Conformational and functional analysis of Apolipoprotein E

载脂蛋白E的构象和功能分析

基本信息

批准号：
10334412
负责人：
Andrea Soranno
金额：
$ 44.45万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
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/10334412
关键词：
Adopted Affinity Alleles Alzheimer&apos s Disease Alzheimer&apos 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

项目摘要

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 等位基因亚型在阿尔茨海默病和大量证据支持蛋白质的构象具有重要作用。然而，关于其构象整体仍然有很多未知之处；全长 ApoE 及其在蛋白质（功能障碍）中的作用，很大程度上是因为其增加的倾向聚集/寡聚和固有的灵活性。在我们的实验室中，我们通过利用最先进的单分子技术克服了这些并发症荧光光谱，我们第一次能够获得该结构的整体单体全长 ApoE4（游离于溶液中，嵌入寡聚物中，并与脂质结合）。数据清楚地表明 ApoE4 采用至少三种不同的构象异构体（之前未识别）这些构象异构体是高度动态的并且可塑性低聚和脂质结合。这些新颖的观察使我们认识到单点突变和小分子可以扰乱有利于/不利于特定“致病”构象的结构整体，而相互作用与配体（例如脂质）改变不同构象异构体之间的平衡，选择特定的我们的方法将使我们能够确定蛋白质功能所需的构象。 ApoE的性质，探讨当前的结构模型，并检验我们的假设，具体目标如下： 1) 确定致病性 ApoE4 内的构象变化和结构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；从合成脂质囊泡到生理脂蛋白的相互作用。全面描述 ApoE 存在的结构构象和动力学脂质的缺失将揭示其在健康和疾病中作用背后的分子机制，为开发新的治疗策略的方法。