INTERACTIONS OF APO A-I WITH LIPIDS AND CELL SURFACES

APO A-I 与脂质和细胞表面的相互作用

• 批准号: 6925494
• 负责人: Michael C. Phillips
• 金额: $ 31.3万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6925494
• 关键词: apolipoproteins; blood lipoprotein metabolism; chemical binding; high density lipoproteins; human tissue; hydropathy; intermolecular interaction; lipid transport; membrane activity; membrane transport proteins; molecular polarity; molecular shape; molecular size; phospholipids; protein binding; protein engineering; protein structure function; protein transport; receptor; scavenger receptor; tissue /cell culture

The goal of this project is to elucidate the molecular mechanisms underlying the functions of apolipoprotein (apo) A-I in reverse cholesterol transport (RCT). Apo A-I is the major protein of plasma high density lipoprotein (HtDL) and the functions of this molecule underlie the anti-atherogenic properties of this lipoprotein. Specific Aim 1 is to define the properties of apo A-I amphipathic alpha-helices required for optimal solubilization of phospholipid (PL), and high affinity binding to lipid and lipoprotein particles of different sizes. A range of engineered apo A-I molecules and synthetic peptides with altered alpha-helix properties will be studied using physical-biochemical methods to establish the quantitative parameters that control lipid binding. Specific Aim 2 is to determine the features of the apo A-I molecule that are critical for it to form nascent (pre-beta) HDL particles by binding PL and cholesterol molecules delivered from a cell plasma membrane via the ATP-binding cassette transporter A1 (ABCA1). The kinetics of PL and cholesterol efflux from ABCA1-expressing fibroblasts and macrophages growing in culture to the engineered apo A-I molecules will be measured; the structures of the nascent HDL particles created in the extracellular medium will also be determined. The cholesterol transport properties of these particles will be explored in collaboration with Project 1. Specific Aim 3 is to define the lipidation of apo A-I and the properties of alpha-helices required for binding to scavenger receptor class B, type I (SR-BI) so that the subsequent selective uptake of HDL lipids into cells is optimal. Different HDL particles containing engineered apo A-I molecules will be incubated with SR-BI-expressing cells to examine how HDL structure modulates lipid transfer to the cells. In collaboration with Project 3, variant apo A-I molecules with altered functionality will be expressed in mice and the effects on HDL metabolism, RCT and atherosclerosis will be determined. The results of this project wilt provide greater understanding of the mechanisms by which apo A-I and HDL protect against premature coronary artery disease.

该项目的目标是阐明载脂蛋白 (apo) A-I 在胆固醇反向转运 (RCT) 中功能的分子机制。 Apo A-I 是血浆高密度脂蛋白 (HtDL) 的主要蛋白质，该分子的功能是该脂蛋白抗动脉粥样硬化特性的基础。具体目标 1 是确定最佳溶解磷脂 (PL) 所需的 apo A-I 两亲性 α 螺旋的特性，以及与不同大小的脂质和脂蛋白颗粒的高亲和力结合。将使用物理生化方法研究一系列工程化的 apo A-I 分子和具有改变的 α 螺旋特性的合成肽，以建立控制脂质结合的定量参数。 具体目标 2 是确定 apo A-I 分子的特征，这些特征对于它通过 ATP 结合盒转运蛋白 A1 (ABCA1) 结合从细胞质膜递送的 PL 和胆固醇分子来形成新生（前β）HDL 颗粒至关重要。 ）。将测量培养物中生长的表达 ABCA1 的成纤维细胞和巨噬细胞的 PL 和胆固醇流出至工程化 apo A-I 分子的动力学；细胞外介质中产生的新生 HDL 颗粒的结构也将被确定。这些颗粒的胆固醇转运特性将与项目 1 合作进行探索。具体目标 3 是确定 apo A-I 的脂化以及与 B 类清道夫受体 I 型 (SR-BI) 结合所需的 α ​​螺旋特性以便随后选择性摄取 HDL 脂质 进入细胞是最佳的。含有工程化 apo A-I 分子的不同 HDL 颗粒将与表达 SR-BI 的细胞一起孵育，以检查 HDL 结构如何调节脂质向细胞的转移。与项目 3 合作，功能改变的变体 apo A-I 分子将在小鼠中表达，并确定其对 HDL 代谢、RCT 和动脉粥样硬化的影响。该项目的结果将使人们更好地了解载脂蛋白 A-I 和 HDL 预防过早冠状动脉疾病的机制。