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Sulfiredoxin, the cysteine sulfinic acid reductase specific to 2-Cys peroxiredoxin: its discovery, mechanism of action, and biological significance

2007; Elsevier BV; Volume: 72; Linguagem: Inglês

10.1038/sj.ki.5002380

1523-1755

Sue Goo Rhee, Woojin Jeong, Tong Shin Chang, Hyun Ae Woo,

Glutathione Transferases and Polymorphisms

Peroxiredoxin (Prx) is a family of bifunctional proteins that exhibit peroxidase and chaperone activities. Prx proteins contain a conserved Cys residue that undergoes a redox change between thiol and disulfide states. 2-Cys Prx enzymes, a subgroup of Prx family, are intrinsically susceptible to reversible hyperoxidation to cysteine sulfinic acid during catalysis. Cysteine hyperoxidation of Prx was shown to result in loss of peroxidase activity and a concomitant gain of chaperone activity. Reduction of sulfinic Prx enzymes, the first known biological example of such a reaction, is catalyzed by sulfiredoxin (Srx) in the presence of ATP. Srx appears to exist solely to support the reversible sulfinic modification of 2-Cys Prx enzymes. Srx specifically binds to 2-Cys Prx enzymes by recognizing several critical surface-exposed residues of the Prxs, and transfer the γ-phosphate of ATP to their sulfinic moiety, using its conserved cysteine as the phosphate carrier. The resulting sulfinic phosphoryl ester is reduced to cysteine after oxidation of four thiol equivalents. Peroxiredoxin (Prx) is a family of bifunctional proteins that exhibit peroxidase and chaperone activities. Prx proteins contain a conserved Cys residue that undergoes a redox change between thiol and disulfide states. 2-Cys Prx enzymes, a subgroup of Prx family, are intrinsically susceptible to reversible hyperoxidation to cysteine sulfinic acid during catalysis. Cysteine hyperoxidation of Prx was shown to result in loss of peroxidase activity and a concomitant gain of chaperone activity. Reduction of sulfinic Prx enzymes, the first known biological example of such a reaction, is catalyzed by sulfiredoxin (Srx) in the presence of ATP. Srx appears to exist solely to support the reversible sulfinic modification of 2-Cys Prx enzymes. Srx specifically binds to 2-Cys Prx enzymes by recognizing several critical surface-exposed residues of the Prxs, and transfer the γ-phosphate of ATP to their sulfinic moiety, using its conserved cysteine as the phosphate carrier. The resulting sulfinic phosphoryl ester is reduced to cysteine after oxidation of four thiol equivalents. Peroxiredoxins (Prxs) are a family of enzymes that reduce hydrogen peroxide (H2O2) and alkyl hydroperoxides to water and alcohol, respectively, with the use of reducing equivalents provided by a physiological thiol like thioredoxin.1.Chae H.Z. Robison K. Poole L.B. et al.Cloning and sequencing of thiol-specific antioxidant from mammalian brain: alkyl hydroperoxide reductase and thiol-specific antioxidant define a large family of antioxidant enzymes.Proc Natl Acad Sci USA. 1994; 91: 7017-7021Crossref PubMed Scopus (701) Google Scholar, 2.Hofmann B. Hecht H.J. Flohe L. Peroxiredoxins.Biol Chem. 2002; 383: 347-364Crossref PubMed Scopus (772) Google Scholar, 3.Rhee S.G. Kang S.W. Chang T.S. et al.Peroxiredoxin, a novel family of peroxidases.IUBMB Life. 2001; 52: 35-41Crossref PubMed Scopus (517) Google Scholar, 4.Wood Z.A. Schroder E. Robin Harris J. et al.Structure, mechanism and regulation of peroxiredoxins.Trends Biochem Sci. 2003; 28: 32-40Abstract Full Text Full Text PDF PubMed Scopus (2114) Google Scholar, 5.Rhee S.G. Chae H.Z. Kim K. Peroxiredoxins: a historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling.Free Radic Biol Med. 2005; 38: 1543-1552Crossref PubMed Scopus (1140) Google Scholar All Prx proteins contain a conserved Cys residue (designated the peroxidatic cysteine, CP), which corresponds to Cys51 in mammalian Prx I and II, located in the N-terminal portion of the molecule. The majority of Prx proteins, including four (Prx I–IV) of six mammalian Prxs, contain an additional conserved Cys residue (the resolving cysteine, CR) in the C-terminal region that corresponds to Cys172 in mammalian Prx I. The Prx enzymes containing two conserved Cys residues are thus called '2-Cys Prx', in contrast with a small number of Prx proteins termed '1-Cys Prx', which contain only the N-terminal-conserved cysteine. In 2-Cys Prx enzymes, the CP is oxidized by H2O2 to cysteine-sulfenic acid (Cys51-SOH), which then reacts with the resolving cysteine (Cys172-SH) of the other subunit to produce an intermolecular