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• W2008861915 abstract "Peroxiredoxins (Prxs) are a group of peroxidases containing a cysteine thiol at their catalytic site. During peroxidase catalysis, the catalytic cysteine, referred to as the peroxidatic cysteine (CP), cycles between thiol (CP-SH) and disulfide (–S–S–) states via a sulfenic (CP-SOH) intermediate. Hyperoxidation of the CP thiol to its sulfinic (CP-SO2H) derivative has been shown to be reversible, but its sulfonic (CP-SO3H) derivative is irreversible. Our comparative study of hyperoxidation and regeneration of Prx I and Prx II in HeLa cells revealed that Prx II is more susceptible than Prx I to hyperoxidation and that the majority of the hyperoxidized Prx II formation is reversible. However, the hyperoxidized Prx I showed much less reversibility because of the formation of its irreversible sulfonic derivative, as verified with CP-SO3H-specific antiserum. In an attempt to identify the multiple hyperoxidized spots of the Prx I on two-dimensional PAGE analysis, an N-acetylated Prx I was identified as part of the total Prx I using anti-acetylated Lys antibody. Using peptidyl-Asp metalloendopeptidase (EC 3.4.24.33) peptide fingerprints, we found that Nα-terminal acetylation (Nα-Ac) occurred exclusively on Prx II after demethionylation. Nα-Ac of Prx II blocks Prx II from irreversible hyperoxidation without altering its affinity for hydrogen peroxide. A comparative study of non-Nα-acetylated and Nα-terminal acetylated Prx II revealed that Nα-Ac of Prx II induces a significant shift in the circular dichroism spectrum and elevation of Tm from 59.6 to 70.9 °C. These findings suggest that the structural maintenance of Prx II by Nα-Ac may be responsible for preventing its hyperoxidation to form CP-SO3H. Peroxiredoxins (Prxs) are a group of peroxidases containing a cysteine thiol at their catalytic site. During peroxidase catalysis, the catalytic cysteine, referred to as the peroxidatic cysteine (CP), cycles between thiol (CP-SH) and disulfide (–S–S–) states via a sulfenic (CP-SOH) intermediate. Hyperoxidation of the CP thiol to its sulfinic (CP-SO2H) derivative has been shown to be reversible, but its sulfonic (CP-SO3H) derivative is irreversible. Our comparative study of hyperoxidation and regeneration of Prx I and Prx II in HeLa cells revealed that Prx II is more susceptible than Prx I to hyperoxidation and that the majority of the hyperoxidized Prx II formation is reversible. However, the hyperoxidized Prx I showed much less reversibility because of the formation of its irreversible sulfonic derivative, as verified with CP-SO3H-specific antiserum. In an attempt to identify the multiple hyperoxidized spots of the Prx I on two-dimensional PAGE analysis, an N-acetylated Prx I was identified as part of the total Prx I using anti-acetylated Lys antibody. Using peptidyl-Asp metalloendopeptidase (EC 3.4.24.33) peptide fingerprints, we found that Nα-terminal acetylation (Nα-Ac) occurred exclusively on Prx II after demethionylation. Nα-Ac of Prx II blocks Prx II from irreversible hyperoxidation without altering its affinity for hydrogen peroxide. A comparative study of non-Nα-acetylated and Nα-terminal acetylated Prx II revealed that Nα-Ac of Prx II induces a significant shift in the circular dichroism spectrum and elevation of Tm from 59.6 to 70.9 °C. These findings suggest that the structural maintenance of Prx II by Nα-Ac may be responsible for preventing its hyperoxidation to form CP-SO3H. Peroxiredoxins (Prxs) 4The abbreviations used are: Prx, peroxiredoxin; hPrx, human peroxiredoxin; rPrx, recombinant peroxiredoxin; CP, peroxidatic cysteine; CR, resolving cysteine; HPLC, high performance liquid