Selective Uptake and Hydrolysis of Cholesteryl Ester by SR-BI

描述（由申请人提供）：I类清道夫受体（SR-BI）是调节HDL-胆固醇代谢的高密度脂蛋白（HDL）受体，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在胆固醇酯（CE）从HDL输送到肝脏进行胆固醇处置中的功能。对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加。目的1将确定SR-BI寡聚体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法来确认活细胞中SR-BI低聚物的存在，并监测配体接合后低聚物形成的变化。在目标2中，将在SR-BI敲除小鼠中寡聚化缺陷突变体SR-BI受体的腺病毒介导的表达后评估SR-BI寡聚化在胆固醇反向转运中的生理相关性。目的2旨在检查HDL和SR-BI之间形成“生产性复合物”（即适当对齐）的分子决定因素，这些决定因素促进HDL-CE的选择性摄取。在目标1中，将设计一系列SR-BI/CD 36嵌合体，以鉴定SR-BI胞外结构域内对HDL-CE选择性摄取至关重要且对“生产性复合物”形成至关重要的区域。在目标2中，位点特异性配体定向交联和质谱法的组合将用于绘制SR-BI和HDL之间相互作用的位点。目的3探讨SR-BI胞外结构域的构象如何影响脂质从HDL向质膜的转移。目标1将使用色氨酸淬灭来检验以下假设：需要SR-BI的疏水区与质膜和/或配体相互作用，以促进有效的脂质转移和胆固醇通量。目标2将确定细胞外半胱氨酸残基在SR-BI功能中的作用，并设计实验以鉴定分子内和分子间二硫键模式。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。 公共卫生相关性：每年死于心脏病的美国人比所有癌症的总和还要多。我们的研究旨在了解我们如何改善胆固醇从体内的清除和降低血浆胆固醇水平。我们的发现将有助于确定治疗心脏病和其他相关并发症的新策略。

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.