Asymmetric Distribution of Cholesterol in Membranes

富含胆固醇的质膜微区、脂筏和小窝，介导高密度脂蛋白逆转胆固醇 运输、信号传递、抗体识别，并作为潜在的生物恐怖毒素、病毒和 寄生虫。虽然胆固醇对脂筏/小窝的功能是必不可少的，但对此几乎一无所知。 胆固醇在脂筏或小窝中的分布、结构和调节。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，以及 抑制高密度脂蛋白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。相比之下，有教养的 原代肝细胞(来自野生型、SCP-2过表达和SCP-2基因靶向的小鼠)为 解决本质上缺乏小窝蛋白-1，但富含 介导胆固醇在质膜和高密度脂蛋白(SRB1、P-gp、ABCA1)之间的转运。我们现在提出四个建议 明确的目标。 目的1.确定存在哪些蛋白质(SRB1、ABCA1、P-gp)介导高密度脂蛋白-胆固醇流出/摄取 在质膜脂筏和小窝，它们之间的相互作用，以及与小窝-1的相互作用。 目的2.研究SCP-2如何与小窝蛋白1相互作用，以及SCP-2是否与脂筏或小窝蛋白结合 调节高密度脂蛋白-胆固醇摄取/流出(SRB-1、ABCA1、P-gp)。 目的3.确定SCP-2的表达是否改变了胆固醇的分布、胆固醇的动力学和结构 纯净的木筏和洞穴。SCP-2过表达的L细胞成纤维细胞和原代培养的SCP-2水平将被修饰 培养的SCP-2肝细胞高表达和基因消融小鼠。 目的4.在活细胞中，确定SCP-2的表达是否改变了胆固醇的分布、结构和动力学 在脂筏或小窝中。 这些结果将提供脂筏中胆固醇组织和动力学的基本、机制细节， SCP-2的调节，木筏和小窝在介导高密度脂蛋白摄取/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因子的潜在干预的见解。

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.