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) from Arabidopsis thaliana and its reversible modification by glutathione. We used purified recombinant cTPI to demonstrate the enzyme sensitivity to inhibition by N-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 by S-glutathionylation. Recombinant cTPI was also sensitive to the oxidized form of glutathione (GSSG) in the micromolar range. Activity of cTPI was restored after reversion of S-glutathionylation by two purified recombinant A. thaliana cytosolic glutaredoxins (GRXs). GRXs-mediated deglutathionylation of cTPI was dependent on a GSH-regenerating system. Analysis of cTPI by mass spectrometry after S-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 significantly S-glutathionylated. cTPI modification with BioGEE was reversed using DTT. Our study provides the first identification of the amino acid residues involved in cTPI S-glutathionylation and supports the hypothesis that this reversible modification could be part of an oxidative stress response pathway.
Highlights
In plant cells, reactive oxygen species (ROS) such as superoxide anion (O2−), hydrogen peroxide (H2O2) and hydroxyl radical (OH) are normal by-products of many cellular processes like photosynthesis (Karpinski et al, 2003; Apel and Hirt, 2004) and aerobic metabolism (Miwa and Brand, 2003)
Protein S-glutathionylation results from the reaction of sulfenic acid thiol with reduced glutathione (GSH) or by thiol disulfide exchange between a reduced thiol group and oxidized glutathione (GSSG) leading to the formation of a mixed disulfide bond between glutathione and the targeted protein (Dalle-Donne et al, 2009; Zaffagnini et al, 2012d)
We demonstrate that S-glutathionylation of cTPI is reversible by A. thaliana glutaredoxin C1 (GRXC1) and glutaredoxin C2 (GRXC2)
Summary
ROS such as superoxide anion (O2−), hydrogen peroxide (H2O2) and hydroxyl radical (OH) are normal by-products of many cellular processes like photosynthesis (Karpinski et al, 2003; Apel and Hirt, 2004) and aerobic metabolism (Miwa and Brand, 2003). Many biotic and abiotic stresses can lead to abnormal accumulation of ROS (Inzé and Montagu, 1995; Mittler, 2002) which can potentially damage cell structures and impair metabolic activities through oxidation of biomolecules such as nucleic acids, fatty acids and proteins (Gill and Tuteja, 2010). Due to their nature, ROS play important roles in plant redox signaling under basal conditions and during stress response by triggering defense mechanisms against oxidative stress (Foyer and Noctor, 2009).
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