Conformational and functional analysis of Apolipoprotein E

摘要 载脂蛋白E(ApoE4)的ε4等位基因亚型在阿尔茨海默病和 心血管病理学。大量证据支持蛋白质的构象是有用的 它对功能和疾病的贡献；然而，关于它的构象集合仍有许多未知 全长载脂蛋白E及其在蛋白质(Dis)功能中的作用，主要是因为它具有更高的 聚合/齐聚和固有的灵活性。 在我们的实验室里，我们已经通过利用最先进的单分子来克服这些并发症 荧光光谱学，我们第一次能够访问 单体全长载脂蛋白E4(游离在溶液中，包埋在低聚物中，并与脂类结合)。我们的预赛 数据清楚地表明，ApoE4至少采用了三种不同的构象-以前未确定- 在平衡中共存。这些构象具有高度的动态和延展性，可用于齐聚和脂类结合。 这些新颖的观察结果让我们假设，单点突变和小分子可以 干扰有利于/不利于特定“致病”构象的结构集合，而相互作用 与配体(如脂类)一起改变不同构象之间的平衡，选择特定的 蛋白质功能所需的构象。我们的方法将允许我们确定构象 ApoE的性质，探索当前的结构模型，并验证我们的假设。具体目标如下： 1)确定致病ApoE4的构象变化和结构域移动，并阐明 点突变和小分子如何调节蛋白质毒性；2)了解脂质的作用机制 相互作用，从合成脂泡到生理脂蛋白。 ApoE存在时的结构构象和动力学的全面描述 而缺乏脂质将有助于阐明其在健康和疾病中的作用背后的分子机制，为 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.