of functionally important cysteine residue(s) in the sarcoplasmic reticulum Ca-ATPase.

Skeletal muscle contraction and relaxation is efficiently modulated through the reaction of reactive oxygen-nitrogen species with sarcoplasmic reticulum protein thiols in vivo. However, the exact locations of functionally important modifications are at present unknown. Here, we determine by HPLC-MS that the modification of one (out of 24) Cys residue of the sarcoplasmic reticulum (SR) Ca-ATPase isoform SERCA1, Cys(349), by peroxynitrite is sufficient for the modulation of enzyme activity. Despite the size and nature of the SR Ca-ATPase, a 110 kDa membrane protein, identification and quantitation of Cys modification was achieved through labeling with 4-(dimethylamino)phenylazophenyl-4'-maleimide (DABMI) and/or N-(2-iodoethyl)trifluoroacetamide (IE-TFA) followed by an exhaustive tryptic digestion and on-line HPLC-UV-electrospray MS analysis. The reaction with IE-TFA generates aminoethylcysteine, a new trypsin cleavage site, which allows the production of specific peptide fragments that are diagnostic for IE-TFA labeling, conveniently identified by mass spectrometry. Exposure of the SR Ca-ATPase to low concentrations (0.1 mM) of peroxynitrite resulted in the fully reversible chemical modification of Cys at positions 344, 349, 471, 498, 525, and 614 (nitrosylation of Cys(344) and Cys(349) was seen), whereas higher concentrations of peroxynitrite (0.45 mM) additionally affected Cys residues at positions 636, 670, and 674. When the SR Ca-ATPase was exposed to 0.45 mM peroxynitrite in the presence of 5.0 mM glutathione (GSH), thiol modification became partially reversible and S-glutathiolation was detected for Cys residues at positions 344, 349, 364, 498, 525, and 614. The extent of enzyme inactivation (determined previously) quantitatively correlated with the loss of labeling efficiency (i) of a single Cys residue and (ii) of the tryptic fragment containing both Cys(344) and Cys(349). Earlier results had shown that the independent selective modification of Cys(344) is functionally insignificant [Kawakita, M., and Yamashita, T. (1987) J. Biochem. (Tokyo) 102, 103-109]. Thus, we conclude that modification of only Cys(349) is responsible for the modulation of the SR Ca-ATPase activity by peroxynitrite.     

S-glutathiolation of Ras mediates redox-sensitive signaling by angiotensin II in vascular smooth muscle cells

Angiotensin II (AII) increases production of reactive oxygen species from NAD(P)H oxidase, a response that contributes to vascular hypertrophy. Here we show in cultured vascular smooth muscle cells that S-glutathiolation of the redox-sensitive Cys(118) on the small GTPase, Ras, plays a critical role in AII-induced hypertrophic signaling. AII simultaneously increased the Ras activity and the S-glutathiolation of Ras (GSS-Ras) detected by biotin-labeled GSH or mass spectrometry. Both the increase in activity and GSS-Ras was labile under reducing conditions, suggesting the essential nature of this thiol modification to Ras activation. Overexpression of catalase, a dominant-negative p47(phox), or glutaredoxin-1 decreased GSS-Ras, Ras activation, p38, and Akt phosphorylation and the induction of protein synthesis by AII. Furthermore, expression of a Cys(118) mutant Ras decreased AII-mediated p38 and Akt phosphorylation as well as protein synthesis. These results show that H(2)O(2) from NAD(P)H oxidase forms GSS-Ras on Cys(118) and increases its activity leading to p38 and Akt phosphorylation, which contributes to the induction of protein synthesis. This study suggests that GSS-Ras is a redox-sensitive signaling switch that participates in the cellular response to AII.     

Irreversible inactivation of magnesium-dependent neutral sphingomyelinase 1 (NSM1) by peroxynitrite,a nitric oxide-derived oxidant

Previous results have indicated that the generation of ceramide by hydrolysis of sphingomyelin by magnesium-dependent neutral sphingomyelinase 1 (NSM1) is reversibly inhibited by hydrogen peroxide (H2O2) and oxidized glutathione (GSSG). This redox-dependent reversible regulation of NSM1 activity has been shown to involve the reversible formation and breakage of disulfide bonds. In this paper, we show that peroxynitrite, a nitric oxide-derived oxidant generated by SIN1, inactivates dose-dependently the NSM1 activity in an irreversible manner. In addition, we show that, in contrast to the reversible inhibition of NSM1 by H2O2 or GSSG which involves the formation of disulfide bonds, irreversible inactivation of this enzyme by peroxynitrite generated from SIN1 is likely due to definitive oxidative thiol modification. These results suggest that depending on the nature of the oxidative stress, the enzymatic activity of NSM1 could be reversibly or irreversibly inactivated.     