disulfide (Figure 1).6.Rhee S.G. Kang S.W. Jeong W. et al.Intracellular messenger function of hydrogen peroxide and its regulation by peroxiredoxins.Curr Opin Cell Biol. 2005; 17: 183-189Crossref PubMed Scopus (612) Google Scholar The CP is sensitive to the oxidation by peroxides because it is surrounded by positively charged amino acid residues, which stabilize the thiolate (Cys-S-) anion. The thiolate anion is more readily oxidized by peroxides than its protonated thiol counterpart (Cys-SH). Reduction of the disulfide intermediate of Prx I–IV is specific, in that it can be achieved by thioredoxin (Trx). Thus, the reducing equivalents for the peroxidase activity of Prx I–IV are ultimately derived from nicotinamide adinine dinucleotide phosphate via thioredoxin reductase and Trx.3.Rhee S.G. Kang S.W. Chang T.S. et al.Peroxiredoxin, a novel family of peroxidases.IUBMB Life. 2001; 52: 35-41Crossref PubMed Scopus (517) Google Scholar, 7.Chae H.Z. Chung S.J. Rhee S.G. Thioredoxin-dependent peroxide reductase from yeast.J Biol Chem. 1994; 269: 27670-27678Abstract Full Text PDF PubMed Google Scholar, 8.Chae H.Z. Uhm T.B. Rhee S.G. Dimerization of thiol-specific antioxidant and the essential role of cysteine 47.Proc Natl Acad Sci USA. 1994; 91: 7022-7026Crossref PubMed Scopus (281) Google Scholar The crystal structures of 2-Cys and 1-Cys Prx enzymes revealed that the CP is located in a small pocket formed by the N- and C-terminal domains of the two subunits.9.Choi H.J. Kang S.W. Yang C.H. et al.Crystal structure of a novel human peroxidase enzyme at 2.0 A resolution.Nat Struct Biol. 1998; 5: 400-406Crossref PubMed Scopus (332) Google Scholar, 10.Hirotsu S. Abe Y. Okada K. et al.Crystal structure of a multifunctional 2-Cys peroxiredoxin heme-binding protein 23 kDa/proliferation-associated gene product.Proc Natl Acad Sci USA. 1999; 96: 12333-12338Crossref PubMed Scopus (235) Google Scholar, 11.Schroder E. Littlechild J.A. Lebedev A.A. et al.Crystal structure of decameric 2-Cys peroxiredoxin from human erythrocytes at 1.7 A resolution.Structure. 2000; 8: 605-615Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 12.Alphey M.S. Bond C.S. Tetaud E. et al.The structure of reduced tryparedoxin peroxidase reveals a decamer and insight into reactivity of 2Cys-peroxiredoxins.J Mol Biol. 2000; 300: 903-916Crossref PubMed Scopus (141) Google Scholar, 13.Wood Z.A. Poole L.B. Hantgan R.R. et al.Dimers to doughnuts: redox-sensitive oligomerization of 2-cysteine peroxiredoxins.Biochemistry. 2002; 41: 5493-5504Crossref PubMed Scopus (297) Google Scholar The reactive cysteine is thus protected from larger oxidant molecules that contain disulfide linkages. The structures also show that the N-terminal conserved cysteine is surrounded by positively charged amino-acid residues, which stabilize the thiolate (Cys-S-) anion. The thiolate anion is more readily oxidized by peroxides than its protonated thiol counterpart (Cys-SH).14.Kim J.R. Yoon H.W. Kwon K.S. et al.Identification of proteins containing cysteine residues that are sensitive to oxidation by hydrogen peroxide at neutral pH.Anal Biochem. 2000; 283: 214-221Crossref PubMed Scopus (244) Google Scholar This provides the mechanistic basis for the observed sensitivity of the active-site cysteine to oxidation by peroxides. Recent kinetic studies with bacterial AhpC and mammalian Prx II indicated that the catalytic efficiency of Prx enzymes (1-4 × 107 M s) is comparable to those of catalase and glutathione peroxidase.15.Parsonage D. Youngblood D.S. Sarma G.N. et al.Analysis of the link between enzymatic activity and oligomeric state in AhpC, a bacterial peroxiredoxin.Biochemistry. 2005; 44: 10583-10592Crossref PubMed Scopus (172) Google Scholar,16.Peskin A.V. Low F.M. Paton L.N. et al.The high reactivity peroxiredoxin 2 with H2O2 is not reflected in its reaction with other oxidants and thiol reagents.J Biol Chem. 2007; 282: 11885-11892Crossref PubMed Scopus (314) Google Scholar On the basis of studies with various biological oxidants, Prx II was suggested to have a tertiary structure that provides a specific environment that makes CP highly reactive with H2O2 while at the same time shielding it from reaction with other thiol oxidants, like chloramines, and alkylating agents like iodoacetamide and N-ethylmaleimide. When Prx-dependent nicotinamide adinine dinucleotide phosphate oxidation was followed spectrophotometrically, the activity of 2-Cys Prxs was observed to decrease gradually with time.17.Yang K.S. Kang S.W. Woo H.A. et al.Inactivation of human peroxiredoxin I