Mechanism of Endocytic Trafficking of Cholesterol

描述（由申请人提供）：高胆固醇血症是一种主要风险 冠状动脉疾病（CAD）和脑血管疾病的发展因素 血管疾病（CVD）。血浆低密度脂蛋白水平升高 (LDL)导致多余的胆固醇在动脉中沉积， 动脉粥样硬化虽然调节LDL摄取和释放的体内平衡机制 胆固醇的从头合成已得到很好的表征，细胞机制 调节低密度脂蛋白胆固醇转运的机制 明白有证据表明NPC 1和HE 1蛋白参与了 一种有效运输内化膜的共同途径 胆固醇的质膜（PM）和ER。我们假设NPC 1 促进囊泡和/或小管的形成， 限制性内体膜含NPC 1的囊泡然后运输到PM或 急诊室运送他们的固醇货物。该项目的目的是确定 固醇运输途径中的分子机制，并测试我们的 关于NPC 1在分类和分布中的作用的假设 内化膜胆固醇这将通过以下方式实现： 具体目标：（1）使用功能性哺乳动物遗传筛选， 细胞内转运受损的中国仓鼠卵巢（CHO）突变体 胆固醇，（2）胆固醇的表征和鉴定 突变CHO细胞系中的运输缺陷，和（3）组成和 含有NPC 1的晚期内体中蛋白质的功能分析 隔室，并检查NPC 1膜囊泡形成是否依赖于 固醇浓度和/或NPC 1中完整的固醇敏感结构域。的 本建议中概述的研究将进一步加深我们对 NPC 1在胆固醇稳态中的关键作用。此外，研究 通过我们的基因筛选和 含有NPC 1的内体区室的组成分析可以 确定甾醇转运途径中的新靶点， CAD和CVD的治疗。

DESCRIPTION (Provided by applicant): Hypercholesterolemia is a major risk factor for the development of coronary artery disease (CAD) and cerebral vascular disease (CVD). Increased plasma levels of low-density lipoprotein (LDL) leads to deposition of excess cholesterol in arteries, initiating atherosclerosis. While homeostatic mechanisms that regulate LDL uptake and de novo synthesis of cholesterol are well characterized, the cellular mechanisms that regulate trafficking of LDL cholesterol after internalization are not well understood. Evidence is emerging that the NPC1 and HE1 proteins participate in a common pathway for the efficient trafficking of internalized membrane cholesterol to the plasma membrane (PM) and to the ER. We hypothesize that NPC1 promotes formation of vesicles and/or tubules through the budding of the limiting endosomal membrane. NPC1-containing vesicles then traffic to the PM or ER to deliver their sterol cargo. The purpose of this project is to identify the molecular machinery in the sterol trafficking pathway and to test our hypothesis regarding this role of NPC1 in the sorting and distribution of internalized membrane cholesterol. This will be achieved by the following specific aims: (1) Use of a functional mammalian genetic screen to isolate Chinese hamster ovary (CHO) mutants with impaired intracellular trafficking of cholesterol, (2) Characterization and identification of the cholesterol trafficking defects in the mutant CHO cell lines, and (3) Compositional and functional analysis of proteins in the NPC1-containing late endosomal compartment, and examination of whether NPC1 membrane vesciulation is dependent on sterol concentrations and/or an intact sterol-sensing domain in NPC1. The studies outlined in this proposal will further our understanding of the critical role of NPC1 in cholesterol homeostasis. Furthermore, study of the function of gene products identified by our genetic screens and by compositional analysis of the NPC1-containing endosomal compartment may identify novel targets within the sterol transport pathway for pharmacologic therapy of CAD and CVD.