title: Dual regulation of cytosolic ascorbate peroxidase (APX) by tyrosine nitration and S-nitrosylation.
abstract: Post-translational modifications (PTMs) mediated by nitric oxide (NO)-derived molecules have become a new area of research, as they can modulate the function of target proteins. Proteomic data have shown that ascorbate peroxidase (APX) is one of the potential targets of PTMs mediated by NO-derived molecules. Using recombinant pea cytosolic APX, the impact of peroxynitrite (ONOO-) and S-nitrosoglutathione (GSNO), which are known to mediate protein nitration and S-nitrosylation processes, respectively, was analysed. While peroxynitrite inhibits APX activity, GSNO enhances its enzymatic activity. Mass spectrometric analysis of the nitrated APX enabled the determination that Tyr5 and Tyr235 were exclusively nitrated to 3-nitrotyrosine by peroxynitrite. Residue Cys32 was identified by the biotin switch method as S-nitrosylated. The location of these residues on the structure of pea APX reveals that Tyr235 is found at the bottom of the pocket where the haem group is enclosed, whereas Cys32 is at the ascorbate binding site. Pea plants grown under saline (150 mM NaCl) stress showed an enhancement of both APX activity and S-nitrosylated APX, as well as an increase of H2O2, NO, and S-nitrosothiol (SNO) content that can justify the induction of the APX activity. The results provide new insight into the molecular mechanism of the regulation of APX which can be both inactivated by irreversible nitration and activated by reversible S-nitrosylation.
authors.affiliation: Área de Bioquímica y Biología Molecular, Departamento de Biología Experimental, Facultad de Ciencias Experimentales, Ed. B3. Campus Universitario "Las Lagunillas" s/n, Universidad de Jaén, E-23071 Jaén, Spain.
authors.forename: Juan C
authors.initials: JC
authors.lastname: Begara-Morales
authors.forename: Beatriz
authors.initials: B
authors.lastname: Sánchez-Calvo
authors.forename: Mounira
authors.initials: M
authors.lastname: Chaki
authors.forename: Raquel
authors.initials: R
authors.lastname: Valderrama
authors.forename: Capilla
authors.initials: C
authors.lastname: Mata-Pérez
authors.forename: Javier
authors.initials: J
authors.lastname: López-Jaramillo
authors.forename: María N
authors.initials: MN
authors.lastname: Padilla
authors.forename: Alfonso
authors.initials: A
authors.lastname: Carreras
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Corpas
authors.forename: Juan B
authors.initials: JB
authors.lastname: Barroso
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D060387:Ascorbate Peroxidases / Q000378:metabolism*; D002853:Chromatography, Liquid; D003600:Cytosol / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006861:Hydrogen Peroxide / Q000378:metabolism; D015227:Lipid Peroxidation / Q000187:drug effects; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015538:Nitrosation / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects; D018532:Pisum sativum / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology; D010455:Peptides / Q000737:chemistry; D030421:Peroxynitrous Acid / Q000494:pharmacology; D010940:Plant Proteins / Q000302:isolation & purification / Q000378:metabolism; D055503:Protein Multimerization / Q000187:drug effects; D011994:Recombinant Proteins / Q000378:metabolism; D026422:S-Nitrosoglutathione / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D013312:Stress, Physiological / Q000187:drug effects; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D010455:Peptides; D010940:Plant Proteins; D011994:Recombinant Proteins; D030421:Peroxynitrous Acid; D014443:Tyrosine; D012965:Sodium Chloride; D026422:S-Nitrosoglutathione; D006861:Hydrogen Peroxide; D060387:Ascorbate Peroxidases
keywords: Ascorbate peroxidase; S-nitrosoglutathione; S-nitrosylation; nitration; nitric oxide; peroxynitrite; reactive nitrogen species; salinity stress.