Mechanisms of phospholipid/cholesterol translocation by ABCA1

ABCA1 的磷脂/胆固醇易位机制

• 批准号: 10711264
• 负责人: Jere P Segrest
• 金额: $ 19.98万
• 依托单位: UNIVERSITY OF CINCINNATI
• 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 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10711264
• 关键词: ATP-Binding Cassette Transporters; Apolipoprotein A-I; Apolipoprotein A-II; Apolipoproteins; Binding; Binding Sites; Cell membrane; Cells; Chemicals; Cholesterol; Clinic; Clinical Pathways; Collaborations; Communication; Computer Models; Cryoelectron Microscopy; Cultured Cells; Data; Detergents; Diabetes Mellitus; Dimerization; Disparate; Extracellular Domain; Failure; Family; Goals; Grain; Heterogeneity; High Density Lipoprotein Cholesterol; High Density Lipoproteins; Homeostasis; Human; Hydrophobicity; In Vitro; Isotopes; Lipids; Lipoproteins; Liposomes; Macrophage; Measures; Mediating; Membrane; Micelles; Modeling; Molecular; Mutagenesis; Nature; Phospholipids; Plasma; Plasma Cells; Point Mutation; Production; Publications; Radiolabeled; Resolution; Scheme; Series; Site; Structure; System; Testing; Validation; Variant; Visualization; Work; cardiovascular disorder risk; crosslink; dimer; experimental study; extracellular; in silico; interdisciplinary approach; lipid transport; molecular dynamics; nanodisk; nanosized; novel; particle; programs; reconstitution; translocase

SUMMARY (Project 3) The ATP-binding cassette transporter A1 (ABCA1) is critical for the production of high-density lipoproteins (HDL) and, in turn, for all the protective functions of that lipoprotein family. Numerous attempts have been made to boost ABCA1-mediated lipid transport activity through various cellular and extracellular mechanisms. For a variety of reasons, few of these have made it into the clinic. We believe that a major factor underlying the failure to effectively exploit the ABCA1 pathway clinically is a fundamental lack of understanding of how the transporter works at the molecular level. Taking a mechanistic and structural approach, both in silico and in wet bench experiments, the main goal of Project 3 is to delineate the molecular mechanism behind ABCA1’s action. Our work in the previous cycle of this Program Project led to a 3-step model that serves as the central hypothesis of this work. Step 1: ABCA1 extracts lipid from the host cell’s outer membrane leaflet and moves it into a hydrophobic tunnel in its extracellular domain (Segrest et al., ABCA1 is an extracellular PL translocase, Na- ture Communications, 2022). Step 2: At a critical concentration of translocated lipid, ABCA1 dimerizes to com- plete the formation of a lipid receptacle that fills with lipid. Step 3: The extracellular lipid receptacle creates a highly specific dimeric binding site for APOA1 so that lipid extruded from the receptacle creates nascent HDL. Our model is supported by MD studies that support the targeting of extracellular leaflet lipid and its accumulation in a remote receptacle, by our refinement of the cryo-EM structure, which now strongly supports lipid extraction from the outer leaflet, and by mutagenesis of key residues in ABCA1 and APOA1 driven by our models. Importantly, our model explains observations that have been used to support both the direct binding and the microsolubiliza- tion mechanisms. Aim 1 will integrate in silico and in vitro approaches to test four key aspects of our model. i) We will experimentally determine if ABCA1 extracts lipids from the extracellular leaflet of the cell membrane and will use MD studies to elucidate whether natural sequence variants in ABCA1 support the outer leaflet lipid extraction model. ii) We will use MD simulations to visualize the effects of adding PLs into ABCA1’s proposed lipid recepta- cle domain and evaluate the receptacle as a potential binding site for amphipathic helical apolipoproteins. iii) We will test the requirement for ABCA1 dimerization via a series of point mutations predicted to alter dimerization. iv) We will model APOA1’s interactions with a ‘clasp’ domain of ABCA1 that we believe is critical for HDL assembly. Aim 2 presents direct evidence for ABCA1 self-interaction that is stable to detergent from cryo-EM data and pro- poses to reconstruct the dimer in bilayers. We will reconstitute self-interacting ABCA1 into unilammelar liposomes and novel oversized nanodisc platforms and characterize by cryo-EM. We will exploit our ABCA1 reconstitution system to perform isotope-assisted chemical crosslinking experiments to: i) test intramolecular contacts predicted, ii) verify that ABCA1 dimerizes, and test predicted intermolecular contacts, and iii) identify sites on both lipid-free and lipid-associated APOA1 and ABCA1 that mediate interactions between the two molecules.

摘要（项目 3） ATP 结合盒转运蛋白 A1 (ABCA1) 对于高密度脂蛋白的产生至关重要 (HDL)，进而研究该脂蛋白家族的所有保护功能。 通过各种细胞和细胞外机制增强 ABCA1 介导的脂质转运活性。 由于多种原因，这些药物很少进入临床，我们认为这是造成这种情况的一个主要因素。 未能在临床上有效利用 ABCA1 通路是因为对 ABCA1 通路如何发挥作用缺乏了解。 转运蛋白在分子水平上发挥作用，在计算机和湿法中均采用机械和结构方法。 实验室实验中，项目 3 的主要目标是描绘 ABCA1 背后的分子机制 我们在该计划项目的上一个周期中的工作形成了一个作为中心的三步模型。 这项工作的假设 步骤 1：ABCA1 从宿主细胞的外膜小叶中提取脂质并移动它。 进入其胞外结构域的疏水通道（Segrest 等人，ABCA1 是一种胞外 PL 转位酶，Na- ture Communications，2022) 步骤 2：在易位脂质的临界浓度下，ABCA1 二聚化。 完成充满脂质的脂质容器的形成第3步：细胞外脂质容器创建一个。 APOA1 的高度特异性二聚体结合位点，以便从容器中挤出的脂质产生新生的 HDL。 该模型得到 MD 研究的支持，这些研究支持细胞外小叶脂质的靶向及其在 远程容器，通过我们对冷冻电镜结构的改进，现在强烈支持从 重要的是，通过我们的模型驱动的 ABCA1 和 APOA1 中关键残基的诱变。 我们的模型解释了用于支持直接结合和微溶解的观察结果 目标 1 将整合计算机和体外方法来测试我们模型的四个关键方面 i) 我们。 将通过实验确定 ABCA1 是否从细胞膜的细胞外小叶中提取脂质，并将 使用 MD 研究来阐明 ABCA1 中的自然序列变异是否支持外叶脂质提取 ii) 我们将使用 MD 模拟来可视化将 PL 添加到 ABCA1 提出的脂质受体中的效果。 cle 结构域并评估受体作为两亲性螺旋载脂蛋白的潜在结合位点 iii) 我们。 将通过一系列预计会改变二聚化的点突变来测试 ABCA1 二聚化的要求 iv) 我们将模拟 APOA1 与 ABCA1 的“扣环”结构域的相互作用，我们认为该结构域对于 HDL 组装至关重要。 目标 2 提供了 ABCA1 自相互作用的直接证据，该自相互作用对来自冷冻电镜数据和亲的洗涤剂稳定。 我们将把自相互作用的 ABCA1 重建成单层脂质体。 以及以冷冻电镜为特征的新型超大纳米圆盘平台，我们将利用我们的 ABCA1 重构。 系统进行同位素辅助化学交联实验：i) 测试预测的分子内接触， ii) 验证 ABCA1 二聚化，并测试预测的分子间接触，以及 iii) 识别无脂质上的位点 与脂质相关的 APOA1 和 ABCA1 介导两个分子之间的相互作用。