title: Caspase-8 mediates mitochondrial release of pro-apoptotic proteins in a manner independent of its proteolytic activity in apoptosis induced by the protein synthesis inhibitor acetoxycycloheximide in human leukemia Jurkat cells.
abstract: The cysteine protease caspase-8 plays an essential role in apoptosis induced by death receptors. The protein synthesis inhibitor acetoxycycloheximide (Ac-CHX) has been previously shown to induce rapid apoptosis mediated by the release of cytochrome c in human leukemia Jurkat cells. In this study, the novel molecular mechanism that links caspase-8 to the mitochondrial release of pro-apoptotic proteins has been identified. Jurkat cells deficient in caspase-8 were more resistant to Ac-CHX than wild-type Jurkat cells and manifested decreased apoptosis induction and caspase activation as well as inefficient release of cytochrome c, Smac/DIABLO, and AIF into the cytosol. In contrast to Fas ligand stimulation, the general caspase inhibitor barely prevented the mitochondrial release of these pro-apoptotic proteins in Ac-CHX-treated cells, suggesting that caspase-8 activity is dispensable for triggering the mitochondrial pathway in Ac-CHX-induced apoptosis. Consistent with this notion, caspase-8-deficient Jurkat cells reconstituted with catalytically inactive caspase-8 became sensitive to Ac-CHX and exhibited apoptosis, caspase activation, the liberation of pro-apoptotic proteins into the cytosol, and Bak conformational change as efficiently as wild-type Jurkat cells. Unlike caspase-3, -6, -7, and -9, a small but significant portion of caspase-8 was found to localize in mitochondria before and after exposure to Ac-CHX. These results clearly demonstrate that caspase-8 is able to mediate the mitochondrial release of pro-apoptotic proteins in a manner independent of its proteolytic activity in Ac-CHX-induced apoptosis.
authors.affiliation: Center for Biological Resources and Informatics and Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501.
authors.forename: Kimiko
authors.initials: K
authors.lastname: Kadohara
authors.forename: Michiko
authors.initials: M
authors.lastname: Nagumo
authors.forename: Shun
authors.initials: S
authors.lastname: Asami
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Tsukumo
authors.forename: Hikaru
authors.initials: H
authors.lastname: Sugimoto
authors.forename: Masayuki
authors.initials: M
authors.lastname: Igarashi
authors.forename: Kazuo
authors.initials: K
authors.lastname: Nagai
authors.forename: Takao
authors.initials: T
authors.lastname: Kataoka
mesh_terms: D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D051017:Apoptosis Regulatory Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D053181:Caspase 8 / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism; D003513:Cycloheximide / Q000031:analogs & derivatives* / Q000494:pharmacology; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D019169:Jurkat Cells; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D011487:Protein Conformation; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000494:pharmacology; D013347:Subcellular Fractions; D014162:Transfection; D051028:bcl-2-Associated X Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D051017:Apoptosis Regulatory Proteins; C494826:BAX protein, human; D011500:Protein Synthesis Inhibitors; D034741:RNA, Small Interfering; D051028:bcl-2-Associated X Protein; D045304:Cytochromes c; D003513:Cycloheximide; D053181:Caspase 8; D020169:Caspases; C004434:acetoxycycloheximide