during catalysis as the result of the oxidation of the catalytic site cysteine to cysteine-sulfinic acid.J Biol Chem. 2002; 277: 38029-38036Crossref PubMed Scopus (372) Google Scholar Such inactivation is consistent with further oxidation of the sulfenic acid moiety of the reaction intermediate by H2O2 to cysteine sulfinic acid (Cys-SO2H) before disulfide formation with Cys172 can occur (Figure 1). We demonstrated that H2O2 alone is not sufficient to cause oxidation of Cys51 to Cys51-SO2H. Rather, all catalytic components (H2O2, thioredoxin, thioredoxin reductase, and nicotinamide adinine dinucleotide phosphate) must be present, indicating that such hyperoxidation occurs only when Prx I is engaged in the catalytic cycle. Prx I was hyperoxidized at a rate of 0.072% per turnover at 30°C. Structural analysis revealed a conformation in which the sulfenic acid moiety of Prx I Cys51 is stabilized and shielded from solvent by forming a salt bridge with Arg127 such that it is impervious to further oxidation to sulfinic acid.11.Schroder E. Littlechild J.A. Lebedev A.A. et al.Crystal structure of decameric 2-Cys peroxiredoxin from human erythrocytes at 1.7 A resolution.Structure. 2000; 8: 605-615Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar However, parts of the Prx protein must undergo major rearrangements in order to accommodate disulfide formation between Cys51-SOH and Cys172. These conformational changes that necessarily accompany the catalytic cycle are expected to deprive the sulfenic acid moiety of shielding and stabilization, increasing its susceptibility to further oxidation. We also showed that sulfinic acid cannot be reduced in vitro by exogenous Trx or dithiothreitol. Thus, it appeared that the catalytic cycle of PrxI allows the occasional hyperoxidation of the Cys51-SOH intermediate to Cys51-SO2H, resulting in enzyme inactivation that cannot be reversed by Trx. Woo et al. investigated the fate of overoxidized (sulfinylated) Prx I using Raw 264.7 mouse macrophage cells that had been metabolically labeled with 35S. The intensity of the 35S-labeled acidic spots corresponding to sulfinylated Prx I and II on two-dimensional gels increased during exposure of cells to H2O2, and then decreased as the intensity of the spots corresponding to the respective reduced forms of the enzymes increased after removal of H2O2 in the presence of the protein synthesis inhibitor cycloheximide.18.Woo H.A. Chae H.Z. Hwang S.C. et al.Reversing the inactivation of peroxiredoxins caused by cysteine sulfinic acid formation.Science. 2003; 300: 653-656Crossref PubMed Scopus (468) Google Scholar This observation led us to propose that the sulfinylation reaction is actually reversible in cells.18.Woo H.A. Chae H.Z. Hwang S.C. et al.Reversing the inactivation of peroxiredoxins caused by cysteine sulfinic acid formation.Science. 2003; 300: 653-656Crossref PubMed Scopus (468) Google Scholar,19.Woo H.A. Kang S.W. Kim H.K. et al.Reversible oxidation of the active site cysteine of peroxiredoxins to cysteine sulfinic acid. Immunoblot detection with antibodies specific for the hyperoxidized cysteine-containing sequence.J Biol Chem. 2003; 278: 47361-47364Crossref PubMed Scopus (203) Google Scholar Until this discovery, it has been thought that hyperoxidiation to sulfinic acid is irreversible process in cells. The enzyme responsible for the reduction of sulfinylated, sulfiredoxin (Srx), was subsequently identified in yeast by Biteau et al.20.Biteau B. Labarre J. Toledano M.B. ATP-dependent reduction of cysteine-sulphinic acid by S. cerevisiae sulphiredoxin.Nature. 2003; 425: 980-984Crossref PubMed Scopus (797) Google Scholar on the basis of the observations that its expression was induced by H2O2 and that deletion of its gene resulted in a reduced tolerance to H2O2. Srx defines a protein family of lower and higher eukaryotes whose members possess a conserved cysteine residue. The fact that Srx is conserved only among eukaryotes is consistent with the observation that prokaryotic Prx enzymes are not susceptible to oxidative inactivation. Studies with the yeast enzyme showed that the reduction of Prx by Srx requires the conserved cysteine of Srx, ATP hydrolysis, Mg2+, and a thiol as an electron donor.20.Biteau B. Labarre J. Toledano M.B. ATP-dependent reduction of cysteine-sulphinic acid by S. cerevisiae sulphiredoxin.Nature. 