Peroxiredoxin Functions as a Peroxidase and a Regulator and Sensor of Local Peroxides
2011; Elsevier BV; Volume: 287; Issue: 7 Linguagem: Inglês
10.1074/jbc.r111.283432
ISSN1083-351X
AutoresSue Goo Rhee, Hyun Ae Woo, In Sup Kil, Soo Han Bae,
Tópico(s)Glutathione Transferases and Polymorphisms
ResumoPeroxiredoxins (Prxs) contain an active site cysteine that is sensitive to oxidation by H2O2. Mammalian cells express six Prx isoforms that are localized to various cellular compartments. The oxidized active site cysteine of Prx can be reduced by a cellular thiol, thus enabling Prx to function as a locally constrained peroxidase. Regulation of Prx via phosphorylation in response to extracellular signals allows the local accumulation of H2O2 and thereby enables its messenger function. The fact that the oxidation state of the active site cysteine of Prx can be transferred to other proteins that are less intrinsically susceptible to H2O2 also allows Prx to function as an H2O2 sensor. Peroxiredoxins (Prxs) contain an active site cysteine that is sensitive to oxidation by H2O2. Mammalian cells express six Prx isoforms that are localized to various cellular compartments. The oxidized active site cysteine of Prx can be reduced by a cellular thiol, thus enabling Prx to function as a locally constrained peroxidase. Regulation of Prx via phosphorylation in response to extracellular signals allows the local accumulation of H2O2 and thereby enables its messenger function. The fact that the oxidation state of the active site cysteine of Prx can be transferred to other proteins that are less intrinsically susceptible to H2O2 also allows Prx to function as an H2O2 sensor. IntroductionAn unusual antioxidant protein, now called peroxiredoxin (Prx), 2The abbreviations used are: PrxperoxiredoxinROSreactive oxygen speciesTrxthioredoxinCPperoxidatic CysCRresolving CysERendoplasmic reticulumSrxsulfiredoxinKOknock-outPTPprotein-tyrosine phosphatasePTKprotein-tyrosine kinaseNoxNADPH oxidasePDIprotein-disulfide isomerasePLA2phospholipase A2. was initially identified on the basis of its capacity to protect proteins from oxidative damage induced by reactive oxygen species (ROS) produced in the presence of DTT. It was named “protector protein” or “thiol-specific antioxidant” before being renamed Prx (1Kim I.H. Kim K. Rhee S.G. Induction of an antioxidant protein of Saccharomyces cerevisiae by O2, Fe3+, or 2-mercaptoethanol.Proc. Natl. Acad. Sci. U.S.A. 1989; 86: 6018-6022Crossref PubMed Scopus (131) Google Scholar, 2Kim K. Kim I.H. Lee K.Y. Rhee S.G. Stadtman E.R. 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Redox Signal. 2011; 15: 781-794Crossref PubMed Scopus (324) Google Scholar). The ROS were generated as the result of the reduction of molecular oxygen by DTT to superoxide and H2O2, which were further reduced to hydroxyl radicals in the presence of trace amounts of contaminating metal (iron or copper) ions (6Kim K. Rhee S.G. Stadtman E.R. Nonenzymatic cleavage of proteins by reactive oxygen species generated by dithiothreitol and iron.J. Biol. Chem. 1985; 260: 15394-15397Abstract Full Text PDF PubMed Google Scholar). Analysis of purified yeast Prx revealed that it did not contain conventional redox centers such as metals, heme, flavin, or selenocysteine. Prx therefore did not resemble any antioxidant known at the time. It was subsequently found that (i) Prx is present in all biological kingdoms from bacteria to mammals; (ii) two cysteine residues, corresponding to Cys47 and Cys170 of yeast Prx, are highly conserved among Prx family members; (iii) Prxs are homodimers arranged in a head-to-tail orientation; and (iv) Cys47–SH of Prx is specifically oxidized by H2O2 to cysteine sulfenic acid (Cys–SOH), which is resolved by reaction with Cys170–SH of the adjacent monomer, resulting in the formation of a disulfide linkage, Cys47–S-S–Cys170 (7Chae H.Z. Uhm T.B. Rhee S.G. Dimerization of thiol-specific antioxidant and the essential role of cysteine 47.Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 7022-7026Crossref PubMed Scopus (280) Google Scholar). The antioxidant function of Prx was attributed to the fact that the disulfide could be reduced by DTT, resulting in completion of a catalytic cycle. Thioredoxin (Trx) was eventually shown to be the biological donor of reducing equivalents for the catalytic function of Prx (8Chae 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), with peroxynitrite (ONOO−) in addition to H2O2 and lipid peroxides also being found to be reduced by Prx enzymes (9Bryk R. Griffin P. Nathan C. Peroxynitrite reductase activity of bacterial peroxiredoxins.Nature. 2000; 407: 211-215Crossref PubMed Scopus (563) Google Scholar). The conserved Cys residue corresponding to Cys47 of yeast Prx was later referred to as the peroxidatic Cys (CP) to reflect its sensitivity to oxidation by peroxides, and the conserved Cys residue corresponding to Cys170 was designated the resolving Cys (CR) (10Wood Z.A. Poole L.B. Karplus P.A. Peroxiredoxin evolution and the regulation of hydrogen peroxide signaling.Science. 2003; 300: 650-653Crossref PubMed Scopus (1129) Google Scholar).Given that H2O2 reacts with the thiolate (deprotonated) form of cysteine, oxidation of cysteine is enhanced by a microenvironment that lowers the normally high pKa (∼8.6) of cysteine thiols to a value below neutral pH. The pKa values of the CP residue of members of the Prx family are in the range of 5–6 (11Hugo M. Turell L. Manta B. Botti H. Monteiro G. Netto L.E. Alvarez B. Radi R. Trujillo M. Thiol and sulfenic acid oxidation of AhpE, the one-cysteine peroxiredoxin from Mycobacterium tuberculosis: kinetics, acidity constants, and conformational dynamics.Biochemistry. 2009; 48: 9416-9426Crossref PubMed Scopus (92) Google Scholar, 12Nelson K.J. Parsonage D. Hall A. Karplus P.A. Poole L.B. Cysteine pKa values for the bacterial peroxiredoxin AhpC.Biochemistry. 2008; 47: 12860-12868Crossref PubMed Scopus (89) Google Scholar, 13Ogusucu R. 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The low pKa alone thus appears to be insufficient to account for the high reactivity of Prx with peroxide. The unusually high activity was recently attributed to the fact that Prx enzymes activate not only the thiolate of CP but also the peroxide substrate via a hydrogen bonding network formed by four amino acids (Pro, Thr, Arg, and Glu, Gln, or His) conserved in the active site of all Prx enzymes (18Hall A. Nelson K. Poole L.B. Karplus P.A. Structure-based insights into the catalytic power and conformational dexterity of peroxiredoxins.Antioxid. Redox Signal. 2011; 15: 795-815Crossref PubMed Scopus (241) Google Scholar).Mechanism of Peroxidase ReactionOn the basis of the location or absence of the CR residue, Prxs are classified into 2-Cys, atypical 2-Cys, and 1-Cys Prx subfamilies (4Chae H.Z. Robison K. Poole L.B. Church G. Storz G. Rhee S.G. 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. U.S.A. 1994; 91: 7017-7021Crossref PubMed Scopus (696) Google Scholar, 5Rhee S.G. Woo H.A. Multiple functions of peroxiredoxins: peroxidases, sensors and regulators of the intracellular messenger H2O2, and protein chaperones.Antioxid. Redox Signal. 2011; 15: 781-794Crossref PubMed Scopus (324) Google Scholar, 19Rhee 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 (1131) Google Scholar). Mammalian cells express six isoforms of Prx: four 2-Cys Prx isoforms (Prxs I–IV), one atypical 2-Cys Prx isoform (Prx V), and one 1-Cys Prx isoform (Prx VI). These isoforms vary in subcellular localization, with Prx I, II, and VI being localized mainly in the cytosol; Prx III being restricted to mitochondria; Prx IV being found predominantly in the endoplasmic reticulum (ER); and Prx V being present in the cytosol, mitochondria, and peroxisomes. As discussed below, however, Prx localization is multifarious, being dependent on the cell type and environment.The peroxidase reaction mechanisms of mammalian Prx enzymes are shown in Fig. 1. As demonstrated first with yeast Prx, the conserved CP–SH of 2-Cys Prx is selectively oxidized by H2O2 to the CP–SOH intermediate. An intermolecular disulfide is then formed with CR–SH and is ultimately reduced by Trx (Fig. 1A). Eukaryotic members of the 2-Cys Prx subgroup are distinct from most prokaryotic members in that they contain two structural motifs (GGLG and YF) whose interaction restricts the ability of CP–SOH to approach CR–SH and thereby hinders disulfide formation (10Wood Z.A. Poole L.B. 