Acute Cadmium Exposure Inactivates Thioltransferase (Glutaredoxin), Inhibits Intracellular Reduction of Protein-glutathionyl-mixed Disulfides, and Initiates Apoptosis

Abstract

Oxidative stress broadly impacts cells, initiating regulatory pathways as well as apoptosis and necrosis. A key molecular event is protein S-glutathionylation, and thioltransferase (glutaredoxin) is a specific and efficient catalyst of protein-SSG reduction. In this study 30-min exposure of H9 and Jurkat cells to cadmium inhibited intracellular protein-SSG reduction, and this correlated with inhibition of the thioltransferase system, consistent with thioltransferase being the primary intracellular catalyst of deglutathionylation. The thioredoxin system contributed very little to total deglutathionylase activity. Thioltransferase and GSSG reductase in situ displayed similar dose-response curves (50% inhibition near 10 M cadmium in extracellular buffer). Acute cadmium exposure also initiated apoptosis, with H9 cells being more sensitive than Jurkat. Moreover, transfection with antisense thioltransferase cDNA was incompatible with cell survival. Collectively, these data suggest that thioltransferase has a vital role in sulfhydryl homeostasis and cell survival. In separate experiments, cadmium inhibited the isolated component enzymes of the thioltransferase and thioredoxin systems, consistent with the vicinal dithiol nature of their active sites: thioltransferase (IC 50 Ϸ 1 M), GSSG reductase (IC 50 Ϸ 1 M), thioredoxin (IC 50 Ϸ 8 M), thioredoxin reductase (IC 50 Ϸ 0.2 M). Disruption of the vicinal dithiol on thioltransferase (via oxidation to C22-SS-C25; or C25S mutation) protected against cadmium, consistent with a dithiol chelation mechanism of inactivation.

Targeted disruption of the glutaredoxin 1 gene does not sensitize adult mice to tissue injury induced by ischemia/reperfusion and hyperoxia

“…8A), indicating that Grx1 is an important cellular mechanism of de-glutathionylation, as shown in several previous studies [21,22,66,[68][69][70][71]. For example, de-glutathionylation of actin in NIH3T3 fibroblast cells, and its polymerization and migration to the cell periphery, in response to EGF-stimulation was shown to be abolished in cells in which Grx1 was knocked down by interference RNA [71].…”

“…As shown in Fig. 8B, the glutathionyl moiety of most of the S-glutathionylated proteins, with the exception of a few high molecular-weight proteins, are removed in wild-type MEFs within one hour after termination of H 2 O 2 treatment, consistent with rapid Grx1-catalyzed de-glutathionylation which occurs within minutes for most cellular proteins [22]. For Grx1-deficient MEFs also, the majority of protein-SSG mixed disulfides were de-glutathionylated at one hour, consistent with a typical non-enzymatic time course.…”

“…This assay of GSH-dependent de-glutathionylation of protein-SSG is highly selective for Grx [22]. Also, the radiolabel assay avoids the multiple flaws associated with the GSSG reductase-coupled spectrophotometric analysis of tissue homogenates, including interference by light scattering and non-specific NADPH oxidase activity.…”

“…The protein-SSG mixed disulfides are reduced efficiently by Grx via a monothiol mechanism, involving a nucleophilic double displacement reaction in which GSH serves to recycle the enzyme [20]. Furthermore, previous tissue and cell culture studies have indicated that glutathione-dependent catalysis by Grx accounts for most of the deglutathionylation activity [21,22]. Therefore, Grx is believed to play a pivotal role in defense against oxidative stress and in redox regulation of cellular function [3].…”

Triosephosphate Isomerases in Italian Ryegrass (Lolium multiflorum): Characterization and Susceptibility to Herbicides

“…Some reports document that TPIs can be regulated by S-glutathionylation, through the formation of a mixed disulfide between SH residues of the protein and glutathione oxidized GSSG (21,22,44). The S-glutathionylation is an antioxidant device that reduces the impact of oxidants on proteins, and at the same time it is also a regulatory system for many enzyme activities (20,(45)(46)(47).…”