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Peroxisome Proliferator-Activated Alpha: Insight into the Structure, Function and Energy Homeostasis

Dhawal P. Oswal Wright State University

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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 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, ,

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 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 endogenous LCFA and LCFA-CoA to hPPARα – Quenching of

intrinsic aromatic amino acid fluorescence ...... 63

Binding of endogenous LCFA and LCFA-CoA to mPPARα – Quenching of

intrinsic aromatic amino acid fluorescence ...... 69

viii

TABLE OF CONTENTS (Continued)

Effect of endogenous fatty acids and fatty acyl-CoAs on hPPARα secondary

structure ...... 75

Effect of endogenous fatty acids and fatty acyl-CoAs on mPPARα secondary

structure ...... 81

Effect of fatty acids and fatty acyl-CoA on transactivation of human and mouse

PPARα-RXRα heterodimers ...... 87

Discussion ...... 91

CHAPTER II ...... 95

Abstract ...... 96

Introduction ...... 97

Materials and Methods ...... 100

Molecular modeling simulations...... 100

Molecular docking simulations ...... 100

Chemicals ...... 102

Purification of Recombinant F272I mutant mPPARα protein ...... 102

Fluorescence based Ligand Binding Assays ...... 102

ix

TABLE OF CONTENTS (Continued)

Secondary Structure Determination: Effect of ligand binding on F272I mPPARα

circular dichroism ...... 103

Mammalian Expression Plasmids ...... 103

Cell culture and Transactivation assays ...... 104

Statistical Analysis ...... 105

Results and Discussion ...... 106

Molecular modeling simulations of hPPARα-LBD and mPPARα-LBD ...... 106

Molecular docking simulations with hPPARα-LBD and mPPARα-LBD ...... 110

Purification of full-length recombinant F272I mPPARα ...... 128

Binding of fluorescent fatty acids and fatty acyl-CoAs to F272I mPPARα ...... 130

Binding of endogenous LCFA and LCFA-CoA to F272I mPPARα ...... 132

Effect of endogenous fatty acids and fatty acyl-CoAs on F272I mPPARα

secondary structure ...... 141

Effect of fatty acids on transactivation of PPARα-RXRα heterodimers ...... 147

x

TABLE OF CONTENTS (Continued)

SUMMARY AND CONCLUSIONS ...... 152

BIBLIOGRAPHY/REFERENCES ...... 164

APPENDIX ...... 199

Abstract ...... 200

Introduction ...... 201

Development of hypothesis ...... 208

Materials and Methods ...... 212

Chemicals ...... 212

Electrophoretic mobility shift assays (EMSA) ...... 212

Cell culture and treatments: ...... 213

RNA isolation and qRT-PCR ...... 213

Western Blotting Analysis ...... 214

Plasmids ...... 215

Transactivation assays ...... 216

Results and Discussion ...... 218

xi

TABLE OF CONTENTS (Continued)

Electrophoretic mobility shift assays (EMSA) ...... 218

Adiponectin expression in HepG2 cells and its regulation by hPPARα ligands 224

Transactivation assays ...... 229

Future directions ...... 237

BIBLIOGRAPHY/REFERENCES ...... 239

LIST OF ABBREVIATIONS ...... 261

xii

LIST OF FIGURES

INTRODUCTION

1. Pre-messenger RNA and domain structure of PPARα ...... 5

2. Illustration of PPAR-RXR heterodimer binding to DNA ...... 10

3. Illustration of “mousetrap” model for PPARα activation ...... 17

4. PPAR mechanism of action ...... 24

5. Genes regulated by PPARα and their role in lipid metabolism ...... 29

CHAPTER I

6. SDS-PAGE and Coomassie blue staining of recombinant hPPARα and mPPARα

proteins ...... 48

7. Binding of fluorescent fatty acid and fatty acyl-CoA to hPPARα...... 51

8. Binding of fluorescent fatty acid and fatty acyl-CoA to mPPARα ...... 53

9. Binding of endogenous LCFA and LCFA-CoA to hPPARα: Displacement of

bound BODIPY C16-CoA ...... 55

10. Binding of endogenous LCFA and LCFA-CoA to mPPARα – Displacement of

bound BODIPY C16-CoA ...... 60

xiii

LIST OF FIGURES (Continued)

