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: 7227105
• 负责人: Daisy Sahoo
• 金额: $ 36.78万
• 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 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7227105
• 关键词: Adenoviruses; Adrenal Glands; Affect; Atherosclerosis; Biliary; Binding; Biological Assay; Biological Models; C-terminal; Carbon; Cell membrane; Cells; Cholesterol; Cholesterol Esters; Cholesterol Homeostasis; Co-Immunoprecipitations; Complex; Cultured Cells; Cyclophosphamide/Fluorouracil/Prednisone; Cytoplasmic Tail; Data; Dimerization; Disruption; Enzymes; Epitopes; Extracellular Domain; Failure; Fatty Acids; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Goals; Grant; Hepatic Tissue; Hepatocyte; High Density Lipoprotein Cholesterol; High Density Lipoproteins; Hormones; Hydrolase; Hydrolysis; Immunofluorescence Immunologic; Insecta; Knockout Mice; Label; Length; Leucine Zippers; Life; Ligand Binding; Ligands; Link; Lipids; Lipoprotein Binding; Liposomes; Liver; Localized; Mammalian Cell; Measures; Mediating; Membrane; Membrane Fluidity; Membrane Microdomains; Metabolism; Methods; Molecular; Monitor; Morphology; Mus; Mutagenesis; Nucleosome Binding Domain; Pathology; Pattern; Peripheral; Phospholipids; Plasmids; Preparation; Process; Property; Regulation; Research; Research Personnel; Role; Role playing therapy; SR-BI receptor; Site; Site-Directed Mutagenesis; Spectrometry, Mass, Electrospray Ionization; Sphingomyelins; Spin Labels; Stearoyl-CoA Desaturase; System; Techniques; Testing; Time; Tissues; Transfection; Tryptophan; Vesicle; Wild Type Mouse; base; cardiovascular disorder prevention; desaturase; design; extracellular; high density lipoprotein receptor; hypercholesterolemia; improved; in vivo; inhibitor/antagonist; insight; mutant; novel; novel therapeutics; oxidized low density lipoprotein; prevent; receptor; receptor function; reconstitution; restoration; reverse cholesterol transport; sterol esterase; tandem mass spectrometry; uptake

The long-term objective of this research is to understand the function of scavenger receptor class B type I (SR-BI) in the delivery of cholesteryl ester (CE) from high density lipoproteins (HDL) to the plasma membrane for its subsequent metabolism. SR-BI is the HDL receptor that regulates HDL cholesterol metabolism and is directly linked to the ability of HDL to be athero-protective. Therefore, understanding how SR-BI delivers HDL-CE to a site in the plasma membrane that allows for its efficient metabolism is key to developing methods for prevention of cardiovascular disease. This proposal consists of three primary objectives that will evaluate the mechanisms of SR-BI-mediated uptake and hydrolysis of HDL-CE. Aim 1 will investigate the structural organization of SR-BI at the plasma membrane. Goal 1 will use fluorescence resonance energy transfer techniques to extend our understanding of the oligomeric organization of SR-BI in intact cells in the presence and absence of ligands. Additionally, mutagenesis studies will help delineate the region within SR-BI that is required for its oligomeric properties. Goal 2 will examine the functional domains in the extracellular domain of SR-BI using tryptophan quenching by spin labeled fatty acids. Aim 2 is designed to test the hypothesis that SR-BI modifies the composition of membrane phospholipids to favour selective uptake of HDL-CE. Goal 1will compare liver/adrenal membrane phospholipid profiles in wild-type and SR-BI knock-out mice using tandem mass spectrometry. Then, we will exploit adenovirus-mediated liver/adrenal expression of SR-BI and its mutants to investigate Ijhe structural regions/functions of SR-BI that are required for changes in phospholipid speciation. Goal 2 will examine the effects of SR-BI-independent alterations in membrane phospholipids on selective uptake efficiency, either by transfection of cells with desaturases/elongases or by reconstitution of SR-BI into liposomes with different ratios of sphingomyelin: cholesterol. Aim 3 will help us understand the mechanisms of HDL-CE delivery and hydrolysis at the plasma membrane. We hypothesize that an accumulation of CE in the plasma membrane will prevent further uptake of HDL-CE. Therefore, in Goal 1, vesicle reconstitution, in addition to the use of a novel fluorescent lipid, will let us measure the accumulation of CE at the plasma membrane in the presence of wild-type or a non-functional SR-BI mutant. Goal 2 will examine the co-localization of hormone-sensitive lipase with SR-BI at the plasma membrane for hydrolysis of HDL-CE in adrenal cells and tissues: Together, these studies will provide new mechanistic information about the role of SR-BI in HDL-CE selective uptake and hydrolysis, and shed new insights into cholesterol metabolism and protection against atherosclerosis.

本研究的长期目标是了解 B 类清道夫受体 I 型的功能 (SR-BI) 将胆固醇酯 (CE) 从高密度脂蛋白 (HDL) 输送至血浆 膜以供其随后的代谢。 SR-BI 是调节 HDL 胆固醇的 HDL 受体 代谢，与 HDL 的动脉粥样硬化保护能力直接相关。因此，了解 SR-BI 如何将 HDL-CE 传递到质膜中的某个位点以实现其高效代谢是关键 开发预防心血管疾病的方法。该提案包括三个主要内容 评估 SR-BI 介导的 HDL-CE 摄取和水解机制的目标。目标1 将研究 SR-BI 在质膜上的结构组织。目标 1 将使用荧光 共振能量转移技术扩展了我们对 SR-BI 寡聚组织的理解 在存在或不存在配体的完整细胞中。此外，诱变研究将有助于描述 SR-BI 内其寡聚特性所需的区域。目标 2 将检查功能 使用自旋标记脂肪酸进行色氨酸淬灭来构建 SR-BI 胞外结构域中的结构域。目标2 旨在测试 SR-BI 修改膜磷脂的组成以有利于 HDL-CE 的选择性摄取。目标 1 将比较野生型的肝脏/肾上腺膜磷脂谱 以及使用串联质谱法的 SR-BI 敲除小鼠。然后，我们将利用腺病毒介导的 SR-BI 及其突变体的肝脏/肾上腺表达，以研究 SR-BI 的结构区域/功能 是磷脂形态变化所必需的。目标 2 将检查独立于 SR-BI 的影响 通过转染细胞来改变膜磷脂对选择性摄取效率的影响 去饱和酶/延长酶或通过将 SR-BI 重建到具有不同比例的鞘磷脂的脂质体中： 胆固醇。目标 3 将帮助我们了解 HDL-CE 传递和水解的机制 质膜。我们假设 CE 在质膜中的积累会阻止 HDL-CE 的进一步摄取。因此，在目标 1 中，除了使用新颖的囊泡重构之外， 荧光脂质，让我们可以在存在以下物质的情况下测量质膜上 CE 的积累 野生型或无功能的 SR-BI 突变体。目标 2 将检查激素敏感的共定位 脂肪酶与质膜上的 SR-BI 一起水解肾上腺细胞和组织中的 HDL-CE：一起， 这些研究将提供有关 SR-BI 在 HDL-CE 选择性摄取中的作用的新机制信息 和水解，并为胆固醇代谢和预防动脉粥样硬化提供了新的见解。