Nitric oxide activates p21ras and leads to the inhibition of endothelial NO synthase by protein nitration

Recent data has indicated that exogenous nitric oxide (NO) has the ability to decrease endogenous NO production by inhibiting the enzyme responsible for its generation, NO synthase (NOS). Our previous studies have indicated that increased generation of reactive oxygen species (ROS) play an important role in the inhibitory event. However, the mechanisms for these effects remain unclear. Previous studies have suggested that NO can activate p21ras. Thus, the objective of this study was to determine whether NO-mediated activation of p21ras is involved in the inhibitory process, and to further elucidate the involvement of ROS. Using primary cultures of ovine pulmonary arterial endothelial cells we demonstrated that the NO donor SpermineNONOate, increased p21ras activity by 2.3-fold compared to untreated cells, and that the farnesyl-transferase inhibitor, alpha-hydroxyfarnesylphosphonic acid, reduced p21ras activity and significantly reduced inhibition of eNOS. The overexpression of p21ras increased, while the overexpression of an NO unresponsive mutant of p21ras (p21ras C118S) reduced, the inhibition of eNOS by NO. Further, we identified an increase in the level of superoxide and peroxynitrite in endothelial cells exposed to NO that was reduced by p21ras C118S transient transfection. Conversely, levels of superoxide and peroxynitrite could be increased by the over expression of wild type p21ras. Similarly, eNOS nitration induced by NO exposure was reduced by p21ras C118S transient transfection, and increased by the overexpression of wild-type p21ras. Finally, results also demonstrated that eNOS itself was a significant producer of superoxide, and that this appeared to be related to a p21ras-dependent increase in phosphorylation of Ser1177. Our results implicate a signaling pathway involving p21ras activation, superoxide generation, and peroxynitrite formation as being important in the NO-mediated inhibition of eNOS.     

The cysteine-rich protein thimet oligopeptidase as a model of the structural requirements for S-glutathiolation and oxidative oligomerization

Thimet oligopeptidase (EP24.15) is a cysteine-rich metallopeptidase containing fifteen Cys residues and no intra-protein disulfide bonds. Previous work on this enzyme revealed that the oxidative oligomerization of EP24.15 is triggered by S-glutathiolation at physiological GSSG levels (10-50 μM) via a mechanism based on thiol-disulfide exchange. In the present work, our aim was to identify EP24.15 Cys residues that are prone to S-glutathiolation and to determine which structural features in the cysteinyl bulk are responsible for the formation of mixed disulfides through the reaction with GSSG and, in this particular case, the Cys residues within EP24.15 that favor either S-glutathiolation or inter-protein thiol-disulfide exchange. These studies were conducted by in silico structural analyses and simulations as well as site-specific mutation. S-glutathiolation was determined by mass spectrometric analyses and western blotting with anti-glutathione antibody. The results indicated that the stabilization of a thiolate sulfhydryl and the solvent accessibility of the cysteines are necessary for S-thiolation. The Solvent Access Surface analysis of the Cys residues prone to glutathione modification showed that the S-glutathiolated Cys residues are located inside pockets where the sulfur atom comes into contact with the solvent and that the positively charged amino acids are directed toward these Cys residues. The simulation of a covalent glutathione docking onto the same Cys residues allowed for perfect glutathione posing. A mutation of the Arg residue 263 that forms a saline bridge to the Cys residue 175 significantly decreased the overall S-glutathiolation and oligomerization of EP24.15. The present results show for the first time the structural requirements for protein S-glutathiolation by GSSG and are consistent with our previous hypothesis that EP24.15 oligomerization is dependent on the electron transfer from specific protonated Cys residues of one molecule to previously S-glutathionylated Cys residues of another one.     

Protein thiols and glutathione influence the nitric oxide-dependent regulation of the red blood cell metabolism.