11. Binding of endogenous LCFA and LCFA-CoA to hPPARα – Quenching of

intrinsic aromatic amino acid fluorescence ...... 64

12. Binding of endogenous LCFA and LCFA-CoA to mPPARα – Quenching of

intrinsic aromatic amino acid fluorescence ...... 71

13. Effect of endogenous fatty acids and fatty acyl-CoAs on hPPARα secondary

structure ...... 77

14. Effect of endogenous fatty acids and fatty acyl-CoAs on mPPARα secondary

structure ...... 83

15. Effect of fatty acids and fatty acyl-CoA on transactivation of human and mouse

PPARα-RXRα heterodimers ...... 89

CHAPTER II

16. Comparison of primary amino acid sequence of human and mouse PPARα ..... 107

17. An overlay of the energy minimized structures of hPPARα-LBD and mPPARα-

LBD ...... 109

18. Validation of molecular docking simulations ...... 111

19. Comparison of molecular docking simulations of C16:0 complexed with

hPPARα-LBD, mPPARα-LBD and F272I mPPARα-LBD ...... 115

20. Energy minimized structures of hPPARα-LBD in complex with palmitoleic acid,

stearic acid and docosahexaenoic acid ...... 117

xiv

LIST OF FIGURES (Continued)

21. Energy minimized structures of mPPARα-LBD in complex with palmitoleic acid,

stearic acid and docosahexaenoic acid ...... 120

22. Energy minimized structures of F272I mPPARα-LBD in complex with

palmitoleic acid, stearic acid and docosahexaenoic acid ...... 123

23. Illustration of saturated LCFA binding to human and mouse PPARα – Importance

of amino acid residues at position 272 and 279 ...... 125

24. Ligand binding pocket volumes for hPPARα-LBD, mPPARα-LBD, F272I

mPPARα-LBD and F272I, M279T mPPARα-LBD ...... 127

25. SDS-PAGE and Coomassie blue staining of purified recombinant mPPARα and

F272I mPPARα ...... 129

26. Binding of fluorescent fatty acid and fatty acyl-CoA to F272I mPPARα ...... 131

27. Binding of endogenous LCFA and LCFA-CoA to F272I mPPARα –

Displacement of bound BODIPY C16-CoA...... 133

28. Binding of endogenous LCFA and LCFA-CoA to F272I mPPARα – Quenching

of intrinsic aromatic amino acid fluorescence ...... 137

29. An overlay of the far UV circular dichroic spectra of hPPARα, mPPARα and

F272I mPPARα ...... 142

30. Effect of endogenous fatty acids and fatty acyl-CoAs on F272I mPPAR

secondary structure ...... 144

xv

LIST OF FIGURES (Continued)

31. Effect of fatty acids and fatty acyl-CoA on transactivation of human, mouse and

F272I mouse PPARα-RXRα heterodimers ...... 149

APPENDIX

32. Primary structure of adiponectin ...... 203

33. Electrophoretic mobility shift assays (EMSA) ...... 222

34. Adiponectin expression in HepG2 cells and its regulation by hPPARα ligands 228

35. The transactivation of adiponectin promoter in COS-7 cells ...... 231

36. Effect of PPARα ligands on the transactivation of adiponectin promoter ...... 233

xvi

LIST OF TABLES

CHAPTER I

I. Affinity of hPPARα for non-fluorescent ligands determined by quenching of

hPPARα aromatic amino acid fluorescence and by displacement of hPPARα-

bound BODIPY C16-CoA ...... 68

II. Affinity of mPPAR for non-fluorescent ligands determined by quenching of

PPAR aromatic amino acid fluorescence and by displacement of mPPAR-

bound BODIPY C16-CoA ...... 74

III. Effect of ligands on the relative proportion of hPPAR secondary structure

determined by circular dichroism spectroscopy ...... 80

IV. Effect of ligands on the relative proportion of mPPAR secondary structure

determined by circular dichroism spectroscopy ...... 86

CHAPTER II

V. Comparison of binding energies for mouse and human PPARα LBD complexed

with LCFA ligands ...... 118

VI. Affinity of F272I mPPAR for non-fluorescent ligands determined by

quenching of PPAR aromatic amino acid fluorescence and by displacement of

F272I mPPAR-bound BODIPY C16-CoA ...... 140

xvii

LIST OF TABLES (Continued)

VII. Effect of ligands on the relative proportion of F272I mPPAR secondary

structure determined by circular dichroism spectroscopy...... 146

xviii

ACKNOWLEDGEMENTS

I would like to thank my advisor Dr. Heather A. Hostetler for the opportunity to work in her lab, for the disciplined training enviorment and for her guidance and patience throughout my dissertation research. I would also like to thank my committee members

Drs. Berberich, Cool, Elased and Prochaska for their time and their invaluable input over all these years. My sincere thanks to Dr. Alter for helpful s