chromatography; CHX, cycloheximide; ABC, ammonium bicarbonate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; DTT, dithiothreitol; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS, mass spectrometry; RPLC/ESI-MS/MS, reverse-phase liquid chromatography/electrospray ionization tandem mass spectrometry; AspN, peptidyl-Asp metalloendopeptidase; ACN, acetonitrile; FBS, fetal bovine serum; Ac, acetylation; rTrx, rat thioredoxin; ACTH, adrenocorticotropic hormone. are a family of peroxidases that possess a conserved cysteine residue at the catalytic site for the reduction of peroxide/peroxynitrite. Using thiol-based reducing equivalents, like thioredoxin, Prxs catalyze the reduction of hydrogen peroxide, alkylhydroperoxides, and peroxynitrite to water, corresponding alcohols, and nitrite, respectively (1Rhee S.G. Chae H.Z. Kim K. Free Radic. Biol. Med. 2005; 38: 1543-1552Crossref PubMed Scopus (1146) Google Scholar, 2Wood Z.A. Schröder E. Harris R.J. Poole L.B. Trends Biochem. Sci. 2003; 28: 32-40Abstract Full Text Full Text PDF PubMed Scopus (2136) Google Scholar, 3Hofmann B. Hecht H.-J. Flohé L. Biol. Chem. 2002; 383: 347-364Crossref PubMed Scopus (774) Google Scholar, 4Chae H.Z. Robison K. Poole L.B. Church G. Storz G. Rhee S.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7017-7021Crossref PubMed Scopus (706) Google Scholar, 5Rhee S.G. Kang S.W. Chang T.-S. Jeong W. Kim K. IUBMB Life. 2001; 52: 35-41Crossref PubMed Scopus (520) Google Scholar, 6Bryk R. Griffin P. Nathan C. Nature. 2000; 407: 211-215Crossref PubMed Scopus (571) Google Scholar, 7Trujillo M. Ferrer-Sueta G. Radi R. Methods Enzymol. 2008; 441: 173-196Crossref PubMed Scopus (61) Google Scholar, 8Trujillo M. Ferrer-Sueta G. Thomson L. Flohé L. Radi R. Subcell. Biochem. 2007; 44: 83-113Crossref PubMed Scopus (112) Google Scholar). Based on the number and location of conserved cysteine residue(s) directly involved in peroxide reduction, the six isotypes of mammalian Prx can be grouped into three distinct subgroups as follows: 2-Cys Prx, atypical 2-Cys Prx, and 1-Cys Prx, (1Rhee S.G. Chae H.Z. Kim K. Free Radic. Biol. Med. 2005; 38: 1543-1552Crossref PubMed Scopus (1146) Google Scholar, 2Wood Z.A. Schröder E. Harris R.J. Poole L.B. Trends Biochem. Sci. 2003; 28: 32-40Abstract Full Text Full Text PDF PubMed Scopus (2136) Google Scholar, 5Rhee S.G. Kang S.W. Chang T.-S. Jeong W. Kim K. IUBMB Life. 2001; 52: 35-41Crossref PubMed Scopus (520) Google Scholar). Human Prx I (hPrx I) and Prx II (hPrx II) are members of the 2-Cys Prx subgroup and thus contain two conserved cysteine residues that are directly involved in peroxidase activity. Cys52 for hPrx I and Cys51 for hPrx II are designated the peroxidatic cysteines (CP). These residues attack the O–O bond of the peroxide (ROOH) substrate to form the product (ROH) and the sulfenic derivative CP-SOH. This sulfenic derivative then forms a disulfide bond with the other conserved cysteine residue, which is referred to as the resolving cysteine (CR; Cys173 in hPrx I and Cys172 in hPrx II). In the case of 2-Cys Prxs, the disulfide partners, CP and CR, reside within different subunits; therefore, the disulfide bond established between CP and CR (CP-S–S-CR) is intermolecular. The reduced thioredoxin molecule is responsible for reducing the CP-S–S-CR disulfide bond to generate sulfhydryls (1Rhee S.G. Chae H.Z. Kim K. Free Radic. Biol. Med. 2005; 38: 1543-1552Crossref PubMed Scopus (1146) Google Scholar, 2Wood Z.A. Schröder E. Harris R.J. Poole L.B. Trends Biochem. Sci. 2003; 28: 32-40Abstract Full Text Full Text PDF PubMed Scopus (2136) Google Scholar, 3Hofmann B. Hecht H.-J. Flohé L. Biol. Chem. 2002; 383: 347-364Crossref PubMed Scopus (774) Google Scholar, 5Rhee S.G. Kang S.W. Chang T.