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

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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.