Chemistry and biology of enzymes in protein glutathionylation

Protein S-glutathionylation is emerging as a central oxidation that regulates redox signaling and biological processes linked to diseases. In recent years, the field of protein S-glutathionylation has expanded by developing biochemical tools for the identification and functional analyses of S-glutathionylation, investigating knockout mouse models, and developing and evaluating chemical inhibitors for enzymes involved in glutathionylation. This review will highlight recent studies of two enzymes, glutathione transferase omega 1 (GSTO1) and glutaredoxin 1 (Grx1), especially introducing their glutathionylation substrates associated with inflammation, cancer, and neurodegeneration and showcasing the advancement of their chemical inhibitors. Lastly, we will feature protein substrates and chemical inducers of LanC-like protein (LanCL), the first enzyme in protein C-glutathionylation.     

Prediction of glutathionylation sites in proteins using minimal sequence information and their experimental validation

S-glutathionylation of proteins plays an important role in various biological processes and is known to be protective modification during oxidative stress. Since, experimental detection of S-glutathionylation is labor intensive and time consuming, bioinformatics based approach is a viable alternative. Available methods require relatively longer sequence information, which may prevent prediction if sequence information is incomplete. Here, we present a model to predict glutathionylation sites from pentapeptide sequences. It is based upon differential association of amino acids with glutathionylated and non-glutathionylated cysteines from a database of experimentally verified sequences. This data was used to calculate position dependent F-scores, which measure how a particular amino acid at a particular position may affect the likelihood of glutathionylation event. Glutathionylation-score (G-score), indicating propensity of a sequence to undergo glutathionylation, was calculated using position-dependent F-scores for each amino-acid. Cut-off values were used for prediction. Our model returned an accuracy of 58% with Matthew’s correlation-coefficient (MCC) value of 0.165. On an independent dataset, our model outperformed the currently available model, in spite of needing much less sequence information. Pentapeptide motifs having high abundance among glutathionylated proteins were identified. A list of potential glutathionylation hotspot sequences were obtained by assigning G-scores and subsequent Protein-BLAST analysis revealed a total of 254 putative glutathionable proteins, a number of which were already known to be glutathionylated. Our model predicted glutathionylation sites in 93.93% of experimentally verified glutathionylated proteins. Outcome of this study may assist in discovering novel glutathionylation sites and finding candidate proteins for glutathionylation.     

Determination of site-specificity of S-glutathionylated cellular proteins

Redox modification by S-glutathionylation is an expanding field within cell signalling research. However, the methods available for analysis of S-glutathionylated proteins in complex mixtures are not sufficiently accurate to specifically and in a high-throughput manner on a structural level establish the effects of S-glutathionylation on the individual proteins. A method has been developed for rapid identification of the S-glutathionylation sites of proteins in diamide-treated ECV304 cells, through tagging of deglutathionylated proteins with a cysteine-reactive biotin-affinity tag, trypsinisation, avidin-affinity purification of tagged peptides, and subsequent analysis by liquid chromatography and quadrupole time-of-flight tandem mass spectrometry. The method has led to identification of the glutathionylation sites of gamma-actin (Cys(217)), heat shock protein 60 (Cys(447)), and elongation factor 1-alpha-1 (Cys(411)). Further developments of accuracy within the field of peptide-affinity capture and mass spectrometry are discussed.     

Galectin-3 S-glutathionylation regulates its effect on adipocyte insulin signaling

