Peroxiredoxins (Prx) are abundant thiol peroxidases that react rapidly with H2O2, constituting an... more Peroxiredoxins (Prx) are abundant thiol peroxidases that react rapidly with H2O2, constituting an important antioxidant defense and acting as sensors and transmitters of H2O2 signals in cells. Certain Prxs are readily inactivated by H2O2 at submillimolar concentrations, whereas others are less sensitive. This oxidative inactivation, or hyperoxidation, occurs when H2O2 further reacts with the Cp-SOH producing Cp-SO2- and/or Cp-SO3-. Since HCO3- accelerates several of the reactions of H2O2 with biothiols, we examined whether HCO3- could increase recombinant human peroxiredoxin 1 (Prx1) hyperoxidation by H2O2. Kinetic studies of NADPH consumption by Prx1 in presence of yeast Trx, yeast Trr and various H2O2 concentrations in the absence and presence of HCO3- (25 mM) provided values of Chyper1% of 335 µM and 110 µM, respectively. Western blot (with an antiPrx-SO3-/SO2- antibody) analyses and Nano-ESI-Q-TOF MS/MS analyses of incubations of Prx1 (2.5 µM) and H2O2 (25 µM) showed higher leve...
Access to protein substrates homogenously modified by ubiquitin (Ub) is critical for biophysical ... more Access to protein substrates homogenously modified by ubiquitin (Ub) is critical for biophysical and biochemical investigations aimed at deconvoluting the myriad biological roles for Ub. Current chemical strategies for protein ubiquitylation, however, employ temporary ligation auxiliaries that are removed under harsh denaturing conditions and have limited applicability. We report an unprecedented aromatic thiol-mediated N-O bond cleavage and its application towards native chemical ubiquitylation with the ligation auxiliary 2-aminooxyethanethiol. Our interrogation of the reaction mechanism suggests a disulfide radical anion as the active species capable of cleaving the N-O bond. The successful semisynthesis of full-length histone H2B modified by the small ubiquitin-like modifier-3 (SUMO-3) protein further demonstrates the generalizability and compatibility of our strategy with folded proteins.
Despite extensive investigation of the irreversible oxidations undergone by proteins in vitro and... more Despite extensive investigation of the irreversible oxidations undergone by proteins in vitro and in vivo, the products formed from the oxidation of Trp residues remain incompletely understood. Recently, we characterized a ditryptophan cross-link produced by the recombination of hSOD1-tryptophanyl radicals generated from attack of the carbonate radical produced during the bicarbonate-dependent peroxidase activity of the enzyme. Here, we examine whether the ditryptophan cross-link is produced by the attack of the carbonate radical on proteins other than hSOD1. To this end, we treated hen egg white lysozyme with photolytically and enzymatically generated carbonate radical. The radical yields were estimated and the lysozyme modifications were analyzed by SDS-PAGE, western blot, enzymatic activity and MS/MS analysis. Lysozyme oxidation by both systems resulted in its inactivation and dimerization. Lysozyme treated with the photolytic system presented monomers oxidized to hydroxy-tryptophan at Trp(28) and Trp(123) and N-formylkynurenine at Trp(28), Trp(62) and Trp(123). Lysozyme treated with the enzymatic system rendered monomers oxidized to N-formylkynurenine at Trp(28). The dimers were characterized as lysozyme-Trp(28)-Trp(28)-lysozyme and lysozyme-Trp(28)-Trp(32)-hSOD1. The results further demonstrate that the carbonate radical is prone to causing biomolecule cross-linking and hence, may be a relevant player in pathological mechanisms. The possibility of exploring the formation of ditryptophan cross-links as a carbonate radical biomarker is discussed.
