Novel Role for Glutathione S-Transferase π

Abstract

Glutathione S-transferase Pi (GSTπ) is a marker protein in many cancers and high levels are linked to drug resistance, even when the selecting drug is not a substrate. S-Glutathionylation of proteins is critical to cellular stress response, but characteristics of the forward reaction are not known. Our results show that GSTπ potentiates S-glutathionylation reactions following oxidative and nitrosative stress in vitro and in vivo. Mutational analysis indicated that the catalytic activity of GST is required. GSTπ is itself redox-regulated. S-Glutathionylation on Cys47 and Cys101 autoregulates GSTπ, breaks ligand binding interactions with c-Jun NH2-terminal kinase (JNK), and causes GSTπ multimer formation, all critical to stress response. Catalysis of S-glutathionylation at low pK cysteines in proteins is a novel property for GSTπ and may be a cause for its abundance in tumors and cells resistant to a range of mechanistically unrelated anticancer drugs. Glutathione S-transferase Pi (GSTπ) is a marker protein in many cancers and high levels are linked to drug resistance, even when the selecting drug is not a substrate. S-Glutathionylation of proteins is critical to cellular stress response, but characteristics of the forward reaction are not known. Our results show that GSTπ potentiates S-glutathionylation reactions following oxidative and nitrosative stress in vitro and in vivo. Mutational analysis indicated that the catalytic activity of GST is required. GSTπ is itself redox-regulated. S-Glutathionylation on Cys47 and Cys101 autoregulates GSTπ, breaks ligand binding interactions with c-Jun NH2-terminal kinase (JNK), and causes GSTπ multimer formation, all critical to stress response. Catalysis of S-glutathionylation at low pK cysteines in proteins is a novel property for GSTπ and may be a cause for its abundance in tumors and cells resistant to a range of mechanistically unrelated anticancer drugs. Glutathione S-transferases (GSTs) 2The abbreviations used are: GST, glutathione S-transferase; JNK, c-Jun NH2-terminal kinase; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; NO, nitric oxide; MEF, mouse embryo fibroblasts; HEK, human embryonic kidney; CDNB, 1-chloro-2,4-dinitrobenzene; PBS, phosphate-buffered saline; WT, wild type; CD, circular dichroism; PABA, O2-{2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl}1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate. are classified as a family of Phase II detoxification enzymes that have classically been described as catalyzing the conjugation of glutathione (GSH) to electrophilic compounds through thioether linkages (1Boyland E. Chasseaud L.F. Biochem. J. 1967; 104: 95-102Crossref PubMed Scopus (268) Google Scholar). The Pi class (GSTπ) is present at high levels in many solid tumors (particularly ovarian, non-small cell lung, breast, liver, pancreas, colon, and lymphomas) and has been indicated in many reports to be overexpressed in drug-resistant tumors (2Tew K.D. Cancer Res. 1994; 54: 4313-4320PubMed Google Scholar, 3Hayes J.D. Pulford D.J. Crit. Rev. Biochem. Mol. Biol. 1995; 30: 445-600Crossref PubMed Scopus (3254) Google Scholar). Although its increased expression was frequently linked with enhancement of drug detoxification, in most instances the selecting drugs were not substrates of GSTπ. This ambiguity and the high prevalence of GSTπ in tumors have intimated cellular functions for the protein that are unrelated to catalytic detoxification. Recently GSTπ has been identified as an endogenous protein binding partner and regulator of c-Jun NH2-terminal kinase (JNK) and peroxiredoxin VI (1-cysPrx) (4Adler V. Yin Z. Fuchs S.Y. Benezra M. Rosario L. Tew K.D. Pincus M.R. Sardana M. Henderson C.J. Wolf C.R. Davis R.J. Ronai Z. EMBO J. 1999; 18: 1321-1334Crossref PubMed Scopus (962) Google Scholar, 5Wang T. Arifoglu P. Ronai Z. Tew K.D. J. Biol. Chem. 2001; 276: 20999-21003Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 6Manevich Y. Feinstein S.I. Fisher A.B. