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。

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.