Nitric oxide (NO) can modulate red blood cell (RBC) glycolysis by translocation of the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPD) (EC 1.2.1.12) from the cytosolic domain of the membrane protein band 3 (cdb3) in the cytosol. In this study we have investigated which NO-reactive thiols might be influencing GAPD translocation and the specific role of glutathione. Two highly reactive Cys residues were identified by transnitrosylation with nitrosoglutathione (GSNO) of cdb3 and GAPD (K(2) = 73.7 and 101.5 M(-1) s(-1), respectively). The Cys 149 located in the catalytic site of GAPD is exclusively involved in the GSNO-induced nitrosylation. Reassociation experiments carried out at equilibrium with preparations of RBC membranes and GAPD revealed that different NO donors may form -SNO on, and decrease the affinity between, GAPD and cdb3. In intact RBC, the NO donors 3-morpholinosydnonimine (SIN-1) and peroxynitrite (ONOO(-)) significantly increased GAPD activity in the cytosol, glycolysis measured as lactate production, and energy charge levels. Our data suggest that ONOO(-) is the main NO derivative able to cross the RBC membrane, leading to GAPD translocation and -SNO formation. In cell-free experiments and intact RBC, diamide (a thiol oxidant able to inhibit GAPD activity) was observed to reverse the effect of SIN-1 on GAPD translocation. The results demonstrate that cdb3 and GAPD contain reactive thiols that can be transnitrosylated mainly by means of GSNO; these can ultimately influence GAPD translocation/activity and the glycolytic flux.     

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.     

Thiol-disulfide exchanges modulate aldo–keto reductase family 1 member B10 activity and sensitivity to inhibitors

The reversible thiol/disulfide exchange is an important regulatory mechanism of protein enzymatic activity. Many protein enzymes are susceptible to S-thiolation induced by reactive oxygen species (ROS); and the glutathione (GSH) and free amino acid cysteine (Cys) are critical cellular thiol anti-oxidants, protecting proteins from irreversible oxidative damage. In this study, we found that aldo–keto reductase family 1 member B10 (AKR1B10) contains 4 Cys residues, i.e., Cys45, Cys187, Cys200, and Cys299. Exposing AKR1B10 to ROS mixtures resulted in significant decrease of its free sulfhydryl groups, up to 40–50% in the presence of physiological thiol cysteine at 0.5 or 1.0 mM; and accordingly, AKR1B10 enzymatic activity was reversibly decreased, in parallel with the oxidation of the sulfhydryl groups. ROS-induced thiolation also affected the sensitivity of AKR1B10 to inhibitors EBPC, epalrestat, and statil. Together our results showed for the first time that AKR1B10's enzymatic activity and inhibitor sensitivity are modulated by thiol/disulfide exchanges.     

Redox proteomics of thiol proteins in mouse heart during ischemia/reperfusion using ICAT reagents and mass spectrometry

There is strong evidence for the involvement of reactive oxygen species in ischemia/reperfusion injury. Although oxidation of individual thiol proteins has been reported, more extensive redox proteomics of hearts subjected to ischemia/reperfusion has not been performed. We have carried out an exploratory study using mass spectrometry with isotope-coded affinity tags (ICAT) aimed at identifying reversible oxidative changes to protein thiols in Langendorff perfused isolated mouse hearts subjected to 20 min ischemia with or without aerobic reperfusion for 5 or 30 min. Reduced thiols were blocked by adding N-ethylmaleimide during protein extraction, then reversibly oxidized thiols in extracts of control perfused and treated hearts were reduced and labeled with the light and heavy ICAT reagents, respectively. Protein extracts were mixed in equal amounts and relative proportions of the isotope-labeled peaks were used to quantify oxidative changes between the control and the treated groups. Approximately 300 peptides with ICAT signatures were reliably identified in each sample, with 181 peptides from 118 proteins common to all treatments. A proportion showed elevated ICAT ratios, consistent with reversible thiol oxidation. This was most evident after early reperfusion, with apparent reversal after longer reperfusion. In comparison, there was gradual accumulation of protein carbonyls and loss of GSH with longer reperfusion. Many of the thiol changes were in mitochondrial proteins, including components of electron transport complexes and enzymes involved in lipid metabolism. The results are consistent with mitochondria being a major site of oxidant generation during early cardiac reperfusion and mitochondrial thiol proteins being targets for oxidation.     

Peroxynitrite-induced tyrosine nitration and inhibition of protein kinase C

Protein kinase C (PKC) is an important intracellular signaling molecule whose activity is essential for a number of aspects of neuronal function including synaptic plasticity. We investigated the regulation of PKC activity by reactive nitrogen species in order to examine whether such species regulate PKC in neurons. Neither autonomous nor cofactor-dependent PKC activity was altered when either hippocampal homogenates or rat brain purified PKC were incubated briefly with three different nitric oxide donor compounds. However, brief incubation of either hippocampal homogenates or purified PKC with peroxynitrite (ONOO(-)) inhibited cofactor-dependent PKC activity in a manner that correlated with the nitration of tyrosine residues on PKC, suggesting that this modification was responsible for the inhibition of PKC. Consistent with this idea, reducing agents had no effect on the inhibition of PKC activity caused by ONOO(-). Because there are numerous PKC isoforms that differ in the composition of the regulatory domain, we studied the effect of ONOO(-) on various PKC isoforms. ONOO(-) inhibited the cofactor-dependent activity of the alpha, betaII, epsilon, and zeta isoforms, indicating that inhibition of enzymatic activity by ONOO(-) was not PKC isoform-specific. We also were able to isolate nitrated PKCalpha and PKCbetaII from ONOO(-)-treated hippocampal homogenates via immunoprecipitation. Collectively, our findings support the hypothesis that ONOO(-) inhibits PKC activity via tyrosine nitration in neurons.     

Reactive nitrogen species derived activation of rat liver microsomal glutathione S-transferase

The effect of reactive nitrogen species on rat liver microsomal glutathione S-transferase (MGST1) was investigated using microsomes and purified MGST1. When microsomes or the purified enzyme were incubated with peroxynitrite (ONOO(-)), the GST activity was increased to 2.5-6.5 fold in concentration-dependent manner and a small amount of the MGST1 dimer was detected. MGST1 activity was increased by ONOO(-) in the presence of high amounts of reducing agents including glutathione (GSH) and the activities increased by ONOO(-) or ONOO(-) plus GSH treatment were decreased by 30-40% by further incubation with dithiothreitol (DTT, reducing disulfide) or by sodium arsenite (reducing sulfenic acid). Furthermore, GSH was detected by HPLC from the MGST1 which was incubated with ONOO(-) plus GSH or S-nitrosoglutathione followed by DTT treatment. In addition, the MGST1 activity increased by nitric oxide (NO) donors such as S-nitrosoglutathione, S-nitrosocysteine or the non-thiol NO donor 1-hydroxy-2-oxo-3 (3-aminopropyl)-3-isopropyl was restored by the DTT treatment. Since DTT can reduce S-nitrosothiol and disulfide bond to thiol, S-nitrosylation and a mixed disulfide bond formation of MGST1 were suggested. Thus, it was demonstrated that MGST1 is activated by reactive nitrogen species through a forming dimeric protein, mixed disulfide bond, nitrosylation and sulfenic acid.     

Mechanism of p21Ras S-nitrosylation and kinetics of nitric oxide-mediated guanine nucleotide exchange

Nitric oxide (NO), a highly reactive redox molecule, can react with protein thiols and protein metal centers to regulate a multitude of physiological processes. NO has been shown to promote guanine nucleotide exchange on the critical cellular signaling protein p21Ras (Ras) by S-nitrosylation of a redox-active thiol group (Cys(118)). This increases cellular Ras-GTP levels in vivo, leading to activation of downstream signaling pathways. Yet the process by which this occurs is not clear. Although several feasible mechanisms for protein S-nitrosylation with NO and NO donating have been proposed, results obtained from our studies suggest that Ras can be S-nitrosylated by direct reaction of Cys(118) with nitrogen dioxide (*NO(2)), a reaction product of NO with O(2), via a Ras thiyl-radical intermediate (Ras-S*). Results from our studies also indicate that Ras Cys(118) can be S-nitrosylated by direct reaction of Cys(118) with a glutathionyl radical (GS*), a reaction product derived from homolytic cleavage of S-nitrosoglutathione (GSNO). Moreover, we present evidence that reaction of GS* with Ras generates a Ras-S* intermediate during GSNO-mediated Ras S-nitrosylation. The Ras-S(*) radical intermediate formed from reaction of the Ras thiol with either *NO(2) or GS*, in turn, reacts with NO to complete Ras S-nitrosylation. NO and GSNO modulate Ras activity by promoting guanine nucleotide dissociation from Ras. Our results suggest that formation of the Ras radical intermediate, Ras-S*, may perturb interactions between Ras and its guanine nucleotide substrate, resulting in enhancement of guanine nucleotide dissociation from Ras.     

