Scavenger Receptor BI in Cellular Cholesterol Metabolism

描述（由申请人提供）：该提案的重点是清道夫受体BI （SR-BI）刺激细胞和脂蛋白之间游离胆固醇（FC）通量的机制。在逆向胆固醇转运（RCT）过程中，从外周细胞去除FC和肝细胞摄取FC和胆固醇酯（CE）分别是第一步和最后一步。在小鼠模型中，SR-BI已被证明参与了这两个步骤，并在防止动脉粥样硬化发展中发挥重要作用。本应用程序有3个特定目的，以评估sr - bi介导的胆固醇通量的机制和生理意义。Aim 1有4个目标，将测试SR-BI在细胞培养和小鼠模型体内的活性。目标1将使用诱变和功能研究来扩展我们对最近发现的SR-BI功能分离（SoF）突变的理解，这些突变失去了SR-BI的一些活性，但没有其他活性。目标2将在体内泡沫细胞模型中测试野生型和SoF突变体刺激FC清除的能力。目标3将测试SR-BI和SoF突变体在小鼠肝脏灌注系统中促进HDL FC和CE摄取、CE水解和FC转运到胆汁的能力。目标4将测试SR-BI/CD36嵌合体和SR-BI SoF突变体通过肾上腺特异性转基因表达恢复SR-BI缺陷小鼠正常肾上腺质膜功能和结构的能力。Aim 2有3个目标，分别检测SR-BI对膜组织的活性和膜组织对SR-BI活性的影响。目标1将使用电子显微镜方法识别SR-BI结构域和活性，这些结构域和活性是在质膜微绒毛延伸上受体聚集所必需的。目标2将检验流体膜结构域促进sr - bi介导的FC通量的假设。这些实验将在细胞中加入具有良好特征的甾醇类似物，以了解sr - bi介导的FC通量是否会通过增加质膜在流体中的比例而不是液体有序状态来增强。目标3将在活细胞中使用膜结构域探针的荧光显微镜来验证SR-BI通过摄取高密度脂蛋白胆固醇来调节膜结构域组织的假设。Aim 3将直接测试SR-BI对FC通量和FC在模型膜中的组织的影响，其中SR-BI被掺入重建的多层和单层大囊泡中。这些研究将为sr - bi介导的胆固醇通量及其在随机对照试验中的作用提供新的重要信息。

DESCRIPTION (provided by applicant): The proposal is focused on the mechanisms by which scavenger receptor BI (SR-BI) stimulates the flux of free cholesterol (FC) between cells and lipoproteins. FC removal from peripheral cells and FC and cholesteryl ester (CE) uptake by liver cells are the first and last steps, respectively, in the process of reverse cholesterol transport (RCT). SR-BI has been shown to participate in both steps and plays an important role in protecting against atherosclerosis development in mouse models. This application has 3 Specific Aims to evaluate the mechanisms and physiological significance of SR-BI-mediated cholesterol flux. Aim 1 has 4 goals that will test SR-BI activities in cell culture and in vivo in mouse models. Goal 1 will use mutagenesis and functional studies to extend our understanding of recently identified SR-BI separation of function (SoF) mutations that have lost some but not other activities of SR-BI. Goal 2 will test wild type and SoF mutants for the ability to stimulate FC clearance in an in vivo foam cell model. Goal 3 will test SR-BI and SoF mutants for the ability to promote HDL FC and CE uptake, CE hydrolysis, and transport of FC to bile using a mouse liver perfusion system. Goal 4 will test SR-BI/CD36 chimeras and SR-BI SoF mutants for the ability to restore normal adrenal plasma membrane function and structure in SR-BI-deficient mice using adrenal-specific transgene expression. Aim 2 has 3 goals that test the activity of SR-BI on membrane organization and test the effect of membrane organization on SR-BI activity. Goal 1 will use electron microscopic approaches to identify SR-BI domains and activities that are necessary for receptor clustering on microvillar extensions of the plasma membrane. Goal 2 will test the hypothesis that fluid membrane domains promote SR-BI-mediated FC flux. These experiments will incorporate well-characterized sterol analogues into cells to ask whether SR-BI-mediated FC flux is enhanced by increasing the fraction of the plasma membrane in the fluid versus liquid-ordered state. Goal 3 will employ fluorescence microscopy with membrane domain probes in living cells to test the hypothesis that SR-BI modulates membrane domain organization via uptake of HDL cholesterol. Aim 3 will directly test the effect of SR-BI on FC flux and the organization of FC in model membranes in which SR-BI is incorporated into reconstituted multilamellar and large unilamellar vesicles. These studies will provide new and important information about SR-BI-mediated cholesterol flux and its role in RCT.