title: The apoptosome pathway to caspase activation in primary human neutrophils exhibits dramatically reduced requirements for cytochrome C.
abstract: Caspase activation is a central event in numerous forms of apoptosis and results in the proteolytic degradation of multiple substrate proteins that contribute to the apoptotic phenotype. An important route to caspase activation proceeds via assembly of the "apoptosome" as a result of the cell stress-associated release of mitochondrial cytochrome c. Previous studies have shown that primary neutrophils are largely incapable of mitochondrial respiration, suggesting that these cells either lack functional mitochondria or possess a defective respiratory chain. This prompted us to examine whether neutrophils retain an intact cytochrome c/apoptotic protease-activating factor 1 (Apaf-1) pathway to caspase activation and apoptosis. We show that primary human neutrophils contain barely detectable levels of cytochrome c as well as other mitochondrial proteins. Surprisingly, neutrophil cell-free extracts readily supported Apaf-1-dependent caspase activation, suggesting that these cells may assemble cytochrome c-independent apoptosomes. However, further analysis revealed that the trace amount of cytochrome c present in neutrophils is both necessary and sufficient for Apaf-1-dependent caspase activation in these cells. Thus, neutrophils have a lowered threshold requirement for cytochrome c in the Apaf-1-dependent cell death pathway. These observations suggest that neutrophils retain cytochrome c for the purpose of assembling functional apoptosomes rather than for oxidative phosphorylation.
authors.affiliation: Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin 2, Ireland.
authors.forename: Brona M
authors.initials: BM
authors.lastname: Murphy
authors.forename: Amanda J
authors.initials: AJ
authors.lastname: O'Neill
authors.forename: Colin
authors.initials: C
authors.lastname: Adrain
authors.forename: R William G
authors.initials: RW
authors.lastname: Watson
authors.forename: Seamus J
authors.initials: SJ
authors.lastname: Martin
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D053477:Apoptotic Protease-Activating Factor 1; D020169:Caspases / Q000378:metabolism*; D002458:Cell Fractionation; D002474:Cell-Free System; D002478:Cells, Cultured; D003574:Cytochrome c Group / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D008928:Mitochondria / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000378:metabolism; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*
chemical_list: C506592:APAF1 protein, human; D053477:Apoptotic Protease-Activating Factor 1; D003574:Cytochrome c Group; D003838:Deoxyadenine Nucleotides; D005456:Fluorescent Dyes; D024101:Mitochondrial Proteins; D011506:Proteins; D020169:Caspases