Nitric oxide-dependent modification of the sarcoplasmic reticulum Ca-ATPase: localization of cysteine target sites

Skeletal muscle contraction and relaxation is modulated through the reaction of sarcoplasmic reticulum (SR) protein thiols with reactive oxygen and nitrogen species. Here, we have utilized high-performance liquid chromatography-electrospray mass spectrometry and a specific thiol-labeling procedure to identify and quantify cysteine residues of the SR Ca-ATPase that are modified by exposure to nitric oxide (NO). NO and/or NO-derived species inactivate the SR Ca-ATPase and modify a broad spectrum of cysteine residues with highest reactivities towards Cys364, Cys670, and Cys471. The selectivity of NO and NO-derived species towards the SR Ca-ATPase thiols is different from that of peroxynitrite. The efficiency of NO at thiol modification is significantly higher compared with that of peroxynitrite. Hence, NO has the potential to modulate muscle contraction through chemical reaction with the SR Ca-ATPase in vivo.     

Oxidation and S-nitrosylation of cysteines in human cytosolic and mitochondrial glutaredoxins: effects on structure and activity

Glutathione (GSH) is the major intracellular thiol present in 1-10-mm concentrations in human cells. However, the redox potential of the 2GSH/GSSG (glutathione disulfide) couple in cells varies in association with proliferation, differentiation, or apoptosis from -260 mV to -200 or -170 mV. Hydrogen peroxide is transiently produced as second messenger in receptor-mediated growth factor signaling. To understand oxidation mechanisms by GSSG or nitric oxide-related nitrosylation we studied effects on glutaredoxins (Grx), which catalyze GSH-dependent thiol-disulfide redox reactions, particularly reversible glutathionylation of protein sulfhydryl groups. Human Grx1 and Grx2 contain Cys-Pro-Tyr-Cys and Cys-Ser-Tyr-Cys active sites and have three and two additional structural Cys residues, respectively. We analyzed the redox state and disulfide pairing of Cys residues upon GSSG oxidation and S-nitrosylation. Cytosolic/nuclear Grx1 was partly inactivated by both S-nitrosylation and oxidation. Inhibition by nitrosylation was reversible under anaerobic conditions; aerobically it was stronger and irreversible, indicating inactivation by nitration. Oxidation of Grx1 induced a complex pattern of disulfide-bonded dimers and oligomers formed between Cys-8 and either Cys-79 or Cys-83. In addition, an intramolecular disulfide between Cys-79 and Cys-83 was identified, predicted to have a profound effect on the three-dimensional structure. In contrast, mitochondrial Grx2 retains activity upon oxidation, did not form disulfide-bonded dimers or oligomers, and could not be S-nitrosylated. The dimeric iron sulfur cluster-coordinating inactive form of Grx2 dissociated upon nitrosylation, leading to activation of the protein. The striking differences between Grx1 and Grx2 reflect their diverse regulatory functions in vivo and also adaptation to different subcellular localization.     

Structural and functional consequences of inactivation of human glutathione S-transferase P1-1 mediated by the catechol metabolite of equine estrogens, 4-hydroxyequilenin

The inactivation mechanism(s) of human glutathione S-transferase P1-1 (hGST P1-1) by the catechol metabolite of Premarin estrogens, 4-hydroxyequilenin (4-OHEN), was (were) studied by means of site-directed mutagenesis, electrospray ionization mass spectrometric analysis, titration of free thiol groups, kinetic studies of irreversible inhibition, and analysis of band patterns on nonreducing sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE). The four cysteines (Cys 14, Cys 47, Cys 101, and Cys 169 in the primary sequence) in hGST P1-1 are susceptible to electrophilic attack and/or oxidative damage leading to loss of enzymatic activity. To investigate the role of cysteine residues in the 4-OHEN-mediated inactivation of this enzyme, one or a combination of cysteine residues was replaced by alanine residues (C47A, C101A, C47A/C101A, C14A/C47A/C101A, and C47A/C101A/C169A mutants). Mutation of Cys 47 decreased the affinity for the substrate GSH but not for the cosubstrate 1-chloro-2,4-dinitrobenzene (CDNB). However, the Cys 47 mutation did not significantly affect the rate of catalysis since V(max) values of the mutants were similar or higher compared to that of wild type. Electrospray ionization mass spectrometric analyses of wild-type and mutant enzymes treated with 4-OHEN showed that a single molecule of 4-OHEN-o-quinone attached to the proteins, with the exception of the C14A/C47A/C101A mutant where no covalent adduct was detected. 4-OHEN also caused oxidative damage as demonstrated by the appearance of disulfide-bonded species on nonreducing SDS--PAGE and protection of 4-OHEN-mediated enzyme inhibition by free radical scavengers. The studies of thiol group titration and irreversible kinetic experiments indicated that the different cysteines have distinct reactivity for 4-OHEN; Cys 47 was the most reactive thiol group whereas Cys 169 was resistant to modification. These results demonstrate that hGST P1-1 is inactivated by 4-OHEN through two possible mechanisms: (1) covalent modification of cysteine residues and (2) oxidative damage leading to proteins inactivated by disulfide bond formation.     

