Heintze Tamara, Wilhelm Denise, Schmidlin Thierry, Hofmann Ute, Zanger Ulrich M, Schwab Matthias, Klein Kathrin
Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.
Eberhard Karls University, Tübingen, Germany.
Front Pharmacol. 2021 Nov 15;12:769703. doi: 10.3389/fphar.2021.769703. eCollection 2021.
cytochrome P450 oxidoreductase (POR) is the obligate electron donor for microsomal cytochrome P450 (CYP) enzymes involved in the biosynthesis of endogenous substances like bile acids and other steroids as well as in the oxidative metabolism of xenobiotics. P450 oxidoreductase also supports other redox enzymes in fatty acid and cholesterol pathways. Recently, we have established CRISPR/Cas9-mediated POR knockdown in a human hepatic cell model, HepaRG, and demonstrated the differential effects of limited POR expression on CYP activity. The aim of the present work was to systematically investigate the impact of POR knockdown with a focus on the expression of ADME (absorption, distribution, metabolism, and excretion) genes and related regulators. Functional consequences have been assessed using quantitative mass spectrometry for targeted metabolomics covering bile acids, and cholesterol and its precursors, and for untargeted proteomics. In addition to the previously described alteration of RNA expression of CYP genes, we showed significant downregulation of transcriptional regulators of drug metabolism and transport, including NR1I3 (CAR), NR1I2 (PXR), NR1H4 (FXR), and NR1H3 (LXRα) in cells with gene disruption. Furthermore, POR knockdown resulted in deregulated bile acid and cholesterol biosynthesis demonstrated by low levels of cholic acid derivates and increased concentrations of chenodeoxycholic acid derivates, respectively. Systemic effects of POR knockdown on global protein expression were indicated by downregulation of several metabolic pathways including lipid metabolism and biological oxidation reactions. The deduced protein network map corroborates CYP enzymes as direct interaction partners, whereas changes in lipid metabolism and homeostasis are the result of indirect effects. In summary, our results emphasize a widespread role of POR in various metabolic pathways and provide the first human data on the effects of diminished POR expression on drug and endogenous metabolism in a genomeedited HepaRG cell model.
烟酰胺腺嘌呤二核苷酸磷酸（NADPH）：细胞色素P450氧化还原酶（POR）是微粒体细胞色素P450（CYP）酶的专一性电子供体，这些酶参与内源性物质如胆汁酸和其他类固醇的生物合成以及外源性物质的氧化代谢。P450氧化还原酶还支持脂肪酸和胆固醇途径中的其他氧化还原酶。最近，我们在人肝细胞模型HepaRG中建立了CRISPR/Cas9介导的POR基因敲低，并证明了POR表达受限对CYP活性的不同影响。本研究的目的是系统地研究POR基因敲低的影响，重点是药物吸收、分布、代谢和排泄（ADME）基因及其相关调节因子的表达。使用定量质谱法对靶向代谢组学（涵盖胆汁酸、胆固醇及其前体）和非靶向蛋白质组学评估了功能后果。除了先前描述的CYP基因RNA表达改变外，我们还发现，在基因破坏的细胞中，药物代谢和转运的转录调节因子，包括NR1I3（组成型雄烷受体，CAR）、NR1I2（孕烷X受体，PXR）、NR1H4（法尼醇X受体，FXR）和NR1H3（肝X受体α，LXRα）显著下调。此外，POR基因敲低分别导致胆酸衍生物水平降低和鹅去氧胆酸衍生物浓度升高，从而证明胆汁酸和胆固醇生物合成失调。POR基因敲低对整体蛋白质表达的系统性影响表现为包括脂质代谢和生物氧化反应在内的几种代谢途径下调。推导的蛋白质网络图证实CYP酶是直接相互作用伙伴，而脂质代谢和稳态的变化是间接效应的结果。总之，我们的结果强调了POR在各种代谢途径中的广泛作用，并提供了首个关于在基因组编辑的HepaRG细胞模型中POR表达降低对药物和内源性代谢影响的人体数据。
