STEROL DEPENDENT REGULATION OF MEVALONATE SYNTHESIS

甲羟戊酸合成的甾醇依赖性调节

• 批准号: 3357650
• 负责人: JOHN A WATSON
• 金额: $ 10.85万
• 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/3357650
• 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.

人们已经认识到，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 强调了实现基本理解的重要性， 类异戊烯代谢调节的分子水平 尤其是胆固醇。 其主要目的是 建议是评估氧化固醇合成和/或其他 血清中的类异戊烯（LDL）胆固醇介导的降低 直接调节细胞甲羟戊酸合成， 间接地。 我们将使用一个突变的CHO细胞系，不能去甲基化4 α-甲基甾醇作为我们的实验模型。 突变 CHO细胞的代谢损伤消除了代谢的复杂性 由4，4-二甲基之间的20多种中间体产生 固醇和胆固醇。 因此，我们的变化，以检测，隔离， 并鉴定由以下物质合成的潜在调节性氧甾醇： （3 H）甲羟戊酸或预形成的（14 C/3 H）胆固醇增加。 我们将执行一项工作计划， 血清介导的氧化固醇合成的动力学评估 开始下调3-羟基，3-甲基戊二酰 辅酶A还原酶/辅酶A合成酶 活动及其各自的营业额。 此外，我们会 评估细胞色素P450依赖性过程对 血清介导的下调级联反应。 潜在的生物合成调节固醇将由 放射性HPLC，分离，纯化并通过GC/MS鉴定。 放射免疫化学技术将用于评估特定的 酶周转 我们希望我们的研究能够产生新的信息， 发生在血清远端的强制性类固醇生成事件 脂蛋白摄取和催化。 这些信息应有助于 作为新一代基本问题的框架， 使用单一激发的完整动物进行处理 胆固醇餐。 最终，我们希望一个基本的 将建立一个知识库，以提供必要的见解 了解血清介导的甲羟戊酸控制 合成.