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For Prx V, the resulting CP–SOH then reacts with CR–SH of the same subunit to form an intramolecular disulfide linkage, which is also reduced by Trx as in 2-Cys Prx (Fig. 1B). In the catalytic cycle of Prx VI, Cys–SOH does not form a disulfide because of the unavailability of another Cys–SH nearby. CP–SOH of oxidized Prx VI is reduced by GSH in the presence of the Pi isoform of GST but not by Trx or glutaredoxin (29Choi H.J. Kang S.W. Yang C.H. Rhee S.G. Ryu S.E. Crystal structure of a novel human peroxidase enzyme at 2.0 Å resolution.Nat. Struct. Biol. 1998; 5: 400-406Crossref PubMed Scopus (328) Google Scholar, 30Montemartini M. Kalisz H.M. Hecht H.J. Steinert P. Flohé L. Activation of active-site cysteine residues in the peroxiredoxin-type tryparedoxin peroxidase of Crithidia fasciculata.Eur. J. Biochem. 1999; 264: 516-524Crossref PubMed Scopus (66) Google Scholar, 34Fisher A.B. 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Chem. 2005; 280: 3125-3128Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar).Reduction of Locally Produced Peroxides by Concerted Action of 2-Cys Prx and SrxThe role of Prx enzymes as peroxidases has been demonstrated by increasing or decreasing their expression levels in various cell lines and evaluating the sensitivity of the cells to oxidative insults. Prx-deficient mice have also been generated by targeting the gene for each isoform. These mice develop to adulthood with no overt phenotype when maintained under normal laboratory conditions. Prx II knock-out (KO) mice subsequently develop hemolytic anemia (38Lee T.H. Kim S.U. Yu S.L. Kim S.H. Park D.S. Moon H.B. Dho S.H. Kwon K.S. Kwon H.J. Han Y.H. Jeong S. Kang S.W. Shin H.S. Lee K.K. Rhee S.G. Yu D.Y. Peroxiredoxin II is essential for sustaining life span of erythrocytes in mice.Blood. 2003; 101: 5033-5038Crossref PubMed Scopus (326) Google Scholar), and ablation of Prx I resulted in spontaneous tumor formation in aging mice in one study (39Neumann C.A. Krause D.S. Carman C.V. Das S. Dubey D.P. Abraham J.L. Bronson R.T. Fujiwara Y. Orkin S.H. Van Etten R.A. Essential role for the peroxiredoxin Prdx1 in erythrocyte antioxidant defense and tumor suppression.Nature. 2003; 424: 561-565Crossref PubMed Scopus (628) Google Scholar). These phenotypes of Prx KO mice may not be related to the peroxidase activity of the targeted protein, however. As described below, certain Prx enzymes also function as local regulators of H2O2, which serves as an intracellular messenger. The tumor suppressor function of Prx I was attributed to such regulation of H2O2 (40Cao J. Schulte J. Knight A. Leslie N.R. Zagozdzon A. Bronson R. Manevich Y. Beeson C. Neumann C.A. Prdx1 inhibits tumorigenesis via regulating PTEN/AKT activity.EMBO J. 2009; 28: 1505-1517Crossref PubMed Scopus (270) Google Scholar, 41Woo H.A. Yim S.H. Shin D.H. Kang D. Yu D.Y. Rhee S.G. Inactivation of peroxiredoxin I by phosphorylation allows localized H2O2 accumulation for cell signaling.Cell. 2010; 140: 517-528Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar).An in vivo antioxidant function of Prx I was recently demonstrated in the livers of ethanol-fed mice (42Bae S.H. Sung S.H. Cho E.J. Lee S.K. Lee H.E. Woo H.A. Yu D.Y. Kil I.S. Rhee S.G. Concerted action of sulfiredoxin and peroxiredoxin I protects against alcohol-induced oxidative injury in mouse liver.Hepatology. 