COMPLEMENT REGULATION BY CLUSTERIN

通过集群蛋白补充监管

• 批准号: 6301645
• 负责人: TUAJUANDA C. JORDAN
• 金额: $ 3.85万
• 依托单位: XAVIER UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6301645
• 关键词: animal tissue; biological signal transduction; clusterin; complement inhibitors; complement pathway; gene deletion mutation; guinea pigs; point mutation; protein purification; protein structure function; site directed mutagenesis

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.

长期目标是开发差异调节末端补体复合物（TCC）的方法，以抑制其细胞溶解活性，而不干扰需要复合物亚溶解插入膜的活性，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。异源二聚体蛋白是抑制流体相中的TCC的异源二聚体蛋白。具体目标1是鉴定负责抑制TCC的簇蛋白序列。实验最初的目的是确定功能序列，特别是那些在簇蛋白的α亚基的羧基末端，使用短和嵌合肽。在这些研究之后，将通过定点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、纯化并测定其抑制TCC的能力。具体目标2是鉴定β亚基内负责其抑制活性的结构域。设计实验以在蛋白质的该区域中产生缺失突变体。然后将蛋白质表达、纯化并分析TCC抑制活性。