-S. Jeong W. Kim K. IUBMB Life. 2001; 52: 35-41Crossref PubMed Scopus (520) Google Scholar, 9Poole L.B. Subcell. Biochem. 2007; 44: 61-81Crossref PubMed Scopus (93) Google Scholar). The CP of eukaryotic 2-Cys Prxs is vulnerable to hyperoxidation, which results in the loss of its peroxidase activity. This feature is referred to as the “floodgate” mechanism, by which Prxs function as a redox sensor for the regulation of cell signaling (10Wood Z.A. Poole L.B. Karplus P.A. Science. 2003; 300: 650-653Crossref PubMed Scopus (1147) Google Scholar, 11Karplus P.A. Hall A. Subcell. Biochem. 2007; 44: 41-60Crossref PubMed Scopus (84) Google Scholar). Hyperoxidation of CP does not occur when the disulfide bond (CP-S–S-CR) is formed. However, the thiol (CP-SH) can be hyperoxidized via the sulfenic (CP-SOH) derivative intermediate in the absence of CP-S-S-CR formation during catalysis (12Yang 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). Two different hyperoxidation products of CP, the reversible sulfinic (CP-SO2H) derivative and the irreversible sulfonic (CP-SO3H) derivative, have been identified. The irreversible CP-SO3H was reported in Tsa1p, a yeast 2-Cys Prx, based on in vivo and in vitro regeneration assay results, and a stronger reactivity to an anti-Tsa1p-SO3H antibody, which exhibits high specificity toward Tsa1p-CP-SO3H relative to Tsa1p-CP-SO2H (13Lim J.C. Choi H.-I. Park Y.S. Nam H.W. Woo H.A. Kwon K.-S. Kim Y.S. Rhee S.G. Kim K. Chae H.Z. J. Biol. Chem. 2008; 283: 28873-28880Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Both forms of hyperoxidized Prxs, CP-SO2H and CP-SO3H, are superimposed on the acidic migrated spot instead of the Prx-SH spot on a two-dimensional polyacrylamide gel because of the introduction of one negative charge by hyperoxidation (12Yang 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, 13Lim J.C. Choi H.-I. Park Y.S. Nam H.W. Woo H.A. Kwon K.-S. Kim Y.S. Rhee S.G. Kim K. Chae H.Z. J. Biol. Chem. 2008; 283: 28873-28880Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 14Woo H.A. Kang S.W. Kim H.K. Yang K.S. Chae H.Z. Rhee S.G. J. Biol. Chem. 2003; 278: 47361-47364Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 15Mitsumoto A. Takanezawa Y. Okawa K. Iwamatsu A. Nakagawa Y. Free Radic. Biol. Med. 2001; 30: 625-635Crossref PubMed Scopus (104) Google Scholar, 16Rabilloud T. Heller M. Gasnier F. Luche S. Rey C. Aebersold R. Benahmed M. Louisot P. Lunardi J. J. Biol. Chem. 2002; 277: 19396-19401Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). The protein sulfinic acid reductase, sulfiredoxin, is responsible for reversing 2-Cys Prx-SO2H to Prx-SH in the presence of ATP and thiol-reducing equivalents like thioredoxin or glutathione (17Wagner E. Luche S. Penna L. Chevallet M. Dorsselaer A.V. Leize-Wagner E. Rabilloud T. Biochem. J. 2002; 366: 777-785Crossref PubMed Google Scholar, 18Woo H.A. Chae H.Z. Hwang S.C. Yang K.S. Kang S.W. Kim K. Rhee S.G. Science. 2003; 300: 653-656Crossref PubMed Scopus (470) Google Scholar, 19Biteau B. Labarre J. Toledano M.B. Nature. 2003; 425: 980-984Crossref PubMed Scopus (801) Google Scholar, 20Budanov A.V. Sablina A.A. Feinstein E. Koonin E.V. Chumakov P.M. Science. 2004; 304: 596-600Crossref PubMed Scopus (625) Google Scholar, 21Chang T.S. Jeong W. Woo H.A. Lee S.M. Park S. Rhee S.G. J. Biol. Chem. 2004; 279: 50994-51001Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 22Rhee S.G. Jeong W. Chang T.S. Woo H.A. Kidney Int. 2007; 106: S3-S8Abstract Full Text Full Text PDF Scopus (126) Google Scholar, 23Jönsson T.J. Lowther W.T. Subcell. Biochem. 2007; 44: 115-141Crossref PubMed Scopus (63) Google Scholar, 24Jönsson T.J. Johnson L.C. Lowther W.T. Nature. 