Caspase activation involves the formation of the aposome, a large (approximately 700 kDa) caspase-activating complex.
In mammals, apoptotic protease-activating factor 1 (Apaf-1), cytochrome c, and dATP activate caspase-9, which initiates the postmitochondrial-mediated caspase cascade by proteolytic cleavage/activation of effector caspases to form active approximately 60-kDa heterotetramers. We now demonstrate that activation of caspases either in apoptotic cells or following dATP activation of cell lysates results in the formation of two large but different sized protein complexes, the "aposome" and the "microaposome". Surprisingly, most of the DEVDase activity in the lysate was present in the aposome and microaposome complexes with only small amounts of active caspase-3 present as its free approximately 60-kDa heterotetramer. The larger aposome complex (M(r) = approximately 700,000) contained Apaf-1 and processed caspase-9, -3, and -7. The smaller microaposome complex (M(r) = approximately 200,000-300,000) contained active caspase-3 and -7 but little if any Apaf-1 or active caspase-9. Lysates isolated from control THP.1 cells, prior to caspase activation, showed striking differences in the distribution of key apoptotic proteins. Apaf-1 and procaspase-7 may be functionally complexed as they eluted as an approximately 200-300-kDa complex, which did not have caspase cleavage (DEVDase) activity. Procaspase-3 and -9 were present as separate and smaller 60-90-kDa (dimer) complexes. During caspase activation, Apaf-1, caspase-9, and the effector caspases redistributed and formed the aposome. This resulted in the processing of the effector caspases, which were then released, possibly bound to other proteins, to form the microaposome complex.
Cain
K
K
Medical Research Council Toxicology Unit, Hodgkin Building, University of Leicester, P. O. Box 138, Lancaster Road, Leicester LE1 9HN, United Kingdom. kc5@le.ac.uk
Brown
D G
DG
Langlais
C
C
Cohen
G M
GM
D017209:Apoptosis / Q000502:physiology*; D053477:Apoptotic Protease-Activating Factor 1; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D002451:Cell Compartmentation; D002458:Cell Fractionation; D004789:Enzyme Activation; D006801:Humans; D009000:Monocytes; D009097:Multienzyme Complexes / Q000737:chemistry; D009842:Oligopeptides / Q000378:metabolism; D010447:Peptide Hydrolases / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
C506592:APAF1 protein, human; D053477:Apoptotic Protease-Activating Factor 1; D009097:Multienzyme Complexes; D009842:Oligopeptides; D011506:Proteins; C120774:aposome; D010447:Peptide Hydrolases; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases; C108711:DEVDase