Scavenger Receptor BI in Cellular Cholesterol Metabolism

描述（由申请人提供）：该提案侧重于清道夫受体BI（SR-BI）刺激细胞和脂蛋白之间游离胆固醇（FC）流动的机制。在胆固醇逆向转运（RCT）过程中，外周细胞对FC的清除以及肝细胞对FC和胆固醇酯（CE）的摄取分别是第一步和最后一步。SR-BI已被证明参与这两个步骤，并在小鼠模型中预防动脉粥样硬化发展中发挥重要作用。本申请具有3个特定目的，以评价SR-BI介导的胆固醇通量的机制和生理学意义。目标1有4个目标，将在细胞培养物和小鼠模型中体内测试SR-BI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，这些突变已经失去了SR-BI的一些但不是其他活性。目标2将测试野生型和SoF突变体在体内泡沫细胞模型中刺激FC清除的能力。目标3将使用小鼠肝脏灌注系统测试SR-BI和SoF突变体促进HDL FC和CE摄取、CE水解以及FC转运至胆汁的能力。目标4将测试SR-BI/CD 36嵌合体和SR-BI SoF突变体使用肾上腺特异性转基因表达在SR-BI缺陷小鼠中恢复正常肾上腺质膜功能和结构的能力。目的2有3个目标，即检测SR-BI对细胞膜结构的影响，以及细胞膜结构对SR-BI活性的影响。目标1将使用电子显微镜的方法来确定SR-BI域和活动，是必要的受体聚集在质膜的微绒毛延伸。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。这些实验将充分表征的甾醇类似物掺入细胞中，以询问SR-BI介导的FC通量是否通过增加流体与液体有序状态中质膜的分数而增强。目标3将在活细胞中使用具有膜结构域探针的荧光显微镜来检验SR-BI通过摄取HDL胆固醇调节膜结构域组织的假设。目的3将直接测试SR-BI对FC通量和模型膜中FC的组织的影响，其中SR-BI被掺入重构的多层和大单层囊泡中。这些研究将为SR-BI介导的胆固醇流动及其在RCT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.