Inhibition of peroxisomal hydroxypyruvate reductase (HPR1) by tyrosine nitration |
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Authors: | Francisco J Corpas Marina Leterrier Juan C Begara-Morales Raquel Valderrama Mounira Chaki Javier López-Jaramillo Francisco Luque José M Palma María N Padilla Beatriz Sánchez-Calvo Capilla Mata-Pérez Juan B Barroso |
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Institution: | 1. Departamento de Bioquímica, Biología Celular y Molecular de Plantas, Estación Experimental del Zaidín (EEZ), CSIC, Apartado 419, E-18080 Granada, Spain;2. Grupo de Señalización Molecular y Sistemas Antioxidantes en Plantas, Unidad Asociada al CSIC (EEZ), Área de Bioquímica y Biología Molecular, Universidad de Jaén, E-23071 Jaén, Spain;3. Instituto de Biotecnología, Universidad de Granada, Spain |
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Abstract: | BackgroundProtein tyrosine nitration is a post-translational modification (PTM) mediated by nitric oxide-derived molecules. Peroxisomes are oxidative organelles in which the presence of nitric oxide (NO) has been reported.MethodsWe studied peroxisomal nitroproteome of pea leaves by high-performance liquid chromatography with tandem mass spectrometry (LC–MS/MS) and proteomic approaches.ResultsProteomic analysis of peroxisomes from pea leaves detected a total of four nitro-tyrosine immunopositive proteins by using an antibody against nitrotyrosine. One of these proteins was found to be the NADH-dependent hydroxypyruvate reductase (HPR). The in vitro nitration of peroxisomal samples caused a 65% inhibition of HPR activity. Analysis of recombinant peroxisomal NADH-dependent HPR1 activity from Arabidopsis in the presence of H2O2, NO, GSH and peroxynitrite showed that the ONOO− molecule caused the highest inhibition of activity (51% at 5 mM SIN-1), with 5 mM H2O2 having no inhibitory effect. Mass spectrometric analysis of the nitrated recombinant HPR1 enabled us to determine that, among the eleven tyrosine present in this enzyme, only Tyr-97, Tyr-108 and Tyr-198 were exclusively nitrated to 3-nitrotyrosine by peroxynitrite. Site-directed mutagenesis confirmed Tyr198 as the primary site of nitration responsible for the inhibition on the enzymatic activity by peroxynitrite.ConclusionThese findings suggest that peroxisomal HPR is a target of peroxynitrite which provokes a loss of function.General significanceThis is the first report demonstrating the peroxisomal NADH-dependent HPR activity involved in the photorespiration pathway is regulated by tyrosine nitration, indicating that peroxisomal NO metabolism may contribute to the regulation of physiological processes under no-stress conditions. |
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Keywords: | HPR hydroxypyruvate reductase GSH reduced glutathione GSNO S-nitrosoglutathione NO nitric oxide ONOO&minus peroxynitrite PTM post-translational modification RNS reactive nitrogen species SIN-1 3-morpholinosydnonimine |
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