ApoE isoform-specific structure: Insights on biology and pathobiology

项目总结 载脂蛋白E(ApoE)中的单个氨基酸差异区分了三种主要的 在大脑中表达时会导致截然不同的功能结果的异构体： 与apoE3相比，apoE4导致阿尔茨海默病(AD)风险增加15倍， 而APOE2似乎具有保护性。大量证据表明， 疾病进展中的蛋白质；然而，载脂蛋白E的异构体依赖的结构特征 仍然难以捉摸，阻碍了我们对apoE4神经毒性机制的理解。 事实上，目前载脂蛋白E的结构模型是从有限数量的 从蛋白质片段中获得的不完全结构在溶液中或在与人工合成的复合体中 脂质体。关于内源性分泌的结构，我们知之甚少 脂蛋白。测试ApoE级联假说(ACH)，这是U19提案的核心焦点， 需要克服这些限制并比较载脂蛋白E亚型的结构差异 当与分泌的脂蛋白和AD相关致病分子如淀粉样蛋白-β结合时 (Aβ)。在这里，我们建议绕过之前的实验障碍，使用一种创新的 结合单分子荧光光谱和低温光谱的多管齐下的方法 电子显微镜(EM)。我们的方法使我们能够访问 APOE以单体、低聚和脂质结合的形式存在，并揭示了多种 平衡构象，这是经典的结构生物学方法看不见的。在……里面 与核心B、单分子测量、负染色和冷冻-EM的合作将是 与分子动力学模拟进行比较和内插以重建原子细节 蛋白质与分泌型脂蛋白和Aβ结合时的模型。在目标1中，我们将确定 人载脂蛋白E/脂蛋白颗粒的异构体特异性结构集成 Knockin鼠来源的星形胶质细胞、小胶质细胞和血管壁细胞(项目2和4) 作为IPSC来源的细胞(核心E)，特定细胞类型的载脂蛋白E小鼠模型(项目3-4)，以及人类 生物群落(核心C和核心D)。在目标2中，我们将评估Aβ之间的相互作用 载脂蛋白E调节其结构系综和寡聚化倾向。这个 拟议的实验将提供apoE的详细原子化结构表示 脂蛋白背景下的异构体，当与Aβ相互作用时，整合生化 核心B的表征和促进对异构体特定功能效应的研究 由项目2-5和核心B-F进行。

PROJECT SUMMARY A single amino acid difference in apolipoprotein E (apoE) distinguishes each of the three major isoforms that lead to dramatically different functional outcomes when expressed in the brain: apoE4 contributes up to a 15 fold risk increase in Alzheimer’s disease (AD) compared to apoE3, whereas apoE2 appears to be protective. A large amount of evidence suggests a direct role of the protein in disease progression; yet, the isoform- dependent structural features of apoE remain elusive, hampering our understanding of the mechanism behind apoE4 neurotoxicity. Indeed, current structural models of apoE have been derived from a limited number of incomplete structures obtained from protein fragments free in solution or in complex with synthetic liposomes. Very little is known about the structure in the context of endogenously-secreted lipoproteins. Testing the ApoE Cascade Hypothesis (ACH), the core focus of this U19 proposal, requires overcoming these limitations and comparing the structural differences of apoE isoforms when bound to secreted lipoproteins and AD-related pathogenic molecules such as amyloid-β (Aβ). Here, we propose to bypass previous experimental obstacles by using an innovative multipronged approach that combines single- molecule fluorescence spectroscopy and cryo- electron microscopy (EM). Our approach enables accessing the conformational ensemble of apoE in its monomeric, oligomeric, and lipid-bound forms and reveals coexistence of multiple conformations in equilibrium, which are invisible to classical methods of structural biology. In collaboration with Core B, single-molecule measurements, negative-stain, and cryo-EM will be compared and interpolated with molecular dynamics simulations to reconstruct atomistic-detailed models of the protein when bound to secreted lipoproteins and Aβ. In Aim 1, we will determine the isoform-specific structural ensemble of apoE/lipoprotein particles obtained from human apoE knockin mouse-derived astrocytes, microglia, and vascular mural cells (Project 2 and 4), as well as iPSC-derived cells (Core E), cell type-specific apoE mouse models (Projects 3-4), and human biospecimens (Core C and D). In aim 2, we will assess how the interplay between Aβ and apoE modulate their structural ensembles and oligomerization propensity. The proposed experiments will provide a detailed atomistic structural representation of apoE isoforms in the context of lipoproteins and while interacting with Aβ, integrating the biochemical characterization of Core B, and facilitating investigations of isoform-specific functional effects conducted by Projects 2-5 and Core B-F.