2008; 451: 98-101Crossref PubMed Scopus (115) Google Scholar). Until now, an intracellular enzymatic regeneration system for Prx-SO3H has not been reported. Because mammalian Prx I and Prx II have been studied independently in a number of different organisms and cultured cells, the comparative biochemical data supporting their distinctive functional identities is still very limited. Recombinant Prx I (rPrx I) showed a 2.6-fold higher specific activity as a peroxidase than the recombinant Prx II (rPrx II) without any obvious catalytic or mechanistic differences (25Chae H.Z. Kim H.J. Kang S.W. Rhee S.G. Diabetes Res. Clin. Pract. 1999; 45: 101-112Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 26Kang S.W. Chae H.Z. Seo M.S. Kim K. Baines I.C. Rhee S.G. J. Biol. Chem. 1998; 273: 6297-6302Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar). Recent competition kinetics studies of hPrx II revealed a rate constant of 1.3 × 107 m–1 s–1, which is fast enough to favor an intracellular hydrogen peroxide target even in competition with catalase or glutathione peroxidase (27Peskin A.V. Low F.M. Paton L.N. Maghzal G.J. Hampton M.B. Winterbourn C.C. J. Biol. Chem. 2007; 282: 11885-11892Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 28Ogusucu R. Rettori D. Munhoz D.C. Netto L.E.S. Augusto O. Free Radic. Biol. Med. 2007; 42: 326-334Crossref PubMed Scopus (158) Google Scholar). The kinetic parameters of the competition assay for hPrx I are still not available. Mammalian Prx I interacts with and regulates a broad spectrum of proteins, such as the Src homology domain 3 of c-Abl (29Wen S.T. Van Etten R.A. Genes Dev. 1997; 11: 2456-2467Crossref PubMed Scopus (239) Google Scholar), the Myc box II (MBII) domain of c-Myc (30Mu Z.M. Yin X.Y. Prochownik E.V. J. Biol. Chem. 2002; 277: 43175-43184Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), the macrophage migration inhibitory factor (MIF, 31), the androgen receptor (32Park S.-Y. Yu X. Ip C. Mohler J.L. Bogner P.N. Park Y.-M. Cancer Res. 2007; 67: 9294-9303Crossref PubMed Scopus (69) Google Scholar), and the apoptosis signal-regulating kinase-1 (ASK-1) (33Kim S.Y. Kim T.J. Lee K.-Y. FEBS Lett. 2008; 582: 1913-1918Crossref PubMed Scopus (109) Google Scholar). The suggested roles of Prx I in interactions with these molecules are those of a tumor repressor, a survival enhancer, and a growth regulator. Although these suggested functions are controversial (34Neumann C.A. Fang Q. Curr. Opin. Pharmacol. 2007; 7: 375-380Crossref PubMed Scopus (111) Google Scholar), all of them can be attributed to the peroxide-scavenging capacity of Prx I (at least in part), except for the enhancement of androgen receptor transactivation (32Park S.-Y. Yu X. Ip C. Mohler J.L. Bogner P.N. Park Y.-M. Cancer Res. 2007; 67: 9294-9303Crossref PubMed Scopus (69) Google Scholar). Prx II interacts with platelet-derived growth factor receptor and functions as a negative regulator for platelet-derived growth factor signaling (35Choi M.H. Lee I.K. Kim G.W. Kim B.U. Han Y.H. Yu D.Y. Park H.S. Kim K.Y. Lee J.S. Choi C. Bae Y.S. Lee B.I. Rhee S.G. Kang S.W. Nature. 2005; 435: 347-353Crossref PubMed Scopus (332) Google Scholar). Prx II also binds to phospholipase D1 (PLD1) and functions as a hydrogen peroxide-stimulated PLD1 signal terminator (36Xiao N. Du G. Frohman M.A. FEBS J. 2005; 272: 3929-3937Crossref PubMed Scopus (21) Google Scholar). Both of these suggested Prx II roles are attributable to the peroxidase activity of Prx II. The major phenotypes of Prx I knock-out mice involve the development of a variety of age-related cancers, hemolytic anemia (37Neumann 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. Nature. 