Steven Berberich (Committee Member), David Cool (Committee Member), Khalid Elased (Committee Member), Heather Hostetler (Advisor), Lawrence Prochaska (Committee Member)
Doctor of Philosophy (PhD)
Peroxisome proliferator-activated receptor alpha (PPAR alpha) belongs to the family of ligand-activated nuclear transcription factors and serves as a lipid sensor to regulate nutrient metabolism and energy homeostasis. The transcriptional activity of PPAR alpha is thought to be regulated by the binding of exogenous ligands (example, fenofibrate, TriCor), as well as endogenous ligands including fatty acids and their derivatives. Although long-chain fatty acids (LCFA) and their thioesters (long-chain fatty acyl-CoA; LCFA-CoA) have been shown to activate PPAR alpha of several species, the true identity of high-affinity endogenous ligands for human PPAR alpha (hPPAR alpha) has been more elusive. This two part dissertation is a structural and functional evaluation of human and mouse PPAR alpha binding to LCFA and LCFA-CoA using biophysical and biochemical approaches of spectrofluorometry, circular dichroism spectroscopy, mutagenesis, molecular modelling and transactivation assays. The first goal of this dissertation was to determine whether LCFA and LCFA-CoA constitute high-affinity endogenous ligands for full-length hPPAR alpha. Data from spectrofluorometry suggests that LCFA and LCFA-CoA serve as physiologically relevant endogenous ligands of hPPAR alpha. These ligands bind hPPAR alpha and induce strong secondary structural changes in the circular dichroic spectra, consistent with the binding of ligand to nuclear receptors. Ligand binding is also associated with activation of hPPAR alpha, as observed in transactivation assays. The second goal of this dissertation was to determine whether there exist species differences for ligand specificity and affinity between hPPAR alpha and mouse PPAR alpha (mPPAR alpha). This is important because despite high amino acid sequence identity (>90 precent), marked differences in PPAR alpha ligand binding, activation and gene regulation have been noted across species. Similar to previous observations with synthetic agonists, we reported differences in ligand affinities and extent of activation between hPPAR alpha and mPPAR alpha in response to saturated long chain fatty acids. In order to determine if structural alterations between the two proteins could account for these differences, we performed in silico molecular modeling and docking simulations. Modeling suggested that polymorphisms at amino acid position 272 and 279 are likely to be responsible for differences in saturated LCFA binding to hPPAR alpha and mPPAR alpha. To confirm these results experimentally, spectrofluorometry based-binding assays, circular dichroism, and transactivation studies were performed using a F272I mutant form of mPPAR alpha. Experimental data correlated with in silico docking simulations, further confirming the importance of amino acid 272 in LCFA binding. Although the driving force for evolution of species differences at this position are yet unidentified, this study enhances our understanding of ligand-induced regulation by PPAR alpha. Apart from demonstrating significant structure activity relationships explaining species differences in ligand binding, data in this dissertation identifies endogenous ligands for hPPAR alpha which will further help delineate the role of PPAR alpha as a nutrient sensor in regulating energy homeostasis.
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