Phosphatidylcholine transfer protein interacts with PPARd to modulate activity

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SUMMARY: In 2030 it is predicted that about half of the USA population will be clinically obese. Obesity can be lethal due to development of co-morbidities such as diabetes, nonalcoholic steatohepatitis, stroke, and heart attack. Recently, peroxisome proliferator-activated receptor δ (PPARδ) agonists have shown great promise in treating obesity and associated comorbidities by: increasing insulin sensitivity, weight loss, endurance, and lipid metabolism, while suppressing proinflammatory pathways, liver fibrosis, smooth muscle cell proliferation, and endothelial cell dysfunction. The endogenous ligand for PPARs is thought to be arachidonic acid, although plenty of studies show PPARs bind and are activated by fatty acids, phosphatidylcholines (PCs), and their metabolites. The mechanism by which PPARs gain access to these lipophilic ligands generated in the cytosol remains unknown. Studies in our lab identified a PPARδ-FABP5-polyunsaturated fatty acids (PUFA) pathway, in which PUFAs are shuttled to the nucleus by FABPs which in turn upregulate PPARδ activity. However, FABP5 only binds a subset of reported PPARδ ligands. To find other candidate lipid transport proteins (LTPs), we performed a protein complementation assay (PCA) between LTPs and PPARs. We uncovered a novel interaction between PPARδ and phosphatidylcholine transfer protein (PC-TP). Preliminary data show that this interaction opposes canonical PPARδ signaling. The overall goal of this proposal is to biochemically and functionally characterize the regulation of PPARδ through its interaction with PC-TP. I hypothesize that certain PC molecular species drive PC-TP translocation to inhibit PPARδ transactivation of genes. In Aim 1, I will use in cell protein-protein interaction assays to test PPARδ association with either WT or mutant PCTP, defective in ligand binding. In tandem, I will test the role of chemical probes known to alter PC-TP/ PPARδ function on this interaction. This analysis will be complimented by lipidomics, specifically interrogating PCs bound to PC-TP taking advantage of conditions known to facilitate complex formation. Lipids bound to PC-TP detected via mass spectrometry will then be tested for their ability to enhance PPARδ binding and suppression. Certain perturbations may allow PC- TP to interact with PPARδ but may lead to an inert complex. To probe this possibility, I will perform luciferase reporter assays and qPCR micro-arrays specifically interrogating PPARδ-controlled genes. In Aim 2, I will define the topographic position of the repressive full length PPARδ and PC-TP complex by combining information obtained from hydrogen deuterium exchange coupled to mass spectrometry and crosslinking mass spectrometry experiments. This analysis will be complimented by determining the stoichiometry, and kinetics of complex formation. Combined, these approaches will functionally and biochemically characterize how PC-TP regulates PPARδ through direct interaction in the hopes of determining a molecular framework of how aberrant lipid levels associated with obesity could affect lipid homeostasis.