<p>A) The body weight loss in G93A rats untreated (n = 11) and treated (n = 9) with tempol;... more <p>A) The body weight loss in G93A rats untreated (n = 11) and treated (n = 9) with tempol; wild-type rats treated with tempol (n = 11) exhibited similar behavior to those not treated (not shown). Female rats received tempol (30 mM) in their drinking water starting at 7 weeks of age. B) The motor scores of the same animals were determined as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055868#s2" target="_blank">Materials and Methods</a>; the values shown correspond to the mean ± standard error; *p<0.05 (two-way ANOVA with Bonferroni post-test). C) The survival analysis of untreated and treated G93A rats. The results are shown as a Kaplan-Meier plot and are significantly different at p<0.05 (log-rank test). The end points of the non-treated and treated rats were 127.6±2.2 and 136.8±4.3 days, respectively.</p
How to cite Complete issue More information about this article Journal's homepage in redalyc... more How to cite Complete issue More information about this article Journal's homepage in redalyc.org Scientific Information System
Recent studies have provided evidence for a dual role of nitric oxide (NO) during murine leishman... more Recent studies have provided evidence for a dual role of nitric oxide (NO) during murine leishmaniasis. To explore this problem, we monitored the formation of NO and its derived oxidants during the course of Leishmania amazonensis infection in tissues of susceptible (BALB/c) and relatively resistant (C57BL/6) mice. NO production was detected directly by low-temperature electron paramagnetic resonance spectra of animal tissues. Both mouse strains presented detectable levels of hemoglobin nitrosyl (HbNO) complexes and of heme nitrosyl and iron-dithiol-dinitrosyl complexes in the blood and footpad lesions, respectively. Estimation of the nitrosyl complex levels demonstrated that most of the NO is synthesized in the footpad lesions. In agreement, immunohistochemical analysis of the lesions demonstrated the presence of nitrotyrosine in proteins of macrophage vacuoles and parasites. Since macrophages lack myeloperoxidase, peroxynitrite is likely to be the nitrating NO metabolite produced ...
Protein-disulfide isomerase (PDI) is a ubiquitous dithiol-disulfide oxidoreductase that performs ... more Protein-disulfide isomerase (PDI) is a ubiquitous dithiol-disulfide oxidoreductase that performs an array of cellular functions, such as cellular signaling and responses to cell-damaging events. PDI can become dysfunctional by post-translational modifications, including those promoted by biological oxidants, and its dysfunction has been associated with several diseases in which oxidative stress plays a role. Because the kinetics and products of the reaction of these oxidants with PDI remain incompletely characterized, we investigated the reaction of PDI with the biological oxidant peroxynitrite. First, by determining the rate constant of the oxidation of PDI's redox-active Cys residues (Cysand Cys) by hydrogen peroxide (= 17.3 ± 1.3 msat pH 7.4 and 25 °C), we established that the measured decay of the intrinsic PDI fluorescence is appropriate for kinetic studies. The reaction of these PDI residues with peroxynitrite was considerably faster (= (6.9 ± 0.2) × 10ms), and both Cys re...
Peroxiredoxins (Prx) are abundant thiol peroxidases that react rapidly with H2O2, constituting an... more Peroxiredoxins (Prx) are abundant thiol peroxidases that react rapidly with H2O2, constituting an important antioxidant defense and acting as sensors and transmitters of H2O2 signals in cells. Certain Prxs are readily inactivated by H2O2 at submillimolar concentrations, whereas others are less sensitive. This oxidative inactivation, or hyperoxidation, occurs when H2O2 further reacts with the Cp-SOH producing Cp-SO2- and/or Cp-SO3-. Since HCO3- accelerates several of the reactions of H2O2 with biothiols, we examined whether HCO3- could increase recombinant human peroxiredoxin 1 (Prx1) hyperoxidation by H2O2. Kinetic studies of NADPH consumption by Prx1 in presence of yeast Trx, yeast Trr and various H2O2 concentrations in the absence and presence of HCO3- (25 mM) provided values of Chyper1% of 335 µM and 110 µM, respectively. Western blot (with an antiPrx-SO3-/SO2- antibody) analyses and Nano-ESI-Q-TOF MS/MS analyses of incubations of Prx1 (2.5 µM) and H2O2 (25 µM) showed higher leve...
Access to protein substrates homogenously modified by ubiquitin (Ub) is critical for biophysical ... more Access to protein substrates homogenously modified by ubiquitin (Ub) is critical for biophysical and biochemical investigations aimed at deconvoluting the myriad biological roles for Ub. Current chemical strategies for protein ubiquitylation, however, employ temporary ligation auxiliaries that are removed under harsh denaturing conditions and have limited applicability. We report an unprecedented aromatic thiol-mediated N-O bond cleavage and its application towards native chemical ubiquitylation with the ligation auxiliary 2-aminooxyethanethiol. Our interrogation of the reaction mechanism suggests a disulfide radical anion as the active species capable of cleaving the N-O bond. The successful semisynthesis of full-length histone H2B modified by the small ubiquitin-like modifier-3 (SUMO-3) protein further demonstrates the generalizability and compatibility of our strategy with folded proteins.
