Glutathionylation Induces the Dissociation of 1-Cys D-peroxiredoxin Non-covalent Homodimer

1-Cys peroxiredoxins (1-Cys Prxs) are antioxidant enzymes that catalyze the reduction of hydroperoxides into alcohols using a strictly conserved cysteine. 1-Cys B-Prxs, homologous to human PrxVI, were recently shown to be reactivated by glutathione S-transferase (GST) π via the formation of a GST-Prx heterodimer and Prx glutathionylation. In contrast, 1-Cys D-Prxs, homologous to human PrxV, are reactivated by the glutaredoxin-glutathione system through an unknown mechanism. To investigate the mechanistic events that mediate the 1-Cys D-Prx regeneration, interaction of the Prx with glutathione was studied by mass spectrometry and NMR. This work reveals that the Prx can be glutathionylated on its active site cysteine. Evidences are reported that the glutathionylation of 1-Cys D-Prx induces the dissociation of the Prx non-covalent homodimer, which can be recovered by reduction with dithiothreitol. This work demonstrates for the first time the existence of a redox-dependent dimer-monomer switch in the Prx family, similar to the decamer-dimer switch for the 2-Cys Prxs. 1-Cys peroxiredoxins (1-Cys Prxs) are antioxidant enzymes that catalyze the reduction of hydroperoxides into alcohols using a strictly conserved cysteine. 1-Cys B-Prxs, homologous to human PrxVI, were recently shown to be reactivated by glutathione S-transferase (GST) π via the formation of a GST-Prx heterodimer and Prx glutathionylation. In contrast, 1-Cys D-Prxs, homologous to human PrxV, are reactivated by the glutaredoxin-glutathione system through an unknown mechanism. To investigate the mechanistic events that mediate the 1-Cys D-Prx regeneration, interaction of the Prx with glutathione was studied by mass spectrometry and NMR. This work reveals that the Prx can be glutathionylated on its active site cysteine. Evidences are reported that the glutathionylation of 1-Cys D-Prx induces the dissociation of the Prx non-covalent homodimer, which can be recovered by reduction with dithiothreitol. This work demonstrates for the first time the existence of a redox-dependent dimer-monomer switch in the Prx family, similar to the decamer-dimer switch for the 2-Cys Prxs. Peroxiredoxins (Prxs) 3The abbreviations used are: Prx, peroxiredoxin; GSH, reduced glutathione; GSSG, oxidized glutathione; GST, glutathione S-transferase; Grx, glutaredoxin; NOE, nuclear Overhauser effect; HSQC, heteronuclear single quantum correlation; DTT, dithiothreitol. 3The abbreviations used are: Prx, peroxiredoxin; GSH, reduced glutathione; GSSG, oxidized glutathione; GST, glutathione S-transferase; Grx, glutaredoxin; NOE, nuclear Overhauser effect; HSQC, heteronuclear single quantum correlation; DTT, dithiothreitol. represent a novel family of peroxidases that reduce hydrogen peroxide and hydroperoxides to water and alcohols using a strictly conserved cysteine (for review, see Refs. 1Wood Z.A. Schro¨der E. Harris J.R. Poole L.B. Trends Biochem. Sci. 2003; 28: 32-40Abstract Full Text Full Text PDF PubMed Scopus (2112) Google Scholar and 2Hofmann B. Hecht H.-J. Flohe´ L. Biol. Chem. 2002; 383: 347-364Crossref PubMed Scopus (772) Google Scholar). When reducing the peroxide substrate, the catalytic cysteine of the Prx is oxidized to a cysteine-sulfenic acid (3Ellis H.R. Poole L.B. Biochemistry. 1997; 36: 15013-15018Crossref PubMed Scopus (210) Google Scholar, 4Choi H.-J. Kang S.W. Yang C.-H. Rhee S.G. Ryu S.