UNIVERSITY OF OKLAHOMA GRADUATE COLLEGE THE LIGAND BINDING PROPERTIES OF THE OXYSTEROL-BINDING PROTEIN FAMILY SUBFAMILY-I A DISSERTATION SUBMITTED TO THE GRADUATE FACULTY in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY By JUAN IGNACIO VIERA NUÑEZ Norman, Oklahoma 2018 THE LIGAND BINDING PROPERTIES OF OXYSTEROL-BINDING PROTEIN FAMILY SUBFAMILY-I A DISSERTATION APPROVED FOR THE DEPARTMENT OF CHEMISTRY AND BIOCHEMISTRY BY THE COMMITTEE CONSISTING OF BY Dr. Anthony Burgett, Chair Dr. Helen Zgurskaya Dr. Christina Bourne Dr. Robert Cichewicz Dr. Laura Bartley Dr. Michael Ihnat © Copyright by JUAN IGNACIO VIERA NUÑEZ 2018 All Rights Reserved. I dedicate this my mom, who raised me and was my first role model. I dedicated this to my family, especially to my sister and my brother, I keep you in my heart. To my nephew, and future generations, I hope my accomplishments inspire you and allow you to succeed. iv Acknowledgments I want to acknowledge the members of the Burgett Lab who are my friends, colleagues and my second family. Thank you, Dr. Sims, Dr. Zgurskaya, Dr. Cichewicz, and Dr. Bourne, who allowed me to use instruments in their labs, which let me accomplish my research. Thank you OU Department of Electronics Shop for helping me keep the scintillation counter alive. v Table of Contents Acknowledgments ............................................................................................................ v List of Tables .................................................................................................................... x List of Figures .................................................................................................................. xi Abstract ........................................................................................................................... xv Chapter 1: OSBP and OSBP-Related Proteins (ORPs) .................................................... 1 1.1 Overview .............................................................................................................. 1 1.2 Overview of the OSBP/ORPs ............................................................................... 2 1.2.1 Overview ..................................................................................................... 2 1.2.2 History of the OSBP and ORPS .................................................................. 2 1.2.3 Properties of OSBP/ORPs ........................................................................... 3 1.2.4 Structural Biology of the Anchoring Domains and Motifs ......................... 4 1.2.5 Structural Biology of the Ligand Binding Domain ..................................... 6 1.3 Known OSBP/ORP Small Molecule Ligands, Oxysterols, and ORPphilin Compounds ................................................................................................... 10 1.3.1 Overview ................................................................................................... 10 1.3.2 Cellular Lipid Classes ................................................................................ 11 1.3.3 Oxysterols .................................................................................................. 14 1.3.4 ORPphilins ................................................................................................ 15 1.4 Other Oxysterol Binding Protein Families ......................................................... 19 1.4.1 Overview ................................................................................................... 19 1.4.2 Hedgehog Signaling Pathways .................................................................. 19 1.4.3 SREBP Pathway ........................................................................................ 20 vi 1.4.4 Liver X Receptors ...................................................................................... 21 1.5 The OSBP/ORPs in Human Disease .................................................................. 21 1.5.1 Overview ................................................................................................... 21 1.5.2 Subfamily-I: OSBP and ORP4 .................................................................. 22 1.5.3 Subfamily-II: ORP1 and ORP2 ................................................................. 23 1.6 Cellular Biology of the OSBP/ORPs .................................................................. 27 1.6.1 Overview ................................................................................................... 27 1.6.2 Subfamily-I: OSBP and ORP4 .................................................................. 27 1.6.3 Subfamily-II: ORP1 and ORP2 ................................................................. 29 1.6.4 Subfamily-III: ORP3, ORP6, and ORP7 ................................................... 30 1.6.5 Subfamily-IV: ORP5 and ORP8 ............................................................... 31 1.6.6 Subfamily-V: ORP9 .................................................................................. 32 1.6.7 Subfamily-VI: ORP10 and ORP11 ........................................................... 33 1.7 Ligand Binding to OSBP/ORPs ......................................................................... 33 1.7.1 Overview ................................................................................................... 33 1.7.2 Cholesterol and Oxysterols ........................................................................ 34 1.7.3 Phospholipids ............................................................................................ 35 1.7.4 ORPphilins and Other Inhibitors ............................................................... 36 Chapter 2: Profiling Ligand Binding to OSBP and ORP4L with Oxysterols and Oxysterol Analogs .............................................................................................. 37 2.1 Abstract ............................................................................................................... 38 2.2 Introduction ........................................................................................................ 39 2.2.1 The Cellular Role of OSBP and ORP4L ................................................... 39 vii 2.2.2 The Therapeutic Potential of Inhibiting OSBP and ORP4L ..................... 40 2.2.3 Current Knowledge on the Ligand Binding Capabilities of OSBP and ORP4L .................................................................................................... 40 2.3 Results and Discussion ....................................................................................... 41 2.3.1 Human OSBP and ORP4L Binds 25-Hydroxycholesterol at Nanomolar Levels ..................................................................................................... 41 2.3.2 ORPphilins: OSW1 and Itraconazole Do Not Bind to OSBP or ORP4L in a Similar Manner ....................................................................................... 42 2.3.3 Oxysterols Reveal hydroxylation and Stereochemistry Affect Competitive Binding to OSBP or ORP4L ................................................................... 45 2.3.4 20(S)-Hydroxycholesterol and 7α,25-Dihydroxycholesterol Induce OSBP Cellular Localization Similar to 25-Hydroxycholesterol ....................... 49 2.3.5 Structure-Activity Relationship of 20-Hydroxycholesterol Analogs to OSBP or ORP4L using the [3H]-25-Hydroxycholesterol Binding Assay ................................................................................................................ 51 2.3.6 Screening Non-oxysterol compounds for OSBP or ORP4L via competitive inhibition of 25-Hydroxycholesterol Binding ........................................ 56 2.3 Conclusion .......................................................................................................... 59 2.4 Experimental Procedures: ................................................................................... 61 2.5.1 Materials and Reagents .............................................................................. 61 2.5.2 Plasmids and Cloning ................................................................................ 61 2.5.3 Tissue Culture ............................................................................................ 62 2.5.4 [3H]-25-Hydroxycholesterol Charcoal/Dextran Binding Assay ................ 64 viii 2.5.5 Western Blot .............................................................................................. 66 2.5.6 General Method for the Synthesis of 20-Hydroxycholesterol Analogs .... 66 Chapter 3: Developing A Systematic Biochemical Approach to Study Ligand Binding to OSBP .............................................................................................................. 68 3.1 Abstract ............................................................................................................... 69 3.2 Introduction ........................................................................................................ 70 3.3 Results and Discussion ....................................................................................... 74 3.3.1 Site-Directed Mutagenesis of OSB ........................................................... 74 3.3.2 Binding Profile of OSBP H522A .............................................................. 76 3.3.3 Binding Assay of OSW-1 Analogs ............................................................ 78 3.3.4 Purification of Overexpressed OSBP .......................................................