title: Pro-caspase-8 is predominantly localized in mitochondria and released into cytoplasm upon apoptotic stimulation.
abstract: The recruitment and cleavage of pro-caspase-8 to produce the active form of caspase-8 is a critical biochemical event in death receptor-mediated apoptosis. However, the source of pro-caspase-8 available for activation by apoptotic triggers is unknown. In human fibroblasts and mouse clonal striatal cells, confocal microscopy revealed that pro-caspase-8 immunofluorescence was colocalized with cytochrome c in mitochondria and was also distributed diffusely in some nuclei. Biochemical analysis of subcellular fractions indicated that pro-caspase-8 was enriched in mitochondria and in nuclei. Pro-caspase-8 was found in the intermembrane space, inner membrane, and matrix of mitochondria after limited digestion of mitochondrial fractions, and this distribution was confirmed by immunogold electron microscopy. Pro-caspase-8 and cytochrome c were released from isolated mitochondria that were treated with an inhibitor of the ADP/ATP carrier atractyloside, which opens the mitochondria permeability transition pore. Release was blocked by the mitochondria permeability transition pore inhibitor cyclosporin A (CsA). After clonal striatal cells were exposed for 6 h to an apoptotic inducer tumor necrosis factor-alpha (TNF-alpha), mitochondria immunoreactive for cytochrome c and pro-caspase-8 became clustered at perinuclear sites. Pro-caspase-8 and cytochrome c levels decreased in mitochondrial fractions and increased, along with pro-caspase-8 cleavage products, in the cytoplasm of the TNF-alpha-treated striatal cells. CsA blocked the TNF-alpha-induced release of pro-caspase 8 but not cytochrome c. Internucleosomal DNA fragmentation started at 6 h and peaked 12 h after TNF-alpha treatment. These results suggest that pro-caspase-8 is predominantly localized in mitochondria and is released upon apoptotic stimulation through a CsA-sensitive mechanism.
authors: Laboratory of Cellular Neurobiology, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts 02129, USA.
authors: Z H
authors: ZH
authors: Qin
authors: Y
authors: Y
authors: Wang
authors: K K
authors: KK
authors: Kikly
authors: E
authors: E
authors: Sapp
authors: K B
authors: KB
authors: Kegel
authors: N
authors: N
authors: Aronin
authors: M
authors: M
authors: DiFiglia
mesh_terms: D017209:Apoptosis; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000032:analysis / Q000378:metabolism*; D002478:Cells, Cultured; D016572:Cyclosporine / Q000494:pharmacology; D003593:Cytoplasm / Q000201:enzymology*; D004792:Enzyme Precursors / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007473:Ion Channels; D008565:Membrane Proteins / Q000502:physiology; D008928:Mitochondria / Q000201:enzymology*; D000226:Mitochondrial ADP, ATP Translocases / Q000502:physiology; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D004792:Enzyme Precursors; D007473:Ion Channels; D008565:Membrane Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D014409:Tumor Necrosis Factor-alpha; D016572:Cyclosporine; D000226:Mitochondrial ADP, ATP Translocases; C505530:CASP8 protein, human; C506433:CASP9 protein, human; C505526:Casp8 protein, mouse; C506434:Casp9 protein, mouse; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases