Proteomic and functional analysis of missense variants of APOE associated with Alzheimer disease risk

项目总结 阿尔茨海默病(AD)是一种进行性神经退行性疾病，是导致 美国的痴呆症。载脂蛋白E基因(APOE)的变异与 AD的发展。在大脑中，载脂蛋白E主要在星形胶质细胞中合成。APOE的主要作用是 将脂肪和胆固醇从星形胶质细胞输送到其他类型的细胞。大多数人类都有一种主要的等位基因 载脂蛋白E、载脂蛋白ε2(R176C)、载脂蛋白ε3(参考等位基因)和载脂蛋白ε4(C130R)。这些错误的变体是 与不同水平的阿尔茨海默病风险相关，其中载脂蛋白Eε4增加疾病风险，而 载脂蛋白ε2可降低AD的风险。最近对100,000多名患者进行的基因分型分析表明，罕见的 错义变异与不同水平的阿尔茨海默病风险相关。其分子机制的研究进展 载脂蛋白E的变异如何改变星形胶质细胞的生理，从而可能导致阿尔茨海默病，目前尚不清楚。至 为了理解这一点，我们之前的工作采用了基于质谱学的热蛋白质组图谱来 确定错义变异导致的蛋白质相互作用的变化。使用酵母作为简化模型，我们 能够显示突变的热蛋白质组图谱以解决亚复杂水平的蛋白质-蛋白质相互作用 错义变异导致蛋白质复合体的破坏。来研究这些罕见的错义变体的作用 在星形胶质细胞的载脂蛋白E方面，我们将采用小鼠原代星形胶质细胞培养，它将产生人的载脂蛋白E 特定的错义变体。首先，我们将确定罕见的错义变体对脂蛋白的影响 功能和星形胶质细胞生理学。我们将确定APOE变体之间的差异 Aβ多肽，脂化状态，以及对星形胶质细胞转录组的影响。第二，我们将确定 使用突变热蛋白质组谱检测APOE变异导致的蛋白质相互作用的变化 热稳定性发生了变化。综上所述，这些数据将扩大我们对误解变异的理解 改变载脂蛋白E的功能，有助于深入了解导致阿尔茨海默氏症的分子机制 疾病。

PROJECT SUMMARY Alzheimer’s disease (AD) is a progressive neurodegenerative disease that is the most common cause of dementia in the United States. Variants in the apolipoprotein E gene (APOE) is strongly associated with development of AD. In the brain, APOE is mostly synthesized in the astrocytes. The primary role of APOE is to deliver lipids and cholesterol from astrocytes to other cell types. Most humans have one of the major allele of APOE, APOE ε2 (R176C), APOE ε3 (reference allele), and APOE ε4 (C130R). These missense variants are associated with different levels of Alzheimer disease risks where APOE ε4 increases the risk of disease whereas APOE ε2 decrease risk of AD. A recent analysis that genotyped over 100,000 patients showed that rare missense variants were associated with different levels of Alzheimer disease risk. The molecular mechanism of how variants in APOE changes astrocyte physiology that could lead to Alzheimer disease is not well known. To attempt to understand this, our previous work adapted mass spectrometry-based thermal proteome profiling to determine changes in protein interaction as a result of missense variants. Using yeast as simplified model, we were able to show mutant thermal proteome profiling to resolve subcomplex level protein-protein interaction disruptions of a protein complexes due to missense variants. To study the role of these rare missense variants on APOE in astrocytes, we will employ an mouse primary astrocyte culture that will generate human APOE with the specific missense variant. First, we will determine the effect of the rare missense variants on the lipoprotein functions and astrocyte physiology. We will determine the differences between the APOE variants of uptake of Aβ peptides, lipidation status, and the effects on the astrocyte transcriptome. Second, we will determine the changes in protein interactions as a result in APOE variants using mutant thermal proteome profiling to detect thermal stability changes. Taken together, these data will expand our understanding of how missense variants change the functions of APOE and help give insight into the molecular mechanisms that result in Alzheimer disease.