Cholesterol Metabolism-Role of Apo A-I and SR-B1

胆固醇代谢-Apo A-I 和 SR-B1 的作用

• 批准号: 6625685
• 负责人: DAVID LEE WILLIAMS
• 金额: $ 37.18万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 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 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6625685
• 关键词: adrenal glands; apolipoproteins; blood lipoprotein metabolism; blood lipoprotein transport; caveolins; cell membrane; cell morphology; cholesterol esters; crosslink; electron microscopy; electrospray ionization mass spectrometry; high density lipoproteins; laboratory mouse; light microscopy; lipid metabolism; liver cells; phospholipids; protein localization; protein structure function; receptor binding; receptor expression; scavenger receptor; steroid biosynthesis; tissue /cell culture

DESCRIPTION (Provided by applicant): The two aims of this proposal are focused on scavenger receptor B1 (SR-BI) and apolipoprotein Al (apoA-I) and their function in the lipoprotein cholesteryl ester (CE) selective uptake pathway. Previous studies with mice and rats and cultured human cells indicate that this pathway plays a major role in the uptake of high density lipoprotein (HDL) CE into the liver and steroidogenic cells. Studies in gene knockout mice show that SR-BI is the receptor responsible for HDL CE selective uptake and that apoA-I is the key HDL ligand for SR-BI. Recent studies indicate that SR-BI has multiple effects on cellular cholesterol metabolism including changes in plasma membrane properties. Aim 1 has four goals in which the mechanisms by which SR-BI alters plasma membrane properties will be investigated. Goal 1 will test the hypothesis that SR-BI is necessary for the formation of microvillar channels and the cell surface localization of HDL particles on steroidogenic cells. These experiments will compare wild type and SR-BI-deficient mice and will use ultrastructural analysis at the electron microscope (EM) level and HDL localization analyses at the light microscopic (LM) and EM levels. Goal 2 will test the role of caveolin-1 in SR-BI-mediated HDL CE selective uptake by comparisons of wild type and caveolin-1-deficient mice. These experiments include morphological analyses and in vivo analysis of HDL CE selective uptake. Goal 3 will test the hypothesis that SR-BI-induced alterations in plasma membrane morphology reflect changes in membrane phospholipids. Tandem mass spectrometry will be used to determine the phospholipid distribution and acyl tail compositions in membranes prepared from two cell types in which SR-BI alters membrane morphology, the mouse adrenal z. fasciculata cell and the insect Sf9 cell. Goal 4 will test the hypothesis the SR-BI is assembled in the plasma membrane of adrenal cells as a homo-oligomeric complex. Aim 2 is focused on the key issue of how SR-BI recognizes its ligand during docking of the HDL particle. These experiments use single cys mutants of apoA-I to provide site-specific chemical cross-linking to SR-BI. Sites of cross-linking in SR-BI will be determined by isolation of SR-BI peptides and identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry. These experiments test the hypothesis that SR-BI recognizes apoA-I via its amphipathic alpha-helical repeat units. Additionally, sites of cross-linking in SR-BI will identify key residues and receptor domains involved in the recognition of HDL particles. These studies will provide new and important information relevant to the role of HDL in protecting against atherosclerotic disease.

描述（由申请人提供）：该提案重点关注两个目标 清道夫受体 B1 (SR-BI) 和载脂蛋白 Al (apoA-I) 及其 在脂蛋白胆固醇酯（CE）选择性摄取途径中发挥作用。 先前对小鼠和大鼠以及培养的人类细胞的研究表明，这 途径在高密度脂蛋白 (HDL) CE 的摄取中起主要作用 进入肝脏和类固醇生成细胞。对基因敲除小鼠的研究表明 SR-BI 是负责 HDL CE 选择性摄取的受体，apoA-I 是 SR-BI 的关键 HDL 配体。最近的研究表明 SR-BI 对细胞胆固醇代谢的多重影响，包括血浆的变化 膜特性。目标 1 有四个目标，其中的机制 将研究 SR-BI 改变质膜特性的情况。目标 1 将进行测试 SR-BI对于微绒毛形成所必需的假设 HDL 颗粒对类固醇生成的通道和细胞表面定位 细胞。这些实验将比较野生型和 SR-BI 缺陷型小鼠 将使用电子显微镜 (EM) 水平和 HDL 的超微结构分析 光学显微镜 (LM) 和电磁水平的定位分析。目标 2 将 测试 Caveolin-1 在 SR-BI 介导的 HDL CE 选择性摄取中的作用 野生型和 Caveolin-1 缺陷型小鼠的比较。这些实验 包括 HDL CE 选择性摄取的形态学分析和体内分析。 目标 3 将检验 SR-BI 诱导血浆改变的假设 膜形态反映了膜磷脂的变化。串联质量 光谱法将用于确定磷脂分布和酰基 由两种细胞类型制备的膜中的尾部成分，其中 SR-BI 改变膜形态，小鼠肾上腺z。束状细胞和 昆虫Sf9细胞。目标 4 将检验 SR-BI 组装于 肾上腺细胞质膜作为同源寡聚复合物。目标2是重点 关于SR-BI在HDL对接过程中如何识别其配体的关键问题 粒子。这些实验使用 apoA-I 的单个 cys 突变体来提供 与 SR-BI 的位点特异性化学交联。 SR-BI 中的交联位点 将通过 SR-BI 肽的分离和鉴定来确定 基质辅助激光解吸电离飞行时间质谱法。 这些实验验证了 SR-BI 通过其识别 apoA-I 的假设 两亲性α螺旋重复单元。此外，交联位点 SR-BI将识别参与该过程的关键残基和受体结构域 HDL 颗粒的识别。这些研究将提供新的、重要的 与 HDL 在预防动脉粥样硬化中的作用相关的信息 疾病。