2003; 425: 980-984Crossref PubMed Scopus (797) Google Scholar Srx has significant sequence and structural similarity to a functionally unrelated protein, ParB, a DNA-binding protein with a helix-turn-helix domain, which is involved in chromosome partitioning in bacteria.21.Basu M.K. Koonin E.V. Evolution of eukaryotic cysteine sulfinic acid reductase, sulfiredoxin (Srx), from bacterial chromosome partitioning protein ParB.Cell Cycle. 2005; 4: 947-952Crossref PubMed Scopus (26) Google Scholar Sequence comparison and phylogenetic analysis of the Srx and ParB protein families suggest that Srx evolved via truncation of ParB, which removed the entire C-terminal half of the protein and a substitution of cysteine for a glutamic acid in a highly conserved structural motif of ParB. The latter substitution apparently created the sulfinic acid reductase catalytic site. The conserved motif around the catalytic cysteine of Srx is particularly remarkable, in that it contains three strictly conserved residues. Evolution of a redox enzyme from a DNA-binding protein, with retention of highly significant sequence similarity, is unusual because it involved a radical change not only of the biological function but also of the biochemical activity of the protein from a sequence-specific DNA-binding protein to a redox enzyme. Chang et al.22.Chang T.S. Jeong W. Woo H.A. et al.Characterization of mammalian sulfiredoxin and its reactivation of hyperoxidized peroxiredoxin through reduction of cysteine sulfinic acid in the active site to cysteine.J Biol Chem. 2004; 279: 50994-51001Crossref PubMed Scopus (306) Google Scholar characterized mammalian orthologs of yeast Srx with an assay based on monitoring of the reduction of sulfinic Prx by immunoblot analysis with antibodies specific for the sulfinic state. Sulfinic reduction by mammalian Srx was found to be a slow process (kcat=0.18/min) that requires ATP hydrolysis. ATP could be efficiently replaced by guanosine 5'-triphosphate, deoxyadenosine triphosphate, or deoxyguanosine triphosphate but not by cytidine triphosphate, uridine 5'-triphosphate, deoxycytidine triphosphate, or deoxythymidine triphosphate. Both glutathione and thioredoxin are potential physiological electron donors for the Srx reaction, given that their Km values (1.8 mM and 1.2 μM, respectively) are in the range of their intracellular concentrations, and the Vmax values obtained with the two reductants were similar. Although its pKa is relatively low (∼7.3), the active site cysteine of Srx remained reduced even when the active site cysteine of most Prx molecules became oxidized. Depletion of human Srx by RNA interference suggested that Srx is responsible for reduction of the Cys-SO2H of Prx in A549 human cells. Hyperoxidation of cysteine to sulfinic acid is not restricted to Prx enzymes.23.Woo H.A. Jeong W. Chang T.S. et al.Reduction of cysteine sulfinic acid by sulfiredoxin is specific to 2-cys peroxiredoxins.J Biol Chem. 2005; 280: 3125-3128Crossref PubMed Scopus (264) Google Scholar Critical cysteine residues of many other proteins, including glyceraldehyde-3-phosphate dehydrogenase, carbonic anhydrase III, metalloproteinases, protein tyrosine phosphatase (PTP) 1B, and the Parkinson disease-associated protein DJ-1, also undergo this modification. In contrast, reduction by Srx appears to be a highly selective process. Among the Prx isoforms, only the sulfinic forms of members of the 2-Cys Prx subgroup (Prx I–IV), not those of members of the atypical 2-Cys or 1-Cys subgroups (Prx V and VI), were found to be reduced by Srx. Moreover, Srx did not act on the sulfinic forms of glyceraldehyde-3-phosphate dehydrogenase or DJ-1 in vitro or in cells. Srx thus appears to exist solely to support the reversible sulfinic modification of specific Prx enzymes. To date, only the 2-Cys Prx isoforms have been shown to cycle between thiol and sulfinic acid states in cells. Non-enzymic reduction of sulfinic acid requires harsh reaction conditions, and the reduction of sulfinic 2-Cys Prx isoforms is the first known biological example of such a reaction. On the basis of the observation that the reduction of sulfinic Prx by yeast Srx is dependent on ATP and Mg2+, Biteau et al.20.Biteau B. Labarre J. Toledano M.B. ATP-dependent reduction of cysteine-sulphinic acid by S. cerevisiae sulphiredoxin.Nature. 2003; 425: 980-984Crossref PubMed Scopus (797) Google Scholar proposed that the first step of the Srx reaction involves phosphorylation of sulfinic acid (Prx-Cys-SO2H) to yield a sulfinic acid phosphoryl ester (Prx-Cys-S(=O)OPO32-), which is reminiscent of the activation of a carboxylic group by phosphorylation in a variety of enzyme systems. The crystal structure of ADP-bound human Srx24.Jonsson T.J. Murray M.S. Johnson L.C. et al.Structural basis for the retroreduction of inactivated peroxiredoxins by human sulfiredoxin.Biochemistry. 