Selective Uptake and Hydrolysis of Cholesteryl Ester by SR-BI

SR-BI 选择性摄取和水解胆固醇酯

• 批准号: 8625808
• 负责人: Daisy Sahoo
• 金额: $ 36.87万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-04-01 至 2015-08-17
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8625808
• 关键词: American; Apolipoprotein A-I; Atherosclerosis; Binding; Biotinylation; CD36 gene; Cell membrane; Cells; Chimera organism; Cholesterol; Cholesterol Esters; Cholesterol Homeostasis; Complex; Coupled; Cysteine; Data; Excision; Extracellular Domain; Fluorescence; Fluorescence Resonance Energy Transfer; Fourier transform ion cyclotron resonance; Goals; Grant; Heart Diseases; Hepatic; High Density Lipoprotein Cholesterol; High Density Lipoproteins; Hydrolysis; Knockout Mice; Laboratories; Lead; Life; Ligand Binding; Ligands; Link; Lipids; Lipoprotein Binding; Liver; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Measures; Mediating; Methods; Molecular; Molecular Conformation; Monitor; Mutation; Pathology; Pattern; Peptide Mapping; Peptides; Physiological; Plasma; Prevention; Property; Proteins; Protocols documentation; Research; Role; SR-BI receptor; Series; Site; Site-Directed Mutagenesis; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectrum Analysis; Stroke; Testing; Tryptophan; Variant; Work; adenoviral-mediated; base; cardiovascular disorder prevention; crosslink; design; dimer; disulfide bond; extracellular; high density lipoprotein receptor; hypercholesterolemia; improved; in vivo; insight; killings; monomer; mutant; novel therapeutics; prevent; public health relevance; receptor; research study; reverse cholesterol transport; scavenger receptor; uptake

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.

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。