Abstract
The protease caspase-9 is activated on the apoptosome, a multiprotein signal transduction platform that assembles in response to mitochondria-dependent apoptosis initiation. Despite extensive molecular research, the assembly of the holo-apoptosome and the process of caspase-9 activation remain incompletely understood. Here, we therefore integrated quantitative data on the molecular interactions and proteolytic processes during apoptosome formation and apoptosis execution and conducted mathematical simulations to investigate the resulting biochemical signaling, quantitatively and kinetically. Interestingly, when implementing the homodimerization of procaspase-9 as a prerequisite for activation, the calculated kinetics of apoptosis execution and the efficacy of caspase-3 activation failed to replicate experimental data. In contrast, assuming a scenario in which procaspase-9 is activated allosterically upon binding to the apoptosome backbone, the mathematical simulations quantitatively and kinetically reproduced all experimental data. These data included a XIAP threshold concentration at which apoptosis execution is suppressed in HeLa cervical cancer cells, half-times of procaspase-9 processing, as well as the molecular timer function of the apoptosome. Our study therefore provides novel mechanistic insight into apoptosome-dependent apoptosis execution and suggests that caspase-9 is activated allosterically by binding to the apoptosome backbone. Our findings challenge the currently prevailing dogma that all initiator procaspases require homodimerization for activation.