2005; 44: 8634-8642Crossref PubMed Scopus (48) Google Scholar and nuclear magnetic resonance structure of ATP-bound human Srx25.Lee D.Y. Park S.J. Jeong W. et al.Mutagenesis and modeling of the peroxiredoxin (Prx) complex with the NMR structure of ATP-bound human sulfiredoxin implicate aspartate 187 of Prx I as the catalytic residue in ATP hydrolysis.Biochemistry. 2006; 45: 15301-15309Crossref PubMed Scopus (20) Google Scholar have been determined. ATP binds, independently of PrxI, to a pocket of Srx, in which the phosphates of ATP interact with Lys61, His100, Gly98, Cys99, and Arg101 of hSrx as shown in Figure 2. Srx binds to 2-Cys Prx enzymes, as evidenced by their co-immunoprecipitation. Srx specifically recognizes members of the 2-Cys Prx subgroup through contacts with several of its surface-exposed amino-acid residues, including Arg51, Asp56, and Asp80 in hSrx. Although ATP binds Srx independently of Prx I, it does not catalyze ATP hydrolysis by itself and has no catalytic residues typical of most ATPase and kinase family proteins. Modeling of Prx complex with ATP-bound Srx indicates that Asp187 of Prx I (Asn186 of Prx II) is in contact with the Srx-bound ATP β- and γ-phosphate groups, and serves as the catalytic residues responsible for ATP hydrolysis in the formation of sulfinic acid phosphoryl ester (Prx-Cys-S(=O)OPO32-). The mechanism for the reduction of sulfinic Prx has been proposed (Figure 3).26.Jeong W. Park S.J. Chang T.S. et al.Molecular mechanism of the reduction of cysteine sulfinic acid of peroxiredoxin to cysteine by mammalian sulfiredoxin.J Biol Chem. 2006; 281: 14400-14407Crossref PubMed Scopus (123) Google Scholar In the mechanism, the thiol group of Cys99 deprotonated as a result of its ionic interaction with the guanidine group of Arg51, and the resulting thiolate anion transfers the γ-phosphate of ATP, probably through formation of a transient thiophosphate intermediate, to the sulfinic acid moiety of PrxI, thereby yielding a sulfinic acid phosphoryl ester. The sulfinic acid moiety is critical for the phosphotransferase reaction, because it promotes the extraction of the phosphate from ATP by the thiolate anion of Cys99 in addition to serving as the acceptor of the phosphate. The sulfinic acid phosphoryl ester is then reductively cleaved by thiols such as reduced glutathione, dithiothreitol, or Trx to produce a disulfide-S-monoxide (Prx-Cys-S(=O)-S-R, where RSH indicates the thiol donor molecule). The disulfide-S-monoxide is further reduced, after oxidation of three thiol equivalents, to Prx-Cys-SH. The proposed reaction mechanism indicates that the conserved cysteine of Srx is critical for sulfinic reductase activity as a result of its phosphate-carrier function, not because of a thioltransferase function, as proposed previously.20.Biteau B. Labarre J. Toledano M.B. ATP-dependent reduction of cysteine-sulphinic acid by S. cerevisiae sulphiredoxin.Nature. 2003; 425: 980-984Crossref PubMed Scopus (797) Google Scholar,27.Guan K.L. Dixon J.E. Evidence for protein-tyrosine-phosphatase catalysis proceeding via a cysteine-phosphate intermediate.J Biol Chem. 1991; 266: 17026-17030Abstract Full Text PDF PubMed Google Scholar It is clear that 2-Cys Prx enzymes are programmed to be inactivated during catalysis, whereas other H2O2-eliminating enzymes, such as catalases and glutathione peroxidases, are insensitive to the oxidative inactivation. Why then do the cells expend resources to produce large amounts of 2-Cys Prx enzymes that are so readily inactivated and bother to reactivate them using an energy-requiring process? Sequence analysis by Wood et al.20.Biteau B. Labarre J. Toledano M.B. ATP-dependent reduction of cysteine-sulphinic acid by S. cerevisiae sulphiredoxin.Nature. 2003; 425: 980-984Crossref PubMed Scopus (797) Google Scholar,28.Wood Z.A. Poole L.B. Karplus P.A. Peroxiredoxin evolution and the regulation of hydrogen peroxide signaling.Science. 2003; 300: 650-653Crossref PubMed Scopus (1139) Google Scholar revealed that eukaryotic 2-cys Prxs contain a Gly–Gly–Leu–Gly conserved motif at their COOH termini and that prokaryotic 2-Cys Prx enzymes, which do not contain the C-terminal GGLG tail of their eukaryotic counterparts, are insensitive to oxidative inactivation. In addition, prokaryotes do not contain Srx. In support of the role of the COOH-terminal tail, yeast 2-Cys Prx enzyme loses its insensitivity to overoxidation when its COOH-terminal tail was truncated.29.Koo K.H. Lee S. Jeong S.Y. et al.Regulation of thioredoxin peroxidase activity by C-terminal truncation.Arch Biochem Biophys. 