Multi-disciplinary Approaches to HDL Structure, Assembly, and Functional Heterogeneity

摘要(满分) 正在开发新的心脏保护药物和准确的心血管风险测量方法 我们认为，这是因为未能正确识别高密度脂蛋白的心脏保护形式 (高密度脂蛋白)和对从动脉壁中清除胆固醇的机制了解不足。这 续订申请将通过七家世界级企业之间持续的动态互动来解决这些问题 从不同但互补的角度研究高密度脂蛋白的科学家。高密度脂蛋白，一个重要的介体 胆固醇的运输是当其最丰富的支架蛋白载脂蛋白A-I(APOA1)相互作用时产生的 结合三磷酸腺苷结合盒转运体A1(ABCA1)。这一过程对于从人体内去除胆固醇至关重要 巨噬细胞。事实上，人类血浆高密度脂蛋白促进胆固醇外流(CEC)的能力 对于心血管疾病(CVD)风险的诊断，而不是传统的高密度脂蛋白胆固醇含量的测量。这个 这项多学科项目拨款的中心假设是ABCA1从事高度具体的 与可能或可能不包含脂质的APOA1颗粒相互作用，产生对心脏具有保护作用的高密度脂蛋白。我们的 目的是从分子水平上了解这一途径以及高密度脂蛋白亚种在脑出血中的作用。 糖尿病的背景。我们将使用三种常见的重叠方法：1)校准离子迁移率 光谱分析将特定的高密度脂蛋白颗粒的大小和数量与糖尿病患者的CEC和心血管疾病风险联系起来， 2)详细的体外机制方法，以解开允许特定脂化高密度脂蛋白颗粒 与ABCA1相互作用，以及3)ABCA1‘S的结构和机制的冷冻EM和计算研究 行动。我们的计划集中在四个地点的三个项目：项目1：超小高密度脂蛋白对心脏的保护 粒子--Jay Heinecke，项目负责人；Karin BornFeldt，Co-I(华盛顿大学)；项目2： ABCA1介导的胆固醇外流至脂化高密度脂蛋白的机制 和PPG首席调查员(辛辛那提大学)；项目3：机制 磷脂/胆固醇转移，ABCA1-Jere Segrest，项目负责人(范德比尔特大学)； Steve Aler，Co-I(阿拉巴马大学伯明翰分校)。此外，四个核心设施将推动科学 NIH资源的协同作用和成本效益利用：核心A：行政-W.肖恩·戴维森，核心领导， 将为该计划提供行政支持。核心B：计算生物学-Jere Segrest，核心 Leader，将执行分子建模、同源建模和双/单态简正模式分析 用于结构研究。核心C：脂蛋白定量和功能--托马斯·维萨尔，核心领导者； 唐崇仁将量化i)高密度脂蛋白颗粒数量(高密度脂蛋白颗粒的大小和浓度)和 Ii)各种HDL和ABCA1突变体的胆固醇外流能力。核心D：载脂蛋白/脂蛋白的产生 -W.Sean Davidson，核心领导人，将为该计划提供高质量的定制载脂蛋白 突变体和脂蛋白样本，都是从人血浆中重组衍生和分离出来的。

SUMMARY (Entire PPG) The development of new cardioprotective drugs and accurate metrics for cardiovascular risk is being hindered, we believe, by the failure to correctly identify the cardioprotective forms of high-density lipoprotein (HDL) and inadequate knowledge about the mechanisms that remove cholesterol from the artery wall. This renewal application will address these issues by continuing dynamic interactions among seven world-class scientists who study HDL from diverse, but complementary, viewpoints. HDL, an important mediator of cholesterol transport, is created when its most abundant scaffold protein, apolipoprotein A-I (APOA1), interacts with ATP-binding cassette transporter A1 (ABCA1). This process is critical for removing cholesterol from macrophages. Indeed, the capacity for human plasma HDL to promote cholesterol efflux (CEC) is more diagnostic for cardiovascular disease (CVD) risk than the traditional measure of HDL’s cholesterol content. The central hypothesis of this multidisciplinary Program Project Grant is that ABCA1 engages in highly specific interactions with APOA1 particles that may or may not contain lipid to produce cardioprotective HDL. Our objective is to derive a molecular understanding of this pathway and the roles played by HDL subspecies in the setting of diabetes. We will use three general overlapping approaches: 1) calibrated ion mobility spectrometry to relate the size and number of specific HDL particles to CEC and CVD risk in diabetic patients, 2) detailed in vitro mechanistic approaches to unravel the factors that allow specific lipidated HDL particles to interact with ABCA1, and 3) cryo-EM and computational studies of the structure and mechanism of ABCA1’s action. Our plan focuses on three Projects at four sites: Project 1: Cardioprotection by extra-small HDL particles – Jay Heinecke, Project Leader; Karin Bornfeldt, Co-I (University of Washington); Project 2: Mechanism of ABCA1-mediated cholesterol efflux to lipidated HDL – W. Sean Davidson, Project Leader and PPG Principal Investigator (University of Cincinnati); Project 3: Mechanisms of phospholipid/cholesterol translocation by ABCA1 – Jere Segrest, Project Leader (Vanderbilt University); Steve Aller, Co-I (University of Alabama at Birmingham). Additionally, four core facilities will drive scientific synergy and cost-effective use of NIH resources: Core A: Administration – W. Sean Davidson, Core Leader, will provide administrative support for the Program. Core B: Computational Biology – Jere Segrest, Core Leader, will perform molecular modeling, homology modeling, and double/single state normal mode analyses for structural studies. Core C: Lipoprotein Quantitation and Function – Tomas Vaisar, Core Leader; Chongren Tang, Co-I, will quantify i) HDL particle number (the sizes and concentrations of HDL particles) and ii) the cholesterol efflux capacity of various HDLs and ABCA1 mutants. Core D: Apo/Lipoprotein Production – W. Sean Davidson, Core Leader, will supply the Program with high-quality apolipoproteins, customized mutants, and lipoprotein samples, both recombinantly derived and isolated from human plasma.