Journal Article

Dual regulation of cytosolic ascorbate peroxidase (APX) by tyrosine nitration and S-nitrosylation

Sánchez-Calvo, Beatriz, Valderrama, Raquel, Mata-Pérez, Capilla, López-Jaramillo, Javier, Carreras, Alfonso, Corpas, Francisco J, Barroso, Juan B, Begara-Morales, Juan C, Chaki, Mounira, Padilla, María N

2014

Overview

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 (150mM 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.

• Publisher: Oxford University Press [etc.],Oxford University Press
• Subject: Amino Acid Sequence / Amino Acids / Amino Acids - metabolism / ascorbate peroxidase / Ascorbate Peroxidases / Ascorbate Peroxidases - metabolism / binding sites / biotin / chemistry / Chloroplasts / Chromatography, Liquid / Cytosol / Cytosol - enzymology / drug effects / Electrophoresis, Polyacrylamide Gel / enzyme activity / Enzymes / enzymology / hydrogen peroxide / Hydrogen Peroxide - metabolism / isolation & purification / Lipid Peroxidation / Lipid Peroxidation - drug effects / Mass Spectrometry / metabolism / Models, Molecular / Molecular Sequence Data / Nitration / nitric oxide / Nitrosation / Nitrosation - drug effects / Oxidative Stress / Oxidative Stress - drug effects / Oxides / peas / Peptides / Peptides - chemistry / Peroxynitrous Acid / Peroxynitrous Acid - pharmacology / pharmacology / Physiological regulation / physiology / Pisum sativum - drug effects / Pisum sativum - enzymology / Pisum sativum - physiology / Plant physiology / Plant Proteins / Plant Proteins - isolation & purification / Plant Proteins - metabolism / Protein Multimerization / Protein Multimerization - drug effects / Proteins / Proteomics / Recombinant Proteins / Recombinant Proteins - metabolism / RESEARCH PAPER / S-Nitrosoglutathione / S-Nitrosoglutathione - pharmacology / Salinity / sodium chloride / Sodium Chloride - pharmacology / Stress, Physiological / Stress, Physiological - drug effects / tyrosine / Tyrosine - metabolism