COMPLEMENT REGULATION BY CLUSTERIN

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The long-term objective is to develop ways to differentially regulate the terminal complement complex (TCC) in order to inhibit its cytolytic activity without interfering with activities that require sub-lytic insertion of the complex into membranes, e.g., in signal transduction This would be helpful in such areas as atherosclerosis and Alzheimer's disease, both of which involve complement-mediated cytolysis. Clusterin is a heterodimeric protein that inhibits the TCC in the fluid phase. Specific Aim 1 is to identify clusterin sequences responsible for its inhibition of the TCC. Experiments are initially designed to identify functional sequences, specifically those at the carboxyl-terminus of clusterin's alpha subunit, using short and chimeric peptides. Subsequent to these studies, point mutations will be introduced into these domains via site-directed mutagenesis to identify functional amino acid residues. The mutant proteins will be expressed, purified, and assayed for their ability to inhibit the TCC. Specific aim 2 is to identify the domains within the beta subunit that are responsible for its inhibitory activity. Experiments are designed to generate deletion mutants in this region of the protein. The proteins will then be expressed, purified, and analyzed for TCC-inhibitory activity.