2011; 53: 945-953Crossref PubMed Scopus (65) Google Scholar). The production of ROS and oxidative stress play a central role in the pathogenesis of alcoholic liver disease. Ethanol consumption induces the accumulation of CYP2E1 (cytochrome P450 2E1), a major contributor to ethanol-induced ROS production in the liver. CYP2E1 catalyzes the oxidation of a wide variety of low molecular weight compounds, including ethanol (43Song B.J. Ethanol-inducible cytochrome P450 (CYP2E1): biochemistry, molecular biology, and clinical relevance: 1996 update.Alcohol. Clin. Exp. Res. 1996; 20: 138A-146ACrossref PubMed Scopus (141) Google Scholar). Elevated CYP2E1 levels alone result in oxidative stress because electron transfer from the donor system (NADPH and NADPH-dependent cytochrome P450 reductase) to CYP2E1 is not perfectly coupled and is therefore leaky (44Rashba-Step J. Turro N.J. Cederbaum A.I. Increased NADPH- and NADH-dependent production of superoxide and hydroxyl radical by microsomes after chronic ethanol treatment.Arch. Biochem. Biophys. 1993; 300: 401-408Crossref PubMed Scopus (134) Google Scholar). Such leaked electrons react with O2 to produce superoxide, which is converted to H2O2 or reacts with nitric oxide to produce peroxynitrite. Ethanol feeding in mice was found to markedly increase the abundance of Srx protein and mRNA in the liver. However, it had no significant effect on the hepatic abundance of the six Prx isoforms. Analysis of Nrf2 KO mice indicated that the ethanol-induced up-regulation of Srx expression is mediated mainly via a pathway dependent on the transcription factor Nrf2 (42Bae S.H. Sung S.H. Cho E.J. Lee S.K. Lee H.E. Woo H.A. Yu D.Y. Kil I.S. Rhee S.G. Concerted action of sulfiredoxin and peroxiredoxin I protects against alcohol-induced oxidative injury in mouse liver.Hepatology. 2011; 53: 945-953Crossref PubMed Scopus (65) Google Scholar).During elimination of peroxides, all 2-Cys Prxs undergo unavoidable hyperoxidation. Among Prxs I-IV, however, only Prx I was found to be hyperoxidized (to a moderate extent) in the livers of ethanol-fed mice, with this effect being markedly enhanced in Srx KO mice (Fig. 2) (42Bae S.H. Sung S.H. Cho E.J. Lee S.K. Lee H.E. Woo H.A. Yu D.Y. Kil I.S. Rhee S.G. Concerted action of sulfiredoxin and peroxiredoxin I protects against alcohol-induced oxidative injury in mouse liver.Hepatology. 2011; 53: 945-953Crossref PubMed Scopus (65) Google Scholar). These observations suggested that Prx I is the most active 2-Cys Prx in elimination of ROS in the livers of ethanol-fed mice and that, despite the elevated expression of Srx in such mice, the capacity of Srx is not sufficient to counteract the hyperoxidation of Prx I that occurs during ROS reduction. The preferential hyperoxidation of Prx I also occurs despite the fact that both Prxs I and II are cytosolic proteins and that Prx II is more prone to hyperoxidation than is Prx I in most cell types (41Woo H.A. Yim S.H. Shin D.H. Kang D. Yu D.Y. Rhee S.G. Inactivation of peroxiredoxin I by phosphorylation allows localized H2O2 accumulation for cell signaling.Cell. 2010; 140: 517-528Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar, 45Seo J.H. Lim J.C. Lee D.Y. Kim K.S. Piszczek G. Nam H.W. Kim Y.S. Ahn T. Yun C.H. Kim K. Chock P.B. Chae H.Z. Novel protective mechanism against irreversible hyperoxidation of peroxiredoxin. Nα-terminal acetylation of human peroxiredoxin II.J. Biol. Chem. 2009; 284: 13455-13465Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). A protease protection assay showed that a large fraction of Prx I, but not of Prx II, is located at the cytoplasmic side of the ER membrane, where CYP2E1 is embedded (42Bae S.H. Sung S.H. Cho E.J. Lee S.K. Lee H.E. Woo H.A. Yu D.Y. Kil I.S. Rhee S.G. Concerted action of sulfiredoxin and peroxiredoxin I protects against alcohol-induced oxidative injury in mouse liver.Hepatology. 2011; 53: 945-953Crossref PubMed Scopus (65) Google Scholar). The selective burden of ROS removal borne by Prx I is thus likely attributable to its proximity to the ROS-generating CYP2E1 (Fig. 2). In addition to inducing the expression of Srx in the liver, ethanol feeding elicited the translocation of some Srx molecules to microsomes. However, the capacity of Srx located near the surface of the ER was not sufficient to fully counteract the hyperoxidation of Prx I, with consequent accumulation of a small amount of Prx I–SO2. Chronic ethanol feeding in Srx KO mice resulted in hyperoxidation of 30–50% of total Prx I, which likely represents virtually all ER-bound Prx I molecules (42Bae S.H. Sung S.H. Cho E.J. Lee S.K. Lee H.E. Woo H.A. Yu D.Y. Kil I.S. Rhee S.G. Concerted action of sulfiredoxin and peroxiredoxin I protects against alcohol-induced oxidative injury in mouse liver.Hepatology. 