STEROL DEPENDENT REGULATION OF MEVALONATE SYNTHESIS

甲羟戊酸合成的甾醇依赖性调节

• 批准号: 3357648
• 负责人: JOHN A WATSON
• 金额: $ 9.45万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-04-01 至 1992-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3357648
• 关键词: HMG coA reductases; cholesterol; cholesterol esters; coronary disorder; cytochrome P450; enzyme inhibitors; enzyme mechanism; fatty acid biosynthesis; gas chromatography mass spectrometry; gel electrophoresis; high performance liquid chromatography; immunochemistry; low density lipoprotein; methylation; mevalonate; oxidoreductase inhibitor; radiotracer; steroid biosynthesis; steroid metabolism; tissue /cell culture

It has been recognized that a major risk factor for coronary heart disease is increased serum cholesterol levels. This finding highlights the importance of achieving a basic understanding, at the molecular level, of the metabolic regulation of isopentenoids in general and cholesterol specifically. The broad aim of this proposal is to assess the role of oxysterol synthesis and/or other isopentenoids in serum (LDL) cholesterol mediated down- regulation of cellular mevalonate synthesis directly and indirectly. We will use a mutant CHO cell line, unable to demethylate 4 alpha-methyl sterols, as our experimental model. The mutant CHO cells' metabolic lesion eliminates the metabolic complexity generated by greater than 20 intermediates between 4,4-dimethyl sterols and cholesterol. As such our changes to detect, isolate, and identify potential regulatory oxysterol(s) synthesized from (3H)mevalonate or preformed (14C/3H)cholesterol are increased. We will pursue a work plan which incorporates the coordinated, kinetic assessment of serum mediated oxysterol synthesis with the onset of the down-regulation for 3-hydroxy, 3-methyl glutaryl coenzyme A (HMG-CoA) reductase/HMG-CoA synthetase activities, and their respective turnover. In addition, we will assess the role of cytochrome P450 dependent processes on the serum mediated, down-regulation cascade. Potential biosynthesized regulatory sterols will be monitored by radio-HPLC, isolated, purified and identified by GC/MS. Radioimmunochemical techniques will be used to assess specific enzyme turnover. We expect our immediate study to yield new information about the obligatory sterologenic events which occur distal to serum lipoprotein uptake and catabolism. Such information should serve as a framework for a new generation of fundamental questions to be addressed with intact animals challenged by a single cholesterol meal. Ultimately we hope that a fundamental knowledge base will be generated to provide the necessary insight for understanding serum mediated control of mevalonate synthesis.

人们已认识到冠心病的主要危险因素 疾病是血清胆固醇水平升高。 这一发现 强调了达成基本理解的重要性， 类戊烯代谢调节的分子水平 一般而言，特别是胆固醇。 本次活动的总体目标 建议评估氧甾醇合成和/或其他物质的作用 血清中的类异戊烯 (LDL) 胆固醇介导的下调 直接调节细胞甲羟戊酸合成 间接地。 我们将使用突变型 CHO 细胞系，无法去甲基化 4 α-甲基甾醇，作为我们的实验模型。 突变体 CHO细胞的代谢损伤消除了代谢复杂性 由超过 20 个 4,4-二甲基之间的中间体生成 甾醇和胆固醇。 因此，我们的改变是检测、隔离、 并鉴定合成的潜在调节性氧甾醇 (3H)甲羟戊酸或预形成的(14C/3H)胆固醇增加。 我们将制定一项工作计划，其中包括协调一致的、 血清介导的氧甾醇合成的动力学评估 3-羟基，3-甲基戊二酰开始下调 辅酶 A (HMG-CoA) 还原酶/HMG-CoA 合成酶 活动及其各自的营业额。 此外，我们将 评估细胞色素 P450 依赖性过程的作用 血清介导的下调级联反应。 潜在的生物合成监管甾醇将由 放射性 HPLC，通过 GC/MS 分离、纯化和鉴定。 放射免疫化学技术将用于评估特定 酶周转。 我们期望我们的立即研究能够产生有关以下方面的新信息： 发生在血清远端的强制性甾醇生成事件 脂蛋白的摄取和分解代谢。 此类信息应服务于 作为新一代基本问题的框架 处理受到单一挑战的完整动物 胆固醇餐。 最终我们希望能够从根本上 将生成知识库以提供必要的见解 用于了解血清介导的甲羟戊酸控制 合成。