2002; 397: 312-318Crossref PubMed Scopus (94) Google Scholar The reversible inactivation of 2-Cys Prx has therefore been suggested to be the result of structural features acquired during evolution. Besides the peroxides produced at basal levels owing to normal metabolism, cells produce H2O2 transiently in response to the activation of various cell-surface receptors. H2O2 thus produced has been increasingly recognized as an important intracellular messenger that modifies protein function through the oxidation of critical cysteine residues of many proteins including PTPs.30.Finkel T. Oxygen radicals and signaling.Curr Opin Cell Biol. 1998; 10: 248-253Crossref PubMed Scopus (1010) Google Scholar, 31.Rhee S.G. Redox signaling: hydrogen peroxide as intracellular messenger.Exp Mol Med. 1999; 31: 53-59Crossref PubMed Scopus (568) Google Scholar, 32.Rhee S.G. Bae Y.S. Lee S.R. et al.Hydrogen peroxide: a key messenger that modulates protein phosphorylation through cysteine oxidation.Sci STKE. 2000; PE1: 2000Google Scholar Tyrosine phosphorylation is governed by the opposing activities of PTPs and protein tyrosine kinases. The PTP family features a common CX5R active site motif. Owing to the invariant arginine, the conserved catalytic cysteine possesses a low pKa and exists as a thiolate anion with enhanced susceptibility to oxidation by H2O2. Oxidation of the essential cysteine abolishes PTP activity and can be reversed by cellular thiols (RSH). Reversible inactivation of several different PTPs was demonstrated in relevant cell types stimulated with platelet-derived growth factor, epidermal growth factor, insulin, extracellular matrix ligand, and B-cell antigen receptor ligand.32.Rhee S.G. Bae Y.S. Lee S.R. et al.Hydrogen peroxide: a key messenger that modulates protein phosphorylation through cysteine oxidation.Sci STKE. 2000; PE1: 2000Google Scholar,33.Tonks N.K. Redox redux: revisiting PTPs and the control of cell signaling.Cell. 2005; 121: 667-670Abstract Full Text Full Text PDF PubMed Scopus (603) Google Scholar Oxidative inactivation of PTPs and increased tyrosine phosphorylation of target proteins were dependent on H2O2 production. Moreover, in tumor necrosis factor α-stimulated cells, inactivation of mitogen-activated protein kinase phosphatases by H2O2 resulted in sustained c-Jun NH2 terminal kinase activation.34.Kamata H. Honda S. Maeda S. et al.Reactive oxygen species promote TNFalpha-induced death and sustained JNK activation by inhibiting MAP kinase phosphatases.Cell. 2005; 120: 649-661Abstract Full Text Full Text PDF PubMed Scopus (1501) Google Scholar Given that H2O2 is readily converted to the toxic hydroxyl radical in the presence of Fe and Cu ions, localized production of H2O2 only where needed for intracellular signaling would appear to be desirable, as is the destruction of H2O2 molecules that diffuse away from this site of action. The removal of H2O2 in cells is mediated predominantly by catalase, glutathione peroxidase (GPx), and Prx. Catalase is localized exclusively in peroxisomes, so that elimination of cytosolic H2O2 by catalase requires its diffusion into these organelles. The major isoform of glutathione peroxidase, glutathione peroxidase1, is largely restricted to the cytosol but is also present in mitochondria. Prx I and II, though their catalytic efficiency is less than that of glutathione peroxidase or catalase by 1–3 orders of magnitude, are abundant in the cytosol, typically constituting 0.1–0.8% of total soluble protein, and exhibit higher affinity toward H2O2 (their Km values for H2O2 are <20 μM).35.Chae H.Z. Kim H.J. Kang S.W. et al.Characterization of three isoforms of mammalian peroxiredoxin that reduce peroxides in the presence of thioredoxin.Diabetes Res Clin Pract. 1999; 45: 101-112Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar Therefore Prx I and II are prime candidates for regulators of H2O2 signaling initiated by cell-surface receptors. Indeed, when overexpressed or partially depleted using RNAi methodology in various cells, Prx enzymes affected the intracellular level of H2O2 produced in the cells stimulated with platelet-derived growth factor or tumor necrosis factor-α, and modulated signaling induced by those ligands, indicating that Prx enzymes serve as component of signaling cascades by removing H2O2.36.Jin D.Y. Chae H.Z. Rhee S.G. et al.Regulatory role for a novel human thioredoxin peroxidase in NF-kappaB activation.J Biol