2011; 53: 945-953Crossref PubMed Scopus (65) Google Scholar).FIGURE 2Antioxidant roles of Prx I, Prx III, and Srx in ethanol-fed mouse liver. EtOH feeding increases the abundance of CYP2E1 in the ER. A large proportion of Prx I is present at the cytosolic side of the ER membrane, where CYP2E1 is located. Prx I is therefore preferentially engaged in the reduction of ROS produced by CYP2E1 and becomes hyperoxidized. Reactivation of such hyperoxidized Prx I requires the action of Srx, the expression of which is greatly increased via an Nrf2- and antioxidant regulatory element (ARE)-dependent pathway in the livers of ethanol-fed mice. Ethanol feeding also increases the abundance of CYP2E1 and the production of ROS in mitochondria, likely resulting in hyperoxidation of Prx III that is reversed in part by translocation of Srx from the cytosol into mitochondria.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Ethanol feeding also increases the abundance of CYP2E1 and the production of ROS in mitochondria (46Cederbaum A.I. Lu Y. Wu D. Role of oxidative stress in alcohol-induced liver injury.Arch. Toxicol. 2009; 83: 519-548Crossref PubMed Scopus (457) Google Scholar), likely resulting in the hyperoxidation of Prx III, a member of the 2-Cys Prx subfamily that is specifically localized to mitochondria (Fig. 2). However, Prx III–SO2 was not detected in the livers of ethanol-fed wild-type mice, probably because increased oxidative stress resulted in the translocation of Srx into mitochondria (47Noh Y.H. Baek J.Y. Jeong W. Rhee S.G. Chang T.S. Sulfiredoxin translocation into mitochondria plays a crucial role in reducing hyperoxidized peroxiredoxin III.J. Biol. Chem. 2009; 284: 8470-8477Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), and the capacity of mitochondrial Srx was then sufficient to counteract the hyperoxidation of Prx III. A small proportion of Prx III was hyperoxidized in the livers of ethanol-fed Srx-deficient mice, suggesting that Prx III is vulnerable to hyperoxidation by ethanol-induced ROS and that Prx III–SO2 accumulates only in the absence of Srx. Mammalian cells also contain a mitochondrion-specific Trx system (48Lee S.R. Kim J.R. Kwon K.S. Yoon H.W. Levine R.L. Ginsburg A. Rhee S.G. Molecular cloning and characterization of a mitochondrial selenocysteine-containing thioredoxin reductase from rat liver.J. Biol. Chem. 1999; 274: 4722-4734Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar), suggesting that Prx III together with mitochondrial Trx might provide a primary line of defense against H2O2 produced by the mitochondrial respiratory chain.Ethanol-induced oxidative damage in the liver includes protein modification such as carbonylation, addition of 4-hydroxynonenal, and nitration of tyrosine to yield 3-nitrotyrosine (49Dey A. Cederbaum A.I. Alcohol and oxidative liver injury.Hepatology. 2006; 43: S63-S74Crossref PubMed Scopus (476) Google Scholar). The pivotal roles of Srx and Prx I in protection of the liver against ethanol-induced oxidative stress were apparent in mice deficient in Srx or Prx I. Subjection of such mice to chronic ethanol feeding thus resulted in more severe oxidative damage to the liver, as revealed by protein carbonylation and by the formation of 4-hydroxynonenal and 3-nitrotyrosine adducts, compared with that observed in ethanol-fed wild-type mice (42Bae S.H. Sung S.H. Cho E.J. Lee S.K. Lee H.E. Woo H.A. Yu D.Y. Kil I.S. Rhee S.G. Concerted action of sulfiredoxin and peroxiredoxin I protects against alcohol-induced oxidative injury in mouse liver.Hepatology. 2011; 53: 945-953Crossref PubMed Scopus (65) Google Scholar).Regulation by Prx I of H2O2 Produced Locally at Plasma MembraneMany mammalian cell types produce H2O2 for the purpose of intracellular signaling in response to
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