Selective Uptake and Hydrolysis of Cholesteryl Ester by SR-BI

描述（由申请人提供）：I型清道夫受体（SR-BI）是一种高密度脂蛋白（HDL）受体，可调节HDL-胆固醇代谢，并与HDL的动脉粥样硬化保护能力直接相关。我们研究的长期目标是了解SR-BI在将胆固醇酯（CE）从HDL传递到肝脏以处理胆固醇的功能。关于SR-BI如何介导HDL-CE递送效率的新见解是开发预防心血管疾病方法的关键。该提案包括三个主要目标，将评估SR-BI在质膜上的结构组织以及SR-BI与HDL的适当对齐如何介导胆固醇向肝脏的通量增强。目的1将确定SR-BI低聚物在体内的生理组织和相关性。目标1将使用双分子荧光互补结合荧光共振能量转移光谱来确认活细胞中SR-BI低聚物的存在，并监测配体接合时低聚物形成的变化。在目标2中，在SR-BI敲除小鼠中，通过腺病毒介导的寡聚化缺陷突变SR-BI受体的表达，将评估SR-BI寡聚化在逆向胆固醇运输中的生理相关性。目的2旨在检查促进HDL- ce选择性摄取的HDL和SR-BI之间“生产性复合物”形成（即正确对齐）的分子决定因素。在目标1中，将设计一系列SR-BI/CD36嵌合体，以确定SR-BI细胞外区域内对HDL-CE选择性摄取和“生产复合物”形成至关重要的区域。在目标2中，将结合位点特异性配体定向交联和质谱法来绘制SR-BI和HDL之间相互作用的位点。目的3将探讨SR-BI胞外结构域的构象如何影响从HDL到质膜的脂质转移。目标1将使用色氨酸猝灭来验证SR-BI疏水区域需要与质膜和/或配体相互作用以促进有效的脂质转移和胆固醇通量的假设。目标2将确定细胞外半胱氨酸残基在SR-BI功能中的作用，并设计实验以确定分子内和分子间的二硫键模式。总之，这些研究将提高我们对SR-BI如何介导HDL-CE选择性摄取效率的理解，并将为胆固醇代谢和预防动脉粥样硬化提供新的见解。

DESCRIPTION (provided by applicant): The class type I scavenger receptor (SR-BI) is the high density lipoprotein (HDL) receptor that regulates HDL- cholesterol metabolism and is directly linked to the ability of HDL to be athero-protective. The long-term objective of our research is to understand the function of SR-BI in the delivery of cholesteryl ester (CE) from HDL to the liver for cholesterol disposal. New insight into how SR-BI mediates the efficiency of HDL-CE delivery is key to developing methods for prevention of cardiovascular disease. This proposal consists of three primary objectives that will evaluate how the structural organization of SR-BI at the plasma membrane and the proper alignment of SR-BI with HDL mediate enhanced cholesterol flux to the liver. Aim 1 will determine the physiological organization and relevance of the SR-BI oligomer in vivo. Goal 1 will use bimolecular fluorescence complementation coupled with fluorescence resonance energy transfer spectroscopy to confirm the presence of SR-BI oligomers in live cells and monitor changes in oligomer formation upon ligand engagement. In Goal 2, the physiological relevance of SR-BI oligomerization in reverse cholesterol transport will be assessed following adenoviral-mediated expression of oligomerization-defective mutant SR-BI receptors in SR-BI knock-out mice. Aim 2 is designed to examine the molecular determinants for "productive complex" formation (i.e. proper alignment) between HDL and SR-BI that promote selective uptake of HDL-CE. In Goal 1, a series of SR-BI/CD36 chimeras will be designed to identify regions within the extracellular domain of SR-BI that are crucial for HDL-CE selective uptake and vital for "productive complex" formation. In Goal 2, the combination of site-specific ligand-directed crosslinking and mass spectrometry will be used to map sites of interaction between SR-BI and HDL. Aim 3 will explore how the conformation of the extracellular domain of SR-BI impacts lipid transfer from HDL to the plasma membrane. Goal 1 will use tryptophan quenching to test the hypothesis that hydrophobic regions of SR-BI are required to interact with the plasma membrane and/or ligand to facilitate efficient lipid transfer and cholesterol flux. Goal 2 will determine the role of extracellular cysteine residues in SR-BI function and experiments are designed to identify intra- and intermolecular disulfide bonding patterns. Together, these studies will improve our understanding of how SR-BI mediates the efficiency of HDL-CE selective uptake and will shed new insights into cholesterol metabolism and protection against atherosclerosis. PUBLIC HEALTH RELEVANCE: Heart disease kills more Americans each year than all cancers combined. Our research is designed to understand how we can improve cholesterol removal from the body and lower plasma cholesterol levels. Our findings will help identify new strategies for treating heart disease and other related complications.