Project 1 - Structural basis of HDL assembly

项目1 - HDL组装的结构基础

基本信息

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
项目类别：
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

项目摘要

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 是主要的 HDL 平台，并作为构象动态支架发挥作用促进许多其他载脂蛋白和脂质重塑因子的相互作用。由此可见假设通过传统的单学科不可能详细了解 HDL 组装方法，但需要综合的多学科团队方法。我们在这个项目中的目标是使用这种方法能够以前所未有的细节了解 HDL 组装的结构基础。我们将结合在计算机方法与更传统的溶液内实验方法：i) 高分辨率质量光谱测定法与硫醇可裂解交联化学相结合（核心 D），ii) 定点诱变（核心 D) 和 iii) 功能研究（核心 C）。为实现这一目标，我们提出三个具体目标：目标 1：三 HDL 组装的早期步骤：i) ApoA-I 单体结构和动力学。我们将通过实验来测试我们的使用交联和定点诱变（核心 D）和功能分析（核心 C）的模型。 ii)ABCA1 结构和动力学。我们将使用计算方法来开发面向细胞外和细胞内的 ABCA1 构象循环的分子基础以及该循环如何产生磷脂泵（核心 B）。使用纯化的哺乳动物细胞衍生的 ABCA1，我们将通过实验测试分别通过质谱-化学交联（核心 C 和 D）和定点模型使用基于细胞的 HDL 功能测定和 HDL 颗粒定量（核心 C）进行诱变（核心 D）。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 对椎间盘膜裂变和脂化机制的影响二聚 apoA-I（核心 B）。诱变将针对 ABCA1 胞外域中的关键赖氨酸残基其中建议发生打褶和 apoA-I 关联。高分辨率电磁断层扫描将用于检查不同脂质成分条件下出芽椎间盘的 3D 图像。目标 2：中间的两个步骤 HDL 组装：i) LCAT 与 apoA-I 和 dHDL 相互作用的模拟（核心 B）。这些模型将是使用交联和诱变进行实验测试（Heinecke）。 ii) 相互作用的模拟 apoA-II 与 apoA-I 和 HDL。这些模型将使用交联（戴维森）进行实验测试。目标 3： HDL 组装的三个后续步骤。 i) ABCG1 结构和动力学。这些模型将使用以下方式开发使用 MODELLER 进行同源建模、双态正则模式分析和 MD 模拟（核心 B）。二） PLTP 及其与 HDL 相互作用的详细分子模型 (Davidson)。这些模型将是使用 MODELLER 的同源建模（核心 B）、功能分析（核心 C）、交叉连接（核心 C 和 D）和高分辨率 EM 断层扫描。 iii) 相互作用的详细分子模型带有 HDL 的 PON-1。 apoA-I（或 Davidson 鉴定的其他结构域）螺旋重复 4 中的 ApoA-I 突变将被重构为dHDL和PON-1活性测定。