title: Mechanisms of nitrosylation and denitrosylation of cytoplasmic glyceraldehyde-3-phosphate dehydrogenase from Arabidopsis thaliana.
abstract: Nitrosylation is a reversible post-translational modification of protein cysteines playing a major role in cellular regulation and signaling in many organisms, including plants where it has been implicated in the regulation of immunity and cell death. The extent of nitrosylation of a given cysteine residue is governed by the equilibrium between nitrosylation and denitrosylation reactions. The mechanisms of these reactions remain poorly studied in plants. In this study, we have employed glycolytic GAPDH from Arabidopsis thaliana as a tool to investigate the molecular mechanisms of nitrosylation and denitrosylation using a combination of approaches, including activity assays, the biotin switch technique, site-directed mutagenesis, and mass spectrometry. Arabidopsis GAPDH activity was reversibly inhibited by nitrosylation of catalytic Cys-149 mediated either chemically with a strong NO donor or by trans-nitrosylation with GSNO. GSNO was found to trigger both GAPDH nitrosylation and glutathionylation, although nitrosylation was widely prominent. Arabidopsis GAPDH was found to be denitrosylated by GSH but not by plant cytoplasmic thioredoxins. GSH fully converted nitrosylated GAPDH to the reduced, active enzyme, without forming any glutathionylated GAPDH. Thus, we found that nitrosylation of GAPDH is not a step toward formation of the more stable glutathionylated enzyme. GSH-dependent denitrosylation of GAPC1 was found to be linked to the [GSH]/[GSNO] ratio and to be independent of the [GSH]/[GSSG] ratio. The possible importance of these biochemical properties for the regulation of Arabidopsis GAPDH functions in vivo is discussed.
authors.affiliation: Laboratory of Plant Redox Biology, Department of Pharmacy and Biotechnology, University of Bologna, Via Irnerio 42, 40126 Bologna, Italy.
authors.forename: Mirko
authors.initials: M
authors.lastname: Zaffagnini
authors.forename: Samuel
authors.initials: S
authors.lastname: Morisse
authors.forename: Mariette
authors.initials: M
authors.lastname: Bedhomme
authors.forename: Christophe H
authors.initials: CH
authors.lastname: Marchand
authors.forename: Margherita
authors.initials: M
authors.lastname: Festa
authors.forename: Nicolas
authors.initials: N
authors.lastname: Rouhier
authors.forename: Stéphane D
authors.initials: SD
authors.lastname: Lemaire
authors.forename: Paolo
authors.initials: P
authors.lastname: Trost
mesh_terms: D017360:Arabidopsis / Q000201:enzymology*; D003593:Cytoplasm / Q000201:enzymology*; D005978:Glutathione / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D010084:Oxidation-Reduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D009569:Nitric Oxide; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D005978:Glutathione
keywords: Arabidopsis; Denitrosylation; Glutathione; Glutathionylation; Glyceraldehyde-3-phosphate Dehydrogenase; Nitrosylation; Redox Signaling; Thioredoxin