-E. Nat. Struct. Biol. 1998; 5: 400-406Crossref PubMed Scopus (332) Google Scholar). The sulfenic acid is then reduced by a thiolcontaining electron donor (1-Cys Prxs) or is involved in intermolecular (typical 2-Cys Prxs) or intramolecular (atypical 2-Cys Prxs) disulfide bridges reduced by disulfide oxidoreductases (1Wood Z.A. Schro¨der E. Harris J.R. Poole L.B. Trends Biochem. Sci. 2003; 28: 32-40Abstract Full Text Full Text PDF PubMed Scopus (2112) Google Scholar, 2Hofmann B. Hecht H.-J. Flohe´ L. Biol. Chem. 2002; 383: 347-364Crossref PubMed Scopus (772) Google Scholar). The involvement of a third cysteine has been very recently proposed for Mycobacterium tuberculosis AhpC and Aeropyrum pernix K1 (5Guimaraes B.G. Souchon H. Honore N. Saint-Joanis B. Brosch R. Shepard W. Cole S.T. Alzari P.M. J. Biol. Chem. 2005; 280: 25735-25742Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 6Mizohata E. Sakai H. Fusatomi E. Terada T. Murayama K. Shirouzu M. Yokoyama S. J. Mol. Biol. 2005; 354: 317-329Crossref PubMed Scopus (46) Google Scholar). According to primary sequences, Prxs can be divided into six groups: A-Prxs (2-Cys Prxs homologous to human PrxII), B-Prxs (1-Cys Prxs homologous to human PrxVI), C-Prxs (Prxs-Q), D-Prxs (type-II Prxs homologous to human PrxV), E-Prxs (bacterial thiol peroxidases), and F-Prxs (Prxs homologous to archaeal A. pernix K1) (2Hofmann B. Hecht H.-J. Flohe´ L. Biol. Chem. 2002; 383: 347-364Crossref PubMed Scopus (772) Google Scholar, 6Mizohata E. Sakai H. Fusatomi E. Terada T. Murayama K. Shirouzu M. Yokoyama S. J. Mol. Biol. 2005; 354: 317-329Crossref PubMed Scopus (46) Google Scholar, 7Trivelli X. Krimm I. Ebel C. Verdoucq L. Prouzet-Maule´on V. Chartier Y. Tsan P. Lauquin G. Meyer Y. Lancelin J.-M. Biochemistry. 2003; 42: 14139-14149Crossref PubMed Scopus (32) Google Scholar, 8Echalier A. Trivelli X. Corbier C. Rouhier N. Walker O. Tsan P. Jacquot J.-P. Aubry A. Krimm I. Lancelin J.-M. Biochemistry. 2005; 44: 1755-1767Crossref PubMed Scopus (45) Google Scholar). The physiological electron donor for 1-Cys Prxs has been identified only recently. Although the reduction of 1-Cys B-Prxs requires glutathionylation mediated by πGST (9Manevich Y. Feinstein S.I. Fisher A.B. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3780-3785Crossref PubMed Scopus (288) Google Scholar, 10Ralat L.A. Manevich Y. Fisher A.B. Colman R.F. Biochemistry. 2006; 45: 360-372Crossref PubMed Scopus (155) Google Scholar), 1-Cys D-Prxs found in plant and pathogenic bacteria were shown to catalyze a Grx/GSH-dependent reduction of hydroperoxides (11Rouhier N. Gelhaye E. Sautie`re P.-E. Brun A. Laurent P. Tagu D. Ge´rard J. de Fay E. Meyer Y. Jacquot J.-P. Plant Physiol. 2001; 127: 1299-1309Crossref PubMed Scopus (189) Google Scholar, 12Rouhier N. Gelhaye E. Jacquot J.-P. J. Biol. Chem. 2002; 277: 13609-13614Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 13Bre´helin C. Meyer E.H. De Souris J.-P. Bonnard G. Meyer Y. Plant Physiol. 2003; 132: 2045-2057Crossref PubMed Scopus (105) Google Scholar, 14Cha M.K. Hong S.K. Lee D.S. Kim I.H. J. Biol. Chem. 2004; 279: 11035-11041Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 15Vergauwen B. Pauwels F. Jacquemotte F. Meyer T.E. Cusanovich M.A. Bartsch R.G. Van Beeumen J.J. J. Biol. Chem. 2001; 276: 20890-20897Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 16Pauwels F. Vergauwen B. Vanrobaeys F. Devreese B. Van Beeumen J.J. J. Biol. Chem. 2003; 278: 16658-16666Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 17Rouhier N. Jacquot J.