20 S Proteasome from Saccharomyces cerevisiae Is Responsive to Redox Modifications and IsS-Glutathionylated

The 20 S proteasome core purified fromSaccharomyces cerevisiae is inhibited by reduced glutathione (GSH), cysteine (Cys), or the GSH precursor γ-glutamylcysteine. Chymotrypsin-like activity was more affected by GSH than trypsin-like activity, whereas the peptidylglutamyl-hydrolyzing activity (caspase-like) was not inhibited by GSH. Cys-sulfenic acid formation in the 20 S core was demonstrated by spectral characterization of the Cys-S(O)-4-nitrobenzo-2-oxa-1,3-diazole adduct, indicating that 20 S proteasome Cys residues might react with reduced sulfhydryls (GSH, Cys, and γ-glutamylcysteine) through the oxidized Cys-sulfenic acid form. S-Glutahionylation of the 20 S core was demonstrated in vitro by GSH-biotin incorporation and by decreased alkylation with monobromobimane. Compounds such asN-ethylmaleimide (-S-sulfhydril H alkylating), dimedone (-SO sulfenic acid H reactant), or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (either -SH or -SOH reactant) highly inhibited proteasomal chymotrypsin-like activity. In vivo experiments revealed that 20 S proteasome extracted from H2O2-treated cells showed decreased chymotrypsin-like activity accompanied byS-glutathionylation as demonstrated by GSH release from the 20 S core after reduction with NaBH4. Moreover, cells pretreated with H2O2 showed decreased reductive capacity assessed by determination of the GSH/oxidized glutathione ratio and increased protein carbonyl levels. The present results indicate that at the physiological level the yeast 20 S proteasome is regulated by its sulfhydryl content, thereby coupling intracellular redox signaling to proteasome-mediated proteolysis. The 20 S proteasome core purified fromSaccharomyces cerevisiae is inhibited by reduced glutathione (GSH), cysteine (Cys), or the GSH precursor γ-glutamylcysteine. Chymotrypsin-like activity was more affected by GSH than trypsin-like activity, whereas the peptidylglutamyl-hydrolyzing activity (caspase-like) was not inhibited by GSH. Cys-sulfenic acid formation in the 20 S core was demonstrated by spectral characterization of the Cys-S(O)-4-nitrobenzo-2-oxa-1,3-diazole adduct, indicating that 20 S proteasome Cys residues might react with reduced sulfhydryls (GSH, Cys, and γ-glutamylcysteine) through the oxidized Cys-sulfenic acid form. S-Glutahionylation of the 20 S core was demonstrated in vitro by GSH-biotin incorporation and by decreased alkylation with monobromobimane. Compounds such asN-ethylmaleimide (-S-sulfhydril H alkylating), dimedone (-SO sulfenic acid H reactant), or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (either -SH or -SOH reactant) highly inhibited proteasomal chymotrypsin-like activity. In vivo experiments revealed that 20 S proteasome extracted from H2O2-treated cells showed decreased chymotrypsin-like activity accompanied byS-glutathionylation as demonstrated by GSH release from the 20 S core after reduction with NaBH4. Moreover, cells pretreated with H2O2 showed decreased reductive capacity assessed by determination of the GSH/oxidized glutathione ratio and increased protein carbonyl levels. The present results indicate that at the physiological level the yeast 20 S proteasome is regulated by its sulfhydryl content, thereby coupling intracellular redox signaling to proteasome-mediated proteolysis. oxidized glutathione reduced cysteine Cys-sulfenic acid Cys-sulfinic acid Cys-sulfonic acid dithionitrobenzoic acid diethylenetriaminepentaacetic acid dithiothreitol γ-glutamylcysteine reduced glutathione monobromobimane 4-methylcoumarin-7-amide 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole N-ethylmaleimide succinyl-Leu-Leu-Val-Tyr-MCA high pressure liquid chromatography analysis of variance The proteasome is an essential proteolytic complex in eukaryotic cells where it is responsible for the degradation of many cellular proteins. It plays an important role in cell-cycle regulation, cell signaling, including apoptosis, and elimination of abnormal proteins generated by mutation (1Coux O. Tanaka K. Goldberg A.F. Annu. Rev. Biochem. 1996; 65: 801-847Crossref PubMed Scopus (2239) Google Scholar, 2Bochtler M. Ditzel L. Groll M. Hatmann C. Huber R. Ann. Rev. Biophys. Biomol. Struct. 1999; 28: 295-317Crossref PubMed Scopus (425) Google Scholar) and oxidative damage (3Giulivi C. Pacifici R.E. Davies K.J.A. Arch. Biochem. Biophys. 