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3780-3785Crossref PubMed Scopus (293) Google Scholar). Moreover, oxidative stress causes increased GSTπ expression, the regulation of which has been identified as a downstream event linked to wild-type p53 function (7Lo H.W. Pleasants L. Cao X. Milas M. Pollock R. Ali-Osman F. Mol. Cancer Res. 2008; 6: 848-850Crossref Scopus (44) Google Scholar). Cellular response to oxidative or nitrosative stress includes S-glutathionylation, a post-translational modification characterized by conjugation of glutathione to low pK cysteine sulfhydryl or sulfenic acid moieties in target proteins. This adds a three-amino acid side chain and introduces a net negative charge (as a consequence of glutamic acid) to the protein (8Shelton M.D. Chock P.B. Mieyal J.J. Antioxid. Redox Signal. 2005; 7: 348-366Crossref PubMed Scopus (326) Google Scholar). Consequently, protection from further oxidative damage and/or alteration of protein conformation affecting function and/or cellular localization occurs. Proteins so far identified that are susceptible to S-glutathionylation can be categorized into six distinct clusters: cytoskeletal, glycolysis/energy metabolism, kinases and signaling pathways, calcium homeostasis, antioxidant enzymes, and protein folding (9Mack J.T. Beljanski V. Soulika A.M. Townsend D.M. Brown C.B. Davis W. Tew K.D. Mol. Cell. Biol. 2007; 27: 44-53Crossref PubMed Scopus (33) Google Scholar). Reversibility of S-glutathionylation spontaneously by GSH or catalytically by glutaredoxin or sulfiredoxin (8Shelton M.D. Chock P.B. Mieyal J.J. Antioxid. Redox Signal. 2005; 7: 348-366Crossref PubMed Scopus (326) Google Scholar, 10Beer S.M. Taylor E.R. Brown S.E. Dahm C.C. Costa N.J. Runswick M.J. Murphy M.P. J. Biol. Chem. 2004; 279: 47939-47951Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar) 3Findlay, V. J., Townsend, D. M., Morris, T. E., Fraser, J. P., He, L., and Tew, K. D. (2006) Cancer Res. 66, 6800–6806 provides the cell with a dynamic cycle of regulatory events. A plausible link between GSTπ and such regulation was provided by the observation that GSTπ could conjugate glutathione to a member of the peroxiredoxin family, peroxiredoxin VI, a non-selenoglutathione-dependent peroxidase that converts lipids and other hydroperoxides to corresponding alcohols (6Manevich Y. Feinstein S.I. Fisher A.B. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3780-3785Crossref PubMed Scopus (293) Google Scholar, 12Ralat L.A. Manevich Y. Fisher A.B. Colman R.F. Biochemistry. 2006; 45: 360-372Crossref PubMed Scopus (158) Google Scholar). The catalytically important cysteine residue on peroxiredoxin VI is sterically inaccessible for GSH within the globular homodimeric complex and GSTπ facilitated transfer of GS- to this site. The resultant activation of the enzyme served a cellular regulatory role through maintaining intracellular H2O2 levels (13Rhee S.G. Science. 2006; 312: 1882-1883Crossref PubMed Scopus (1718) Google Scholar), particularly with respect to antioxidant protection of cell membranes. Moreover, S-glutathionylation of protein-tyrosine phosphatase (PTP1B) or protein-disulfide isomerase inactivates these enzymes and can have impact on kinase-mediated proliferative events and protein folding (14Townsend D.M. Findlay V.J. Fazilev F. Ogle M. Fraser J. Saavedra J.E. Ji X. Keefer L.K. Tew K.D. Mol. Pharmacol. 2006; 69: 501-508Crossref PubMed Scopus (89) Google Scholar, 15Townsend D.M. Mol. Interv. 2007; 7: 313-324Crossref PubMed Scopus (173) Google Scholar). 3Findlay, V. J., Townsend, D. M., Morris, T. E., Fraser, J. P., He, L., and Tew, K. D. (2006) Cancer Res. 66, 6800–6806 Both endogenous and exogenous reactive oxygen and nitrogen species generation can cause S-glutathionylation of certain targeted proteins. For the present studies, we developed multiple model systems of oxidative and nitrosative stress to investigate the role of GSTs in S-glutathionylation reactions, using oxidized glutathione (GSSG) and two agents, NOV-002 and PABA/NO. NOV-002 is a mimetic of oxidized glutathione and causes oxidative stress induction (16Townsend D.M. He L. Hutchens S. VandenBerg T.E. Pazoles C.J. Tew K.D. Cancer Res. 2008; 68: 2870-2877Crossref PubMed Scopus (73) Google Scholar). PABA/NO (O2-{2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl}1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate) (17Saavedra J.E. Srinivasan A. Buzard G.S. Davies K.M. Waterhouse D.J. Inami K. Wilde T.C. Citro M.L. Cuellar M. Deschamps J.R. Parrish D. Shami P.J. Findlay V.J. Townsend D.M. Tew K.D. Singh S. Jia L. Ji X. Keefer L.K. J. Med. Chem. 2006; 49: 1157-1164Crossref PubMed Scopus (81) Google Scholar) is a prodrug, which releases nitric oxide (NO) and activates stress response pathways that involve JNK, eventually leading to apoptosis (14Townsend D.M. Findlay V.J. Fazilev F. Ogle M. Fraser J. Saavedra J.E. Ji X. Keefer L.K. Tew K.D. Mol. Pharmacol. 2006; 69: 501-508Crossref PubMed Scopus (89) Google Scholar, 18Findlay V.J. Townsend D.M. Saavedra J.E. Buzard G.S. Citro M.L. Keefer L.K. Ji X. Tew K.D. Mol. Pharmacol. 2004; 65: 1070-1079Crossref PubMed Scopus (109) Google Scholar). Elevated levels of NO provide the primary source of reactive nitrogen species and can alter protein function directly through post-translational modifications (nitration/nitrosylation) or indirectly through interactions with oxygen, superoxide, thiols, and heavy metals, the products of which can lead to S-glutathionylation. Recognition that S-glutathionylation of proteins and its cycling is important in regulating critical cellular events means that there is a need to define both the forward and reverse components of the cycle. Until this time, the forward reaction of S-glutathionylation has been tacitly assumed to occur spontaneously. Our present study shows for the first time that GSTπ catalyzes protein S-glutathionylation in vivo and is contributory to its high expression levels in many drug-resistant tumors. Materials and Cell Lines—Generation of GSTπ wild-type and knock-out mice (19Henderson C.J. Smith A.G. Ure J. Brown K. Bacon E.J. Wolf C.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5275-5280Crossref PubMed Scopus (348) Google Scholar), extraction of the mouse embryo fibroblasts (MEF) cells, and details of the transfected NIH3T3 cell lines were described previously (20Rosario L.A. O'Brien M.L. Henderson C.J. Wolf C.R. Tew K.D. Mol. Pharmacol. 2000; 58: 167-174Crossref PubMed Scopus (51) Google Scholar, 21Ruscoe J.E. Rosario L.A. Wang T. Gate L. Arifoglu P. Wolf C.R. Henderson C.J. Ronai Z. Tew K.D. J. Pharmacol. Exp. Ther. 2001; 298: Google Scholar). Cell lines were in and at and GSH and were from PABA/NO was as previously described (14Townsend D.M. Findlay V.J. Fazilev F. Ogle M. Fraser J. Saavedra J.E. Ji X. Keefer L.K. Tew K.D. Mol. Pharmacol. 2006; 69: 501-508Crossref PubMed Scopus (89) Google Scholar). NOV-002 was provided by GSTπ GSTπ was using from a from cells using the following and The were into the of for expression and into the of for expression in and were to by using the by The catalytically was by a by The of the was by Cell cells were transfected with human wild-type or the catalytically GSTπ using the were by cells with the GSTπ enzyme activity was with the as previously described E. Chasseaud L.F. Mol. Biol. Google Scholar). cells were and of protein reaction The reaction was in with GSH and The at was for in a The of conjugation of GSH to was from the of The for the conjugate at in was used to the were and with Cell were in and with phosphatase and and a were for on by for and for at in the were with the using as a of protein were on proteins were Proteins were binding was by the in and for were with the indicated at with for and with the to peroxidase for The were and developed with The were with a and with a The were in a The of was using and as in to and was used to S-glutathionylation of GSTπ in cells with of PABA/NO. Proteins from cell were and to as described were with and with both and protein as described with an was developed with and both from to the were with and with wild-type and knock-out mice and were in the of the of was provided in with the in the for the and of were with PABA/NO or NOV-002 by was in by at time The was and protein were and for cysteine of of and was by Proteins were in with and on the range from to For the the were on of were and to was by at the of the of was using from the for protein of in