Despite extensive investigation of the irreversible oxidations undergone by proteins in vitro and... more Despite extensive investigation of the irreversible oxidations undergone by proteins in vitro and in vivo, the products formed from the oxidation of Trp residues remain incompletely understood. Recently, we characterized a ditryptophan cross-link produced by the recombination of hSOD1-tryptophanyl radicals generated from attack of the carbonate radical produced during the bicarbonate-dependent peroxidase activity of the enzyme. Here, we examine whether the ditryptophan cross-link is produced by the attack of the carbonate radical on proteins other than hSOD1. To this end, we treated hen egg white lysozyme with photolytically and enzymatically generated carbonate radical. The radical yields were estimated and the lysozyme modifications were analyzed by SDS-PAGE, western blot, enzymatic activity and MS/MS analysis. Lysozyme oxidation by both systems resulted in its inactivation and dimerization. Lysozyme treated with the photolytic system presented monomers oxidized to hydroxy-tryptophan at Trp(28) and Trp(123) and N-formylkynurenine at Trp(28), Trp(62) and Trp(123). Lysozyme treated with the enzymatic system rendered monomers oxidized to N-formylkynurenine at Trp(28). The dimers were characterized as lysozyme-Trp(28)-Trp(28)-lysozyme and lysozyme-Trp(28)-Trp(32)-hSOD1. The results further demonstrate that the carbonate radical is prone to causing biomolecule cross-linking and hence, may be a relevant player in pathological mechanisms. The possibility of exploring the formation of ditryptophan cross-links as a carbonate radical biomarker is discussed.
<p>A) The body weight loss in G93A rats untreated (n = 11) and treated (n = 9) with tempol;... more <p>A) The body weight loss in G93A rats untreated (n = 11) and treated (n = 9) with tempol; wild-type rats treated with tempol (n = 11) exhibited similar behavior to those not treated (not shown). Female rats received tempol (30 mM) in their drinking water starting at 7 weeks of age. B) The motor scores of the same animals were determined as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055868#s2" target="_blank">Materials and Methods</a>; the values shown correspond to the mean ± standard error; *p<0.05 (two-way ANOVA with Bonferroni post-test). C) The survival analysis of untreated and treated G93A rats. The results are shown as a Kaplan-Meier plot and are significantly different at p<0.05 (log-rank test). The end points of the non-treated and treated rats were 127.6±2.2 and 136.8±4.3 days, respectively.</p
How to cite Complete issue More information about this article Journal's homepage in redalyc... more How to cite Complete issue More information about this article Journal's homepage in redalyc.org Scientific Information System
Recent studies have provided evidence for a dual role of nitric oxide (NO) during murine leishman... more Recent studies have provided evidence for a dual role of nitric oxide (NO) during murine leishmaniasis. To explore this problem, we monitored the formation of NO and its derived oxidants during the course of Leishmania amazonensis infection in tissues of susceptible (BALB/c) and relatively resistant (C57BL/6) mice. NO production was detected directly by low-temperature electron paramagnetic resonance spectra of animal tissues. Both mouse strains presented detectable levels of hemoglobin nitrosyl (HbNO) complexes and of heme nitrosyl and iron-dithiol-dinitrosyl complexes in the blood and footpad lesions, respectively. Estimation of the nitrosyl complex levels demonstrated that most of the NO is synthesized in the footpad lesions. In agreement, immunohistochemical analysis of the lesions demonstrated the presence of nitrotyrosine in proteins of macrophage vacuoles and parasites. Since macrophages lack myeloperoxidase, peroxynitrite is likely to be the nitrating NO metabolite produced ...
Protein-disulfide isomerase (PDI) is a ubiquitous dithiol-disulfide oxidoreductase that performs ... more Protein-disulfide isomerase (PDI) is a ubiquitous dithiol-disulfide oxidoreductase that performs an array of cellular functions, such as cellular signaling and responses to cell-damaging events. PDI can become dysfunctional by post-translational modifications, including those promoted by biological oxidants, and its dysfunction has been associated with several diseases in which oxidative stress plays a role. Because the kinetics and products of the reaction of these oxidants with PDI remain incompletely characterized, we investigated the reaction of PDI with the biological oxidant peroxynitrite. First, by determining the rate constant of the oxidation of PDI's redox-active Cys residues (Cysand Cys) by hydrogen peroxide (= 17.3 ± 1.3 msat pH 7.4 and 25 °C), we established that the measured decay of the intrinsic PDI fluorescence is appropriate for kinetic studies. The reaction of these PDI residues with peroxynitrite was considerably faster (= (6.9 ± 0.2) × 10ms), and both Cys re...
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