title: Glutathione dependence of caspase-8 activation at the death-inducing signaling complex.
abstract: Apoptosis triggered by the death receptor CD95 (APO-1 or Fas) is pivotal for the homeostasis of the immune system. We investigated differential effects of glutathione depletion on CD95-triggered apoptosis in T and B cell lines as well as the glutathione dependence of caspase-8 activation. In B lymphoblastoid SKW6.4 cells, CD95-mediated apoptosis was prevented upstream of caspase-8 activation and caspase-3-like activity after acute glutathione depletion by diethyl maleate or cis-chloro-dinitrobenzene. Immunoprecipitation of the death-inducing signaling complex (DISC) revealed that the DISC was still formed in the glutathione-depleted state. The first cleavage step of procaspase-8 activation at the DISC, however, was inhibited. Accordingly, under cell-free conditions, radiolabeled procaspase-8 was processed at the immunoprecipitated DISC only after the addition of exogenous dithiothreitol or reduced glutathione. We also observed suppression of CD95-mediated apoptosis in glutathione-depleted CEM and H9 cells. Notably, Jurkat cells still died upon CD95 engagement under this condition, displaying incomplete nuclear fragmentation and a partial switch to necrosis; this may be explained by reduced cytochrome c/dATP-mediated caspase activation observed in cytosol from glutathione-depleted Jurkat cytosol. Our data indicate that the activation of caspase-8 at the DISC and hence CD95-mediated apoptosis induction shows a cell-specific requirement for intracellular glutathione.
authors.affiliation: Biochemical Pharmacology, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany. hentzeh@imcb.nus.edu.sg
authors.forename: Hannes
authors.initials: H
authors.lastname: Hentze
authors.forename: Ingo
authors.initials: I
authors.lastname: Schmitz
authors.forename: Markus
authors.initials: M
authors.lastname: Latta
authors.forename: Andreas
authors.initials: A
authors.lastname: Krueger
authors.forename: Peter H
authors.initials: PH
authors.lastname: Krammer
authors.forename: Albrecht
authors.initials: A
authors.lastname: Wendel
mesh_terms: D017209:Apoptosis; D015153:Blotting, Western; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D016923:Cell Death; D002460:Cell Line; D002474:Cell-Free System; D003600:Cytosol / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D011233:Precipitin Tests; D011485:Protein Binding; D014176:Protein Biosynthesis; D015398:Signal Transduction; D013997:Time Factors; D019014:fas Receptor / Q000096:biosynthesis
chemical_list: D019014:fas Receptor; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D005978:Glutathione