title: Cytosolic Triosephosphate Isomerase fromArabidopsis thalianaIs Reversibly Modified by Glutathione on Cysteines 127 and 218.
abstract: In plant cells, an increase in cellular oxidants can have multiple effects, including the promotion of mixed disulfide bonds between glutathione and some proteins (S-glutathionylation). The present study focuses on the cytosolic isoform of the glycolytic enzyme triosephosphate isomerase (cTPI) fromArabidopsis thalianaand its reversible modification by glutathione. We used purified recombinant cTPI to demonstrate the enzyme sensitivity to inhibition byN-ethylmaleimide, hydrogen peroxide and diamide. Treatment of cTPI with diamide in the presence of reduced glutathione (GSH) led to a virtually complete inhibition of its enzymatic activity byS-glutathionylation. Recombinant cTPI was also sensitive to the oxidized form of glutathione (GSSG) in the micromolar range. Activity of cTPI was restored after reversion ofS-glutathionylation by two purified recombinantA. thalianacytosolic glutaredoxins (GRXs). GRXs-mediated deglutathionylation of cTPI was dependent on a GSH-regenerating system. Analysis of cTPI by mass spectrometry afterS-glutathionylation by GSSG revealed that two Cys residues (Cys127 and Cys218) were modified by glutathione. The role of these two residues was assessed using site-directed mutagenesis. Mutation of Cys127 and Cys218 to Ser separately or together caused different levels of decrease in enzyme activity, loss of stability, as well as alteration of intrinsic fluorescence, underlining the importance of these Cys residues in protein conformation. Comparison of wild-type and mutant proteins modified with biotinyl glutathione ethyl ester (BioGEE) showed partial binding with single mutants and total loss of binding with the double mutant, demonstrating that both Cys residues were significantlyS-glutathionylated. cTPI modification with BioGEE was reversed using DTT. Our study provides the first identification of the amino acid residues involved in cTPIS-glutathionylation and supports the hypothesis that this reversible modification could be part of an oxidative stress response pathway.
authors.affiliation: Institut de Recherche en Biologie Végétale, Département de sciences biologiques, Université de Montréal Montréal, QC, Canada.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Dumont
authors.affiliation: Morden Research and Development Centre, Agriculture and Agri-Food Canada Morden, MB, Canada.
authors.forename: Natalia V
authors.initials: NV
authors.lastname: Bykova
authors.affiliation: Institut de Recherche en Biologie Végétale, Département de sciences biologiques, Université de Montréal Montréal, QC, Canada.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Pelletier
authors.affiliation: Institut de Recherche en Biologie Végétale, Département de sciences biologiques, Université de Montréal Montréal, QC, Canada.
authors.forename: Sonia
authors.initials: S
authors.lastname: Dorion
authors.affiliation: Institut de Recherche en Biologie Végétale, Département de sciences biologiques, Université de Montréal Montréal, QC, Canada.
authors.forename: Jean
authors.initials: J
authors.lastname: Rivoal
keywords: Arabidopsis thaliana; S-glutathionylation; cysteine; cytosolic triosephosphate isomerase; deglutathionylation; glutaredoxin; redox post-translational modification