Apoptosis or programmed cell death is a crucial process for the development and maintenance of a functional immune system. Apoptosis depends upon a family of cysteine proteases (caspases) whose activation is subject to both negative and positive regulatory mechanisms. Recently we discovered and have partially characterized a novel pro-apoptotic caspase adaptor protein (PACAP) that interacts with two initiator caspases, caspase-2 and -9. To date we have observed the following: when PACAP is overexpressed in cell lines, apoptosis is induced and the NF-kappaB pathway is activated. Support for a role for PACAP as a regulatory protein derives from the observations that the transcription of endogenously expressed PACAP in primary human B cells is rapidly and dramatically down regulated after B-cell activation and upregulated during apoptosis. Moreover PACAP forms complexes with Bcl10, a protein linked to NF-kappaB activation in some cell types and to a variety of human malignancies. Thus it is our hypothesis that PACAP may serve to direct extracellular signals to either casp-2 or -9 apoptotic pathways and may further regulate other intracellular pathways activated during the apoptotic pathway. To test this hypothesis we propose three Specific Aims that utilize molecular and cellular biological, immunological and biochemical approaches to further define the properties of PACAP in the context of physiologically relevant cellular models where casp-2 and caspase-9 play essential roles in cell death pathways. The proposed Specific Aims are: 1. To provide a detailed biochemical analysis of PACAP interactions; 2. To provide a comprehensive definition of the components that comprise the PACAP apoptotic signaling pathway; and 3. To provide further identification of other potential regulators of PACAP mediated intracellular signaling with an emphasis on the NF-kappaB pathway. The proposed studies are designed to provide fundamental information about this newly discovered protein that serves as a regulator of apoptotic pathways linked to both caspases-2 and-9. The long term goals of the proposed studies are to enhance our abilities to understand basic processes leading to human diseases linked to a dysfunctional immune system.
