Asymmetric Distribution of Cholesterol in Membranes

描述（由申请人提供）：质膜富含胆固醇的微结构域，脂筏和小泡，介导HDL逆向胆固醇运输，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。虽然胆固醇对脂筏/小泡功能至关重要，但关于脂筏或小泡中胆固醇的分布、结构和调节几乎一无所知。初步数据显示，甾醇载体蛋白-2 （SCP-2）在体外和体内结合小窝蛋白-1，改变脂质筏/小窝胆固醇结构域，抑制富含小窝蛋白-1的细胞（l细胞成纤维细胞）高密度脂蛋白介导的胆固醇外排。相比之下，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，用于解决本质上缺乏小窝蛋白-1但富含介导胆固醇在质膜和HDL （SRB1、P-gp、ABCA1）之间转运的蛋白质的细胞中的胆固醇膜动力学问题。我们现在提出四个具体目标。目的1。确定哪些蛋白（SRB1, ABCA1, P-gp）介导高密度脂蛋白胆固醇外排/摄取存在于质膜脂筏和小泡中，它们之间的相互作用，以及与小泡蛋白-1的相互作用。目标2。检查SCP-2如何与小窝蛋白-1相互作用，以及SCP-2是否结合脂质筏或小窝蛋白介导hdl -胆固醇摄取/排出（srb1, ABCA1, P-gp）。目标3。确定SCP-2表达是否改变了纯化木筏和小泡中的胆固醇分布、胆固醇动态和结构。SCP-2水平将通过SCP-2过表达的l细胞成纤维细胞和来自SCP-2过表达和基因消融小鼠的原代培养肝细胞进行修饰。目标4。在活细胞中，确定SCP-2的表达是否会改变脂筏或小泡中的胆固醇分布、结构和动力学。结果将提供胆固醇组织和脂质筏动力学的基本机制细节，SCP-2的调节，脂质筏与小泡在介导hdl -胆固醇摄取/排出中的不同作用，以及对代谢性疾病、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.