2003; 424: 561-565Crossref PubMed Scopus (643) Google Scholar), and dramatic shifts in subcellular reactive oxygen species localization (38Egler R.A. Fernandes E. Rothermund K. Sereika S. de Souza-Pinto N. Jaruga P. Dizdaroglu M. Prochownik E.V. Oncogene. 2005; 24: 8038-8050Crossref PubMed Scopus (184) Google Scholar). Prx II knock-out mice exhibit splenomegaly and a lack of tumor development in any cell type or tissue (39Lee 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. Blood. 2003; 101: 5033-5038Crossref PubMed Scopus (334) Google Scholar). Until now, the protein molecule that interacts with Prx I and Prx II has not been characterized, and there is no indication of a heterodimer between Prx I and Prx II. Despite their similar peroxide-scavenging capacities, it is reasonable to conclude that the Prx I and Prx II are unable to compensate for each other in terms of physiological roles. There are several examples of tissue- or cell type-specific expression patterns, such as exclusive Prx I expression in astrocytes and Leydig cells and Prx II expression in neurons and Sertoli cells (40Sarafian T.A. Verity M.A. Vinters H.V. Shih C.C. Shi L. Ji X.D. Dong L. Shau H. J. Neurosci. Res. 1999; 56: 206-212Crossref PubMed Scopus (104) Google Scholar, 41Lee K. Park J.-S. Kim Y.-J. Lee Y.S. Hwang T.S. Kim D.-J. Park E.-M. Park Y.-M. Biochem. Biophys. Res. Commun. 2002; 296: 337-342Crossref PubMed Scopus (66) Google Scholar); however, Prx I and Prx II are coexpressed in the majority of mammalian cells and tissues, suggesting distinguished biochemical characteristics of their cellular regulatory mechanisms. Recently, the unique presence of Cys83 in hPrx I, which contributes to the stability of the dimer-dimer interface and suppresses local unfolding, has been claimed to be prone to overoxidation of Prx I (42Lee W. Choi K.-S. Tiddell J. Ip C. Ghosh D. Park J.-H. Park Y.-M. J. Biol. Chem. 2007; 282: 22011-22022Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). The contribution of the dimer-decamer interconversion to the regulation of Prx I activity has also been reported (43Matsumura T. Okamoto K. Iwahara S. Hori H. Takahashi Y. Nishino T. Abe Y. J. Biol. Chem. 2008; 283: 284-293Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In this study, we confirmed that hPrx II was more susceptible to hyperoxidation as well as more prone to regeneration than hPrx I in HeLa cells. We also found that the difficulty in regenerating hPrx I was caused by irreversible sulfonic (CP-SO3H) hyperoxidation. Using AspN (EC 3.4.24.33) peptide fingerprints, we identified the Nα-terminal acetylation exclusively on hPrx II. In addition, we provide evidence for the structural maintenance offered by Nα-terminal acetylation of hPrx II, which possibly contributes to preventing irreversible overoxidation of CP-SO3H. Preparation of Proteins—hPrx II was purified from human red blood cells (Red Cross Blood Center, Chonnam National University, Gwangju, Korea) via combined HPLC column chromatography methods as described previously (44Chae H.Z. Kang S.W. Rhee S.G. Methods Enzymol. 1999; 300: 219-226Crossref PubMed Scopus (202) Google Scholar). rPrx I and rPrx II were purified from Escherichia coli cells overexpressing each protein by previously described methods (25Chae H.Z. Kim H.J. Kang S.W. Rhee S.G. Diabetes Res. Clin. Pract. 1999; 45: 101-112Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). Thioredoxin reductase was purified from rat liver as described previously (45Luthman M. Holmgren A. Biochemistry. 1982; 21: 6628-6633Crossref PubMed Scopus (510) Google Scholar) or purchased from Sigma. Recombinant rat thioredoxin (rTrx) was purified from the E. coli overexpression system by a previously described method (44Chae H.Z. Kang S.W. Rhee S.G. Methods Enzymol. 