of and of were through a to both GSH and of was to of in a of at The at of was for and an of to a of the of glutathione was to the to that was not with the of The S.E. of at of GSTπ in of GSTπ S-glutathionylation on enzyme was by circular were on a using a GSTπ in were at using a were in the with of of time of and The of the was to the protein acid and into was on an using and were and GSTπ in and with PABA/NO and GSH were The was to an of protein and were from of the GSTπ was by with PABA/NO and GSH for in at PABA/NO and GSH were by through with was by the for at were for The are of at were for between the and with using of GSTπ on S-Glutathionylation following and of S-glutathionylation is and critical to cellular stress 15Townsend D.M. Mol. Interv. 2007; 7: 313-324Crossref PubMed Scopus (173) Google Scholar). The of this reaction has not been as or the first model from GSTπ wild-type and mice were used to the of S-glutathionylation following to that oxidative or nitrosative the we a and in protein modification following oxidative (GSSG) and nitrosative stress in proteins were in or The of from was and the to was using The of S-glutathionylation a for of an The may be by the of PABA/NO The of S-glutathionylation are described by for both and cells corresponding to the of S-glutathionylation. The S-glutathionylation of cellular proteins in cells is and when with the cells, that levels of S-glutathionylation may be or by other GSTπ to its and The of S-glutathionylation shows the of a of this The of GSTπ results in of this The of S-glutathionylation was to for PABA/NO A is that the of S-glutathionylation may to a of stress or a of glutathione expression S-glutathionylation of proteins. embryo cells from or were with of or PABA/NO for Proteins were by and S-glutathionylation by with The of the S-glutathionylation reaction were using a to for following or PABA/NO for and stress by PABA/NO to S-glutathionylation of multiple of which were identified in a (14Townsend D.M. Findlay V.J. Fazilev F. Ogle M. Fraser J. Saavedra J.E. Ji X. Keefer L.K. Tew K.D. Mol. Pharmacol. 2006; 69: 501-508Crossref PubMed Scopus (89) Google Scholar). protein S-glutathionylation proteins from to was following a PABA/NO there were in with cells S-glutathionylation of proteins in cells was at cells levels of proteins at this time stress by a and S-glutathionylation of a protein that we previously identified as (14Townsend D.M. Findlay V.J. Fazilev F. Ogle M. Fraser J. Saavedra J.E. Ji X. Keefer L.K. Tew K.D. Mol. Pharmacol. 2006; 69: 501-508Crossref PubMed Scopus (89) Google Scholar). S-glutathionylation of was at was in and at with for globular to S-Glutathionylation of its and both stress and the of in or shows that in there is a in of both stress and with are with S-glutathionylation of cellular as a consequence of GSTπ. of an following and and mice were used as an in vivo model to S-glutathionylation of and proteins. were with a of PABA/NO or NOV-002 NOV-002 to S-glutathionylation of in in a and S-Glutathionylation of was in following The of following PABA/NO and NOV-002 at was in mice with of in with cysteine and was used to proteins following PABA/NO and NOV-002 in and mice corresponding with protein sulfhydryl cysteine was following PABA/NO and NOV-002 in the of mice sulfhydryl modification in the was for the are with analysis a of cellular proteins following PABA/NO oxidized glutathione to S-glutathionylation of were in the from mice following Our that GSTπ expression protein S-glutathionylation in vivo. The of GSTπ for S-Glutathionylation in model was to the activity of GSTπ is for S-glutathionylation cells were transfected with wild-type or a catalytically P. R. J. Biochem. 