Glutathionylation of human thioredoxin: A possible crosstalk between the glutathione and thioredoxin systems

Abstract

To identify proteins undergoing glutathionylation (formation of protein-glutathione mixed disulfides) in human T cell blasts, we radiolabeled the glutathione pool with 35 S, exposed cells to the oxidant diamide, and analyzed cellular proteins by two-dimensional electrophoresis. One of the proteins undergoing glutathionylation was identified by molecular weight, isoelectric point, and immunoblotting as thioredoxin (Trx). Incubation of recombinant human Trx with glutathione disulfide or S-nitrosoglutathione led to the formation of glutathionylated Trx, identified by matrixassisted laser desorption ionization-time-of-flight mass spectrometry. The glutathionylation site was identified as Cys-72. Glutathionylation of rhTrx abolished its enzymatic activity as insulin disulfide reductase in the presence of NADPH and Trx reductase. Activity was, however, regained with sigmoidal kinetics, indicating a process of autoactivation due to the ability of Trx to deglutathionylate itself. These data suggest that the intracellular glutathione͞glutathione disulfide ratio, an indicator of the redox state of the cell, can regulate Trx functions reversibly through thiol-disulfide exchange reactions. T hioredoxin (Trx; ref. 1), a ubiquitous redox protein, is an essential cofactor electron donor for ribonucleotide reductase, but also has many other cellular functions, including regulation of transcription factors, apoptosis, and antioxidant activity and can act exogenously as a redox active growth factor (1, 2). The catalytic activity of Trx resides in its active site where the two redox active Cyss (Cys-31 and Cys-34 in human Trx) undergo reversible oxidation͞reduction. In addition to the conserved Cys residues in the active site, three additional structural Cys residues (Cys-61, Cys-68, and Cys-72) are present in the structure of human Trx.The present paper describes the experiments that led us to the conclusion that Trx can undergo glutathionylation in T cells exposed to oxidative stress. Protein glutathionylation, the formation of a disulfide between a Cys in a protein and the Cys in the tripeptide glutathione (GSH), is a modification that can be induced in cells by oxidative stress. Glutathionylation can occur by direct oxidation of a protein and GSH, by a thiol-disulfide exchange between a protein Cys and oxidized glutathione (GSSG), and also with the intermediacy of S-nitrosoglutathione (GSNO; refs. 3 and 4). Glutathionylation of proteins is reversible, as those proteins can be reduced by glutaredoxins (5, 6), and the process serves to regulate protein functions by the redox state of the cell (i.e., by the GSSG͞GSH ratio; refs. 7-9).To demonstrate the glutathionylation of Trx, we first labeled the intracellular GSH pool of the T-cell blasts with [ 35 S]Cys and analyzed the cell lysate by two dimensional electrophoresis under nonreducing conditions. A spot was found in the autoradiographic protein map with IP͞M r corresponding to those of Trx. In the second part of the study, we incubated recombinant human Trx in the presence or ...

Radical-free biology of oxidative stress

“…Irreversible modification of C73 with acrolein or 4-hydroxynonenal creates a form that inhibits Trx reductase-1 (TrxR1) and induces pathogenic phenotype (54). This Cys is also a site for GS-ylation and protein-protein disulfide formation, suggesting that physiological regulation could occur through this Cys (23). The remaining Cys residues, C62 and C69, form a second disulfide under oxidative stress conditions (189).…”

Redox-based regulation of signal transduction: Principles, pitfalls, and promises

“…Intriguingly, in addition to Grx itself, Trx and PDI are targets for S-glutathionylation [109,110]. S-glutathionylation of Trx at cysteine 72, abolished the disulfide reductase activity of Trx [109], whereas S-nitrosylation of cysteine 63 was reported to increase Trx activity [107]. S-nitrosylation of cysteine 72 has been recently implicated in transnitrosation of caspases, but did not seem to affect Trx activity [111,112].…”

“…Furthermore, Trx [107,108] and protein disulfide isomerase (PDI) (see below) are also targets for S-nitrosylation. Intriguingly, in addition to Grx itself, Trx and PDI are targets for S-glutathionylation [109,110]. S-glutathionylation of Trx at cysteine 72, abolished the disulfide reductase activity of Trx [109], whereas S-nitrosylation of cysteine 63 was reported to increase Trx activity [107].…”

Glutathionylation of chloroplast thioredoxin f is a redox signaling mechanism in plants

“…This modification notably occurs in response to enhanced production of reactive oxygen species (ROS) and͞or increases in oxidized glutathione (GSSG). Glutathionylation can protect proteins from irreversible oxidation and͞or modulate their activity (12)(13)(14)(15)(16)(17). Despite its theoretical appeal as a mechanism transmitting oxidative signals under stress, very little is known about glutathionylation in plants.…”