Protein-S-glutathionylation promotes redox signaling in physiological and oxidative distress conditions. Galectin-3 (Gal-3) promotes insulin resistance by down-regulating adipocyte insulin signaling, however, its S-glutathionylation and significance is not known. In this context, we report reversible S-glutathionylation of Gal-3. Site-directed mutagenesis established Gal-3 Cys187 as the putative S-glutathionylation site. Glutathionylated Gal-3 prevents Gal-3(WT)-Insulin Receptor interaction and facilitates insulin-induced murine adipocyte p-IRS1(tyr895) and p-AKT(ser473) signaling and glucose uptake in a Gal-3 Cys187 glutathionylation dependent manner in murine adipocytes, as assessed by Western blotting and 2-NBDG uptake assay respectively. Pre-glutathionylated Gal-3 at Cys187 resisted irreversible oxidation by H₂O₂. M2 macrophages showed enhanced Gal-3 S-glutathionylation when compared to M1 phenotype. Serum and stromal vascular fraction (SVF) isolated from control mice showed increased Gal-3 S-glutathionylation as compared to db/db mice. A significant increase in Gal-3 S-glutathionylation was observed in metformin-treated db/db mice when compared to db/db mice alone. Similar to murine, enhanced Gal-3 S-glutathionylation is observed in primary human monocyte derived M2 macrophages when compared to the M1 macrophage phenotype and Gal-3 regulates primary human adipocyte insulin signaling in a glutathionylation dependent manner. Collectively, we identified Gal-3 S-glutathionylation as a protective phenomenon, which relieves its inhibitory effect on adipocyte insulin signaling.     

The Effect of Oxidant and the Non-Oxidant Alteration of Cellular Thiol Concentration on the Formation of Protein Mixed-Disulfides in HEK 293 Cells

Cellular molecules possess various mechanisms in responding to oxidant stress. In terms of protein responses, protein S-glutathionylation is a unique post-translational modification of protein reactive cysteines forming disulfides with glutathione molecules. This modification has been proposed to play roles in antioxidant, regulatory and signaling in cells under oxidant stress. Recently, the increased level of protein S-glutathionylation has been linked with the development of diseases. In this report, specific S-glutathionylated proteins were demonstrated in human embryonic kidney 293 cells treated with two different oxidative reagents: diamide and hydrogen peroxide. Diamide is a chemical oxidizing agent whereas hydrogen peroxide is a physiological oxidant. Under the experimental conditions, these two oxidants decreased glutathione concentration without toxicity. S-glutathionylated proteins were detected by immunoblotting and glutathione concentrations were determined by high performance liquid chromatography. We further show the effect of alteration of the cellular thiol pool on the amount of protein S-glutathionylation in oxidant-treated cells. Cellular thiol concentrations were altered either by a specific way using buthionine sulfoximine, a specific inhibitor of glutathione biosynthesis or by a non-specific way, incubating cells in cystine-methionine deficient media. Cells only treated with either buthionine sulfoximine or cystine-methionine deficient media did not induce protein S-glutathionylation, even though both conditions decreased 65% of cellular glutathione. Moreover, the amount of protein S-glutathionylation under both conditions in the presence of oxidants was not altered when compared to the amount observed in regular media with oxidants present. Protein S-glutathionylation is a dynamic reaction which depends on the rate of adding and removing glutathione. Phenylarsine oxide, which specifically forms a covalent adduct with vicinal thiols, was used to determine the possible role of vicinal thiols in the amount of glutathionylation. Our data shows phenylarsine oxide did not change glutathione concentrations, but it did enhance the amount of glutathionylation in oxidant-treated cells.     

Thioredoxins, glutaredoxins, and glutathionylation: new crosstalks to explore

Oxidants are widely considered as toxic molecules that cells have to scavenge and detoxify efficiently and continuously. However, emerging evidence suggests that these oxidants can play an important role in redox signaling, mainly through a set of reversible post-translational modifications of thiol residues on proteins. The most studied redox system in photosynthetic organisms is the thioredoxin (TRX) system, involved in the regulation of a growing number of target proteins via thiol/disulfide exchanges. In addition, recent studies suggest that glutaredoxins (GRX) could also play an important role in redox signaling especially by regulating protein glutathionylation, a post-translational modification whose importance begins to be recognized in mammals while much less is known in photosynthetic organisms. This review focuses on oxidants and redox signaling with particular emphasis on recent developments in the study of functions, regulation mechanisms and targets of TRX, GRX and glutathionylation. This review will also present the complex emerging interplay between these three components of redox-signaling networks.Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

S-Glutathionylation of p47phox sustains superoxide generation in activated neutrophils

