Publication Date
2017
Document Type
Dissertation
Committee Members
Gerald Alter (Committee Member), Katherine Excoffon (Committee Member), Heather Hostetler (Advisor), Ashot Kozak (Committee Member), Lawrence Prochaska (Committee Member), Nicholas Reo (Committee Member), Stanley Dean Rider, Jr. (Advisor)
Degree Name
Doctor of Philosophy (PhD)
Abstract
LXRs, LXRa (NR1H3) and LXRß (NR1H2), are ligand-activated transcription factors that are members of the nuclear receptor superfamily. Oxysterols and nonsteroidal synthetic compounds bind directly to LXRs and influence the expression of LXR dependent genes. The use of murine models and LXR-selective agonists have established the important role of LXRs as sterol sensors that govern the absorption, transport, and catabolism of cholesterol. Upon activation, these receptors have been shown to increase reverse cholesterol transport from the macrophage back to the liver to aid in the removal of excess cholesterol. Not surprisingly, LXR dysregulation is a feature of several human diseases, including metabolic syndrome. Due to their roles in the regulation of lipid and cholesterol metabolism, LXRs are potentially attractive pharmaceutical targets. As ligand binding and dimerization play pivotal roles in modulating LXR activity, the identification of novel ligands and requirements for LXR dimerization can potentially aid the drug development process. Herein, using a variety of biophysical assays, including fluorescence based assays coupled with in silico molecular modeling, I have identified medium chain fatty acids and/or their metabolites as the novel endogenous agonists of LXRa. There is mounting evidence that ligand induced dimerization regulates the transcriptional output of nuclear receptors. Thus, it is important to identify factors that modulate protein-protein interactions. This work demonstrated that (a) LXRa binds PPARa with a high affinity (low nanomolar concentration), (b) ligands for LXRa alter the binding dissociation constant values of LXRa-PPARa interaction, and (c) ligand binding induces conformational changes in the dimer secondary structure. Furthermore, site-directed mutagenesis investigated the strength of individual contributions of residues located in the ligand binding domain to dimerization and transactivation properties of LXRa. Data herein highlight the importance of hydrophobic interactions and salt bridges at the interface, and suggest that key interface residues are required for the ligand-dependent activation of LXRa in a promoter specific manner. Mutagenesis of LXRa L414 to an arginine revealed the importance of this site in dimerization, specifically with RXRa. This work showed that this particular mutation specifically abolished dimerization with RXRa. Taken together, this study provided insights into the functional roles of fatty acids as novel LXRa ligands and the effects mutations may have in modulating molecular interactions and activity profile of LXRa.
Page Count
206
Department or Program
Biomedical Sciences
Year Degree Awarded
2017
Copyright
Copyright 2017, some rights reserved. My ETD may be copied and distributed only for non-commercial purposes and may not be modified. All use must give me credit as the original author.
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.
