Project 1 - Structural basis of HDL assembly

我们假设apoA-I是主要的高密度脂蛋白平台，并作为构象动态支架发挥作用 这促进了许多其他载脂蛋白和脂质重塑因子的相互作用。它是由此而来的 假设通过传统的单一学科不可能详细了解高密度脂蛋白汇编 方法，但需要一个综合的多学科团队方法。我们在这个项目中的目标是使用 这种方法以前所未有的详细程度理解高密度脂蛋白汇编的结构基础。我们将结合在一起 更传统的溶液中实验方法的硅胶方法：i)高分辨质量 光谱结合硫醇可裂解的交联化学(核心D)，II)定点突变(核心 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：目标1：三 高密度脂蛋白组装的早期步骤：i)ApoA-I单体结构和动力学。我们将试验性地测试我们的 使用交联和定点突变的模型(核心D)和功能分析(核心C)。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的构象和分子基础，以及这个构象循环是如何产生 磷脂泵(B芯)。使用纯化的哺乳动物细胞来源的ABCA1，我们将在实验中测试 分别通过质谱学-化学交联法(C和D芯法)和定点定向法建立模型 诱变(核心D)使用基于细胞的高密度脂蛋白功能分析和高密度脂蛋白颗粒定量(核心C)。Iii) 新生高密度脂蛋白组装的物理基础。我们将通过MD模拟来检验单层褶皱假说， 例如，研究溶酶PC和游离脂肪酸对盘膜分裂和脂化机制的影响。 二聚体apoA-I(核心B)。突变将针对ABCA1胞外区的关键赖氨酸残基 其中褶皱和载脂蛋白A-I结合被提议发生。高分辨率电磁断层扫描将用于 在不同的脂肪成分条件下检查萌芽盘的3D图像。目标2：两个中间步骤 高密度脂蛋白组装：i)模拟LCAT与apoA-I和dHDL(核心B)的相互作用。模特们将会是 使用交联剂和诱变剂进行实验测试(Heinecke)。二)对以下各项相互作用的模拟 载脂蛋白A-II含有载脂蛋白A-I和高密度脂蛋白。这些模型将使用交联剂(Davidson)进行实验测试。目标3： 高密度脂蛋白汇编中的三个后续步骤。I)abcg1结构和动力学。这些模型将使用以下工具开发 使用Modeler的同源建模、双态简正模式分析和MD模拟(核心B)。Ii) PLTP的详细分子模型及其与高密度脂蛋白的相互作用(Davidson)。模特们将会是 使用模型器(核心B)的同源建模、功能分析(核心C)、交叉分析进行实验测试 连接(C和D岩心)和高分辨率电磁层析成像。三)相互作用的详细分子模型 采用高密度脂蛋白的PON-1。ApoA-I(或Davidson确定的其他结构域)螺旋重复序列4的apoA-I突变将 重组为dHDL和PON-1活性测定。

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.