1994; PubMed Scopus Google Scholar) of GSTπ. the model a GST was used to the activity cells GST activity of cells were The activity of GST was in cells as a consequence of a negative catalytic activity of GST is to the activity can be to other of GST present in This model was used to that GSTπ activity the and in protein S-glutathionylation The that at the of proteins following was in cells as with and were to The of S-glutathionylation in this model shows as in The of GSTπ S-glutathionylation. in S-glutathionylation were following a of PABA/NO the levels of S-glutathionylation were in cells and cells were to and The cells S-glutathionylation that were a negative of the model with that of the may be a property to S-glutathionylation in of S-glutathionylation on GST activity The the of in a of GSTπ through S-Glutathionylation in and in have that oxidative stress and of GSTπ (4Adler V. Yin Z. Fuchs S.Y. Benezra M. Rosario L. Tew K.D. Pincus M.R. Sardana M. Henderson C.J. Wolf C.R. Davis R.J. Ronai Z. EMBO J. 1999; 18: 1321-1334Crossref PubMed Scopus (962) Google Scholar). the of S-glutathionylation reactions, we that GSTπ itself nitrosative stress and this modification to its and GSTπ is in cells with PABA/NO for Our show that a in of cells both and GSTπ present study we used and primary for GSTπ and The and and of and in The GSTπ has a when with GST that a of GSTπ The was using The the of and in The of with is increased and with is to PABA/NO S-glutathionylation of GSTπ is in the cells the catalytically not that GSTπ can catalytically in of this S-Glutathionylation of Cys47 and Cys101 of GSTπ of GSTπ that Cys47 and Cys101 were by of GSH The of S-glutathionylation on the catalytic activity of GSTπ were in cells and in vitro using protein The GST activity of GSTπ or or of GSTπ was with wild-type of The cysteine are not in the catalytic and modification may activity through in protein we the of S-glutathionylation of GSTπ on its The of GSTπ was to and with a in the of the protein on the of GSTπ X. M. R. P. Biochemistry. PubMed Scopus Google Scholar), the sulfhydryl of Cys47 is to Consequently, with the of the the of GSTπ was its S-glutathionylation that of the protein in to with S-glutathionylation. a of protein protein S-glutathionylation of from a through Cys47 S-glutathionylation This is with that analysis shows an of GSTπ S-glutathionylation on its The cellular catalytic activity and of nitrosative S-glutathionylation or of Cys47 and Cys101 were in The activity of GSTπ in the of cells transfected with was of that for cells are with activity for that the catalytic activity of GSTπ in with oxidative or nitrosative stress are for S-glutathionylation of multiple target proteins. cells levels of proteins or cells cells, interactions between GSTπ and are important and both in an oxidative stress to of the complex and activation of both proteins (4Adler V. Yin Z. Fuchs S.Y. Benezra M. Rosario L. Tew K.D. Pincus M.R. Sardana M. Henderson C.J. Wolf C.R. Davis R.J. Ronai Z. EMBO J. 1999; 18: 1321-1334Crossref PubMed Scopus (962) Google Scholar, 5Wang T. Arifoglu P. Ronai Z. Tew K.D. J. Biol. Chem. 2001; 276: 20999-21003Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). of in cells that Cys101 in GSTπ is critical for these interactions and sulfiredoxin can the of proteins S.M. Taylor E.R. Brown S.E. Dahm C.C. Costa N.J. Runswick M.J. Murphy M.P. J. Biol. Chem. 2004; 279: 47939-47951Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, J.J. Pharmacol. 2007; 7: PubMed Scopus Google Scholar, M.J. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). PABA/NO S-glutathionylation of GSTπ in vitro catalytically was to the that the modification was not cause this of the cysteine residue in sulfiredoxin in to GSTπ. Our that S-glutathionylation of Cys47 and/or Cys101 of GSTπ with complex with other proteins. this on the the may be an alteration in cellular signaling events. the that GSTπ is of the proteins in many tumors ovarian, non-small cell lung, breast, colon, and K.D. Biochem. Pharmacol. 2007; PubMed Scopus Google for the protein has Our present results are the first to show that is a catalytic of the forward reaction of S-glutathionylation of to the regulation of a S-glutathionylation cycle. levels of GSTπ have been with of such as and T. K. K. Cancer Res. Google Scholar) and GST can be as a Although GSTπ knock-out mice are are susceptible to certain (19Henderson C.J. Smith A.G. Ure J. Brown K. Bacon E.J. Wolf C.