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.

该项目的目的是阐明载脂蛋白（APO）A-I在反向胆固醇转运（RCT）中的功能的分子机制。 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的抗毒素特性。具体目的1是定义最佳磷脂（PL）溶解所需的Apo A-I两亲α螺旋的特性，并与不同大小的脂质和脂蛋白颗粒的高亲和力结合。将使用物理生物化学方法研究一系列工程的APO A-I分子和具有改变α-螺旋特性的合成肽，以建立控制脂质结合的定量参数。 具体目的2是确定Apo A-I分子的特征，对于通过ATP结合质膜传递的PL和胆固醇分子，通过ATP结合盒纸盒A1（ABCA1）结合了PL和胆固醇分子（ABCA1）。将测量来自ABCA1的成纤维细胞和巨噬细胞在培养物中生长到工程的Apo A-I分子中的PL和胆固醇外排的动力学；还将确定在细胞外培养基中产生的新生HDL颗粒的结构。这些颗粒的胆固醇传输特性将与项目1合作探索。具体目的3是定义APO A-I的脂化以及与Scavenger受体B类，I型（SR-BI）结合所需的α-螺旋的性能 进入细胞是最佳的。将与SR-BI-BI表达细胞一起孵育的不同HDL颗粒，以检查HDL结构如何调节脂质转移到细胞。与项目3合作，将在小鼠中表达具有改变功能的变体A-I分子，并确定对HDL代谢，RCT和动脉粥样硬化的影响。该项目的结果枯萎提供了对Apo A-I和HDL预防过早冠状动脉疾病的机制的更多了解。