Project 1 - Structural basis of HDL assembly

项目1 - HDL组装的结构基础

• 批准号: 9073920
• 负责人: Jere P Segrest
• 金额: $ 22.03万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-15 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9073920
• 关键词: ATP binding cassette transporter 1; Anti-Inflammatory Agents; Anti-inflammatory; Apolipoprotein A-I; Apolipoprotein A-II; Apolipoproteins; Atherosclerosis; Biological Assay; Biological Process; Biology; Blood Vessels; Cations; Cells; Chemicals; Chemistry; Cholesterol; Complex; Computer Simulation; Computers; Computing Methodologies; Consensus; Coronary Arteriosclerosis; Environment; Evolution; Extracellular Domain; Future; Genetic; Heart Diseases; High Density Lipoprotein Cholesterol; High Density Lipoproteins; Homology Modeling; Learning; Lipids; Lipoproteins; Liquid substance; Low-Density Lipoproteins; Mammalian Cell; Mass Spectrum Analysis; Membrane; Metabolic Pathway; Modeling; Molecular; Molecular Conformation; Molecular Models; Mutagenesis; Mutation; Myocardial Infarction; Nucleosome Core Particle; Osteichthyes; Pharmaceutical Preparations; Phosphatidylcholine-Sterol O-Acyltransferase; Phospholipids; Program Research Project Grants; Proteins; Pump; Resolution; Risk; Role; Shapes; Site-Directed Mutagenesis; Structure; Sulfhydryl Compounds; Testing; Three-Dimensional Image; Time; Tube; Tweens; atherogenesis; base; cardiovascular risk factor; crosslink; density; drug development; extracellular; innovation; interdisciplinary approach; molecular modeling; monolayer; monomer; multidisciplinary; nanoscale; novel; particle; prevent; programs; reconstitution; scaffold; simulation; sound; tomography

We hypothesize that apoA-I is the major HDL platform and functions as a conformationally dynamic scaffold that facilitates the interaction of a host of other apolipoproteins and lipid remodeling factors. It follows from this hypothesis that a detailed understanding of HDL assembly is not possible through traditional unidisciplinary approaches but requires an integrated multidisciplinary team approach. Our objective in this project is to use that approach to understand, in unprecedented detail, the structural basis for HDL assembly. We will combine in silico approaches with more traditional in-solution experimental approaches: i) high resolution mass spectrometry combined with thiol-cleavable cross-linking chemistry (Core D), ii) site-directed mutagenesis (Core D), and iii) functional studies (Core C). To achieve this objective, we propose three specific aims: Aim 1: Three early steps in HDL assembly: i) ApoA-I monomer structure and dynamics. We will experimentally test our models using cross-linking and site-directed mutagenesis (Core D) and assays of function (Core C). ii) ABCA1 structure and dynamics. We will use computational methods to develop the extracellular- and intracellular-facing conformations and the molecular basis for this conformational cycle of ABCA1 and how this cycle produces a phospholipid pump (Core B). Using purified and mammalian cell-derived ABCA1, we will experimentally test the models by, respectively, mass spectroscopy-chemical cross-linking (Cores C and D) and site-directed mutagenesis (Core D) using cell-based assays of HDL function and quantification of HDL particles (Core C). iii) Physical basis for assembly of nascent HDL. We will test the monolayer pleating hypothesis by MD simulations, examining, for example, the effects of lysoPC and FFA on disc-membrane fission and mechanisms of lipidation of dimeric apoA-I (Core B). Mutagenesis will target key Lys residues in the extracellular domain of ABCA1 where pleating and apoA-I association is propose to occur. High resolution EM tomography will be used to examine 3D images of budding discs under various conditions of lipid composition. Aim 2: Two middle steps in HDL assembly: i) Simulations of the interactions of LCAT with apoA-I and dHDL (Core B). The models will be experimentally tested using cross-linking and mutagenesis (Heinecke). ii) Simulations of the interactions of apoA-II with apoA-I and HDL. The models will be experimentally tested using cross-linking (Davidson). Aim 3: Three later steps in HDL assembly. i) ABCG1 structure and dynamics. The models will be developed using homology modeling with MODELLER, double state normal mode analysis and MD simulations (Core B). ii) Detailed molecular models of PLTP and its interactions with HDL (Davidson). The models will be experimentally tested using homology modeling with MODELLER (Core B), assays of function (Core C), cross- linking (Cores C and D) and high resolution EM tomography. iii) Detailed molecular models of the interactions of PON-1 with HDL. ApoA-I mutations in helical repeat 4 in apoA-I (or other domains identified by Davidson) will be reconstituted as dHDL and PON-1 activity determined.

我们假设apoA-I是HDL的主要平台，并作为构象动态支架发挥作用 其促进许多其它载脂蛋白和脂质重塑因子的相互作用。由此可见 假设HDL组装的详细了解是不可能通过传统的单学科 但需要一个综合的多学科团队的方法。我们在这个项目中的目标是使用 这种方法可以以前所未有的细节来理解HDL组装的结构基础。我们将联合收割机 采用更传统的溶液中实验方法的硅方法：i）高分辨率质量 光谱法结合硫醇可裂解交联化学（核心D），ii）定点诱变（核心D）， D）和iii）功能研究（核心C）。为了实现这一目标，我们提出了三个具体目标： HDL组装的早期步骤：i）ApoA-I单体结构和动力学。我们将通过实验测试我们的 使用交联和定点诱变（核心D）和功能测定（核心C）的模型。ii）ABCA 1 结构和动力学。我们将使用计算方法来开发细胞外和细胞内的面向 构象和ABCA 1的这种构象循环的分子基础，以及这种循环如何产生 磷脂泵（核心B）。使用纯化的和哺乳动物细胞衍生的ABCA 1，我们将实验性地测试 分别通过质谱化学交联（核心C和D）和定点 使用基于细胞的HDL功能测定和HDL颗粒的定量（核心C），对突变（核心D）进行分析。（三） 新生HDL组装的物理基础。我们将通过MD模拟来检验单层褶皱假设， 例如，检查lysoPC和FFA对盘膜分裂和脂质化机制的影响 二聚体apoA-I（核心B）。突变将靶向ABCA 1胞外结构域中的关键Lys残基 其中提出发生褶皱和apoA-I缔合。高分辨率电磁层析成像将用于 检查在脂质组成的各种条件下的芽盘的3D图像。目标2：两个中间步骤 HDL组装：i）LCAT与apoA-I和dHDL（核心B）的相互作用的模拟。模特们将在 使用交联和诱变进行实验测试（Heinecke）。（二）模拟下列物质的相互作用： apoA-II与apoA-I和HDL。将使用交联（Davidson）对模型进行实验测试。目标3： HDL装配的三个后续步骤。ABCG 1的结构和动力学。这些模型将使用 使用MODELLER的同源性建模、双态正常模式分析和MD模拟（核心B）。ii）第二阶段 PLTP及其与HDL相互作用的详细分子模型（Davidson）。模特们将在 使用MODELLER（核心B）的同源建模、功能测定（核心C）、交叉- 连接（核心C和D）和高分辨率EM断层扫描。iii）以下物质相互作用的详细分子模型 PON-1与HDL。apoA-I（或Davidson鉴定的其他结构域）中螺旋重复序列4中的ApoA-I突变将 作为dHDL和PON-1活性测定的重构。