Cellular Cholesterol Movement and Homeostasis

细胞胆固醇运动和稳态

• 批准号: 7028896
• 负责人: YVONNE LANGE
• 金额: $ 25.29万
• 依托单位: RUSH UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-06-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7028896
• 关键词: biosynthesis; cell membrane; cholesterol; clinical research; endoplasmic reticulum; enzyme activity; erythrocyte membrane; fibroblasts; high density lipoproteins; high throughput technology; homeostasis; human tissue; lipid bilayer membrane; molecular film; monomer; phosphatidylcholines; phospholipids; protein kinase C; small molecule; sphingomyelins; sterols; tissue /cell culture

DESCRIPTION (provided by applicant): A simple new mechanistic hypothesis which could lead to an understanding of how cells sense and respond homeostatically to their cholesterol will be explored. The pool of cholesterol in the endoplasmic reticulum (ER) controls cell cholesterol accretion by modulating the activity of several downstream proteins embedded therein. The ER cholesterol pool is itself regulated by the delivery of cholesterol from the bulk pool in the plasma membrane (PM). A novel concept recently suggested by Harden McConnell is that PM cholesterol makes stoichiometric complexes with phospholipids. PM cholesterol in excess of this equivalence point is monomeric and has a high escape potential. It could therefore redistribute to the ER, setting the cholesterol level therein, and thus directing ER homeostatic activity. Human erythrocyte membranes will be used to establish this hypothesis. The pool of active cholesterol monomers will be characterized under various conditions. For example, modulation of this pool by the intercalation of other sterols, lipids and small water-soluble effectors as well as by bilayer lipid scrambling will be examined. Artificial sterol-containing lipid monolayers will be used to elucidate the molecular basis of red cell behavior. The findings with red cells will be applied to cultured human fibroblasts to test whether the ER cholesterol pool size is set by plasma membrane cholesterol monomer activity in these cells. Whether efflux of plasma membrane cholesterol to high-density lipoproteins depends on active cholesterol monomers will be evaluated. An alternative hypothesis will also be tested, namely, that plasma membrane cholesterol level is signaled to the ER by cholesterol-dependent raft formation. Whether the distribution of cholesterol monomers between the PM and intracellular membranes is governed by passive equilibration or active regulation will be tested using blockers of protein kinase C activity. Whether phosphatidylcholine biosynthesis in the ER is regulated by cholesterol monomer activity in the plasma membrane and whether the abundance of PM sphingomyelin sets the level of PM cholesterol will also be tested. Finally, high throughput multi-well plate assays for active cholesterol monomers in the PM of human fibroblasts and red cells will be used to screen for small molecules that can decrease cell cholesterol and thus serve as potential therapeutic agents against atherosclerosis.

描述(由申请人提供)： 将探索一种简单的新机制假说，它可能导致对细胞如何感知并对其胆固醇做出稳态反应的理解。内质网(ER)中的胆固醇池通过调节嵌入其中的几种下游蛋白的活性来控制细胞胆固醇的积累。内质网胆固醇池本身受质膜(PM)中散装池中胆固醇输送的调节。哈登·麦康奈尔最近提出了一个新的概念，即PM胆固醇与磷脂形成化学计量络合物。PM胆固醇超过这个等价点是单体，具有很高的逃逸潜力。因此，它可以重新分配到内质网，设定其中的胆固醇水平，从而指导内质网的稳态活动。人类红细胞膜将被用来建立这一假说。活性胆固醇单体池将在不同条件下进行表征。例如，通过其他甾醇、脂类和小的水溶性效应物的嵌入以及双层脂类的扰乱来调节这一池。含有人工类固醇的脂单分子膜将用于阐明红细胞行为的分子基础。这一发现将应用于培养的人成纤维细胞，以测试内质网胆固醇池的大小是否由这些细胞中的质膜胆固醇单体活性决定。质膜胆固醇向高密度脂蛋白的外流是否取决于活性的胆固醇单体将被评估。还将检验另一种假说，即质膜胆固醇水平通过胆固醇依赖的RAFT形成向内质网发出信号。胆固醇单体在质膜和细胞内膜之间的分布是由被动平衡还是主动调节决定的，将使用蛋白激酶C活性阻滞剂进行测试。内质网中磷脂酰胆碱的生物合成是否受质膜上胆固醇单体活性的调节，以及PM鞘磷脂的丰富是否设定PM胆固醇的水平也将被测试。最后，人成纤维细胞和红细胞质膜中活性胆固醇单体的高通量多孔板分析将被用于筛选可以降低细胞胆固醇从而作为潜在的抗动脉粥样硬化治疗剂的小分子。