1999; 300: 219-226Crossref PubMed Scopus (202) Google Scholar). Cell Culture and Regeneration Assay—HeLa cells were obtained from the American Type Culture Collection. Cells were cultured in Dulbecco's modified Eagle's media (WelGENE, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS, WelGENE, Daegu, Korea). For the hyperoxidation of Prxs, designated concentrations of hydrogen peroxide in phosphate-buffered saline (PBS) were applied to the cells for 10 min after removal of the serum-containing media and two washes with PBS. For the regeneration of Prxs, after removal of hydrogen peroxide with two additional PBS washes, cells were incubated in fresh 10% FBS-containing media in the presence of 10 μg/ml cycloheximide (CHX) for the designated time periods. Cells were harvested via the addition of 10% trichloroacetic acid immediately after two washings with PBS. Harvested cells were then ultrasonicated for 3 min, and the protein precipitates were washed with 100% acetone, followed by air drying. Dried cell lysates were then solubilized in lysis buffer (40 mm Tris, 9 m urea, and 4% CHAPS). Two-dimensional SDS-PAGE—Cell lysates or purified proteins were mixed with rehydration buffer (8 m urea, 2% CHAPS, 0.5% immobilized pH gradient buffer, 20 mm DTT, and 0.005% bromphenol blue) and loaded onto immobilized pH gradient strips (pH 3–10 or pH 4–7, 7 cm; Amersham Biosciences). Isoelectric focusing was carried out in four steps as follows: 50 V, 12 h; 500 V, 0.5 h; 1000 V, 0.5 h; and 8000 V for a total of 20,000 V-h. After reduction and alkylation, second dimensional electrophoresis was conducted on a 12% SDS-polyacrylamide gel using an SE-260 vertical unit (Amersham Biosciences). In-gel Digestion of Protein Spots on Two-dimensional Polyacrylamide Gel with AspN Endopeptidase—Excised spots from a two-dimensional polyacrylamide gel were washed with distilled water, 50% acetonitrile (ACN), and 100 mm ammonium bicarbonate (ABC, pH 8.5) in microcentrifuge tubes. After 45 min of reduction with a reducing agent (10 mm DTT in a 100 mm ABC, pH 8.5), each piece of the gel was alkylated with 55 mm iodoacetamide in a 100 mm ABC (pH 8.5) for 30 min and then fully dried in a SpeedVac. In-gel digestion of the protein was performed using 12.5 μl of AspN endoproteinase (10 ng/μl, Sigma) in a 100 mm ABC (pH 8.5) for 12 h at 37 °C. MALDI-TOF-MS Analysis—Mass analysis of AspN digests was performed on a Voyager-DE STR MALDI-TOF mass spectrometer (Perceptive Biosystems). In-gel digested samples were eluted from gels by 50% ACN and 5% formic acid. Each sample was then dissolved with 50% ACN and 0.1% trifluoroacetic acid and mixed with 10 mg/ml α-cyano-4-hydroxycinnamic acid (Sigma), dissolved in 0.1% trifluoroacetic acid and 50% ACN. Samples were spotted on a sample plate, and mass spectra were obtained in the positive-ion reflector mode with delayed extraction. Calibration was performed using angiotensin I (1296.79 Da), adrenocorticotropic hormone (ACTH clip 1–17, 2093.01 Da), and ACTH clip 18–39 (2465.20 Da) as external standards. Nanoflow Reversed Phase Liquid Chromatography-Tandem Mass Spectrometry—The samples were prepared as described for MALDI-TOF-MS analysis. The extracted peptides were separated and analyzed via nanoflow reverse phase liquid chromatography/electrospray ionization tandem mass spectrometry (RPLC/ESI-MS/MS) to determine their amino acid sequences. The Ultimate nano-RPLC system was used in combination with an autosampler (Famos) and a precolumn switching device (Switchos). The samples were loaded onto a Zorbax 300SB-C18 trap column (5 μm, 10 × 0.2 mm; Agilent) and washed with the loading solvent (0.1% formic acid, flow rate, 4 μl/min) for 10 min to remove salts. Subsequently, flow paths were transferred to the analytical Zorbax 300SB-C18 column (5 μm, 150 × 0.075 mm; Agilent) using a Switchos II column switching device. The nano-flow eluted at a flow rate of 200 nl/min using a 90-min gradient elution from 0% solvent A to 32% solvent B (solvent A, 0.1% formic acid with 5% ACN; solvent B, 0.1% formic acid with 90% ACN). The column outlet was coupled directly to the nano-ESI source, which was interfaced with the QSTAR Mass spectrometer (Applied Biosystems). The nano RPLC/ESI-MS/MS, running on the QSTAR instrument, was acquired in “Information-dependent Acquisition” mode. The data acquisition time was set to 3 s per spectrum over an m/z range of 400–1500 Da for nano-LC-MS/MS analyses. Data base searches were performed on the National Center for Biotechnology Information (NCBI) nonredundant data base using the MASCOT software package (Matrix Sciences) and were confirmed manually. Peroxidase Activity Assay—The peroxidase activity of Prxs was measured by monitoring the rate of NADPH oxidation at 340 nm in the presence of the thioredoxin system. Reaction mixtures of 150 μl (final volume) contained 50 mm Hepes-NaOH, pH 7.0, 0.2 mm NADPH, 1.1 μm rTrx, 0.12 μm of thioredoxin reductase, and 0.15 μm of each Prx. After adjusting the reaction mixture temperature to 30 °C, reactions were initiated immediately via the addition of hydrogen peroxide. The rate of NADPH oxidation was monitored via the loss of absorbance at 340 nm using a Jasco V-530 UV-visible spectrophotometer equipped with a thermostatic cell holder. An initial linear portion of the absorbance change (10 s) was used for the calculation of peroxidase activity. Preparation of hPrx II-SO2H and hPrx II-SO3H in Vitro—Reaction mixtures of 500 μl (final volume) contained 50 mm Hepes-NaOH, pH 7.0, 2 mm DTT, 1 mm EDTA, 0.4 μm rTrx, and 2 μm hPrx II. After preincubation at 37 °C, reactions were initiated via the addition of hydrogen peroxide. At 60 s after the start of each reaction, 0.1% trifluoroacetic acid (final concentration) was added to stop the reaction. For the preparation of hPrx II-SO2H or hPrx II-SO3H, 100 μm or 3 mm of hydrogen peroxide concentration was used, respectively. The hyperoxidation status was validated by mass spectrometric analysis. The reaction mixtures were separated by reverse phase HPLC with both spectrophotometric and mass spectrometric detection (Agilent Technology, Series 1100 MSD) using a Vydac narrow bore C18 column (Vydac 218TP5205) as described previously (46Taggart C. Cervantes-Laurean D. Kim G. McElvaney N.G. Wehr N. Moss J. Levine R.L. J. Biol. Chem. 2000; 275: 27258-27265Abstract Full Text Full Text PDF PubMed Google Scholar). Reaction mixtures of 20 μl were routinely analyzed, and the data were collected in a mass unit range of 21,700–22,500 Da. CD Analysis—hPrx II and rPrx II proteins were dialyzed against 20 mm sodium phosphate (pH 7.0) containing 100 mm NaCl, with the final protein concentration adjusted to 0.15 mg/ml. CD measurements were performed using a J-715 spectropolarimeter (Jasco) equipped with a PTC-348WI Peltier thermostat (Jasco). For the standard scan, the far-UV was scanned from 260 to 190 nm at 25 °C in a cell with a path length of 0.1 cm. To investigate the stability of the protein, thermal denaturation was monitored from 25 to 90 °C at 222 nm. The constant heating rate was 1 °C/min and the CD signal was recorded at 0.5 °C increments. The denatured fraction (FDX) at each temperature was defined as Fd = (Yn – Y)/(Yn – Yd), where Yn and Yd are the base lines of native protein and denatured proteins, respectively. These values were obtained from the extrapolation