ApoE isoform-specific structure: Insights on biology and pathobiology

项目摘要 载脂蛋白E（apoE）中的一个氨基酸差异将三种主要的 当在大脑中表达时，导致显著不同的功能结果的同种型： 与apoE 3相比，apoE 4导致阿尔茨海默病（AD）的风险增加高达15倍， 而apoE 2似乎具有保护作用。大量证据表明， 疾病进展中的蛋白质;然而，apoE的亚型依赖性结构特征 仍然难以捉摸，阻碍了我们对apoE 4神经毒性机制的理解。 事实上，目前apoE的结构模型是从有限数量的 从游离在溶液中或与合成的蛋白质复合物中的蛋白质片段获得的不完整结构 脂质体。关于内源性分泌的背景下的结构知之甚少， 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apoE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，我们建议通过使用一种创新的方法绕过以前的实验障碍。 多管齐下的方法，结合单分子荧光光谱和低温， 电镜（EM）。我们的方法能够访问构象合奏 apoE以其单体、寡聚体和脂质结合形式存在，并揭示了多种apoE的共存。 平衡构象，这是结构生物学的经典方法不可见的。在 与核心B合作，单分子测量，负染色和冷冻EM将是 与分子动力学模拟进行比较和插值， 蛋白质与分泌的脂蛋白和Aβ结合时的模型。在目标1中，我们将确定 从人apoE获得apoE/脂蛋白颗粒的异构体特异性结构集合 敲入小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），以及 作为iPSC衍生的细胞（Core E），细胞类型特异性apoE小鼠模型（项目3-4），和人 生物标本（核心C和D）。在目标2中，我们将评估Aβ与Aβ之间的相互作用， 和apoE调节它们的结构集合和寡聚化倾向。的 所提出的实验将提供一个详细的apoE原子结构表示 在脂蛋白的背景下，在与Aβ相互作用的同时，整合生物化学 核心B的表征，并促进异构体特异性功能效应的研究 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.