ApoE isoform-specific structure: Insights on biology and pathobiology

ApoE 亚型特异性结构：生物学和病理学的见解

基本信息

批准号：
10407943
负责人：
Andrea Soranno
金额：
$ 53.74万
依托单位：
MAYO CLINIC JACKSONVILLE
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-06-01 至 2026-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10407943
关键词：
Affinity Age-associated memory impairment Aging Alzheimer&apos s Disease Amino Acids Amyloid beta-Protein Apolipoprotein E Astrocytes Automobile Driving Binding Biochemical Biological Assay Biology Blood Vessels Brain Bypass Cells Collaborations Complement Complex Cryoelectron Microscopy Data Disease Progression Equilibrium Event Fluorescence Fluorescence Spectroscopy Foundations Human Investigation Knock-in Mouse Lead Length Libraries Light Lipid Binding Lipids Lipoproteins Liposomes Measurement Methods Microglia Modeling Molecular Molecular Conformation Negative Staining Pathogenicity Pathology Peptides Physiological Property Protein Fragment Protein Isoforms Proteins Risk Role Structural Models Structure Testing Time Work apolipoprotein E-3 apolipoprotein E-4 brain cell cell type experimental study functional outcomes induced pluripotent stem cell innovation insight molecular dynamics mouse model neurotoxicity novel strategies particle single molecule structural biology

项目摘要

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.

项目摘要载脂蛋白E（APOE）的单个氨基酸差异可区分这三个主要在大脑中表达时会导致截然不同的功能结果的同工型：与APOE3相比而APOE2似乎受到保护。大量证据表明疾病进展的蛋白质；但是，APOE的同工型依赖性结构特征保持难以捉摸，阻碍了我们对APOE4神经毒性背后机制的理解。实际上，当前的APOE结构模型是从有限数量的从溶液中的蛋白质片段获得的不完整结构或合成脂质体。在内源分泌的背景下，对结构知之甚少脂蛋白。测试APOE级联假设（ACH），该U19提案的核心重点，需要克服这些局限性并比较APOE同工型的结构差异当与分泌的脂蛋白和广告相关的致病分子（例如淀粉样蛋白-β）结合时（Aβ）。在这里，我们建议通过使用创新范围绕过以前的实验障碍将单分子荧光光谱和冷冻的多收益方法结合在一起电子显微镜（EM）。我们的方法使访问构象合奏 ApoE以其单体，低聚和脂质结合形式，并揭示了多个的共存相等的构象，这是结构生物学的经典方法不可见的。在与核心B，单分子测量，负污染和冷冻EM的合作将是比较并与分子动力学模拟进行了插值，以重建原子量蛋白质的模型与分泌的脂蛋白和Aβ结合时。在AIM 1中，我们将确定从人ApoE获得的ApoE/脂蛋白颗粒的同工型特异性结构集合敲击小鼠衍生的星形胶质细胞，小胶质细胞和血管壁画细胞（项目2和4）如IPSC衍生的细胞（核心E），细胞类型特异性APOE鼠标模型（项目3-4）和人类生物测量（Core C和D）。在AIM 2中，我们将评估Aβ之间的相互作用如何 ApoE调节其结构合奏和低聚的希望。这拟议的实验将提供APOE的详细原子结构表示在脂蛋白的背景下以及与Aβ相互作用的同时，整合生化核心B的表征，并支持对同工型特异性功能效应的研究由项目2-5和核心B-F进行。