Wright State University CORE Scholar Browse all Theses and Dissertations Theses and Dissertations 2014 Peroxisome Proliferator-Activated Receptor Alpha: Insight into the Structure, Function and Energy Homeostasis Dhawal P. Oswal Wright State University Follow this and additional works at: https://corescholar.libraries.wright.edu/etd_all Part of the Biomedical Engineering and Bioengineering Commons Repository Citation Oswal, Dhawal P., "Peroxisome Proliferator-Activated Receptor Alpha: Insight into the Structure, Function and Energy Homeostasis" (2014). Browse all Theses and Dissertations. 1361. https://corescholar.libraries.wright.edu/etd_all/1361 This Dissertation is brought to you for free and open access by the Theses and Dissertations at CORE Scholar. It has been accepted for inclusion in Browse all Theses and Dissertations by an authorized administrator of CORE Scholar. For more information, please contact [email protected]. PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR ALPHA: INSIGHT INTO THE STRUCTURE, FUNCTION AND ENERGY HOMEOSTASIS A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy By Dhawal P. Oswal B.Pharm., Pune University, 2007 M.S., Wright State University, 2009 _________________________________________ 2014 Wright State University COPYRIGHTS BY DHAWAL P. OSWAL 2014 ABSTRACT Oswal, Pravin Dhawal Ph.D., Biomedical Sciences Ph.D. program, Department of Biochemistry and Molecular Biology, Wright State University, 2014. Peroxisome proliferator-activated receptor alpha: Insight into the structure, function and energy homeostasis Peroxisome proliferator-activated receptor alpha (PPARα) 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 PPARα 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 PPARα of several species, the true identity of high-affinity endogenous ligands for human PPARα (hPPARα) has been more elusive. This two part dissertation is a structural and functional evaluation of human and mouse PPARα 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 hPPARα. Data from spectrofluorometry suggests that LCFA and LCFA-CoA serve as physiologically relevant endogenous ligands of hPPAR. These ligands bind hPPARα and induce strong secondary structural changes in the circular dichroic spectra, consistent with the binding iv of ligand to nuclear receptors. Ligand binding is also associated with activation of hPPARα, 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 hPPARα and mouse PPARα (mPPARα). This is important because despite high amino acid sequence identity (>90%), marked differences in PPARα 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 hPPARα and mPPARα 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 hPPARα and mPPARα. To confirm these results experimentally, spectrofluorometry based-binding assays, circular dichroism, and transactivation studies were performed using a F272I mutant form of mPPARα. 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 PPARα. Apart from demonstrating significant structure activity relationships explaining species differences in ligand binding, data in this dissertation identifies endogenous ligands for hPPAR which will further help delineate the role of PPAR as a nutrient sensor in regulating energy homeostasis. v TABLE OF CONTENTS INTRODUCTION..............................................................................................................1 Peroxisome proliferator-activated receptors (PPAR) .......................................................2 PPARα: Structure ............................................................................................................4 The A/B region .......................................................................................................6 DNA binding domain (DBD) or C domain .............................................................7 Hinge region or D domain ....................................................................................11 Ligand binding domain or E/F domain .................................................................11 PPARα: Mode of action ................................................................................................14 Conformational changes .......................................................................................14 Coactivators and corepressors ...............................................................................18 Cellular localization and chain of events ..............................................................21 PPARα: Ligands, physiological role and knockout mice phenotype ............................25 Ligands ..................................................................................................................25 Physiological role of PPARα in lipid metabolism ................................................27 Physiological role of PPARα in lipoprotein metabolism ......................................30 vi TABLE OF CONTENTS (Continued) Physiological role of PPARα in inflammation .....................................................30 PPARα knockout mice model ................................................................................31 HYPOTHESIS..................................................................................................................32 Development of Hypothesis ...........................................................................................32 Hypothesis ......................................................................................................................35 CHAPTER I .....................................................................................................................37 Abstract ..........................................................................................................................38 Introduction ....................................................................................................................39 Materials and Methods ...................................................................................................41 Chemicals ..............................................................................................................41 Purification of Recombinant PPARα protein ......................................................41 Direct Fluorescent Ligand Binding Assays ..........................................................42 Displacement of Bound Fluorescent BODIPY C16-CoA by Non-fluorescent Ligands:..................................................................................................................43 Quenching of PPARα Aromatic Amino Acid Residues by Non-fluorescent Ligands ..................................................................................................................43 vii TABLE OF CONTENTS (Continued) Secondary Structure Determination Effect of ligand binding on PPARα Circular Dichroism ..............................................................................................................44 Mammalian Expression Plasmids .........................................................................45 Cell culture and Transactivation assays ................................................................45 Statistical Analysis .................................................................................................46 Results ............................................................................................................................47 Full-length hPPARα and mPPARα protein purification .......................................47 Binding of fluorescent fatty acid and fatty acyl-CoA to PPARα ..........................49 Binding of endogenous LCFA and LCFA-CoA to hPPARα – Displacement of bound BODIPY C16-CoA ....................................................................................54 Binding of endogenous LCFA and LCFA-CoA to mPPARα – Displacement of bound BODIPY C16-CoA ....................................................................................59 Binding of