Post-translational modifications (PTMs) induced conformational changes of proteins can cause their activation or inactivation. Neutrophils clear pathogen through phagocytosis and oxidative burst generation, while participate in inflammation through sustained and uncontrolled generation of ROS. In activated PMNs, cytosolic NOX-2 subunit p47phox following phosphorylation interacts with p67phox, p40phox and along with Rac2 translocate to the membrane. Phosphorylation of p47phox subunit occurs in both short spurts as well as sustained ROS generation, suggesting towards the unidentified molecular mechanism(s) driving these two diverse outcomes by various stimuli. The present study demonstrates that in PMA or NO treated neutrophils a subunit of NOX2, p47phox gets glutathionylated to sustain ROS generation along with a decrease in catalase, Grx-1 activity and change in GSH/GSSG ratio. Surprisingly, fMLP treated cells neither showed sustained ROS production nor glutathionylation of p47phox. S-Glutathionylation was always secondary to phosphorylation of p47phox and inhibition of glutathionylation did not alter phosphorylation but specifically impaired sustained ROS production. Interestingly, forced S-glutathionylation of p47phox converted the fMLP induced ROS generation into sustained release of ROS. We then identified the glutathionylation susceptible cysteine residues of p47phox by LC-MS/MS with IAM switch mapping. Site-directed mutagenesis of cysteine residues further mitigated p47phox S-glutathionylation. Thus, we demonstrate that p47phox S-glutathionylation plays an essential key role in the sustained ROS generation by human neutrophils.     

The structure of the thioredoxin-triosephosphate isomerase complex provides insights into the reversible glutathione-mediated regulation of triosephosphate isomerase

Protein-protein interactions are crucial for many biological functions. The redox interactome encompasses numerous weak transient interactions in which thioredoxin plays a central role. Proteomic studies have shown that thioredoxin binds to numerous proteins belonging to various cellular processes, including energy metabolism. Thioredoxin has cross talk with other redox mechanisms involving glutathionylation and has functional overlap with glutaredoxin in deglutathionylation reactions. In this study, we have explored the structural and biochemical interactions of thioredoxin with the glycolytic enzyme, triosephosphate isomerase. Nuclear magnetic resonance chemical shift mapping methods and molecular dynamics-based docking have been applied in deriving a structural model of the thioredoxin-triosephosphate isomerase complex. The spatial proximity of active site cysteine residues of thioredoxin to reactive thiol groups on triosephosphate isomerase provides a direct link to the observed deglutathionylation of cysteine 217 in triosephosphate isomerase, thereby reversing the inhibitory effect of S-glutathionylation of triosephosphate isomerase.     

S-glutathionylation in protein redox regulation

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.     

Oxidative stress induced S-glutathionylation and proteolytic degradation of mitochondrial thymidine kinase 2

Protein glutathionylation in response to oxidative stress can affect both the stability and activity of target proteins. Mitochondrial thymidine kinase 2 (TK2) is a key enzyme in mitochondrial DNA precursor synthesis. Using an antibody specific for glutathione (GSH), S-glutathionylated TK2 was detected after the addition of glutathione disulfide (GSSG) but not GSH. This was reversed by the addition of dithiothreitol, suggesting that S-glutathionylation of TK2 is reversible. Site-directed mutagenesis of the cysteine residues and subsequent analysis of mutant enzymes demonstrated that Cys-189 and Cys-264 were specifically glutathionylated by GSSG. These cysteine residues do not appear to be part of the active site, as demonstrated by kinetic studies of the mutant enzymes. Treatment of isolated rat mitochondria with hydrogen peroxide resulted in S-glutathionylation of added recombinant TK2. Treatment of intact cells with hydrogen peroxide led to reduction of mitochondrial TK2 activity and protein levels, as well as S-glutathionylation of TK2. Furthermore, the addition of S-glutathionylated recombinant TK2 to mitochondria isolated from hydrogen peroxide-treated cells led to degradation of the S-glutathionylated TK2, which was not observed with unmodified TK2. S-Glutathionylation on Cys-189 was responsible for the observed selective degradation of TK2 in mitochondria. These results strongly suggest that oxidative damage-induced S-glutathionylation and degradation of TK2 have significant impact on mitochondrial DNA precursor synthesis.     

