Mechanism of Endocytic Trafficking of Cholesterol

描述(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)和脑血管疾病发展的因素 血管疾病(CVD)。血浆低密度脂蛋白水平升高 (低密度脂蛋白)导致过量胆固醇沉积在动脉中，引发 动脉硬化。而调节低密度脂蛋白摄取和降解的动态平衡机制 胆固醇的新合成被很好地表征，其细胞机制 在内化后调节低密度脂蛋白运输的方法不是很好 明白了。有证据表明，NPC1和HE1蛋白参与了 内化膜高效转运的共同途径 胆固醇到质膜(PM)和内质网。我们假设NPC1 通过发芽促进囊泡和/或小管的形成 限制内胚体膜。然后，将含有NPC1的囊泡传输到PM或 急诊室运送他们的类固醇货物。该项目的目的是确定 类固醇运输途径中的分子机制，并测试我们的 关于NPC1在分类和分布中的作用的假说 内化的膜胆固醇。这将通过以下方式实现 具体目的：(1)利用哺乳动物的功能基因筛选分离 中国仓鼠卵巢(CHO)突变体细胞内转运受损 胆固醇，(2)胆固醇的表征和鉴定 突变的CHO细胞系中的运输缺陷，以及(3)组成和 含NPC1的晚期内体蛋白质的功能分析 隔室，并检查NPC1膜囊泡是否依赖 与NPC1中的类固醇浓度和/或完整的类固醇敏感结构域有关。这个 这项建议中概述的研究将进一步加深我们对 NPC1在胆固醇稳态中的关键作用。此外，还研究了该系统的性能 基因产物的功能由我们的基因筛查和鉴定 含有NPC1的内体隔室的成分分析可能 在甾醇转运途径中寻找新的药理靶点 冠心病和心血管疾病的治疗。

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.