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）对于产生高密度脂蛋白至关重要 （HDL）又是该脂蛋白家族的所有受保护功能。许多尝试 通过各种细胞和细胞外机制来增强ABCA1介导的脂质转运活性。 由于多种原因，其中很少有人进入诊所。我们认为，是 临床上未能有效探索ABCA1途径是对如何了解如何 转运蛋白在分子水平上工作。采用机械和结构方法，包括硅和湿 台式实验，项目3的主要目标是描述ABCA1背后的分子机制 行动。我们在该计划项目的上一个周期中的工作导致了一个三步模型，该模型是中央 这项工作的假设。步骤1：ABCA1从宿主细胞的外膜传单中提取脂质并将其移动 进入其细胞外域中的疏水隧道（Segrest等人，ABCA1是一个细胞外PL易位酶，Na- Ture Communications，2022）。步骤2：在临界脂质的临界浓度下，ABCA1二聚体 请形成充满脂质的脂质受体。步骤3：细胞外脂质受体创建 ApoA1的高度特异性二聚体结合位点，因此从受体挤出的脂质会产生新生的HDL。我们的 MD研究支持模型，该研究支持靶向细胞外叶脂质及其在A中的积累 远程受体，通过我们对低温EM结构的改进，现在强烈支持从 通过我们的模型驱动的ABCA1和APOA1中的关键保留诱变的外部小叶。重要的是， 我们的模型解释了已用于支持直接结合和微纤维纤维的观察结果 机制。 AIM 1将集成在计算机和体外方法中，以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中的自然序列变体是否支持外部小叶脂质提取 模型。 ii）我们将使用MD模拟可视化将PLS添加到ABCA1提出的脂质收据中的效果 - 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 将通过预测会改变二聚化的一系列点突变来测试ABCA1二聚化的需求。 iv） 我们将建模APOA1与ABCA1的“扣子”域的交互作用，我们认为这对于HDL组装至关重要。 AIM 2提供了ABCA1自我相互作用的直接证据，可以从低温EM数据中确定并提供稳定 摆姿势重建双层中的二聚体。我们将将自我互动ABCA1重新建立为Unilammelar脂质体 和新颖的超大纳米盘平台，并以冰冻EM为特征。我们将利用我们的ABCA1重组 执行同位素辅助化学交联实验的系统：i）测试分子内接触的预测， ii）验证ABCA1二聚体并测试预测的分子间接触，iii）识别两个无脂质的位点 以及与脂质相关的APOA1和ABCA1，介导两个分子之间的相互作用。