-P. FEBS Lett. 2003; 554: 149-153Crossref PubMed Scopus (29) Google Scholar). Reactivation of 1-Cys B-Prx was demonstrated to occur by heterodimerization of the Prx and πGST, followed by Prx glutathionylation and the formation of an intermolecular disulfide between the Prx and πGST. The disulfide is then reduced by GSH, regenerating an active 1-Cys B-Prx (9Manevich Y. Feinstein S.I. Fisher A.B. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3780-3785Crossref PubMed Scopus (288) Google Scholar, 10Ralat L.A. Manevich Y. Fisher A.B. Colman R.F. Biochemistry. 2006; 45: 360-372Crossref PubMed Scopus (155) Google Scholar). Conversely, the mechanism of reduction of 1-Cys D-Prxs has not yet been clearly demonstrated. The sulfenic acid might be attacked by Grx reduced by GSH or might be attacked by GSH, forming a mixed disulfide bridge reduced by the Grx (12Rouhier N. Gelhaye E. Jacquot J.-P. J. Biol. Chem. 2002; 277: 13609-13614Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 16Pauwels F. Vergauwen B. Vanrobaeys F. Devreese B. Van Beeumen J.J. J. Biol. Chem. 2003; 278: 16658-16666Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 18Rouhier N. Gama F. Wingsle G. Gelhaye E. Gans P. Jacquot J.-P. Biochem. Biophys. Res. Commun. 2006; 341: 1300-1308Crossref PubMed Scopus (11) Google Scholar). To investigate the mechanistic events that mediate the 1-Cys D-Prx regeneration, we report here a mass spectrometry and NMR study of the interaction of glutathione with 1-Cys D-Prx. This work shows that the Prx catalytic cysteine is directly glutathionylated by reaction with glutathione, and reveals that the glutathionylation of the 1-Cys D-Prx catalytic cysteine induces the dissociation of the non-covalent "perpendicular-type" homodimer into a monomer. The Prx dimerization can be reversibly achieved by the reduction of the mixed disulfide bond with dithiothreitol. These results report for the first time the existence of a dimer-monomer switch in the Prx family, similar to the decamer-dimer switch described for the 2-Cys Prxs, and give new insight into the quaternary structure modulation of the peroxiredoxins. Protein Samples—Samples of [U-15N]Prx, C76A [U-15N]Prx, and U-15N, 13C, 50% [2H]Prx were produced as previously described (19Bouillac S. Rouhier N. Tsan P. Jacquot J.-P. Lancelin J.-M. J. Biomol. NMR. 2004; 30: 105-106Crossref PubMed Scopus (6) Google Scholar). Populus tremula D-Prx NMR samples were prepared at pH 7.2 in 50 mm phosphate buffer, 10% D2O, 0.02% NaN3. Mass Spectrometry—Mass spectra were acquired on an API 300 triple quadrupole instrument equipped with an electrospray ion source (Sciex; Toronto, CA). For mass spectrometry-compatible non-denaturing conditions, the [15N]Prx sample was submitted to seven dilution-concentration steps in a pH 7 40 mm ammonium acetate buffer using centrifugal concentrators (Nanosep). Mass spectra were acquired with a Prx sample at a concentration of 250 μm. Thereafter, GSSG was added to reach a final concentration of 2.5 mm corresponding to a 10 m excess. Mass spectra were recorded after 2 h and overnight incubation at 4 °C. NMR Spectrometry—NMR experiments were performed at 28 °C on a Varian INOVA Unity 600 spectrometer fitted with a normal triple resonance (HCN) probe and a z-field gradient coil. 15N HSQC and 15N relaxation experiments were acquired with 512 complex points and a spectral width of 10000 Hz in F2 (1HN) and 128 complex points, 2200 Hz in F1 (15NH). Triple resonance experiments from the Varian Protein Pack were recorded with 512 complex points in F3, 40 complex points in F2, and 80 or 70 complex points for 13Cα, 13CO, and 13Cαβ F1 