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Amplifying the Death Signal

S-glutathionylation of Fas promotes clustering and activation of this death receptor. Binding of the Fas ligand (FasL) to the death receptor Fas triggers the formation of a death-inducing signaling complex that leads to apoptosis. Conditions that produce oxidative stress can sensitize cells to die. Anathy et al. show that Fas is modified by the formation of a sulfide bond with glutathione (called PSSG for protein S-S glutathione). Immunoblot analysis showed that, in response to Fas, PSSG were detected before the formation of reactive oxygen species generated by activation of NADPH oxidases, and PSSG formation was not prevented by pharmacologically inhibiting NADPH oxidase activity. Instead, PSSG appeared to depend on a decrease in the thiol transferase glutaredoxin 1 (Grx1), which catalyzes the reduction of PSSG, restoring the cysteine to the protein. Cells respond to Fas by activating caspase-8 and -3, and addition of the caspase inhibitor ZVAD-FMK prevented the formation of PSSG and Fas-SSG (glutathionylated Fas) and the decrease in Grx1. Fas-SSG, after application of FasL, was detected by immunoprecipitating with antibodies against glutathione and then immunoblotting for Fas. In vitro assays showed that Grx1 was a substrate for caspase-3 and -8, with caspase-3 showing greater activity. Surprisingly, activation of caspase-3 in the absence of Fas ligation did not trigger degradation of Grx1 or Fas-SSG. Cells in which Grx1 was reduced by silencing RNA (siRNA) or cells genetically deficient in Grx1 showed increased sensitivity to the apoptotic effects of FasL, as well as greater abundance and faster formation of Fas-SSG. Overexpression of Grx1 reduced Fas-SSG formation, decreased the Fas-mediated activation of caspases, and enhanced cell survival. Fas forms multimeric complexes and interacts with lipid rafts. Anathy et al. show that, in response to FasL, Fas-SSG accumulates in lipid rafts and in a high molecular weight complex, both of which were decreased in cells overexpressing Grx1. Mutation analysis showed that Cys294 (mouse) was the site of S-glutathionylation and, when this site was mutated to alanine and the protein was expressed in cells lacking wild-type Fas, FasL-induced caspase activation and cell death were largely blocked. Thus, caspase-mediated degradation of Grx1 allows Fas to become S-glutathionylated, which increases its accumulation in lipid rafts and strengthens the death signal. V. Anathy, S. W. Aesif, A. S. Guala, M. Havermans, N. L. Reynaert, Y.-S. Ho, R. C. Budd, Y. M. W. Janssen-Heininger, Redox amplification of apoptosis by caspase-dependent cleavage of glutaredoxin 1 and S-glutathionylation of Fas. J. Cell Biol. 184, 241–252 (2008). [Abstract] [Full Text]