Apoptotic cell death is essential for normal development and maintenance of normal tissue homeostasis. There is growing evidence that dysregulation of apoptosis may lead to several human diseases including cancer, autoimmune disorders and degenerative neuronal diseases such as Alzheimer's and Parkinson's diseases. Emerging evidence suggests that protease activation might be the central mechanism leading to the execution of apoptosis. Unlike in C. elegans where CED-3 cysteine protease is the major executioner of apoptosis, recent observations suggest that execution of apoptosis in higher eukaryotes may involve multiple cysteine proteases related to CED-3 and ICE. These proteases, might be components of an amplifiable apoptotic protease cascade similar to the cascade of complement activation. The number of cysteine proteases is still growing, however their mechanism of activation, role in apoptosis and relevant apoptotic substrates are still largely unknown. To isolate and characterize novel cysteine proteases we developed a PCR technique to enrich for DNA sequences which encode the highly conserved pentapeptides QACRG and GSWFI/GSWYI present in CED-3/ICE-like apoptotic cysteine proteases. We were able to clone several CED-3/ICE-like proteases that could be components of the molecular mechanism of vertebrate apoptosis. Consequently, it is proposed to overexpress full length, and truncated and mutated derivatives of these cysteine proteases in Spodoptera frugiperda (Sf9) cells and in E. coli. This will avail large quantities of these proteins for functional and structural studies. Mammalian apoptotic cysteine proteases cloned in our laboratory, will be characterized. Their genes will be analyzed for tissue specific and temporal expression and the presence of alternatively spliced isoforms. Potential substrates and inhibitors of these proteases will be identified using biochemical and molecular biology techniques. It is anticipated that these studies will contribute to elucidation of the mechanism of activation of this important class of proteases. This will enhance the efforts to identify their relevant endogenous substrates and to design specific drugs that will regulate their activity. In addition to the value which will be gained from understanding the molecular mechanism of apoptosis in development and homeostasis, the proposed studies will also generate significant knowledge that could be applied in the treatment of many human diseases such as cancer and many other degenerative diseases.
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