Conformational and functional analysis of Apolipoprotein E

载脂蛋白E的构象和功能分析

• 批准号: 10088363
• 负责人: Andrea Soranno
• 金额: $ 44.45万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10088363
• 关键词: Adopted; Affinity; Alleles; Alzheimer' s Disease; Alzheimer' s disease risk; Apolipoprotein E; Binding; Binding Proteins; Biochemical; Cardiovascular Pathology; Cardiovascular system; Carrier Proteins; Cerebrospinal Fluid; Cholesterol Homeostasis; Complex; Data; Disease; Equilibrium; Exhibits; Fluorescence Resonance Energy Transfer; Fluorescence Spectroscopy; Health; Heterogeneity; Late Onset Alzheimer Disease; Length; Ligands; Light; Link; Lipid Binding; Lipids; Lipoproteins; Methods; Modeling; Molecular; Molecular Conformation; Movement; Pathogenicity; Physiological; Plasma; Play; Point Mutation; Property; Protein Conformation; Protein Fragment; Protein Isoforms; Proteins; Risk; Role; Structural Models; Structure; Testing; Thinking; Time; Toxic effect; Variant; Vesicle; apolipoprotein E-3; base; cardiovascular disorder risk; conformer; experimental study; flexibility; genetic risk factor; interest; lipid transport; monomer; nanodisk; nanosecond; novel; novel strategies; novel therapeutic intervention; particle; protein function; recruit; single molecule; single-molecule FRET; small molecule; structural biology

ABSTRACT The ε4-allele isoform of apolipoprotein E (ApoE4) plays a key-role in Alzheimer's disease and cardiovascular pathologies. A large body of evidence support that conformations of the protein are instrumental in its contribution to function and disease; yet, much remains unknown about the conformational ensemble of full-length ApoE and its role in protein (dis)function, largely because of its elevated propensity for aggregation/oligomerization and inherent flexibility. In our lab, we have overcome these complications by harnessing state-of-the-art single-molecule fluorescence spectroscopy and, for the first time, we are able to access the structural ensemble of the monomeric full-length ApoE4 (free in solution, embedded in oligomers, and bound to lipids). Our preliminary data clearly indicate that ApoE4 adopts at least three distinct conformers – previously unidentified – that coexist in equilibrium. These conformers are highly dynamic and malleable to oligomerization and lipid binding. These novel observations led us to hypothesize that single-point mutations and small molecules can perturb the structural ensemble favoring/disfavoring specific “pathogenic” conformations, whereas interaction with ligands (e.g. lipids) alters the equilibrium between the different conformers selecting for specific conformations that are required for protein function. Our approach will allow us to determine the conformational properties of ApoE, probe current structural models, and test our hypothesis. The specific aims are as follows: 1) determine the conformational changes and domain movements within the pathogenic ApoE4 and elucidate how point mutations and small molecules modulate protein toxicity; 2) understand the mechanism of lipid interactions, from synthetic lipid vesicles to physiological lipoproteins. A comprehensive description of the structural conformations and dynamics of ApoE in the presence and absence of lipids will shed light on the molecular mechanism behind its role in health and disease, paving the way to developing new therapeutic strategies.

抽象的 载脂蛋白E（APOE4）的ε4-折叠同工型在阿尔茨海默氏病和 心血管病理。大量的证据支持，构造蛋白质是有用的 在对功能和疾病的贡献中；然而，关于构象的合奏仍然不明 全长APOE及其在蛋白质（DIS）功能中的作用，主要是因为其对 聚集/低聚和继承灵活性。 在我们的实验室中，我们通过利用最先进的单分子来克服这些并发症 荧光光谱法，我们首次能够访问 单体全长APOE4（在溶液中免费，嵌入在低聚物中，与脂质结合）。我们的初步 数据清楚地表明APOE4至少采用了至少三个不同的构象异构体（以前未知） 同等的共存。这些构象异构体具有高度动态性，并且具有寡聚化和脂质结合。 这些新颖的观察结果使我们假设单点突变和小分子可以 扰动结构合奏偏爱/不利于特定的“致病性”构象，而相互作用 配体（例如脂质）改变了选择特定的不同构象体之间的等效物 蛋白质功能所需的构象。我们的方法将使我们能够确定构象 APOE的特性，探针当前的结构模型，并检验我们的假设。具体目的如下： 1）确定致病性APOE4内的构象变化和域运动并阐明 点突变和小分子如何调节蛋白质毒性； 2）了解脂质的机制 相互作用，从合成脂质蔬菜到物理脂蛋白。 在存在下对APOE的结构构象和动态的全面描述 缺少脂质会阐明其在健康和疾病中作用的分子机制，铺路 制定新的治疗策略的方式。