Members of the family of ICE-like proteases (caspases) play a central and evolutionarily conserved role in the execution of apoptosis. Several mammalian caspases have been identified and cloned recently in the P.I.'s laboratory. These include CPP32, Mch2, Mch3, Mch5, Mch6 and Mih1. Unlike in C. elegans where CED-3 caspase is the major executioner of apoptosis, recent observations suggest that execution of apoptosis in higher eukaryotes may involve the coordinated action of multiple caspases. These proteases might be components of an amplifiable apoptotic protease cascade similar to the cascade of complement activation. Nevertheless, it is not yet clear whether all or few of these caspases play a role in the mammalian apoptotic cascade. In addition, it is not known how the proenzymes of caspases are activated and what triggers their activation when the cell enters the committed phase of apoptosis. One possibility is the existence of upstream cell death effectors or activators that interact directly with one or more of these procaspases or indirectly with components of the caspase activation pathway. This is supported by recent observations that proMch4, proMch5 and pro-ICH-1 contain long N-terminal prodomains capable of interaction with the apoptotic adapter molecules FADD/Mort1 and CRADD/RAIDD, respectively. These prodomains may also interact with other novel homologous death effector proteins in the FAS/TNFR1 apoptotic pathway and possibly in other apoptotic pathways. ProMch6 also contains a long prodomain that is highly homologous to the CED-3 prodomain, suggesting that its mechanism of activation and interactions might be similar to that of CED-3 in the developmental cell death pathway. Consequently, experiments are proposed to examine the role of the prodomain of upstream caspases (Mch4, Mch5, Mch6 and ICH-1) in their mechanism of activation. The apoptotic potential and interactions of these proteases with cell death effectors such as FADD and CRADD, or inhibitors such as inhibitors of apoptosis (IAPs) and Bcl2 family members, will also be examined. Experiments are also proposed to identify and clone novel death effectors or inhibitors that might interact with the upstream proteases. The glucocorticoid and Fas receptor models of apoptosis are proposed to study the involvement and activation of caspases in vivo. It is anticipated that these studies will contribute to elucidation of the mechanism of activation of this important class of proteases. This could lay the foundation for new chemotherapeutic approaches in cancer and other degenerative diseases where cell death is involved in the pathologic process.
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