Differential cellular proteomics of human apolipoprotein E (APOE) and determination of its interaction with mitochondria-associated membranes and proteins

载脂蛋白E（APOE）传统上已知其在受体介导的脂质转运中的作用，以及其人类亚型APOE 4的疾病风险关联，尽管病理分子机制仍在研究中。新的数据表明，载脂蛋白E及其亚型可能调节线粒体蛋白的表达和功能。在初步的免疫共沉淀实验中，我们鉴定了线粒体蛋白BCKDHA（支链酮酸脱氢酶E1亚单位α）作为载脂蛋白E潜在的蛋白质-蛋白质相互作用伙伴。（按降序排列）：APOE-BCKDHA相互作用的验证及其生物学重要性的评价，APOE在MAM相关ER膜（MAM）上积累的功能特征，以及对APOE亚型应答的差异蛋白质组学和蛋白水解片段化的研究。这些次级项目将独立执行，这样，即使其中一个次级项目，特别是“高风险”工作包出现意外结果，也能顺利完成整个项目。 基于我们的假设，即APOE与BCKDHA相互作用，BCKDHA是线粒体基质中支链氨基酸脱氢酶复合物（BCKD）的成员，我们将使用原位方法验证相互作用，研究潜在的线粒体靶向和易位机制，并确定在APOE及其亚型存在和不存在的情况下BCKD酶活性。将通过超离心分离和免疫印迹研究APOE在MAM的定位，并在存在APOE同种型的情况下进行功能检查（例如使用标记酶活性）。我们设想确定APOE是否在MAM的组装或活性中起功能性作用。为此，将进行应用siRNA敲低内源性APOE和其他MAM蛋白的机制研究。将使用基于定量液相色谱-质谱（LC-MS）的蛋白质组学研究主要代谢途径中涉及的蛋白质相对丰度的潜在变化。将使用基于LC-MS的N-和C-末端组学方法分析细胞内APOE蛋白水解加工。此外，我们从过去进行的研究中全面收集了冷冻组织，其中APOE靶向替代小鼠已接受不同的饮食方案。这些研究使得能够检查差异蛋白质组学、蛋白水解APOE片段化或BCKD活性响应于APOE基因型和体内饮食因素的调节。总的来说，我们希望在拟议的项目中获得的结果，以解开APOE的新的未知功能，这可能有助于了解与不同的人类APOE亚型相关的不同疾病风险的分子机制。

The apolipoprotein E (APOE) is traditionally known for its role in receptor-mediated lipid transport but also for the disease-risk association of its human isoform APOE4, although pathological molecular mechanisms are still under investigation. Novel data suggest that APOE and its isoforms may modulate mitochondrial protein expression and function. In preliminary co-immunoprecipitation experiments we identified the mitochondrial protein BCKDHA (Branched chain keto acid dehydrogenase E1 subunit alpha) as potential protein-protein-interaction partner of APOE.The present project proposal comprises three subprojects with different risk categories (listed in descending order): the validation of the APOE-BCKDHA-interaction and evaluation of its biological importance, the functional characterization of the accumulation of APOE at mitochondria-associated ER-membranes (MAM), and the investigation of differential proteomics and proteolytic fragmentation in response to the APOE isoforms. The subprojects will be executed independently, which allows successful completion of the whole project per se in the event of unexpected results of one of the subprojects, particularly of the “high-risk” work packages. Based on our hypothesis that APOE interacts with BCKDHA, which is a member of the branched chain amino acid dehydrogenase enzyme complex (BCKD) in the mitochondrial matrix, we will validate the interaction using an in situ approach, investigate potential mitochondrial targeting and translocation mechanisms and determine BCKD enzyme activity in the absence and presence of APOE and its isoforms. Localization of APOE at MAM will be studied by ultracentrifugation separation and immunoblotting and functionally examined (e.g. using marker enzyme activity) in the presence of APOE isoforms. We envisage determining whether APOE plays a functional role in the assembly or activity of MAM. To this end, mechanistic studies applying siRNA to knock down endogenous APOE and other MAM proteins will be carried out. Potential changes in the relative abundance of proteins involved in major metabolic pathways will be investigated using quantitative liquid chromatography-mass spectrometry (LC-MS) based proteomics. Intracellular APOE proteolytic processing will be analyzed using LC-MS based N- and C-terminomics methods. Furthermore, we have a comprehensive collection of frozen tissue from studies conducted in the past, where APOE targeted replacement mice have been subjected to diverse dietary regimens. These studies enable the examination of the modulation of differential proteomics, proteolytic APOE fragmentation or BCKD activity in response to APOE genotype and dietary factors in vivo. Overall, we expect the results obtained in the proposed project to unravel novel yet unknown functions of APOE, which may help to understand molecular mechanisms underlying the varying disease risk associated with the different human APOE isoforms.