dimensions. All spectra were processed using NMRPipe scripts (20Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11517) Google Scholar) and were analyzed with NMRView software (21Johnson B.A. Blevins R.A. J. Biomol. NMR. 1994; 4: 603-614Crossref PubMed Scopus (2676) Google Scholar). NMR Titrations—Stock solutions of GSH and GSSG were prepared in the same buffer as the protein. Small aliquots of 10 μl of GSH or GSSG were added to a NMR tube containing 550 μl of 0.8 or 0.2 mm [15N]Prx. One-dimensional 1H and two-dimensional 1H-15N HSQC spectra were recorded at each titration point. Other conditions of glutathionylation were tested on 0.2 mm protein samples. 1-Cys D-Prx was incubated with various GSH/GSSG ratios (from 1:0.1 to 1:5) combined with oxidants (2 mm H2O2 or4mm diamide) (22Michelet L. Zaffagnini M. Marchand C. Collin V. Decottignies P. Tsan P. Lancelin J.-M. Trost P. Miginiac-Maslow M. Noctor G. Lemaire S.D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 16478-16483Crossref PubMed Scopus (160) Google Scholar). For the C76A mutant, small aliquots of 10 μl of GSSG were added to a sample containing 550 μl of 0.2 mm C76A [15N]Prx up to a final concentration of 7 mm GSSG. NMR Assignment—Triple-resonance experiments were recorded on a 0.5 mm 15N, 13C, 50% [2H]Prx sample in the presence of 15 mm GSSG. The sequence-specific backbone resonance assignment of the protein was achieved using the following experiments: HNCO, HNCA, HN(CO)CA, and CBCA-(CO)NH. The backbone resonance assignment was confirmed with a 1H-15N nuclear Overhauser effect spectroscopy-heteronuclear single quantum correlation spectrum recorded with 150 ms of mixing time. Relaxation Rate Measurements—Classical pulse sequences were used to measure longitudinal (R1) and transverse (R2) relaxation rates and 1H-15N steady-state heteronuclear NOE. NMR relaxation experiments were recorded at 28 °C on a 0.8 mm [15N]Prx sample. The relaxation experiments were also performed in the presence of 8 mm at 28 °C and 15 mm GSSG at 38 °C for a comparison with previously reported data (8Echalier A. Trivelli X. Corbier C. Rouhier N. Walker O. Tsan P. Jacquot J.-P. Aubry A. Krimm I. Lancelin J.-M. Biochemistry. 2005; 44: 1755-1767Crossref PubMed Scopus (45) Google Scholar). For R1 measurements of the native Prx, spectra were recorded with inversion recovery delays of 20, 200, 400, 600, 1000, 1200, and 1800 ms. Experiments were duplicated at 20, 400, and 1200 ms. For R1 measurements in the presence of 8 or 15 mm GSSG, delays of 20, 100, 200, 400, 600, 800, 1000, and 1200 ms were used, and experiments were duplicated at 100 and 400 ms. For R2 measurements, spectra were recorded at Carr-Purcell-Meiboom-Gill delays of 10, 30, 50, 70, 90, 110, and 130 ms, and spectra were duplicated at 50 and 110 ms. In both R1 and R2 experiments, the recycle delay was 4 s, and the number of transients used was 8 or 28 in the presence or not of GSSG, respectively. The heteronuclear NOE was determined from spectra recorded in the presence and absence of a 1H presaturation period of 3 s within a total recycle delay of 5 s between acquisitions. The number of transients used was 140. For determination of R1 and R2 relaxation rates, all resonance intensities were fitted as a single exponential function of the relaxation delay in NMRView (21Johnson B.A. Blevins R.A. J. Biomol. NMR. 1994; 4: 603-614Crossref PubMed Scopus (2676) Google Scholar). The steady-state NOE values were determined in NMRView from the ratio of peak intensities obtained with and without 1H saturation. Prx Mass Spectrometry in the Presence of a 10 m Excess of GSSG—D-Prxs, which represent peroxiredoxins homologous to human PrxV, were previously shown to form non-covalent dimers in the reduced native state (7Trivelli X. Krimm I. Ebel C. Verdoucq L. Prouzet-Maule´on V. Chartier Y. Tsan P. Lauquin G. Meyer Y. Lancelin J.