Proteomic identification and quantification of S-glutathionylation in mouse macrophages using resin-assisted enrichment and isobaric labeling

S-Glutathionylation (SSG) is an important regulatory posttranslational modification on protein cysteine (Cys) thiols, yet the role of specific cysteine residues as targets of modification is poorly understood. We report a novel quantitative mass spectrometry (MS)-based proteomic method for site-specific identification and quantification of S-glutathionylation across different conditions. Briefly, this approach consists of initial blocking of free thiols by alkylation, selective reduction of glutathionylated thiols, and covalent capture of reduced thiols using thiol affinity resins, followed by on-resin tryptic digestion and isobaric labeling with iTRAQ (isobaric tags for relative and absolute quantitation) for MS-based identification and quantification. The overall approach was initially validated by application to RAW 264.7 mouse macrophages treated with different doses of diamide to induce glutathionylation. A total of 1071 Cys sites from 690 proteins were identified in response to diamide treatment, with ~90% of the sites displaying >2-fold increases in SSG modification compared to controls. This approach was extended to identify potential SSG-modified Cys sites in response to H₂O₂, an endogenous oxidant produced by activated macrophages and many pathophysiological stimuli. The results revealed 364 Cys sites from 265 proteins that were sensitive to S-glutathionylation in response to H₂O₂ treatment, thus providing a database of proteins and Cys sites susceptible to this modification under oxidative stress. Functional analysis revealed that the most significantly enriched molecular function categories for proteins sensitive to SSG modifications were free radical scavenging and cell death/survival. Overall the results demonstrate that our approach is effective for site-specific identification and quantification of SSG-modified proteins. The analytical strategy also provides a unique approach to determining the major pathways and cellular processes most susceptible to S-glutathionylation under stress conditions.     

Reversible S-glutathionylation of Cys 374 regulates actin filament formation by inducing structural changes in the actin molecule

S-glutathionylation, the reversible formation of mixed disulphides of cysteinyl residues in target proteins with glutathione, occurs under conditions of oxidative stress; this could be a posttranslational mechanism through which protein function is regulated by the cellular redox status. A novel physiological relevance of actin polymerization regulated by glutathionylation of Cys(374) has been recently suggested. In the present study we showed that glutathionylated actin (GS-actin) has a decreased capacity to polymerize compared to native actin, filament elongation being the polymerization step actually inhibited. Actin polymerizability recovers completely after dethiolation, indicating that S-glutathionylation does not induce any protein denaturation and is therefore a reversible oxidative modification. The increased exposure of hydrophobic regions of protein surface observed upon S-glutathionylation indicates changes in actin conformation. Structural alterations are confirmed by the increased rate of ATP exchange as well as by the decreased susceptibility to proteolysis of the subtilisin cleavage site between Met(47) and Gly(48), in the DNase-I-binding loop of the actin subdomain 2. Structural changes in the surface loop 39-51 induced by S-glutathionylation could influence actin polymerization in view of the involvement of the N-terminal portion of this loop in intermonomer interactions, as predicted by the atomic models of F-actin.     

S-Glutathionylation of the Na,K-ATPase Catalytic α Subunit Is a Determinant of the Enzyme Redox Sensitivity

Na,K-ATPase is highly sensitive to changes in the redox state, and yet the mechanisms of its redox sensitivity remain unclear. We have explored the possible involvement of S-glutathionylation of the catalytic α subunit in redox-induced responses. For the first time, the presence of S-glutathionylated cysteine residues was shown in the α subunit in duck salt glands, rabbit kidneys, and rat myocardium. Exposure of the Na,K-ATPase to oxidized glutathione (GSSG) resulted in an increase in the number of S-glutathionylated cysteine residues. Increase in S-glutathionylation was associated with dose- and time-dependent suppression of the enzyme function up to its complete inhibition. The enzyme inhibition concurred with S-glutathionylation of the Cys-454, -458, -459, and -244. Upon binding of glutathione to these cysteines, the enzyme was unable to interact with adenine nucleotides. Inhibition of the Na,K-ATPase by GSSG did not occur in the presence of ATP at concentrations above 0.5 mm. Deglutathionylation of the α subunit catalyzed by glutaredoxin or dithiothreitol resulted in restoration of the Na,K-ATPase activity. Oxidation of regulatory cysteines made them inaccessible for glutathionylation but had no profound effect on the enzyme activity. Regulatory S-glutathionylation of the α subunit was induced in rat myocardium in response to hypoxia and was associated with oxidative stress and ATP depletion. S-Glutathionylation was followed by suppression of the Na,K-ATPase activity. The rat α2 isoform was more sensitive to GSSG than the α1 isoform. Our findings imply that regulatory S-glutathionylation of the catalytic subunit plays a key role in the redox-induced regulation of Na,K-ATPase activity.     

