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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. More recent mechanistic studies have shown that PXR is modulated by multiple PTMs. Herein we provide the first investigation of the role of acetylation in modulating PXR activity. Through LC–MS/MS analysis, we identified lysine 109 (K109) in the hinge as PXR's major acetylation site. Using various biochemical and cell-based assays, we show that PXR's acetylation status and transcriptional activity are modulated by E1A binding protein (p300) and sirtuin 1 (SIRT1). Based on analysis of acetylation site mutants, we found that acetylation at K109 represses PXR transcriptional activity. The mechanism involves loss of RXRα dimerization and reduced binding to cognate DNA response elements. This mechanism may represent a promising therapeutic target using modulators of PXR acetylation levels.

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The pregnane X receptor (PXR) is a key xenobiotic receptor that regulates the expression of numerous drug-metabolizing enzymes. Some posttranslational mechanisms modulate its transcriptional activity. Although several kinases have been shown to directly phosphorylate this receptor, little is known about phosphorylation sites of PXR. In the present work, we examined T248, Y249 and T422 putative phosphorylation sites determined based on in silico consensus kinase site prediction analysis. T248 and T422 residues are critical for the interaction of the PXR ligand-binding domain and the activation function-2 (AF2) domain. Site-directed mutagenesis analysis was performed to generate phospho-deficient and phospho-mimetic mutants. We examined transactivation activity of the PXR mutants in gene reporter assays, formation of PXRmutant/RXRα heterodimer, binding of PXR mutants to the CYP3A4 gene response element DR3 and CYP3A4 expression in HepG2 cells after expression of the mutants. We found that T248D mutant activated CYP3A4 transactivation constitutively regardless of the presence or absence of a ligand. Contrary, T248V mutant exhibited low basal and ligand-inducible transactivation capacity as compared to wild-type PXR. Dose-response analysis revealed reduced ligand-dependent transactivation potency of PXR Y249D mutant. Transactivation of the CYP3A4 promoter was abolished with T422A/D mutants. All PXR mutants formed heterodimer with RXRα at a similar level to that observed with wild-type PXR. The ability to bind to DNA in vitro was substantially decreased in case of T248D, T422D and T248V mutants. Our data thus indicate that phosphorylation of T248, Y249 and T422 residues may be critical for the both basal and ligand-activated function of PXR.