of the linear-regression of data points from 25 to 45 °C and from 75 to 90 °C, respectively. Y is the experimental CD signal at each temperature, and the apparent melting temperature (Tm) values were determined as the temperature at Fd = 0.5. hPrx II Is More Sensitive than hPrx I to Hyperoxidation and Regeneration in HeLa Cells—The sensitivities to hyperoxidation and regeneration of hPrx I and hPrx II were compared in oxidatively stressed HeLa cells based on their migration shifts on two-dimensional polyacrylamide gels. hPrx II hyperoxidized more easily than hPrx I in hydrogen peroxide-treated HeLa cells. More than 80% of hPrx II was hyperoxidized by 10 min of exposure to 50 μm hydrogen peroxide in HeLa cells, whereas less than 40% of hPrx I was hyperoxidized under the same conditions (Fig. 1A). Most of the hPrx I that appeared at the acidic migrated location under normal culture conditions (Fig. 1A) was supposed to be modified; however, we found that it also contained a reduced form (CP-SH) as well as a covalently modified hPrx I. The hyperoxidized form of this modified hPrx I migrated at a position indicated by an asterisk (see Fig. 1A). This observation is in agreement with the previous report that some reduced hPrx I from HeLa cells co-migrates with hyperoxidized hPrx I (47Chevallet M. Wagner E. Luche S. van Dorsselaer A. Leize-Wagner E. Rabilloud T. J. Biol. Chem. 2003; 278: 37146-37153Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Therefore, hyperoxidized hPrx I was calculated as the decrease in intensity of the reduced spot relative to control, plus the relative increase in the spot as designated by an asterisk (see Fig. 1A). To hyperoxidize greater than 80% of hPrx I in HeLa cells, treatment with more than 100 μm hydrogen peroxide was needed. Hyperoxidation of hPrx II was mostly reversible with up to 0.5 mm hydrogen peroxide, as seen by the migration back to the reduced spots in the presence of CHX during regeneration (Fig. 1A). At the acidic migration spots after regeneration, the irreversible portion of the hyperoxidized hPrx I was elevated in a hydrogen peroxide concentration-dependent manner (Fig. 1A). Irreversibly hyperoxidized hPrx I appeared in the 50 μm hydrogen peroxide-treated sample. The majority of hPrx I oxidized by 0.5 mm hydrogen peroxide was irreversible for up to 12 h of regeneration (Fig. 1A). Evaluation through direct visualization of protein spots on two-dimensional polyacrylamide gels with silver staining resulted in a pattern comparable with those of the immunoblots (Fig. 1B). Cell death or detachment of cells was not observed in our experimental condition. N-Acetylation Was Found Exclusively on Acidic Migrated hPrx I—Oxidation induced further acidic migration of acidic migrated hPrx I (Fig. 1A), suggesting the possibility of a post-translational modification other than hyperoxidation. The criteria for modifications were the gain of one negative charge or the loss of one positive charge without affecting mobility on the SDS-PAGE. N-Acetylation was evaluated because hPrxs I and II were recently identified as targets of histone deacetylase 6 (48Parmigiani R.B. Xu W.S. Venta-Perez G. Erdjument-Bromage H. Yaneva M. Tempst P. Marks P.A. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 9633-9638Crossref PubMed Scopus (251) Google Scholar), and this modification met the criteria for modification. Acidic" @default.
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• W2008861915 date "2009-05-01" @default.
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• W2008861915 title "Novel Protective Mechanism against Irreversible Hyperoxidation of Peroxiredoxin" @default.
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