-M. Biochemistry. 2003; 42: 14139-14149Crossref PubMed Scopus (32) Google Scholar, 8Echalier A. Trivelli X. Corbier C. Rouhier N. Walker O. Tsan P. Jacquot J.-P. Aubry A. Krimm I. Lancelin J.-M. Biochemistry. 2005; 44: 1755-1767Crossref PubMed Scopus (45) Google Scholar, 23Sarma G.N. Nickel C. Rahlfs S. Fischer M. Becker K. Karplus P.A. J. Mol. Biol. 2005; 346: 1021-1034Crossref PubMed Scopus (82) Google Scholar). Only D-Prxs found in Prx-Grx hybrid proteins exhibit higher molecular mass species (24Kim S.J. Woo J.R. Hwang Y.S. Jeong D.G. Shin D.H. Kim K. Ryu S.E. J. Biol. Chem. 2003; 278: 10790-10798Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Electrospray ionization mass spectra of the 1-Cys D-Prx carried out in non-denaturing conditions (ammonium acetate buffer, pH 7) are presented in Fig. 1. As illustrated by Fig. 1A, the 1-Cys D-Prx is detected at a concentration of 250 μm, both as a dimeric and monomeric species. When incubated during 2 h at 4 °C with a 10 m excess of GSSG (data not shown), the peak intensities corresponding to the dimeric form strongly decrease, whereas charge states corresponding to monomeric structures of the Prx are predominant. These charge states correspond both to the native form of the Prx and to the Prx shifted by a mass increment assigned to the addition of a glutathione overnight incubation with GSSG, the Prx is only detected as a and as a species The and species correspond to non-covalent are not the These mass spectrometry results that the Prx is glutathionylated in the presence of oxidized GSSG and that the glutathionylation of the Prx induces the dissociation of the non-covalent Prx NMR in the Presence of a 10 m Excess of of GSSG with the Prx was studied by NMR The spectra of the 1-Cys D-Prx and the Prx in the presence of GSSG are in Fig. As illustrated in Fig. the Prx NMR spectrum is strongly in the presence of a 10 m excess of GSSG conditions similar to the mass spectrometry The addition of 15 mm the Prx spectrum in with the mass spectrometry that the NMR spectrum are to the disulfide bridge formation between the Prx and GSSG. The longitudinal relaxation R1 with the of which is directly to molecular To the R1 with GSSG, NMR relaxation experiments were recorded with a 0.8 mm Prx sample without GSSG and in the presence of 8 mm GSSG. The addition of GSSG induces a in the 15N longitudinal relaxation R1 from to This clearly shows that the molecular of the protein in the presence of GSSG and the mass spectrometry Prx with spectra been recorded on a [15N]Prx sample in which GSSG concentration was from to Fig. and the spectrum obtained after the addition of 8 mm GSSG and 15 mm GSSG, respectively. comparison of Fig. and that both spectra are as shown in Fig. and Prx a on the HSQC spectrum with 8 mm GSSG. When GSSG concentration 15 a single NMR is for each and The addition of the NMR spectrum to the in the presence of 15 mm GSSG or whereas the assigned to the native reduced protein in the spectrum Prx in glutathionylation through as the reaction of reduced GSH with oxidized cysteine or the reaction D. R. A. R. I. J. Mol. 2004; PubMed Scopus Google Scholar). various conditions for Prx glutathionylation been tested by [15N]Prx samples with GSH/GSSG ratios (from 1:0.1 to 1:5) and oxidants as hydrogen peroxide or (22Michelet L. Zaffagnini M. Marchand C. Collin V. Decottignies P. Tsan P. Lancelin J.