14-3-3 Sigma Interacts with Liver X Receptor Beta Emily Ann Jackson Louisiana State University and Agricultural and Mechanical College, [email protected]

Louisiana State University LSU Digital Commons LSU Master's Theses Graduate School 2010 14-3-3 sigma interacts with liver X receptor beta Emily Ann Jackson Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_theses Recommended Citation Jackson, Emily Ann, "14-3-3 sigma interacts with liver X receptor beta" (2010). LSU Master's Theses. 4251. https://digitalcommons.lsu.edu/gradschool_theses/4251 This Thesis is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Master's Theses by an authorized graduate school editor of LSU Digital Commons. For more information, please contact [email protected]. 14-3-3 SIGMA INTERACTS WITH LIVER X RECEPTOR BETA A Thesis Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Master of Science in The Department of Biological Sciences by Emily Ann Jackson B.S., Louisiana State University, 2004 May 2010 ACKNOWLEDGEMENTS I would like to thank God for blessing me with the strength, knowledge, and patience to get me through this journey. I would like to acknowledge my family, Brenda W. Jackson, for praying for me and instilling the morals and values necessary for me to succeed in life, my sister and niece, Earmer M. Jackson and Jeanette E. Jackson, for being very supportive throughout my life. Also, I would like to acknowledge my family and friends for their constant support and encouraging words. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS…………………………………………………………....................ii LIST OF TABLES………………………………………………………………………………..iv LIST OF FIGURES……………………………………………………………………………….v LIST OF ABBREVIATIONS…………………………………………………………………….vi ABSTRACT…………………………………………………………………………………….viii CHAPTER 1. GENERAL INTRODUCTION……………………………………………………1 1.1 Nuclear Receptors……………………………………………………………………..1 1.2 Liver X Receptor……………………………………………………………………..12 1.3 14-3-3 Proteins……………………………………………………………………….18 1.4 Cell Lines…………………………………………………………………………….20 CHAPTER 2. MATERIALS AND METHODS………………………………………………...22 2.1 Cells and Expression Constructs………………………………………………..……22 2.2 Expression and Purification of Proteins…………………...…………………………23 2.3 In vitro Binding Assay……………………………………………………………….23 2.4 Transactivation Assays ……………………………………………………………...24 2.5 Electrophoretic Mobility Shift Assay ……………………………………………….24 2.6 Microscopy…………………………………………………………………………..25 2.7 Permeabilization Experiments……………………………………………………….26 CHAPTER 3. 14-3-3 INTERACTS WITH LIVER X RECEPTOR BUT NOT LIVER X RECEPTOR 3.1 Introduction…………………………………………………………………………..27 3.2 Results………………………………………………………………………………..29 3.3 Discussion……………………………………………………………………………34 CHAPTER 4.THE MECHANISM OF NUCLEAR EXPORT OF LIVER X RECEPTOR α AND LIVER X RECEPTOR β REQUIRES ENERGY AND IS CRM-1 INDPENDENT 4.1 Introduction…………………………………………………………………………..41 4.2 Results………………………………………………………………………………..41 4.3 Discussion……………………………………………………………………………42 REFERENCES…………………………………………………………………………………..45 APPENDIX. PERMISSIONS……………………………………………………………………57 VITA……………………………………………………………………………………………..83 iii LIST OF TABLES 1.1 Nuclear Receptor involvement in Physiology………………………………………………...2 1.2 A Proposed Nomenclature for Nuclear Receptors…………………………………………….8 1.3 LXR Target Genes and Their Function……………………………………………………...16 iv LIST OF FIGURES Figure 1.1 Human Nuclear Receptors Classes…………………………………………………….4 Figure 1.2 General structures of NRs……………………………………………………………10 Figure 1.3 Schematic of a Model for Directional Nucleocytoplasmic Transport Import or Export of Receptor-Cargo Complexes through Nuclear Pore Complex………………………………..11 Figure 1.4 The mechanism of transcriptional activity by LXRs…………………………………13 Figure 1.5 Liver X Receptor natural and synthetic ligands……………………………………...14 Figure 3.1 GST, GST 14-3-3ζ, GST-LXRα, and GST-LXRβ are being expressed in E.coli…...30 Figure 3.2 LXRβ but not LXRα interacts with 14-3-3 …………………………………………31 Figure 3.3 LXRα and LXRβ interact with RXR…………………………………………………31 Figure 3.4 LXRβ interacts with 14-3-3 ……………………………………………………...