REGULATION OF HMG COA REDUCTASE GENE BY CHOLESTEROL

胆固醇对 HMG COA 还原酶基因的调节

• 批准号: 2224091
• 负责人: Timothy F Osborne
• 金额: $ 17.22万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-01 至 1997-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2224091
• 关键词: DNA binding protein; Escherichia coli; HMG coA reductases; SDS polyacrylamide gel electrophoresis; chimeric proteins; cholesterol; complementary DNA; gene expression; genetic regulation; hamsters; homeostasis; lipid metabolism; molecular cloning; northern blottings; nucleic acid probes; nucleic acid structure; protein sequence; protein structure function; recombinant proteins; site directed mutagenesis; tissue /cell culture

The applicant has identified a protein, Red 25, that binds to a site in the promoter for HMG CoA reductase required for feedback regulation by cholesterol. Since HMG CoA reductase is the rate controlling enzyme of cholesterol biosynthesis the study of a protein involved in its regulation is essential for understanding the molecular mechanisms involved in maintaining cholesterol homeostasis. The long-term plan is to characterize the Red 25 regulatory protein in detail and determine its exact role in modulating expression of the HMG CoA reductase gene. This information will be used in the future to study the role of Red 25 in cholesterol homeostasis and determine if it is involved in diseases of cholesterol accumulation such as coronary artery disease (CAD). The experiments described in this proposal are to isolate cDNA clones that encode the Red 25 protein and to initiate a thorough study of its mode of action. The protein will be expressed from the cDNA in E. coli for the production of large amounts for both functional studies and to prepare antisera directed against Red 25. Hybridization probes prepared from the cDNA will be used along with the Red 25 antisera to study the pattern of Red 25 mRNA and protein expression in whole animals and in cell lines that can be manipulated experimentally to modulate HMG CoA reductase expression over a 100 fold range. In the future, the human gene for Red 25 will be cloned and the segregation of mutant alleles will be followed in families with a genetic tendency to develop coronary artery disease. The identification of functional domains by the experiments described in the present proposal will be compared to the location of human mutations of the Red 25 gene to understand how a mutation that results in a specific effect in the laboratory assays correlates to a perturbation of cholesterol balance in humans.

申请人已经确定了一种蛋白质红色25，该蛋白与位点结合 通过 胆固醇。 由于HMG COA还原酶是控制酶的速率 胆固醇生物合成的研究 调节对于理解分子机制至关重要 参与维持胆固醇稳态。 长期计划是 详细描述红色25调节蛋白并确定其 在调节HMG COA还原酶基因表达中的确切作用。 这 将来将使用信息来研究红色25的作用 胆固醇稳态并确定它是否参与 胆固醇积累，例如冠状动脉疾病（CAD）。 该提案中描述的实验是分离cDNA克隆 这编码红色25蛋白，并启动对其的彻底研究 行动方式。 该蛋白质将从大肠杆菌中的cDNA表达 用于生产大量功能研究和 准备针对红色25的抗血清。制备的杂交探针 来自cDNA将与红色25抗血清一起研究 红色25 mRNA和蛋白质表达的模式在整个动物和 可以通过实验操纵以调节HMG COA的细胞系 还原酶在100倍范围内的表达。 将来，红色25的人类基因将被克隆，并且 在具有遗传的家族中，将遵循突变等位基因的分离 发展冠状动脉疾病的趋势。 识别 通过本提案中描述的实验功能域 将与红色25基因的人类突变的位置进行比较 了解突变如何导致特定作用 实验室测定与胆固醇平衡的扰动有关 人类。