Asymmetric Distribution of Cholesterol in Membranes

描述（由申请方提供）：质膜富含胆固醇的微区、脂筏和小窝介导HDL反向胆固醇转运、信号传导、抗体识别，并作为潜在生物恐怖毒素、病毒和寄生虫的入口。虽然胆固醇对于脂筏/小窝功能是必不可少的，但关于胆固醇在脂筏或小窝中的分布、结构和调节几乎一无所知。初步数据显示，固醇载体蛋白-2（SCP-2）在体外和体内结合小窝蛋白-1，改变脂筏/小窝胆固醇结构域，并抑制HDL介导的胆固醇从富含小窝蛋白-1的细胞（L-细胞成纤维细胞）流出。相反，培养的原代肝细胞（来自野生型、SCP-2过表达和SCP-2基因靶向小鼠）提供了一种模型，用于解决细胞中胆固醇膜动力学，该细胞基本上缺乏小窝蛋白-1，但富含介导质膜和HDL之间胆固醇转运的蛋白质（SRB 1、P-gp、ABCA 1）。我们现在提出四个具体目标。目标1.确定哪些蛋白质（SRB 1，ABCA 1，P-gp）介导HDL-胆固醇流出/摄取存在于质膜脂筏与小窝中，它们之间的相互作用，以及与小窝蛋白-1的相互作用。目标二。检查SCP-2如何与小窝蛋白-1相互作用，以及SCP-2是否结合介导HDL-胆固醇摄取/流出的脂筏或小窝蛋白（SRB 1，ABCA 1，P-gp）。目标3：解决SCP-2表达是否改变纯化筏和小窝中的胆固醇分布、胆固醇动力学和结构。SCP-2水平将用SCP-2过表达的L细胞成纤维细胞和SCP-2过表达和基因消融小鼠的原代培养肝细胞进行调节。目标4。在活细胞中，确定SCP-2的表达是否会改变脂筏或小窝中的胆固醇分布、结构和动力学。结果将提供基本的，在脂筏中的胆固醇组织和动力学的机制细节，SCP-2的调节，筏与小窝在介导HDL-胆固醇摄取/流出中的不同作用，以及对代谢疾病，病原体进入和生物恐怖剂的潜在干预的见解。

DESCRIPTION (provided by applicant): Plasma membrane cholesterol-rich microdomains, lipid rafts and caveolae, mediate HDL reverse cholesterol transport, signaling, antibody recognition, and serve as entry portals for potential bioterror toxins, viruses, and parasites. While cholesterol is essential for lipid raft/caveolae function, almost nothing is known regarding distribution, structure, and regulation of cholesterol in lipid rafts or caveolae. Preliminary Data show sterol carrier protein-2 (SCP-2) binds caveolin-1 in vitro and in vivo, alters lipid raft/caveolae cholesterol domains, and inhibits HDL-mediated cholesterol efflux from cells rich in caveolin-1 (L-cell fibroblasts). In contrast, cultured primary hepatocytes (from wild-type, SCP-2 overexpressing, and SCP-2 gene targeted mice) provide a model for addressing cholesterol membrane dynamics in cells essentially deficient in caveolin-1, but rich in proteins that mediate cholesterol transport between plasma membrane and HDL (SRB1, P-gp, ABCA1). We now propose four specific aims. Aim 1. Determine which proteins (SRB1, ABCA1, P-gp) mediating HDL-cholesterol efflux/uptake are present in plasma membrane lipid rafts versus caveolae, interactions between them, and interactions with caveolin-1. Aim 2. Examine how SCP-2 interacts with caveolin-1, and whether SCP-2 binds lipid raft or caveolae proteins mediating HDL-cholesterol uptake/efflux (SRB 1, ABCA1, P-gp). Aim 3. Resolve if SCP-2 expression alters cholesterol distribution, cholesterol dynamics, and structure in purified rafts and caveolae. SCP-2 level will be modified with SCP-2 overexpressing L-cell fibroblasts and primary cultured hepatocytes from SCP-2 overexpressed and gene-ablated mice. Aim 4. In living cells, determine if SCP-2 expression alters cholesterol distribution, structure, and dynamics in lipid rafts or caveolae. Results will provide fundamental, mechanistic details of cholesterol organization and dynamics in lipid rafts, regulation by SCP-2, differential roles of rafts versus caveolae in mediating HDL-cholesterol uptake/efflux, and insights on potential intervention in metabolic disease, pathogen entry, and bioterror agents.