Apoptosis is a program of cellular suicide wherein individual cells are removed from the midst of a living tissue without destroying overall tissue architecture. Although the convergence of multiple signaling pathways determines whether a cell will live or die, once the decision to die is made, most cells activate a family of lethal proteases called caspases. By cleaving key intracellular substrates, these enzymes package cellular contents for their orderly elimination by phagocytosis. Caspases can be inhibited by the binding of a class of proteins known as lAPs (inhibitors of apoptosis). In turn, the lAPs can be antagonized by pro-apoptotic IBM (IAP binding motif) proteins that displace lAPs from their caspase binding sites. One such IBM protein, isolated from Drosophila Melanogaster, is the pro-apoptotic protein, Reaper. Reaper not only releases lAPs from caspases, but also promotes IAP ubquitination and degradation, at least in part, through activation of an lAP-intrinsic E3 ubiquitin ligase activity. Although the IBM motif, present at the extreme Nterminus of Reaper, is required for induction of IAP auto-ubiquitination, an additional domain in Reaper, (the GH3 domain) is required for both Reaper-stimulated IAP degradation and localization of Reaper to mitochondria. In a Reaper mutant lacking the GH3 domain, both mitochondria! localization and the ability to induce IAP degradation can be rectified by appending a heterologous mitochondrial targeting sequence. These observations suggest that mitochondrial localization of Reaper and its ability to induce IAP degradation are in some way linked. Upon expression in vertebrate cells, Reaper binds not only lAPs, but also a 150kD protein called Scythe, which participates in Reaper-induced release of mitochondrial cytochrome c to the cytoplasm. The Scythe/Reaper interaction and consequent cytochrome c release is critical for Reaper-mediated caspase activation in vertebrate systems, though its role in Drosophila cell death has not yet been examined. The goals of this proposal are to understand how Reaper functions at mitochondria to promote IAP degradation in both fly and vertebrate cells, to understand how Scythe participates in Reaper-mediated alterations of mitochondria in vertebrate cells, to identify a vertebrate modulator of Scythe function and to determine whether Scythe is also important for fly cell apoptosis. In aggregate, these experiments should not only help to reconcile the effects of Reaper in fly and vertebrate systems, but should also lead to a detailed picture of how the various functions of Reaper contribute to the induction of full-blown apoptosis. These studies will employ both fly and vertebrate systems, including a powerful in vitro reconstitution system derived from Xenopus eggs that allows the recapitulation of apoptotic signaling in vitro. ? ? ? ?
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