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5275-5280Crossref PubMed Scopus (348) Google Scholar) and GSTπ knock-out mouse embryo fibroblasts have wild-type J.E. Rosario L.A. Wang T. Gate L. Arifoglu P. Wolf C.R. Henderson C.J. Ronai Z. Tew K.D. J. Pharmacol. Exp. Ther. 2001; 298: Google Scholar). The of GSTπ with proliferative pathways is not by the detoxification of the the catalytic activity of GSTπ is for of a of identified particularly anticancer drugs. The of GSTπ frequently for drug (2Tew K.D. Cancer Res. 1994; 54: 4313-4320PubMed Google Scholar), even when the selecting drug is not a for thioether with of interactions between GSTπ and regulatory kinases (4Adler V. Yin Z. Fuchs S.Y. Benezra M. Rosario L. Tew K.D. Pincus M.R. Sardana M. Henderson C.J. Wolf C.R. Davis R.J. Ronai Z. EMBO J. 1999; 18: 1321-1334Crossref PubMed Scopus (962) Google Scholar) have identified a role for GSTπ in stress response pathways and provide a for its high Our present are novel in that provide a further important cellular to which GSTπ The S-glutathionylation of target proteins is both in cells oxidative stress and in and cellular localization of proteins. S-Glutathionylation occur in the of GSTπ the and may be as a the of S-glutathionylation is in the of GSTπ. Our show that GSTπ results in stress and oxidative and nitrosative stress for mouse embryonic fibroblasts in This is to to the cell the of response to stress is to be an important of cell such a response may be The catalytic of GSTπ is for the S-glutathionylation reaction to For the catalytic of GSTπ, the of the of GSH is through of a by P. R. J. Biochem. 1994; PubMed Scopus Google Scholar) and this the of the thioether with electrophilic on of the residue with the S-glutathionylation this the of a between the of to GSTπ, and the sulfhydryl acid) of of the target protein to be a activity of GSTπ. The that the cysteine of GSTπ are of consequence to the S-glutathionylation but are in protein complex with and the of for the protein The two drugs used in these have distinct NOV-002 provides a of oxidized glutathione (GSSG) and can alter the intracellular through modification of (16Townsend D.M. He L. Hutchens S. VandenBerg T.E. Pazoles C.J. Tew K.D. Cancer Res. 2008; 68: 2870-2877Crossref PubMed Scopus (73) Google Scholar). PABA/NO is a that releases nitric oxide L.K. Rev. Pharmacol. PubMed Scopus Google Scholar). The of is the of reactive oxygen species and alteration in in a response. NOV-002 is as a that This is that oxidative stress is linked with in cell or Although pathways kinase and have been as in the reactive oxygen of cells of Z. T. M. Biol. 2007; PubMed Scopus Google Scholar), the of these pathways in cell is to be are a of instances and may For S-glutathionylation of a of that are critical to kinase have been S. Chock P.B. Biochemistry. 1999; PubMed Scopus Google Scholar). a of proteins that are are frequently in regulatory pathways D.M. Mol. Interv. 2007; 7: 313-324Crossref PubMed Scopus (173) Google Scholar), further the of GSTπ in the forward The S-glutathionylation cycle may to be as critical in cellular as the by kinases or The S-glutathionylation of GSTπ in in its can be as a of in as a consequence of a of in the protein with impact on and GSTπ has two and The is from the sulfhydryl of S-glutathionylation can M.J. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). The of GSTπ with PABA/NO and GSH in a in which with S-glutathionylation of This Cys47 residue of GSTπ could oxidized to sulfenic or of which the or sulfenic of Cys47 are most susceptible to S-glutathionylation. show that GSTπ used in this study is corresponding with analysis the of GSTπ shows a of to a a this S-glutathionylation causes in GSTπ and and interactions with the target protein for catalytic S-glutathionylation. in response to reactive species by or cells a and S-glutathionylation of target proteins. The of protein S-glutathionylation is by the of a catalytically GSTπ. Catalysis of this post-translational modification by a of GS- and the low pK cysteine of target proteins is a novel property for GSTπ and is an important forward reaction in the S-glutathionylation cycle. with the functions to GSTπ, high expression levels in cells may be a of the pathways that can be to the the and and at the of This was in a with by of from the of the for