Selective Uptake and Hydrolysis of Cholesteryl Ester by SR-BI

 描述(申请人提供)：清道夫受体BI型(SR-BI)和高密度脂蛋白的受体-配体复合体负责通过反向胆固醇运输(RCT)将胆固醇从体内处置出去，在防止功能障碍的高密度脂蛋白形成和动脉粥样硬化方面至关重要。我们研究的长期目标是了解调节SR-BI介导的胆固醇酯(CE)从高密度脂蛋白到肝脏排泄的机制。这项提议包括两个主要目标，这两个目标将检验总的假设，即通过RCT的胆固醇流量取决于“功能”的高密度脂蛋白颗粒与高度结构的SR-BI的寡聚体组装的接触。目的1旨在确定SR-BI跨膜(TM)结构域的寡聚组织及其在促进高密度脂蛋白-CE选择性摄取中的作用。首先，我们将使用前沿的核磁共振策略来确定SR-BI的TM结构域的高分辨率结构和二聚化性质。接下来，我们将设计SR-BI的结构导向突变体，以确定在选择性摄取高密度脂蛋白-CE过程中，配体诱导的SR-BI的TM结构域的邻近和取向变化的要求。综上所述，这些研究将检验这一假设，即SR-BI的TM结构域的结构组织和构象灵活性在介导高密度脂蛋白-CE选择性摄取到质膜中起关键作用。在目标2中，我们将使用一系列基于细胞的分析和体内实验来确定受损的高密度脂蛋白/SR-BI相互作用的下游后果。首先，我们将确定氧化修饰的高密度脂蛋白阻止高密度脂蛋白-CE有效选择性摄取的机制。其次，我们将确定SR-BI缺陷是否会在体内产生功能障碍的高密度脂蛋白颗粒。最后，我们将确定人类SCARB1(人类SR-BI基因)突变对RCT和动脉粥样硬化的影响。这组实验将帮助我们检验这一假说，即破坏性的受体/配体相互作用通过SR-BI介导的选择性摄取和RCT阻止高密度脂蛋白的清除，并导致功能障碍的高密度脂蛋白颗粒的形成，从而促进动脉粥样硬化的形成。总之，这些研究将确定(I)高密度脂蛋白诱导的与SR-BI寡聚复合体相关的构象变化如何促进选择性摄取过程，以及(Ii)受损的受体/配体相互作用如何阻碍选择性摄取过程并降低RCT，导致功能障碍的、致动脉粥样硬化的高密度脂蛋白颗粒的形成。我们预计这些研究的发现将有助于确定治疗高胆固醇血症及其相关病理(如动脉粥样硬化)的新治疗策略。

 DESCRIPTION (provided by applicant): The receptor-ligand complex of scavenger receptor class B type I (SR-BI) and HDL is responsible for cholesterol disposal from the body via reverse cholesterol transport (RCT) and is critical in the prevention of dysfunctional HDL formation and atherosclerosis. The long-term objective of our research is to understand the mechanisms that regulate SR-BI-mediated delivery of cholesteryl ester (CE) from HDL to the liver for excretion. This proposal consists of two primary objectives that will test the overall hypothesis that cholesterol flux via RCT is dependent on the engagement of "functional" HDL particles with an oligomeric assembly of highly- structured SR-BI. Aim 1 is designed to determine the oligomeric organization of the transmembrane (TM) domains of SR-BI and their role in facilitating the selective uptake of HDL-CE. First, we will use cutting-edge NMR strategies to determine the high-resolution structures and dimerization properties of the TM domains of SR-BI. Next, we will design structure-guided mutants of SR-BI to define the requirement of ligand-induced changes in proximity and orientation of the TM domains of SR-BI in the selective uptake of HDL-CE. Together, these studies will test the hypothesis that the structural organization and conformational flexibility of the TM domains of SR-BI are critical in mediating the selective uptake of HDL-CE into the plasma membrane. In Aim 2, we will use a series of cell-based assays and in vivo experiments to determine the downstream consequences of an impaired HDL/SR-BI interaction. First, we will determine the mechanisms by which oxidatively-modified HDL prevents efficient selective uptake of HDL-CE. Second, we will determine whether SR-BI deficiency produces dysfunctional HDL particles in vivo. Finally, we will determine the effects of human mutants of SCARB1 (the human SR-BI gene) on RCT and atherosclerosis. This set of experiments will help us test the hypothesis that disruptive receptor/ligand interactions prevent HDL clearance via SR-BI-mediated selective uptake and RCT, and result in the formation of dysfunctional HDL particles that promote atherogenesis. Together, these studies will identify (i) the mechanisms by which HDL-induced conformational changes associated with the SR-BI oligomeric complex facilitate the selective uptake process and (ii) how impaired receptor/ligand interactions impede the selective uptake process and decrease RCT, resulting in the formation of dysfunctional, atherogenic HDL particles. We anticipate the findings from these studies will help identify novel therapeutic strategies for treating hypercholesterolemia and its associated pathologies such as atherosclerosis.