Interestingly, addition of free cytochrome c to extracts containing mitochondria also greatly accelerates apoptosis, consistent with the idea that release of cytochrome c from mitochondria is a rate limiting step in this process. Taken together these data suggest that Reaper might induce downstream release of cytochrome c from mitochondria, thereby triggering caspase activation. To determine whether Reaper can indeed induce mitochondrial cytochrome c release, Reaper was incubated in unfractionated Xenopus egg extracts. Aliquots were assayed at various times for cytochrome c release and caspase activity. Although the exact timing of apoptotic events varies between extracts, Reaper accelerates both the release of cytochrome c from mitochondria and the consequent activation of DEVD-cleaving caspases.
Many questions remain to be resolved.
Apoptosis is an essential process in the development and homeostasis of all metazoans. In conjunction with other observations, these results reveal how Smac may relieve IAP inhibition of caspase-9 activity.