Selective Uptake and Hydrolysis of Cholesteryl Ester by SR-BI

描述(申请人提供)：第I类清道夫受体(SR-BI)是一种高密度脂蛋白(HDL)受体，调节高密度脂蛋白-胆固醇代谢，并与高密度脂蛋白具有抗动脉粥样硬化的能力直接相关。我们研究的长期目标是了解SR-BI在将胆固醇酯(CE)从高密度脂蛋白输送到肝脏以处理胆固醇方面的功能。对SR-BI如何调节高密度脂蛋白-CE传递效率的新见解是开发预防心血管疾病的方法的关键。这项建议由三个主要目标组成，将评估SR-BI在质膜上的结构组织以及SR-BI与高密度脂蛋白的适当对齐如何介导增加的胆固醇流向肝脏。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和荧光共振能量转移光谱来确认活细胞中SR-BI寡聚体的存在，并监测配体结合时寡聚体形成的变化。在目标2中，在腺病毒的介导下，在SR-BI基因敲除小鼠中表达有寡聚缺陷的SR-BI受体后，将评估SR-BI寡聚在胆固醇反向运输中的生理学相关性。AIM 2旨在研究在高密度脂蛋白和SR-BI之间形成“生产性复合体”(即适当的排列)的分子决定因素，促进对高密度脂蛋白-CE的选择性摄取。在目标1中，将设计一系列SR-BI/CD36嵌合体，以确定SR-BI胞外区域内对高密度脂蛋白-CE选择性摄取至关重要的区域，以及对“生产性复合体”形成至关重要的区域。在目标2中，将使用特定位点配体定向交联和质谱学相结合的方法来绘制SR-BI和高密度脂蛋白之间的相互作用位点。目的3将探索SR-BI胞外区的构象如何影响从高密度脂蛋白到质膜的脂质转移。目标1将使用色氨酸猝灭来验证这样的假设，即SR-BI的疏水区需要与质膜和/或配体相互作用，以促进有效的脂质转移和胆固醇流动。目标2将确定细胞外半胱氨酸残基在SR-BI功能中的作用，实验旨在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如何调节高密度脂蛋白-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。 与公共健康相关：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提高体内胆固醇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.