Development, characterization, and use of a novel yeast expression system to identify inhibitors of the caspase-3 cell death protease

Apoptosis plays an essential role in the normal development and homeostasis of higher organisms. Apoptosis is also a defense strategy used by host cells to destroy pathogen-infected cells. A new family of cytoplasmic cysteine proteases, termed caspases, is responsible for carrying out the morphological and biochemical events that occur during apoptosis. Caspases forge a pathway to cell death through their cleavage of key regulatory and structural proteins in the cell. Organisms have developed counter mechanisms to block the activity of this family of cell death proteases. Recently, a new family of genes has been discovered which encode proteins referred to as IAPs (Inhibitor of Apoptosis Proteins) that inhibit apoptosis by directly inhibiting the proteolytic activity of distinct caspase family members. More specifically, the mammalian c-IAP-1, c-IAP-2, and XIAP proteins were shown to be direct in vitro inhibitors of caspases-3, -7, and -9. Experimental studies have revealed that among the caspase family, caspase-3 is arguably the one caspase responsible for mediating the majority of critical substrate cleavages that occur during the death of the cell. In light of this result, this dissertation has focused on the development and application of a novel caspase-3 expression system in yeast that would facilitate the uncovering of new protein inhibitors to the caspase-3 cell death protease. Despite several attempts, the novel caspase-3 yeast expression system was unable to uncover new protein inhibitors to the caspase-3 protease. However I used the system to characterize previous in vitro protease/inhibitor relationships between caspase-3 and the IAPs, specifically XIAP, c-IAP1, and c-IAP2. The caspase-3 yeast expression system faithfully mimicked previous in vitro associations between caspase-3 and the IAPs. This system also revealed that Survivin, a poorly defined member of the IAP family, is a poor inhibitor of caspase-3, and probably targets some other component of the cell death pathway in the process of inhibiting apoptosis in mammalian cells. I believe the caspase-3 yeast expression system is a powerful tool for isolating and characterizing potent inhibitors to the caspase-3 protease. Identifying new caspase-3 inhibitors may facilitate our understanding of how caspase-3 activity is regulated during development and disease.