Glutathionylation in the photosynthetic model organism Chlamydomonas reinhardtii: a proteomic survey

Protein glutathionylation is a redox post-translational modification occurring under oxidative stress conditions and playing a major role in cell regulation and signaling. This modification has been mainly studied in nonphotosynthetic organisms, whereas much less is known in photosynthetic organisms despite their important exposure to oxidative stress caused by changes in environmental conditions. We report a large scale proteomic analysis using biotinylated glutathione and streptavidin affinity chromatography that allowed identification of 225 glutathionylated proteins in the eukaryotic unicellular green alga Chlamydomonas reinhardtii. Moreover, 56 sites of glutathionylation were also identified after peptide affinity purification and tandem mass spectrometry. The targets identified belong to a wide range of biological processes and pathways, among which the Calvin-Benson cycle appears to be a major target. The glutathionylation of four enzymes of this cycle, phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase, ribose-5-phosphate isomerase, and phosphoglycerate kinase was confirmed by Western blot and activity measurements. The results suggest that glutathionylation could constitute a major mechanism of regulation of the Calvin-Benson cycle under oxidative stress conditions.     

Glutathionylation of chikungunya nsP2 protein affects protease activity

BackgroundChikungunya fever is an emerging disease caused by the chikungunya virus and is now being spread worldwide by the mosquito Aedes albopictus. The infection can cause a persistent severe joint pain and recent reports link high levels of viremia to neuropathologies and fatalities. The viral protein nsP2 is a multifunctional enzyme that plays several critical roles in virus replication. Virus infection induces oxidative stress in host cells which the virus utilizes to aid viral propagation. Cellular oxidative stress also triggers glutathionylation which is a post-translational protein modification that can modulate physiological roles of affected proteins.MethodsThe nsP2 protease is necessary for processing of the virus nonstructural polyprotein generated during replication. We use the recombinant nsP2 protein to measure protease activity before and after glutathionylation. Mass spectrometry allowed the identification of the glutathione-modified cysteines. Using immunoblots, we show that the glutathionylation of nsP2 occurs in virus-infected cells.ResultsWe show that in virus-infected cells, the chikungunya nsP2 can be glutathionylated and we show this modification can impact on the protease activity. We also identify 6 cysteine residues that are glutathionylated of the 20 cysteines in the protein.ConclusionsThe virus-induced oxidative stress causes modification of viral proteins which appears to modulate virus protein function.General significanceViruses generate oxidative stress to regulate and hijack host cell systems and this environment also appears to modulate virus protein function. This may be a general target for intervention in viral pathogenesis.     

Effect of S-nitrosoglutathione on renal mitochondrial function: a new mechanism for reversible regulation of manganese superoxide dismutase activity?

