COMPLEMENT REGULATION BY CLUSTERIN

长期目标是开发方法来差异调节末端补体复合物（TCC），以抑制其细胞溶解活性，而不干扰需要将复合物亚溶解插入膜中的活动，例如在信号转导中。这将有助于动脉粥样硬化和阿尔茨海默病等领域，这两种疾病都涉及补体介导的细胞溶解。聚簇素是一种异二聚体蛋白，在流体阶段抑制TCC。具体目的1是鉴定簇蛋白序列负责其抑制TCC。实验最初设计用于识别功能序列，特别是在聚簇蛋白α亚基的羧基端，使用短肽和嵌合肽。在这些研究之后，将通过位点定向诱变将点突变引入这些结构域，以鉴定功能性氨基酸残基。突变蛋白将被表达、纯化，并检测其抑制TCC的能力。具体目标2是确定β亚基内负责其抑制活性的结构域。实验旨在产生该蛋白质区域的缺失突变体。然后将蛋白质表达、纯化并分析tcc抑制活性。

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.