INTERACTIONS OF APO A-I WITH LIPIDS AND CELL SURFACES

本研究旨在阐明载脂蛋白A-I在胆固醇逆向转运中的分子机制。载脂蛋白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性质的基础。具体目标1是定义磷脂（PL）的最佳溶解以及与不同大小的脂质和脂蛋白颗粒的高亲和力结合所需的apo A-I两亲性α-螺旋的性质。将使用物理-生化方法研究一系列工程化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apo A-I分子通过结合PL和胆固醇分子形成新生（前β）HDL颗粒至关重要，所述胆固醇分子通过ATP结合盒转运蛋白A1（ABCA 1）从细胞质膜递送。将测量PL和胆固醇从培养物中生长的表达ABCA 1的成纤维细胞和巨噬细胞流出至工程化apo A-I分子的动力学;还将确定细胞外培养基中产生的新生HDL颗粒的结构。这些颗粒的胆固醇转运特性将与项目1合作进行探索。具体目标3是确定载脂蛋白A-I的脂化和与清道夫受体B类I型（SR-BI）结合所需的α-螺旋的性质，以便随后选择性摄取HDL脂质 细胞是最佳的。将含有工程化apo A-I分子的不同HDL颗粒与SR-BI表达细胞一起孵育，以检查HDL结构如何调节脂质转移至细胞。与项目3合作，将在小鼠中表达功能改变的变体apo A-I分子，并确定对HDL代谢、RCT和动脉粥样硬化的影响。该项目的结果将提供对apo A-I和HDL预防早发性冠状动脉疾病的机制的更深入的理解。

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.