Conformational and functional analysis of Apolipoprotein E

摘要 载脂蛋白E（ApoE 4）的ε4等位基因亚型在阿尔茨海默病中起关键作用， 心血管疾病大量的证据支持蛋白质的构象是工具性的 在其功能和疾病的贡献;然而，许多仍然未知的构象合奏 全长ApoE及其在蛋白质（功能障碍）中的作用，主要是因为其增加的倾向性， 聚合/低聚和固有的灵活性。 在我们的实验室里，我们通过利用最先进的单分子技术 荧光光谱，并为第一次，我们能够访问的结构合奏的 单体全长ApoE 4（在溶液中游离，包埋在寡聚体中，并与脂质结合）。我们的初步 数据清楚地表明，ApoE 4采用了至少三种不同的构象-以前未鉴定-， 平衡共存。这些构象异构体是高度动态的，并且对于寡聚化和脂质结合具有延展性。 这些新的观察使我们假设单点突变和小分子可以 干扰有利于/不利于特定的“致病”构象的结构系综，而相互作用 与配体（如脂质）的结合改变了不同构象之间的平衡， 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，探测当前的结构模型，并验证我们的假设。具体目标如下： 1)确定致病性ApoE 4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）了解脂质代谢的机制 从合成脂质囊泡到生理脂蛋白的相互作用。 ApoE的结构构象和动力学的全面描述， 而脂质的缺乏将揭示其在健康和疾病中作用背后的分子机制， 开发新的治疗策略的方法。

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.