Chem. 1997; 272: 30952-30961Crossref PubMed Scopus (387) Google Scholar, 37.Kang S.W. Chae H.Z. Seo M.S. et al.Mammalian peroxiredoxin isoforms can reduce hydrogen peroxide generated in response to growth factors and tumor necrosis factor-alpha.J Biol Chem. 1998; 273: 6297-6302Crossref PubMed Scopus (616) Google Scholar, 38.Zhang P. Liu B. Kang S.W. et al.Thioredoxin peroxidase is a novel inhibitor of apoptosis with a mechanism distinct from that of Bcl-2.J Biol Chem. 1997; 272: 30615-30618Crossref PubMed Scopus (336) Google Scholar, 39.Kwon J. Lee S.R. Yang K.S. et al.Reversible oxidation and inactivation of the tumor suppressor PTEN in cells stimulated with peptide growth factors.Proc Natl Acad Sci USA. 2004; 101: 16419-16424Crossref PubMed Scopus (515) Google Scholar, 40.Veal E.A. Findlay V.J. Day A.M. et al.A 2-Cys peroxiredoxin regulates peroxide-induced oxidation and activation of a stress-activated MAP kinase.Mol Cell. 2004; 15: 129-139Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar The messenger function of H2O2 probably requires that its concentration increase rapidly above a certain threshold, a requirement that is likely met through protection of the generated H2O2 molecules from destruction by cytosolic Prx isoforms. The built-in susceptibility of 2-Cys Prxs might represent such signaling function. However, experimental validation of such speculation is yet to come. Unlike in mammalian cells, the signaling function of sulfinylation cycle is better understood in lower eukaryotes. Recent studies suggest that in Schizosaccharomyces pombe, the reversible cysteine overoxidation of the 2-Cys Prx, Tpx1, represents a redox switch that allows the yeast to induce distinct transcriptional responses to low and high levels of H2O2.41.Bozonet S.M. Findlay V.J. Day A.M. et al.Oxidation of a eukaryotic 2-Cys peroxiredoxin is a molecular switch controlling the transcriptional response to increasing levels of hydrogen peroxide.J Biol Chem. 2005; 280: 23319-23327Crossref PubMed Scopus (130) Google Scholar,42.Vivancos A.P. Castillo E.A. Biteau B. et al.A cysteine-sulfinic acid in peroxiredoxin regulates H2O2-sensing by the antioxidant Pap1 pathway.Proc Natl Acad Sci USA. 2005; 102: 8875-8880Crossref PubMed Scopus (197) Google Scholar In S. pombe, the transcriptional response of cells to low levels of H2O2 is dependent on the transcriptional factor Pap1 (homologue of mammalian c-Jun), whereas the activation of Atf1 transcriptional factor via sty1 (homologue of mammalian mitogen-activated protein kinase) plays a more important role at higher levels of H2O2.43.Quinn J. Findlay V.J. Dawson K. et al.Distinct regulatory proteins control the graded transcriptional response to increasing H(2)O(2) levels in fission yeast Schizosaccharomyces pombe.Mol Biol Cell. 2002; 13: 805-816Crossref PubMed Scopus (167) Google Scholar In response to H2O2, Pap1 forms an intramolecular disulfide bond between Cys501 and Cys523, which masks the accessibility of the nuclear exporter Crm1 to the C-terminal nuclear export signal, and results in accumulation of Pap1 in the nucleus and in Pap1-dependent gene expression.44.Castillo E.A. Ayte J. Chiva C. et al.Diethylmaleate activates the transcription factor Pap1 by covalent modification of critical cysteine residues.Mol Microbiol. 2002; 45: 243-254Crossref PubMed Scopus (78) Google Scholar,45.Vivancos A.P. Castillo E.A. Jones N. et al.Activation of the redox sensor Pap1 by hydrogen peroxide requires modulation of the intracellular oxidant concentration.Mol Microbiol. 2004; 52: 1427-1435Crossref PubMed Scopus (93) Google Scholar As the H2O2-induced oxidation of Pap1 is initiated by forming an intermolecular disulfide between the NH2-terminal catalytic cysteine, Cys 48, and Pap1 Cys501 or Cys523, overoxidized TPx1 cannot carry out the TPx1-Pap1 redox relay. Accordingly, at high concentrations of H2O2, Pap1 activation is prevented as the result of the oxidation of TPx1 cysteine to cysteine sulfinic acid, and instead the sty1–Atf1 pathway is triggered, which is essential for the expression of Srx in response to high levels of H2O2. The expression of Srx leads to the reactivation of Tpx1 and subsequently reduction of H2O2 levels, allowing TPx1 to relay redox signal to Pap1. Hence, the reversible sulfinylation of TPx1 acts as a redox switch determining the balance of activation of two independent transcriptional factors Pap1 and Atf1. A novel molecular chaperone function, which is independent of peroxidase activity, has been proposed for yeast cytosolic Prx (cPrx) and mammalian Prx