…32 Figure 3.5 Schematic presentations of C-terminal truncations of LXRβ………………………..32 Figure 3.6 155CFP-LXR but not 105CFP-LXR binds 14-3-3ζ………………………………34 Figure 3.7 14-3-3ζ does not affect LXRβ binding to DNA……………………………………...35 Figure 3.8 14-3-3ζ decreases the transcriptional activity of LXR ……………………………..36 Figure 3.9 PKA or PKC activators had no effect on the transcriptional activity of LXRβ with or without 14-3-3……………………………………………………………………………………37 Figure 3.10 14-3-3ζ did not affect the localization of LXR ……………………………………39 Figure 4.1 Nuclear export of LXRα requires energy but not Crm-1…………………………….43 Figure 4.2 Nuclear export of LXRβ requires energy but not Crm-1…………………………….44 v LIST OF ABBREVIATIONS NR- nuclear receptor DBD- DNA binding domain LBD- ligand binding domain LXR- liver x receptor NR1H2-nuclear receptor subfamily 1, group H, member 2 (LXRβ) NR1H3-nuclear receptor subfamily 1, group H, member 3 (LXRα) ABCA1- ATP binding cassette subfamily A member 1 SREBP-1C- sterol regulatory element binding protein 1C FAS- fatty acid synthase AR- androgen receptor ER- estrogen receptor GR- glucocorticoid receptor MR- mineralcorticoid receptor PR- progesterone receptor FXR- farnesoid X receptor PPAR- peroxisome proliferator-activated receptor RAR- retinoid receptor TR- thyroid hormone receptor VDR- vitamin D receptor Rev-erb- reverse oreientation c-erb ROR- retinoic acid receptor-related orphan receptor FOXO- forkhead box vi LDL- low density lipoproteins HDL- high density lipoproteins FDA-U.S. Food and Drug Administration HNF-4- hepatocyte nuclear factor 4 SF-1- steroidogenic factor SHP- small heterodimeric partner RIP140- receptor interacting protein 140 NCoR- nuclear receptor corepressor SMRT- silencing mediator of retinoic acid and thyroid hormone receptor PBS- phosphate buffer saline RCT- reverse cholesterol transport vii ABSTRACT Atherosclerosis is the leading cause of mortality in developed countries accounting for 50% of all deaths. Atherosclerosis develops when macrophages in the artery wall accumulate large amounts of cholesterol via uptake of oxidized low density lipoproteins (LDL), causing a negative effect on cholesterol metabolism. Thus, the development of atherosclerosis can be inhibited by increasing cholesterol efflux, which can be achieved by activating ATP binding cassette (ABC) transporters in macrophages. In particular, ABCA1 mediates reverse cholesterol transport to the liver via high density lipoproteins (HDL) and therefore is an attractive molecular target for raising HDL levels and protecting against atherosclerosis. ABCA1 gene expression is known to be regulated by various transcription factors, such as liver X receptor (LXR). LXRs are transcription factors that are activated via binding of ligands, which are oxysterols. Activated LXRs bind to promoter regions at specific sequences known as LXR response elements (LXRE) and regulate genes for cholesterol metabolism and transport, as well as for lipogenesis. Synthetic LXR ligands might be useful for treatment of atherosclerosis if they did not induce lipogenesis, which can lead to an accumulation of cholesterol via activation of steroid response element binding protein (SREBP)-1c in the liver. To develop selective treatments for atherosclerosis, we need to understand mechanisms of selective gene regulation. LXRs occur as two isotypes, LXRα (NR1H3) and LXRβ (NR1H2). Both isotypes regulate genes encoding proteins involved in cholesterol metabolism and transport, as well as in lipogenesis. However, knock-out studies have shown that LXRβ activation results in more effective gene activation in the periphery, such as in macrophages, which can promote cholesterol efflux. The mechanism of this LXR isotype- selectivity is poorly understood. In this document, we will show how protein-protein viii interactions affect LXR and LXR function and explore the mechanism of nuclear export of LXR and LXR . ix CHAPTER 1 GENERAL INTRODUCTION 1.1 Nuclear Receptors 1.1.1 Introduction Nuclear receptors are a large superfamily of transcription factors that regulate genes important to physiological functions such as homeostasis, reproduction, and development in target cells (Table 1.1). Nuclear receptor activity can be modified by binding to ligands, typically small biologically active compounds such as hormones and lipids. A few well known nuclear receptor ligands are hormones such as steroids, retinoids, thyroid hormone, and vitamin D3. A large number of nuclear receptors have been identified by amino acid sequence similarity to known receptors, but some have no identified natural ligand and are referred to as nuclear orphan receptors. Nuclear receptors are found within the cytoplasm and nucleus of the cell where they can directly regulate target genes by binding to a specific sequence on DNA, known as the response element, usually found within the promoter region of the target gene. Each response element consists of two half-sites of a consensus sequence which can be oriented as direct repeats or indirect repeats which allows nuclear receptors to bind as a monomer, homodimer, or heterodimer (Giguere 1999). The expression of a large number of genes is regulated by nuclear receptors. Many of these regulated genes are associated with major diseases such as cancer, diabetes, obesity, and atherosclerosis, which explain why nuclear

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