INTERACTIONS OF APO A-I WITH LIPIDS AND CELL SURFACES

本项目的目的是阐明载脂蛋白A-I在胆固醇反向转运(RCT)中作用的分子机制。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的基础。具体目的1是确定apoA-I两亲性α-螺旋的性质，这是磷脂(PL)最佳增溶以及与不同大小的脂和脂蛋白颗粒高亲和力结合所需的。将使用物理-生物化学方法研究一系列工程设计的载脂蛋白A-I分子和具有改变的α-螺旋性质的合成肽，以建立控制脂质结合的定量参数。 具体目标2是确定载脂蛋白A-I分子的特征，这些特征对于它通过三磷酸腺苷结合盒转运体A1(ABCA1)与细胞膜上传递的磷脂酶和胆固醇分子结合，形成新生的(前β)高密度脂蛋白颗粒至关重要。将测量从培养中生长的表达ABCA1的成纤维细胞和巨噬细胞向工程apo A-I分子流出PL和胆固醇的动力学；还将确定在细胞外介质中产生的新生高密度脂蛋白颗粒的结构。这些颗粒的胆固醇运输特性将与项目1合作探索。具体目标3是定义载脂蛋白A-I的脂化以及与清道夫受体B类，I型(SR-BI)结合所需的α-螺旋的性质，以便随后选择性地摄取高密度脂蛋白 进入细胞是最理想的。不同的含有工程载脂蛋白A-I分子的高密度脂蛋白颗粒将与SR-BI表达细胞孵育，以研究高密度脂蛋白结构如何调节脂质转移到细胞。在与项目3的合作中，将在小鼠身上表达功能改变的不同载脂蛋白A-I分子，并将确定其对高密度脂蛋白代谢、RCT和动脉粥样硬化的影响。该项目的结果将有助于更好地理解载脂蛋白A-I和高密度脂蛋白预防过早冠状动脉疾病的机制。

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.