II.46.Jang H.H. Lee K.O. Chi Y.H. et al.Two enzymes in one; two yeast peroxiredoxins display oxidative stress-dependent switching from a peroxidase to a molecular chaperone function.Cell. 2004; 117: 625-635Abstract Full Text Full Text PDF PubMed Scopus (609) Google Scholar,47.Moon J.C. Hah Y.S. Kim W.Y. et al.Oxidative stress-dependent structural and functional switching of a human 2-Cys peroxiredoxin isotype II that enhances HeLa cell resistance to H2O2-induced cell death.J Biol Chem. 2005; 280: 28775-28784Crossref PubMed Scopus (247) Google Scholar This chaperone activity appears to correlate with the oligomerization status of the enzyme. The 2-Cys Prx enzymes are obligate homodimers containing two identical active sites and form high-molecular-weight toroid structures comprises five dimers linked together by hydrophobic interactions. The decameric form appears to aggregate further to form higher-molecular-weight complexes. A critical factor determining the dimer–decamer equilibrium is the redox state of the catalytic cysteine, with the sulfinic enzymes favoring decameric forms and the disulfide enzymes existing mainly as dimers. At low concentrations of H2O2 generated under normal conditions, 2-Cys Prxs form predominantly low-molecular-weight oligomeric protein structures.46.Jang H.H. Lee K.O. Chi Y.H. et al.Two enzymes in one; two yeast peroxiredoxins display oxidative stress-dependent switching from a peroxidase to a molecular chaperone function.Cell. 2004; 117: 625-635Abstract Full Text Full Text PDF PubMed Scopus (609) Google Scholar Prxs in these forms possess dual activities: in addition to removing low levels of H2O2, they also appear to protect proteins from denaturation. However, under conditions of oxidative stress, the 2-Cys Prxs rapidly undergo structural changes such that the low-molecular-weight forms are converted into high-molecular-weight complexes, which act as a superchaperone. In the presence of Srx1, which can reduce Cys-sulfinic acid to Cys-thiol protein, the dissociation of the high-molecular-weight complexes into low-molecular-weight species occurs on removal of H2O2. This change turns on the peroxidase function of Prx and lowers the chaperone activity to the basal level. These results suggest that, in addition to the signaling function, the reversible sulfinylation of 2-Cys Prx enzymes regulate the chaperone function of Prx enzymes (Figure 4). Considerable in vivo evidence supports the notion that the chaperone function of 2-Cys Prxs is functionally important in protecting cells from oxidative stress. For example, in yeast cPrxI significantly prevented oxygen radical-mediated denaturation and aggregation of α-synuclein, which is a key component of Lewy bodies found in the brains of patients with Parkinson's disease and Alzheimer's disease.48.Kim K.S. Choi S.Y. Kwon H.Y. et al.Aggregation of alpha-synuclein induced by the Cu,Zn-superoxide dismutase and hydrogen peroxide system.Free Radic Biol Med. 2002; 32: 544-550Crossref PubMed Scopus (56) Google Scholar Apart from the physiological stress-resistance endowed by 2-Cys Prxs, a large number of papers have reported that the expression of 2-Cys Prxs is closely associated with a variety of human diseases, such as neurodegenerative disorders, Alzheimer's disease, Down syndrome, thyroid tumors, breast cancer, lung cancer, and tumor protection.28.Wood Z.A. Poole L.B. Karplus P.A. Peroxiredoxin evolution and the regulation of hydrogen peroxide signaling.Science. 2003; 300: 650-653Crossref PubMed Scopus (1139) Google Scholar In particular, transgenic mice lacking PrxI have a shortened lifespan because they develop hemolytic anemia and several malignant cancers.49.Neumann C.A. Krause D.S. Carman C.V. et al.Essential role for the peroxiredoxin Prdx1 in erythrocyte antioxidant defence and tumour suppression.Nature. 2003; 424: 561-565Crossref PubMed Scopus (635) Google Scholar These results strongly suggest that the 2-Cys Prxs, like the sHSPs, may help to protect host cells against various diseases. In conclusion, the dual functions of 2-Cys Prxs in modulating H2O2 concentration and preventing protein aggregation may play pivotal roles in cellular response to pathogens and external stresses. Sue Goo Rhee serves as a member of a paid advisory board to Lab Frontier, which produces antibodies, and is supported by Korean Government Grant FPR-0502. The other authors have stated they have nothing to disclose. This work was supported by Grant FPR0502-470 of the 21C Frontier Proteomics Projects from Korean Ministry of Science and Technology.