Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota (W.C.); Stowers Institute for Medical Research, Kansas City, Missouri (E.A.); Department of Bioinformatics, University of Georgia, Ethan, Georgia (X.S.); and Kansas City University of Medicine and Biosciences, Joplin, Missouri (B.T., H.C.-C., J.L.S.).
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota (W.C.); Stowers Institute for Medical Research, Kansas City, Missouri (E.A.); Department of Bioinformatics, University of Georgia, Ethan, Georgia (X.S.); and Kansas City University of Medicine and Biosciences, Joplin, Missouri (B.T., H.C.-C., J.L.S.)
J Pharmacol Exp Ther. 2020 Jun;373(3):370-380. doi: 10.1124/jpet.119.264762. Epub 2020 Mar 23.
The pregnane X receptor (PXR), or nuclear receptor (NR) 1I2, is a ligand-activated NR superfamily member that is enriched in liver and intestine in mammals. Activation of PXR regulates the expression of genes encoding key proteins involved in drug metabolism, drug efflux, and drug transport. Recent mechanistic investigations reveal that post-translational modifications (PTMs), such as phosphorylation, play a critical role in modulating the bimodal function of PXR-mediated transrepression and transactivation of target gene transcription. Upon ligand binding, PXR undergoes a conformational change that promotes dissociation of histone deacetylase-containing multiprotein corepressor protein complexes while simultaneously favoring recruitment histone acetyl transferase-containing complexes. Here we describe a novel adenoviral vector used to deliver and recover recombinant human PXR protein from primary cultures of hepatocytes. Using liquid chromatography and tandem mass spectrometry we report here that PXR is phosphorylated at amino acid residues threonine 135 (T135) and serine 221 (S221). Biochemical analysis reveals that these two residues play an important regulatory role in the cycling of corepressor and coactivator multiprotein complexes. These data further our foundational knowledge regarding the specific role of PTMs, namely phosphorylation, in regulating the biology of PXR. Future efforts are focused on using the novel tools described here to identify additional PTMs and protein partners of PXR in primary cultures of hepatocytes, an important experimental model system. SIGNIFICANCE STATEMENT: Pregnane X receptor (PXR), or nuclear receptor 1I2, is a key master regulator of drug-inducible CYP gene expression in liver and intestine in mammals. The novel biochemical tools described in this study demonstrate for the first time that in cultures of primary hepatocytes, human PXR is phosphorylated at amino acid residues threonine 135 (T135) and serine 221 (S221). Moreover, phosphorylation of PXR promotes the transrepression of its prototypical target gene CYP3A4 through modulating its interactions with coregulatory proteins.
妊娠相关 X 受体（PXR），也称为核受体（NR）1I2，是一种配体激活的 NR 超家族成员，在哺乳动物的肝脏和肠道中丰富表达。PXR 的激活调节编码参与药物代谢、药物外排和药物转运的关键蛋白的基因表达。最近的机制研究揭示，翻译后修饰（PTMs），如磷酸化，在调节 PXR 介导的靶基因转录的反式阻遏和反式激活的双峰功能中起着关键作用。配体结合后，PXR 发生构象变化，促进包含组蛋白去乙酰化酶的多蛋白核心抑制蛋白复合物的解离，同时有利于包含组蛋白乙酰转移酶的复合物的募集。在这里，我们描述了一种新的腺病毒载体，用于从原代培养的肝细胞中递送和回收重组人 PXR 蛋白。我们在这里通过液相色谱和串联质谱报告，PXR 在氨基酸残基苏氨酸 135（T135）和丝氨酸 221（S221）处发生磷酸化。生化分析表明，这两个残基在核心抑制蛋白和共激活蛋白多蛋白复合物的循环中发挥重要的调节作用。这些数据进一步加深了我们对 PTMs（即磷酸化）在调节 PXR 生物学方面的具体作用的基础认识。未来的工作重点是使用这里描述的新工具，在原代培养的肝细胞中鉴定 PXR 的其他 PTMs 和蛋白伴侣，这是一个重要的实验模型系统。意义陈述：妊娠相关 X 受体（PXR），或核受体 1I2，是哺乳动物肝脏和肠道中药物诱导的 CYP 基因表达的关键主调控因子。本研究中描述的新型生化工具首次证明，在原代肝细胞培养物中，人 PXR 在氨基酸残基苏氨酸 135（T135）和丝氨酸 221（S221）处发生磷酸化。此外，PXR 的磷酸化通过调节其与共调节蛋白的相互作用，促进其原型靶基因 CYP3A4 的反式阻遏。
