COMPLEMENT REGULATION BY CLUSTERIN

长期的目标是开发差异调节末端补体复合体(TCC)的方法，以便在不干扰需要将复合体亚裂解插入膜的活性的情况下抑制其细胞溶解活性，例如在信号转导方面，这将有助于动脉粥样硬化和阿尔茨海默病等涉及补体介导的细胞溶解的领域。Clusterin是一种异源二聚体蛋白，在液体相中抑制TCC。具体目的1是确定与其抑制TCC有关的聚集素序列。实验最初是为了识别功能序列，特别是那些位于聚集素α亚单位的羧基末端的序列，使用短肽和嵌合肽。在这些研究之后，将通过定点突变将点突变引入这些结构域，以确定功能氨基酸残基。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亚基中负责其抑制活性的结构域。实验的目的是在蛋白质的这个区域产生缺失突变体。然后，这些蛋白将被表达、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.