Identification and Characterization of Human Inhibitor of Apoptosis (IAP) Associacted Proteins

Erwin Schrödinger Fellowship J 1990 Inhibitor of Apoptosis (IAP) associated proteins Christian STEHLIK 09.10.2000 Apoptosis is a process that is essential for normal develpoment and homeostasis of the body. Aberrant suppression of this process plays an important role in the pathogenesis of a variety of diseases. Elevated levels of the cellular "inhibitor of apoptosis proteins" (IAPs) have been detected in cancers. IAPs represent a family of evolutionary conserved antiapoptotic proteins, whitch function at a central point in the apoptotic cascade by inhibiting caspases, the cell death proteases responsible for apoptosis. Our laboratory has found a correlation between elevated expression of cIAP-1 and in vitro chemoresistance to diverse anti-cancer drugs in the NCI 60 cell line panel. cIAP- 1, and the closely related cIAP-2 protein, are unique among all the members of the IAP family in that they contain CARDs (Caspase Recruitment Domains), which have been proposed to play regulatory roles in apoptosis via homophilic protein interactions with other CARD-containing proteins, such as pro-caspase-regulators. CARD- carrying proteins have also been implicated inactivation proteins, including cIAP-1 and cIAP-2. The first aim is to screen for potential IAP inhibitors using a functional genetic screen in yeast. Secondly, to functionally characterize, with respect to participation in apoptosis regulation and NF-kB activation, a novel discovered CARD protein, referred to as CHI-1 (CARD with Homology to cIAP-1/-2), displaying ~40-50% amino-acid sequence identity with the CARDs of cIAP-1 and cIAP-2. This high degree of similarity suggests that CHI-1 represents a potential binding partner of cIAP-1 and cIAP-2 via CARD-CARD interactions, thus making it a candidate IAP-regulatory protein. Indeed, initial in vitro binding studies revealed binding of CHI-1-CARD to cIAP1 as well as two other CARD proteins, Nod1 and Cardiak. Preliminary data suggest that CHI-1 maybe downregulated in human cancer cell lines compared to normal tissue, suggesting that alterations in its expression may accompany the pathogenesis of cancer. The elucidation of novel apoptosis regulators could potentially provide insights leading to new strategies for the treatment of cancer, using this new protein as target for small-molecule drug discovery, or as tool for cancer gene therapy, antisense oligonucleotide therapeutics or other approaches.