The principal way in which cytochrome c in the cytosol activates apoptosis is by binding Apaf-1, the mammalian homolog of CED-4 (see Figure 21-35, right). In the absence of cytochrome c, monomeric Apaf-1 is bound to dATP. After binding cytochrome c, Apaf-1 cleaves its bound dATP into dADP and P_i and undergoes a dramatic assembly into a disk-shaped heptamer, a 1.4-MDa wheel of death called the apoptosome (Figure 21-41). The apoptosome serves as an activation machine for the initiator caspase caspase-9, which is monomeric in the inactive state. Initiator caspases must be sensitive to activation signals, yet should not be activatable in an irreversible manner because accidental activation would lead to an undesirable snowball effect and rapid cell death. Significantly, caspase-9 does not require cleavage to become activated, but rather is activated by dimerization following binding to the apoptosome. Caspase-9 then cleaves multiple molecules of effector caspases, such as caspase-3 (see Figures 21-35 and 21-40), leading to their activation and subsequent destruction of cell proteins and cell death. The three-dimensional structure of the corresponding nematode CED-4 apoptosome (Figure 21-41c) shows how two CED-3 procaspases bind adjacent to each other on the inside of the funnel-shaped octamer; these molecules then activate each other by dimerization and proteolytic conversion. The structure of the CED-4 apoptosome also provides a model for the as yet unknown three-dimensional structure of the corresponding mammalian apoptosome (Figure 21-41b, right).

In mammals and flies, but not in nematodes, apoptosis is regulated by several other proteins (see Figure 21-35, right). XIAP, one member of a family of inhibitor of apoptosis proteins (IAPs), provides another way to restrain both initiator and effector caspases. XIAP has three N-terminal BIR domains; the one termed BIR2 binds to and inhibits two effector caspases, caspase-3 and caspase-7, while BIR3 binds to and inhibits initiator caspase-9. (Other members of the IAP family inhibit apoptosis induced by TNFα; see Figure 21-42 below.) The inhibition of caspases by IAPs, however, creates a problem when a cell needs to undergo apoptosis. Mitochondria enter the picture once again, since they are the source of a family of proteins, called SMAC/DIABLOs, that inhibit IAPs. Assembly of Bax or Bak oligomers (see Figure 21-40) leads to the release of SMAC/DIABLOs, as well as cytochrome c, from mitochondria. SMAC/DIABLOs then bind to XIAP in the cytosol, thereby blocking XIAP from binding to caspases. By relieving XIAP-mediated inhibition, SMAC/DIABLOs promote caspase activity and cell death.