Udhane Sameer S, Parween Shaheena, Kagawa Norio, Pandey Amit V
Department of Pediatric Endocrinology, Diabetology and MetabolismUniversity Children's Hospital Bern, Bern, Switzerland.
Department of Clinical Research, University of BernBern, Switzerland.
Front Pharmacol. 2017 Aug 25;8:580. doi: 10.3389/fphar.2017.00580. eCollection 2017.
All cytochromes P450s in the endoplasmic reticulum rely on P450 oxidoreductase (POR) for their catalytic activities. Mutations in POR cause metabolic disorders of steroid hormone biosynthesis and affect certain drug metabolizing P450 activities. We studied mutations A115V, T142A, Q153R identified in the flavin mononucleotide (FMN) binding domain of POR that interacts with partner proteins and P284L located in the hinge region that is required for flexibility and domain movements in POR. Human wild-type (WT) and mutant POR as well as CYP3A4 and CYP19A1 proteins in recombinant form were expressed in bacteria, and purified proteins were reconstituted in liposomes for enzyme kinetic assays. Quality of POR protein was checked by cytochrome c reduction assay as well as flavin content measurements. We found that proteins carrying mutations A115V, T142A located close to the FMN binding site had reduced flavin content compared to WT POR and lost almost all activity to metabolize androstenedione via CYP19A1 and showed reduced CYP3A4 activity. The variant P284L identified from apparently normal subjects also had severe loss of both CYP19A1 and CYP3A4 activities, indicating this to be a potentially disease causing mutation. The mutation Q153R initially identified in a patient with disordered steroidogenesis showed remarkably increased activities of both CYP19A1 and CYP3A4 without any significant change in flavin content, indicating improved protein-protein interactions between POR Q153R and some P450 proteins. These results indicate that effects of mutations on activities of individual cytochromes P450 can be variable and a detailed analysis of each variant with different partner proteins is necessary to accurately determine the genotype-phenotype correlations of POR variants.
内质网中的所有细胞色素P450都依赖于P450氧化还原酶（POR）来发挥其催化活性。POR的突变会导致类固醇激素生物合成的代谢紊乱，并影响某些参与药物代谢的P450活性。我们研究了在POR的黄素单核苷酸（FMN）结合域中鉴定出的与伴侣蛋白相互作用的突变A115V、T142A、Q153R，以及位于POR灵活性和结构域运动所需的铰链区的P284L。人野生型（WT）和突变型POR以及重组形式的CYP3A4和CYP19A1蛋白在细菌中表达，纯化后的蛋白重新组装到脂质体中用于酶动力学分析。通过细胞色素c还原试验以及黄素含量测定来检查POR蛋白的质量。我们发现，与WT POR相比，携带位于FMN结合位点附近的突变A115V、T142A的蛋白黄素含量降低，并且几乎丧失了通过CYP19A1代谢雄烯二酮的所有活性，同时CYP3A4活性也降低。从表面正常的受试者中鉴定出的变体P284L也严重丧失了CYP19A1和CYP3A4的活性，表明这是一个潜在的致病突变。最初在一名类固醇生成紊乱患者中鉴定出的突变Q153R显示CYP19A1和CYP3A4的活性显著增加，而黄素含量没有任何显著变化，这表明POR Q153R与某些P450蛋白之间的蛋白质-蛋白质相互作用得到了改善。这些结果表明，突变对单个细胞色素P450活性的影响可能各不相同，对每个与不同伴侣蛋白的变体进行详细分析对于准确确定POR变体的基因型-表型相关性是必要的。