-M. Trost P. Miginiac-Maslow M. Noctor G. Lemaire S.D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 16478-16483Crossref PubMed Scopus (160) Google Scholar). In each the NMR spectra similar to that obtained with a 10 m excess of GSSG Fig. to on the NMR time in the 1-Cys D-Prx cysteine at site and cysteine in the To the cysteine involved in the disulfide bridge the titration was carried out with the similar to of the Prx were in the that the catalytic cysteine and not is involved in the reaction Prx NMR in the Presence of 15 mm spectrum shown in Fig. was assigned by triple resonance According to the from the backbone D.S. Biochemistry. PubMed Scopus Google Scholar) and the structure the containing the active cysteine is in the glutathionylated The the of the is as from the whereas in the to in the native are not which is of a the NMR The between both Prx are at the as illustrated in Fig. and and and Prx in the Presence of 15 mm 600 NMR relaxation experiments are presented in Fig. For the 15N R1 and R2 and the 1H-15N NOE values recorded for the native reduced protein are In the presence of GSSG, the values for the 15N R1 and R2 are and respectively. The is at 38 a in with the for the native dimeric protein at 28 °C and at 38 °C (8Echalier A. Trivelli X. Corbier C. Rouhier N. Walker O. Tsan P. Jacquot J.-P. Aubry A. Krimm I. Lancelin J.-M. Biochemistry. 2005; 44: 1755-1767Crossref PubMed Scopus (45) Google The of is in with values from the or from for a monomeric Prx the of the Prx is similar to the correlation time on proteins with molecular similar to that of the monomeric Prx M. J. Biomol. NMR. 1998; PubMed Scopus (45) Google Scholar). The relaxation experiments clearly that the protein is monomeric in the presence of 15 mm GSSG. As shown in Fig. the reducing the dimeric Prx form the protein is incubated with 8 mm GSSG, whereas a small of protein in the monomeric state after incubation with in the presence of 15 mm GSSG This that the spectrum in Fig. are not to the disulfide bridge formation between Prx and GSSG and that the GSSG the dissociation through non-covalent to the Prx, a not by As illustrated in 3 and in the time are also in the monomeric The relaxation experiments that and both containing are very in the monomeric whereas are in the dimeric protein 3 and cysteine can by forming mixed with protein thiol or with molecular mass as glutathione, the concentration of which in D. R. A. R. I. J. Mol. 2004; PubMed Scopus Google Scholar, P. S. J. Biochem. PubMed Scopus Google Scholar, P. Res. 2005; PubMed Scopus Google Scholar). a is to a or to protein function D. R. A. R. I. J. Mol. 2004; PubMed Scopus Google Scholar, P. S. J. Biochem. PubMed Scopus Google Scholar, P. Res. 2005; PubMed Scopus Google Scholar). The Prx glutathionylation the formation of or 2-Cys Prx can be reduced by B. J. 2003; PubMed Scopus Google Scholar, Jeong W. S.J. Yang Rhee S.G. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar, Jeong W. Woo Lee S. Rhee S.G. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). The glutathionylation of Prxs has been conditions in M. H. M. S. I. M. V. M. F. E. A. J. E. P. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar, M. H. M. S. P. I. J. E. P. 2003; PubMed Scopus Google Scholar, T. Biochemistry. PubMed Scopus Google Scholar) and has been recently reported as an for 1-Cys B-Prx (9Manevich Y. Feinstein S.I. Fisher A.B. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3780-3785Crossref PubMed Scopus (288) Google Scholar, 10Ralat L.A. Manevich Y. Fisher A.B. Colman R.F. Biochemistry. 2006; 45: 360-372Crossref PubMed Scopus (155) Google Scholar). The reduction of 1-Cys B-Prxs (Prxs homologous to human was shown to glutathionylation mediated by πGST through the formation of a heterodimer between Prx and πGST (9Manevich Y. Feinstein S.I. Fisher A.B. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3780-3785Crossref PubMed Scopus (288) Google Scholar, 10Ralat L.A. Manevich Y. Fisher A.B. Colman R.F. Biochemistry. 2006; 45: 360-372Crossref PubMed Scopus (155) Google Scholar). reduction system is used by 1-Cys D-Prxs found in plant and pathogenic bacteria (Prxs homologous to human PrxV), which catalyze a Grx/GSH-dependent reduction of hydroperoxides (11Rouhier N. Gelhaye E. Sautie`re P.-E. Brun A. Laurent P. Tagu D. Ge´rard J. de Fay E. Meyer Y. Jacquot J.-P. Plant Physiol. 2001; 127: 1299-1309Crossref PubMed Scopus (189) Google Scholar, 12Rouhier N. Gelhaye E. Jacquot J.-P. J. Biol. Chem. 2002; 277: 13609-13614Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 13Bre´helin C. Meyer E.H. De Souris J.-P. Bonnard G. Meyer Y. Plant Physiol. 2003; 132: 2045-2057Crossref PubMed Scopus (105) Google Scholar, 14Cha M.K. Hong S.K. Lee D.S. Kim I.H. J. Biol. Chem. 2004; 279: 11035-11041Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 15Vergauwen B. Pauwels F. Jacquemotte F. Meyer T.E. Cusanovich M.A. Bartsch R.G. Van Beeumen J.J. J. Biol. Chem. 2001; 276: 20890-20897Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 16Pauwels F. Vergauwen B. Vanrobaeys F. Devreese B. Van Beeumen J.J. J. Biol. Chem. 2003; 278: 16658-16666Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 17Rouhier N. Jacquot J.-P. FEBS Lett. 2003; 554: 149-153Crossref PubMed Scopus (29) Google Scholar, 18Rouhier N. Gama F. Wingsle G. Gelhaye E. Gans P. Jacquot J.-P. Biochem. Biophys. Res. Commun. 2006; 341: 1300-1308Crossref PubMed Scopus (11) Google Scholar). The mechanism of 1-Cys D-Prx is F. Vergauwen B. Vanrobaeys F. Devreese B. Van Beeumen J.J. J. Biol. Chem. 2003; 278: 16658-16666Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 18Rouhier N. Gama F. Wingsle G. Gelhaye E. Gans P. Jacquot J.-P. Biochem. Biophys. Res. Commun. 2006; 341: 1300-1308Crossref PubMed Scopus (11) Google Scholar). The mass spectrometry and NMR presented here that the catalytic cysteine of the 1-Cys D-Prx can directly with glutathione, in with the that the 1-Cys D-Prx from was as a glutathionylated protein F. Vergauwen B. Vanrobaeys F. Devreese B. Van Beeumen J.J. J. Biol. Chem. 2003; 278: 16658-16666Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The for on the NMR spectra that the Prx glutathionylation is not in the Prx glutathionylation in be the mass spectrometry and NMR a mechanism for the 1-Cys D-Prx can be proposed In the reduction of the D-Prx sulfenic acid the formation of a mixed disulfide with glutathione, reduced in by the Grx In the Grx with a Prx monomeric glutathionylated form and a similar to that of πGST for 1-Cys B-Prx the between 1-Cys B-Prxs and 1-Cys D-Prxs is glutathionylation B-Prxs not directly with The mass spectrometry and NMR presented here that the glutathionylation of the 1-Cys D-Prx catalytic cysteine induces the dissociation of the into monomer. In as illustrated in Fig. NMR experiments that the Prx dissociation is combined with as the and and This demonstrates that a new Prx is obtained the protein is All Prxs a which of a by that the Prxs can form of with a or to the in Refs. 8Echalier A. Trivelli X. Corbier C. Rouhier N. Walker O. Tsan P. Jacquot J.