Asymmetric Distribution of Cholesterol in Membranes

描述(申请人提供)：质膜富含胆固醇的微域、脂筏和小凹，介导高密度脂蛋白的反向胆固醇运输、信号传递、抗体识别，并作为潜在的生物恐怖毒素、病毒和寄生虫的进入门户。虽然胆固醇对脂筏/小窝的功能是必不可少的，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。初步研究表明，类固醇携带蛋白-2在体内外与小凹-1结合，改变脂筏/小窝胆固醇结构域，抑制高密度脂蛋白介导的胆固醇从富含小窝-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基因靶向的小鼠)提供了一个模型，用于研究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之间运输的蛋白质(SRB1、P-gp、ABCA1)的胆固醇膜动力学。我们现在提出四个具体目标。目的1.确定介导高密度脂蛋白-胆固醇流出/摄取的蛋白质(SRB1、ABCA1、P-gp)存在于质膜脂筏和小窝中，以及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与小窝蛋白-1的相互作用，以及SCP-2是否与介导高密度脂蛋白摄取/外流的脂筏或小窝蛋白(SRB-1、ABCA1、P-gp)结合。目的3.确定SCP-2表达是否改变了纯化的排泄物和小窝中的胆固醇分布、胆固醇动力学和结构。SCP-2过表达的L细胞成纤维细胞和SCP-2基因去除小鼠的原代培养的肝细胞将改变SCP-2的水平。目的4.在活细胞中，确定SCP-2的表达是否改变了脂筏或小窝中胆固醇的分布、结构和动力学。这些结果将提供胆固醇在脂筏中的组织和动力学的基本的、机械的细节，SCP-2的调节，脂筏和小窝在调节高密度脂蛋白-胆固醇摄取/流出中的不同作用，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。

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.