1994; 311: 329-341Crossref PubMed Scopus (142) Google Scholar, 4Berlett B.S. Stadtman E.R. J. Biol. Chem. 1997; 272: 20313-20316Abstract Full Text Full Text PDF PubMed Scopus (2809) Google Scholar, 5Ullrich O. Reinheckel T. Sitte N. Hass R. Grune T. Davies K.J.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6223-6228Crossref PubMed Scopus (212) Google Scholar). In recent years, many publications have reported the reversibleS-glutathionylation of a discrete number of proteins (6Claiborne A. Mallett T.C. Yeh J.I. Luba J. Parsonage D. Adv. Prot. Chem. 2001; 58: 215-276Crossref PubMed Scopus (137) Google Scholar). Protein S-glutathionylation seems to play an essential role in redox regulation. This form of regulation has direct effects on both enzyme activity and the ability of transcription and replication factors to bind DNA targets. The mechanism of protein glutathionylation has evolved in recent years from the strict belief that this event would take place solely when intracellular GSSG1 levels increased upon oxidative stress, with the formation of mixed disulfides between protein Cys-SH residues and GSSG (7Gilbert H.F. Methods Enzymol. 1995; 251: 8-28Crossref PubMed Scopus (497) Google Scholar, 8Thomas J.A. Poland B. Honzatko R. Arch. Biochem. Biophys. 1995; 319: 1-9Crossref PubMed Scopus (365) Google Scholar), to the present and more complete understanding of protein sulfhydryl chemistry and evidence showing formation of protein-Cys-SOH derivatives (9Ellis H.R. Poole L.B. Biochemistry. 1997; 36: 15013-15018Crossref PubMed Scopus (210) Google Scholar) that are prone toS-glutathionylation by reduced GSH (10Barrett W.C. DeGnore J.P. Keng Y.F. Zhang Z.Y. Yim M.B. Chock P.B. J. Biol. Chem. 1999; 274: 34543-34546Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). Either individual enzyme activity or global cellular responses can be rapidly controlled by the oxidation of protein-Cys-SH residues (reviewed in Ref. 6Claiborne A. Mallett T.C. Yeh J.I. Luba J. Parsonage D. Adv. Prot. Chem. 2001; 58: 215-276Crossref PubMed Scopus (137) Google Scholar) generating Cys-SOH, Cys-SO2H, and Cys-SO3H acid forms (11Yang K.S. Kang S.W. Woo H.A. Hwang S.C. Chae H.Z. Kim K. Rhee S.G. J. Biol. Chem. 2002; 277: 38029-38036Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar), where Cys-SOH is susceptible toS-thionylation and reversibly reduced to Cys-SH (11Yang K.S. Kang S.W. Woo H.A. Hwang S.C. Chae H.Z. Kim K. Rhee S.G. J. Biol. Chem. 2002; 277: 38029-38036Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, 12Benitez L.V. Allison W.S. Arch. Biochem. Biophys. 1973; 159: 89-96Crossref PubMed Scopus (4) Google Scholar, 13Allison W.S. Acc. Chem. Res. 1976; 9: 293-299Crossref Scopus (251) Google Scholar, 14Claiborne A. Yeh J.I. Mallett T.C. Luba J. Crane E.J. Charrier V. Parsonage D. Biochemistry. 1999; 38: 15407-15416Crossref PubMed Scopus (462) Google Scholar). Cys-SOH formation and S-glutathionylation during enzyme catalysis and redox signaling are novel cofactors in the context of redox regulation (14Claiborne A. Yeh J.I. Mallett T.C. Luba J. Crane E.J. Charrier V. Parsonage D. Biochemistry. 1999; 38: 15407-15416Crossref PubMed Scopus (462) Google Scholar). In a recent publication (15Demasi M. Shringarpure R. Davies K.J.A. Arch. Biochem. Biophys. 2001; 389: 254-263Crossref PubMed Scopus (65) Google Scholar) it was demonstrated that the reduced and oxidized forms of GSH modulate the chymotrypsin-like activity of purified 20 S proteasome extracted from mammalian cells. In the present report we show that the activity of the 20 S proteasome purified from the yeast Saccharomyces cerevisiae is also sensitive to GSH, though in a different way from that observed in the mammalian proteasome. The 20 S proteasome extracted from yeast is inhibited by reduced GSH and S-glutathionylated in vitro, as well as in vivo, when cells are submitted to oxidative challenge. Considering that the proteasome plays important role in cell signaling regulation by hydrolysis of many proteins involved in cascade events of the cellular regulatory pathways, it is not surprising that its activity may be regulated by its Cys-SH residues redox status. Diethylenetriaminepentaacetic acid (DTPA), dimedone (5,5-dimethyl-1,3-cyclo-hexanedione), dinitrophenylhydrazine, dithionitrobenzoic acid (DTNB),N-ethylmaleimide (NEM), fluorogenic substrates succinyl-Leu-Leu-Val-Tyr-MCA (s-LLVY-MCA) andt-butoxycarbonyl-Gly-Lys-Arg-MCA, γ-glutamylcysteine (GC), and streptavidin immobilized on 4% beaded agarose were purchased from Sigma. The fluorogenic substrate carbobenzoxy-Leu-Leu-Glu-MCA, the proteasome inhibitors and and monobromobimane were purchased from was purchased from were of and the was purified with the S. was from and from was by of This has the 20 S proteasome with the and a R. S. R. D. J. J. Biol. PubMed Scopus Google Scholar). were in and yeast or as at with cells were at the of the at of cells were at than cells were to the in Ref. R. S. R. D. J. J. Biol. PubMed Scopus Google The 20 S proteasome from was purified by on and a in an The was to a R. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar), that was of a and the was in the of the protein liquid chromatography The 20 S proteasome core from the with a was purified by chromatography when by chromatography was a to a to the The proteasome was from the with from of were by the degradation of the fluorogenic and by with were and in a by was to the in Ref. R. S. R. D. J. J. Biol. PubMed Scopus Google with the immobilized on agarose The 20 S proteasome was from the agarose when by with the to the The were by and by on a to the in Ref. R. M. J. Biol. Chem. Full Text PDF PubMed Google The different 20 S proteasome were for the The results vivo and in to the from we not with from not 20 S proteasome or cell were at in 20 and to as was by the of of the are in the to the The was by of was at at from the substrates was from a of purified 20 S proteasome were with in experiments was or as and the was for at in the of with or in H2O2 was by of and with through of the 20 S proteasome were for or for hydrolysis after determination of protein of the 20 S core with the as the agarose were to the reduction was with the 20 S proteasome to the complex in the of 20 in for at the was the was for GSH determination and the were with The when was with the 20 S proteasome to the GSH was extracted by the cell in of and of The was for 20 at in a and at in a This was and the were GSH, as well as was to a (15Demasi M. Shringarpure R. Davies K.J.A. Arch. Biochem. Biophys. 2001; 389: 254-263Crossref PubMed Scopus (65) Google Scholar). The determination was by with in the of glutathione and for GSSG were for with after the to GSH from 20 S proteasome was as 20 S proteasome by was with as The was through a The was to with was at and the was to to the of glutathione and GSH was increased in the of glutathione and to in the of GSH was from of reduced GSH was by the between GSH and GSH-biotin was as in Ref. T. Biochemistry. PubMed Scopus Google GSH was with at ratio in for at The was by the of with H2O2-treated or 20 S proteasome was with GSH-biotin for 20 at GSH-biotin was by through a by in protein was in mixed with and for at The were by of in and the protein was from the by for in and by at for The were submitted to after determination of protein of the cell were to an of after the by a of of the were on and at for were were at formation in the proteins was by with and by as in Ref. J.A. Stadtman E.R. Methods Enzymol. 1994; PubMed Scopus Google Protein was with 20 S proteasome core from S. cerevisiae was for hydrolysis with the substrate in the of GSH, and Cys GSSG a on 20 S proteasomal activity at GSH or Cys inhibited the proteasomal activity at The GSH precursor also inhibited chymotrypsin-like activity in a to that by Cys not The trypsin-like activity by the hydrolysis of the fluorogenic was decreased in the of GSH not The trypsin-like activity than of the chymotrypsin-like activity the were for both The activity by the hydrolysis of the fluorogenic was not affected by of the sulfhydryl (GSH, or at or by GSSG not by results was the reduced form of GSH and and not inhibited 20 S proteasomal activity. was that Cys-SH residues in the 20 S proteasome are oxidized to the Cys-SOH susceptible to S-glutathionylation by GSH, as (6Claiborne A. Mallett T.C. Yeh J.I. Luba J. Parsonage D. Adv. Prot. Chem. 2001; 58: 215-276Crossref PubMed Scopus (137) Google Scholar, R. J. Biol. Chem. Full Text PDF PubMed Google Scholar), not by to the this purified 20 S proteasome were in the of and The was to the and the of the may protein in to oxidation and of derivatives Stadtman E.R. Arch. Biochem. Biophys. 1996; PubMed Scopus Google Scholar). in the of or sulfhydryl oxidation to Cys-SOH may though oxidation of Cys-SOH to and Cys-SO3H is (11Yang K.S. Kang S.W. Woo H.A. Hwang S.C. Chae H.Z. Kim K. Rhee S.G. J. Biol. Chem. 2002; 277: 38029-38036Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar). to 20 S proteasome with H2O2 in the of decreased chymotrypsin-like activity to of the level whereas when H2O2 was in the of the activity was reduced to not 20 S proteasome was pretreated with H2O2 the chymotrypsin-like activity was more affected by GSH In after by GSH at as as and GSH at than that In GSSG not 20 S proteasome activity results are in with in the In to proteasomal activity by Moreover, it the proteasome to GSH incorporation after H2O2 20 S proteasome were with by with the chymotrypsin-like activity was more sensitive to GSH when with not with In this proteasomal activity was decreased to and in the of or whereas when the GSH were chymotrypsin-like activity was decreased to and Cys-SOH present in 20 S proteasome was reduced to Cys-SH by with the of of Cys-SOH to or Cys-SO3H by H2O2 to in the when the 20 S core was pretreated with by GSH was observed in its activity proteasome reduced by was not inhibited by GSSG not as be GSSG is to react with sulfhydryl to form mixed disulfides (7Gilbert H.F. Methods Enzymol. 1995; 251: 8-28Crossref PubMed Scopus (497) Google Scholar, 8Thomas J.A. Poland B. Honzatko R. Arch. Biochem. Biophys. 1995; 319: 1-9Crossref PubMed Scopus (365) Google Scholar). S might be from with GSSG by by that of S core residues is responsible for the of chymotrypsin-like activity, we this activity in the of Cys-SH and Cys-SOH such as and observed that chymotrypsin-like activity was inhibited and by and Cys-SH whereas is both Cys-SH and This might at in the is more than in of chymotrypsin-like activity by 20 S proteasomal activity was by with not to release (9Ellis H.R. Poole L.B. Biochemistry. 1997; 36: 15013-15018Crossref PubMed Scopus (210) Google Scholar), this that was by Cys of Cys-SH and Cys-SOH on proteasomal 20 S proteasome was as for at in the of or at the by a at with to the hydrolysis or dimedone were with H2O2 and as and by with dimedone and the with the fluorogenic The hydrolysis is and are of in a The 20 S proteasome was as for at in the of or at the by a at with to the hydrolysis or dimedone were with H2O2 and as and by with dimedone and the with the fluorogenic The hydrolysis is and are of The Cys-SOH dimedone (9Ellis H.R. Poole L.B. Biochemistry. 1997; 36: 15013-15018Crossref PubMed Scopus (210) Google Scholar) when with the at the when purified 20 S proteasome were pretreated with to with we observed increased proteolytic with the observed in the of by dimedone in dimedone decreased chymotrypsin-like activity to of that observed in the results reported indicate that in Cys residues of the 20 S core its activity, at the chymotrypsin-like activity, is the of its (1Coux O. Tanaka K. Goldberg A.F. Annu. Rev. Biochem. 1996; 65: 801-847Crossref PubMed Scopus (2239) Google Scholar). It seems that Cys residues in the 20 S proteasome be reduced as as to activity. residues to to that Cys residues in the 20 S proteasome are oxidized to Cys-SOH, 20 S proteasome were with This with as well as with of Cys-SOH and Cys-SH can be by (9Ellis H.R. Poole L.B. Biochemistry. 1997; 36: 15013-15018Crossref PubMed Scopus (210) Google Scholar). The was generated by 20 S proteasome with by This showed at whereas the purified 20 S core not oxidized by H2O2 the with a at also 20 S proteasome with the Methods Enzymol. 1995; 251: PubMed Scopus Google Scholar). 20 S proteasome were with or GSH. and GSH were and the were with were by after of showed at as high as whereas showed reduced 20 S proteasome mixed disulfides not with This that GSH not play the role of a as GSH was the protein that the of GSH on proteasomal activity is of 20 S proteasome were with the GSH-biotin to the in the T. Biochemistry. PubMed Scopus Google Scholar). This the direct determination of protein can be by the streptavidin 20 S proteasome with H2O2 increased GSH incorporation The protein after protein from the was in pretreated with Protein from and H2O2-treated after from the streptavidin was and of protein with GSH-biotin was the in both This is direct that 20 S proteasome is susceptible toS-glutathionylation by of GSH to a of S-glutathionylated 20 S proteasome was in the of its Cys residues were oxidized to Cys-SOH It be that the with GSH-biotin was The was to after with and of GSH-biotin to the 20 S core from the the results in not to many or of the 20 S core were to the proteasome after with GSH-biotin and to the streptavidin to many and would be the proteasome of and H2O2-treated from the agarose was not to be on the by This is an that or might be Chymotrypsin-like activity of 20 S proteasome an was in the of GSH and for to its the yeast 20 S proteasome. The proteasome was not affected by GSH and was inhibited by In the of mammalian 20 S both GSH and GSSG at the chymotrypsin-like activity, whereas it was inhibited by (15Demasi M. Shringarpure R. Davies K.J.A. Arch. Biochem. Biophys. 