Protein glutathionylation regulates FasL‐induced apoptosis.

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Apoptosis is a basic homeostatic mechanism involved in a variety physiological processes and whose deregulation leads to the progression of several human diseases. It is now clear that changes in the cellular redox homeostasis regulate apoptosis. However, the molecular mechanisms involved have remained largely elusive. Recently, we demonstrated that reduced glutathione (GSH) depletion regulates apoptosis independent of the excessive accumulation of ROS and widespread oxidative stress (J Biol Chem. 2006, 281:29542; J Biol Chem. 2007, 282:30452; J Biol Chem. 2008, Oct 21, Epub) suggesting a direct role of GSH homeostasis in the redox‐dependent regulation of apoptosis. To evaluate potential mechanisms in this process, we studied the role of protein glutathionylation (a reversible oxidative posttranslational modification mediated by changes in GSH homeostasis) on the progression of apoptosis by FasL. FasL induced a dose‐dependent increase in protein glutathionylation in Jurkat cells measured using an anti‐glutathionylated (P‐SSG) residue antibody by western‐immunoblots. FasL‐induced protein glutathionylation was shown to be prevented by inhibition of GSH depletion with high extracellular GSH medium, which suggests that GSH depletion promotes protein glutathionylation during apoptosis. Immunoprecipitation assays demonstrate that FasL induced a dose‐dependent glutathionylation of execution caspases suggesting a regulatory role for protein glutathionylation on caspase activation. GSH depletion is paralleled by a switch in the GSH/glutathione disulfide (GSSG) ratio in favor to an increase in GSSG. Thus, we decided to study if GSSG mediates protein glutathionylation of execution caspases. Using mass spectrometry analysis we demonstrated that in in vitro cell‐free assays, GSSG was able to induce execution caspase glutathionylation at specific cysteinyl moieties with low pKa values distinct from the active site of caspases. In vitro gluathionylation of caspases by GSSG was reversed under reducing conditions (DTT) and in the presence of high GSH concentrations. These results demonstrate that FasL‐induced GSH depletion mediates an increase in glutathionylation of selective proteins. They also suggest that the reduction in GSH/GSSG ratio mediates execution caspase glutathionylation during apoptosis. We propose that GSH homeostasis regulates apoptosis via protein glutathionylation.