Mitochondria are at the heart of all cellular processes as they provide the majority of the energy needed for various metabolic processes. Nitric oxide has been shown to have numerous roles in the regulation of mitochondrial function. Mitochondria have enormous pools of glutathione (GSH≈5–10 mM). Nitric oxide can react with glutathione to generate a physiological molecule, S-nitrosoglutathione (GSNO). The impact GSNO has on mitochondrial function has been intensively studied in recent years, and several mitochondrial electron transport chain complex proteins have been shown to be targeted by GSNO. In this study we investigated the effect of GSNO on mitochondrial function using normal rat proximal tubular kidney cells (NRK cells). GSNO treatment of NRK cells led to mitochondrial membrane depolarization and significant reduction in activities of mitochondrial complex IV and manganese superoxide dismutase enzyme (MnSOD). MnSOD is a critical endogenous antioxidant enzyme that scavenges excess superoxide radicals in the mitochondria. The decrease in MnSOD activity was not associated with a reduction in its protein levels and treatment of NRK cell lysate with dithiothreitol (a strong sulfhydryl-group-reducing agent) restored MnSOD activity to control values. GSNO is known to cause both S-nitrosylation and S-glutathionylation, which involve the addition of NO and GS groups, respectively, to protein sulfhydryl (SH) groups of cysteine residues. Endogenous GSH is an essential mediator in S-glutathionylation of cellular proteins, and the current studies revealed that GSH is required for MnSOD inactivation after GSNO or diamide treatment in rat kidney cells as well as in isolated kidneys. Further studies showed that GSNO led to glutathionylation of MnSOD; however, glutathionylated recombinant MnSOD was not inactivated. This suggests that a more complex pathway, possibly involving the participation of multiple proteins, leads to MnSOD inactivation after GSNO treatment. The major highlight of these studies is the fact that dithiothreitol can restore MnSOD activity after GSNO treatment. To our knowledge, this is the first study showing that MnSOD activity can be reversibly regulated in vivo, through a mechanism involving thiol residues.     

Structural biology of cellular machines

Multi-component macromolecular machines contribute to all essential biological processes, from cell motility and signal transduction to information storage and processing. Structural analysis of assemblies at atomic resolution is emerging as the field of structural cell biology. Several recent studies, including those focused on the ribosome, the acrosomal bundle and bacterial flagella, have demonstrated the ability of a hybrid approach that combines imaging, crystallography and computational tools to generate testable atomic models of fundamental biological machines. A complete understanding of cellular and systems biology will require the detailed structural understanding of hundreds of biological machines. The realization of this goal demands a concerted effort to develop and apply new strategies for the systematic identification, isolation, structural characterization and mechanistic analysis of multi-component assemblies at all resolution ranges. The establishment of a database describing the structural and dynamic properties of protein assemblies will provide novel opportunities to define the molecular and atomic mechanisms controlling overall cell physiology.     

Identification of S-glutathionylated cellular proteins during oxidative stress and constitutive metabolism by affinity purification and proteomic analysis

Redox modification of proteins is proposed to play a central role in regulating cellular function. However, high-throughput techniques for the analysis of the redox status of individual proteins in complex mixtures are lacking. The aim was thus to develop a suitable technique to rapidly identify proteins undergoing oxidation of critical thiols by S-glutathionylation. The method is based on the specific reduction of mixed disulfides by glutaredoxin, their reaction with N-ethylmaleimide-biotin, affinity purification of tagged proteins, and identification by proteomic analysis. The method unequivocally identified 43 mostly novel cellular protein substrates for S-glutathionylation. These include protein chaperones, cytoskeletal proteins, cell cycle regulators, and enzymes of intermediate metabolism. Comparisons of the patterns of S-glutathionylated proteins extracted from cells undergoing diamide-induced oxidative stress and during constitutive metabolism reveal both common protein substrates and substrates failing to undergo enhanced S-glutathionylation during oxidative stress. The ability to chemically tag, select, and identify S-glutathionylated proteins, particularly during constitutive metabolism, will greatly enhance efforts to establish posttranslational redox modification of cellular proteins as an important biochemical control mechanism in coordinating cellular function.     

In situ detection of S-glutathionylated proteins following glutaredoxin-1 catalyzed cysteine derivatization

S-glutathionylation is rapidly emerging as an important post-translational modification, responsible for transducing oxidant signals. However, few approaches are available that allow visualization of glutathione mixed disulfides in intact cells. We describe here a glutaredoxin1-dependent cysteine derivatization and labeling approach, in order to visualize S-glutathionylation patterns in situ. Using this new method, marked S-glutathionylation was observed in epithelial cells, which was predominant at membrane ruffles. As expected, the labeling intensity was further enhanced in response to bolus oxidant treatments, or in cells overexpressing Nox1 plus its coactivators. In addition, manipulation of endogenous levels of glutaredoxin-1 via RNAi, or overexpression resulted in altered sensitivity to H2O2 induced formation of glutathione mixed disulfides. Overall, the derivatization approach described here preferentially detects S-glutathionylation and provides an important means to visualize this post-translational modification in sub-cellular compartments and to investigate its relation to normal physiology as well as pathology.