2001; 389: 254-263Crossref PubMed Scopus (65) Google Scholar). Moreover, 20 S mammalian proteasomal activity was inhibited by the the yeast 20 S core is inhibited by reduced GSH and is inhibited by whereas the proteasome is not affected by GSH form and is affected by of Cys on proteasome proteasome was purchased from was for or at in the of GSH or at the by a at with determination was and are of in a proteasome was purchased from was for or at in the of GSH or at the by a at with determination was and are of results indicate a role of Cys residues the 20 S core its activity. Cys and mammalian 20 S and reduced Cys residues are the of GSH on proteasome activity is to Cys belief is that the 20 S proteasome core has evolved as an important of cellular redox by in its activity by in its Cys the cellular to redox was to for in vivo 20 S was that proteasome would be sensitive to intracellular reductive more as the cell reductive is This might be an way for cells to the signaling to oxidative this we cells with H2O2 the hydrolysis of the fluorogenic substrate was in of 20 S proteasome from the cell with the and in the cell after 20 S proteasome The 20 S complex to agarose was for the hydrolysis of the fluorogenic and after in the of 20 to the by mixed disulfides or to Cys-SOH to can also carbonyl or by the oxidation of acid residues to derivatives E.R. Annu. Rev. Biochem. PubMed Scopus Google Scholar). in was to oxidized Cys residues the 20 S thereby proteasome sulfhydryls and GSH release from the GSH was in the after reduction with as and The reductive cellular capacity was by the determination of the also cell and the formation of carbonyl protein as a of oxidative activity and redox upon of 20 S proteasome are as of as S proteasome was purified from the cell by and for and after reduction with NaBH4. cell are as of as was in the cell of the 20 S proteasome after with was from of 20 S proteasome after reduction with as in carbonyl protein from cell was with results are of to were in to of by a with H2O2 to the at the were for cell The cells were by and with 20 S proteasome by reduction with and of and oxidized GSH from 20 S and carbonyl proteins are and not are as of as 20 S proteasome was purified from the cell by and for and after reduction with was in the cell of the 20 S proteasome after with GSH was from of 20 S proteasome after reduction with as in The protein from cell was with results are of to in a were in to of by a with H2O2 to the at the were for cell The cells were by and with 20 S proteasome by reduction with and of and oxidized GSH from 20 S and carbonyl proteins are and not H2O2 to a in the formation of protein carbonyl when to the at a of at this oxidative was accompanied by decreased reductive capacity of the as by the to with that in cells. the proteasomal activity decreased with and was after reduction with to of of the fluorogenic in the increased by in the T. Reinheckel T. Davies K.J.A. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, T. Reinheckel T. Davies K.J.A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, T. Sitte N. O. U. Davies K.J.A. Grune T. Biochem. J. PubMed Scopus Google Scholar) show that mammalian cells oxidative show increased proteolysis. The that increased is of increased proteasomal activity such as that in vitro when oxidized substrates show increased hydrolysis levels by purified 20 S proteasome from mammalian cells (3Giulivi C. Pacifici R.E. Davies K.J.A. Arch. Biochem. Biophys. 1994; 311: 329-341Crossref PubMed Scopus (142) Google Scholar). results show that yeast 20 S proteasome is highly affected by oxidative whereas hydrolysis not on proteasome is increased The for the of proteasome activity after is the reduction of Cys-SOH or to oxidative are to reduction by formation of carbonyl and J.A. Stadtman E.R. Methods Enzymol. 