-P. Aubry A. Krimm I. Lancelin J.-M. Biochemistry. 2005; 44: 1755-1767Crossref PubMed Scopus (45) Google Scholar and 23Sarma G.N. Nickel C. Rahlfs S. Fischer M. Becker K. Karplus P.A. J. Mol. Biol. 2005; 346: 1021-1034Crossref PubMed Scopus (82) Google Scholar). The is in and whereas the is found in D-Prx and in and or were in M. tuberculosis J. S. J. M.K. Kim I.H. Shin W. 2005; PubMed Scopus Google Scholar), whereas C-Prxs were as S. Kim Kim M. T. J. Mol. Biol. 2005; 346: PubMed Scopus Google Scholar). The redox-dependent of the between the Prx In 2-Cys the intermolecular disulfide bridge formation the which into oxidized dimers S. Y. K. N. H. T. T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, E. N. Full Text Full Text PDF PubMed Scopus Google Scholar, E. J. Mol. Biol. PubMed Scopus Google Scholar, Z.A. Poole L.B. Karplus P.A. Biochemistry. 2002; PubMed Scopus Google Scholar). The is by the of the and is followed by of at the S. Y. K. N. H. T. T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, E. N. Full Text Full Text PDF PubMed Scopus Google Scholar, E. J. Mol. Biol. PubMed Scopus Google Scholar, Z.A. Poole L.B. Karplus P.A. Biochemistry. 2002; PubMed Scopus Google Scholar). In in the of from the in and and been S. Y. K. N. H. T. T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, E. N. Full Text Full Text PDF PubMed Scopus Google Scholar, E. J. Mol. Biol. PubMed Scopus Google Scholar, Z.A. Poole L.B. Karplus P.A. Biochemistry. 2002; PubMed Scopus Google Scholar). the of the 1-Cys D-Prx is as shown by NMR the are conserved in 1-Cys D-Prxs that the of the 1-Cys D-Prx events similar to for 2-Cys Prxs. In contrast, the formation of an intramolecular disulfide bridge not the dissociation of the the of which is to the of by an involved in with an of the Z.A. Poole L.B. Karplus P.A. Biochemistry. 2002; PubMed Scopus Google Scholar). The work reported here shows that the D-Prxs are not which with the small of (8Echalier A. Trivelli X. Corbier C. Rouhier N. Walker O. Tsan P. Jacquot J.-P. Aubry A. Krimm I. Lancelin J.-M. Biochemistry. 2005; 44: 1755-1767Crossref PubMed Scopus (45) Google Scholar, J. S. J. Kim Shin W. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). As previously the Prx dimerization through the to the active site in the reduced active protein and the of the Prx the peroxide H.-J. Kang S.W. Yang C.-H. Rhee S.G. Ryu S.-E. Nat. Struct. Biol. 1998; 5: 400-406Crossref PubMed Scopus (332) Google Scholar, Z.A. Poole L.B. Karplus P.A. Biochemistry. 2002; PubMed Scopus Google Scholar, D. D.S. G.N. Z.A. Karplus P.A. Poole L.B. Biochemistry. 2005; 44: PubMed Scopus Google Scholar). The redox-dependent modulation of the 1-Cys D-Prx quaternary structure between and reported here is similar to the decamer-dimer switch described for 2-Cys Prxs. In both the the Prx is oxidized through the formation of a disulfide bond the catalytic cysteine. Although the glutathionylation of Prxs has been reported in M. H. M. S. I. M. V. M. F. E. A. J. E. P. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar, M. H. M. S. P. I. J. E. P. 2003; PubMed Scopus Google Scholar, T. Biochemistry. PubMed Scopus Google Scholar), the of the by glutathionylation is reported for the first time in the Prx To Prxs can directly with glutathione Prxs experiments be carried out to the structure of the glutathionylated Prx and the GSH

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.