Protein S-glutathiolation triggered by decomposed S-nitrosoglutathione

Recombinant human brain calbindin D(28K) (rHCaBP), human Cu,Zn-superoxide dismutase (HCuZnSOD), rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and bovine serum albumin (BSA) were found to be S-glutathiolated in decomposed S-nitrosoglutathione (GSNO) solutions. Tryptic or Glu-C digestion and MALDI-TOF MS analyses of the digests are consistent with S-thiolation of Cys111 and Cys187 of HCuZnSOD and rHCaBP, respectively, upon exposure to decomposed GSNO. GAPDH activity analysis reveals that S-glutathiolation most likely occurs on the active site Cys149, and the single free Cys34 is assumed to be the site of S-glutathiolation in BSA. The yields of S-glutathiolation of rHCaBP, GAPDH, and BSA were much higher than those of HCuZnSOD. The latter is limited by the accessibility of Cys111 to the glutathiolating reagent in the HCuZnSOD dimer. Unlike decomposed GSNO, fresh GSNO, reduced glutathione (GSH), and oxidized glutathione (GSSG) are not efficient S-glutathiolating agents for the proteins examined here. On the basis of analysis by mass spectrometry and UV-visible absorption, GSNO decomposition in the dark at room temperature yields glutathione disulfide S-oxide [GS(O)SG], glutathione disulfide S-dioxide (GSO(2)SG), and GSSG as products. GS(O)SG is the efficient protein S-glutathiolating agent in GSNO solutions, not GSNO, which does not carry out efficient S-glutathiolation of rHCaBP, HCuZnSOD, or GAPDH in vitro. A hydrolysis pathway yielding GSOH and nitroxyl (HNO/NO(-)) as intermediates is proposed for GSNO decomposition in the dark. This is based on inhibition of GSNO breakdown by dimedone, a reagent specific for sulfenic acids, and on nitroxyl scavenging by metmyoglobin. The results presented here are contrary to numerous reports of protein S-thiolation by low-molecular weight S-nitrosothiols.     

Actin S-glutathionylation: evidence against a thiol-disulphide exchange mechanism

Many proteins, including actin, are targets for S-glutathionylation, the reversible formation of mixed disulphides between protein cysteinyl thiol groups and glutathione (GSH) that can be induced in cells by oxidative stress. Proposed mechanisms of protein S-glutathionylation follow mainly two distinct pathways. One route involves the initial oxidative modification of a reduced protein thiol to an activated protein, which may then react with GSH to the mixed disulphide. The second route involves the oxidative modification of GSH to an activated form such as glutathione disulphide (GSSG), which may then react with a reduced protein thiol, yielding the corresponding protein mixed disulphide. We show here that physiological levels of GSSG induce a little extent of actin S-glutathionylation. Instead, actin with the exposed cysteine thiol activated by diamide or 5,5'-dithiobis(2-nitrobenzoic acid) reacts with physiological levels of GSH, incorporating about 0.7 mol GSH/mol protein. Differently, an extremely high concentration of GSSG induces an increased level of S-glutathionylation that causes a 50% inhibition in actin polymerization not reversed by dithiotreitol. In mammalian cells, GSH is present in millimolar concentrations and is in about 100-fold excess over GSSG. The high concentration of GSSG required for obtaining a significant actin S-glutathionylation as well as attendant irreversible changes in protein functions make unlikely that actin may be S-glutathionylated by a thiol-disulphide exchange mechanism within the cell.