<p> Pregnane X receptor (PXR) is a major transcriptional regulator of xenobiotic metabolism and transport pathways in the liver and intestines, which are critical for protecting organisms against potentially harmful xenobiotic and endobiotic compounds. Inadvertent activation of drug metabolism pathways through PXR is known to contribute to drug resistance, adverse drug-drug interactions, and drug toxicity in humans. In both humans and rodents, PXR has been implicated in non-alcoholic fatty liver disease, diabetes, obesity, inflammatory bowel disease, and cancer. Because of PXR's important functions, it has been a therapeutic target of interest for a long time.</p><p> Recent mechanistic studies have shown that PXR is modulated by multiple PTMs. In this thesis work, we conducted the first detailed examination of PXR regulation by acetylation. We found that PXR is efficiently acetylated <i> in vitro</i> and <i>in vivo</i> in multiple cell lines (293T, HepG2, LS174T). Acetylation and deacetylation are mediated by p300 and SIRT1, respectively. We found that PXR is directly acetylated by p300 at K109 by LC-MS/MS analysis. The K109Q acetylation mimicking mutant displayed reduced transcriptional activity and reduced ability to induce <i>cyp3A4</i> target gene mRNA and protein compared to the WT and the K109R acetylation-defective mutant. The diminished activity of the K109Q mutant appears to be due to impaired heterodimerization with RXRa and impaired binding of the PXR-RXRa heterodimer to DNA response elements. Furthermore, PXR acetylation appears to have an effect at the phenotypic level, in that pharmacological modulation of PXR acetylation levels can modulate its lipogenic function in mouse primary hepatocytes independent of a ligand. Moreover, the K109Q mutant displays impaired chemoprotective function based on morphological assessment of cells overexpressing K109Q and challenged with indomethacin, suggesting that K109 acetylation downregulates PXR's chemoprotective and perhaps anti-apoptotic functions, although this must be explored further. Notably, the K109R mutant displayed the WT phenotype, further supporting that acetylation itself, not just any arbitrary mutation confers the effect. Altogether, the data suggests that acetylation at K109 represents an overall "loss of function" effect on PXR activity. Implications of our findings are discussed in the context of known roles for PXR in transcription, health, and disease.</p>
| Identifer | oai:union.ndltd.org:PROQUEST/oai:pqdtoai.proquest.com:10019210 |
| Date | 05 March 2016 |
| Creators | Pasquel, Danielle R. |
| Publisher | Yeshiva University |
| Source Sets | ProQuest.com |
| Language | English |
| Detected Language | English |
| Type | thesis |
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