1994; PubMed Scopus Google Scholar), in this the acid are not as for Cys-SOH and GSH release from the 20 S core after reduction with is evidence that proteasomal activity might be regulated by S-glutathionylation intracellular oxidative and decreased reductive by a reduced ratio upon H2O2 cell results in vivo after cell with H2O2 indicate that of reductive cellular to and protein carbonyl is with of 20 S proteasomal activity and with its are an of redox of 20 S proteasome activity. It is that many proteins are S-glutathionylated in to intracellular redox (6Claiborne A. Mallett T.C. Yeh J.I. Luba J. Parsonage D. Adv. Prot. Chem. 2001; 58: 215-276Crossref PubMed Scopus (137) Google Scholar, T. Biochemistry. PubMed Scopus Google Scholar, M. M. S. M. V. M. A. J. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar) (reviewed in Ref. Biophys. Res. 1-9Crossref PubMed Scopus Google Scholar). Cys-SOH is an form involved in redox regulation and catalysis by protein sulfhydryl (reviewed in for protein Cys-SOH have (11Yang K.S. Kang S.W. Woo H.A. Hwang S.C. Chae H.Z. Kim K. Rhee S.G. J. Biol. Chem. 2002; 277: 38029-38036Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, 14Claiborne A. Yeh J.I. Mallett T.C. Luba J. Crane E.J. Charrier V. Parsonage D. Biochemistry. 1999; 38: 15407-15416Crossref PubMed Scopus (462) Google Scholar) S-glutathionylation would be of the protein activity. is a and is evidence that GSH release is controlled by C. Methods Enzymol. 2002; PubMed Scopus Google Scholar). In this we the mechanism by 20 S proteasome is in results indicate that during oxidative Cys residues the 20 S proteasome core might be oxidized to Cys-SOH, reversibly by and after cellular from oxidative the in we that to GSH was of 20 S that the of the yeast is GSH was not in 20 S proteasome from of the of the for GSH The GSH from the 20 S core is a of 20 S proteasome at the physiological level is highly Cys residues in the core are prone to S-glutathionylation during is that S-glutathionylation of the 20 S proteasome is to the oxidation of to the the results the of GSH and GSSG on proteasomal activity from yeast and and M. to results (15Demasi M. Shringarpure R. Davies K.J.A. Arch. Biochem. Biophys. 2001; 389: 254-263Crossref PubMed Scopus (65) Google Scholar) for mammalian 20 S it is that the 20 S proteasome to in redox In this it is important to that the mammalian has or proteins as and its cells is it is in the and S. Tanaka K. J. J. Biochem. J. PubMed Scopus Google Scholar). the the yeast proteasome is with the degradation of proteins and the cell and not more than in the with the J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). It is to that GSH yeast cells is not it has demonstrated that GSH is the mammalian at as high as in the M. Res. 1997; 36: PubMed Scopus Google Scholar), its the yeast is not is the of yeast cells mammalian was S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. C. J. M. C. J. M. A. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) whereas on yeast is activity is to direct GSH its the would be evidence of GSH in the Considering proteasome and its role in the yeast as of GSH yeast cells is an important to the important in the S. Tanaka K. J. J. Biochem. J. PubMed Scopus Google Scholar, J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar) is that yeast proteasome is by the S regulatory in to the that its the mammalian 20 S is in the S regulatory In it is that the yeast proteasome is with the S this not the that the by 20 S proteasome is regulated of protein or substrate by the S proteasome. results that redox regulation by glutathionylation is important in vivo of redox regulation have for M. M. S. M. V. M. A. J. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar, J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, Biol. 2002; Scholar). results to that 20 S proteasomal activity can be by S-glutathionylation through the increased of would be in signaling 20 S proteasome would the hydrolysis of proteins responsible for redox signaling, proteins are of the redox of the cell N. 2001; PubMed Scopus (137) Google Scholar) as demonstrated in mammalian are by the proteasome C. C. M. 2001; PubMed Scopus Google Scholar). The of protein S-glutathionylation in redox signaling is the of oxidation of the Cys to or protein by mechanism might in to the mechanism proteasome-mediated The between proteasome glutathionylation and of substrates to be