COMPLEMENT REGULATION BY CLUSTERIN

通过集群蛋白补充监管

• 批准号: 6450662
• 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: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6450662
• 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，而不干扰需要将复合物亚溶解插入膜的活动，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氏病等领域，这两个领域都涉及补体介导的细胞溶解作用。 Clusterin 是一种异二聚体蛋白，可抑制液相中的 TCC。具体目标 1 是鉴定负责抑制 TCC 的凝聚素序列。实验最初设计为使用短肽和嵌合肽来鉴定功能序列，特别是凝聚素α亚基羧基末端的序列。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、纯化并分析其抑制 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。实验旨在在该蛋白质区域产生缺失突变体。然后蛋白质将被表达、纯化并分析 TCC 抑制活性。