Drug Metabolizing Enzymes In Humans

在一个新的项目中，酵母和哺乳动物双杂交筛选表明，Med 25是介体复合物的可变成员，是一种HNF 4a结合蛋白。 我们已经证明Med 25对于RNA聚合酶II的募集是重要的，以选择HNF 4a激活的启动子集，例如重要的药物代谢基因细胞色素P450 2C 9（CYP 2C 9）和CYP 3A 4。 我们假设这涉及Med 25和HNF 4之间的直接相互作用，以改变CYP 2C 9基因的染色质构象，使其处于转录活性状态。 构象变化需要被募集到靶基因的酶修饰组蛋白。 组蛋白修饰包括组蛋白N-末端尾部上赖氨酸和精氨酸氨基酸的甲基化或乙酰化。 例如，组蛋白3赖氨酸4二甲基化（H3 K4 me 2）与基因激活相关，而组蛋白3赖氨酸27三甲基化（H3 K27 me 3）是基因沉默的标志物。 在这项研究中，我们使用HepG 2细胞来确定Med 25在HNF 4a依赖性CYP 2C 9表达的表观遗传调控中的作用。 我们进行了染色质免疫沉淀，以确定组蛋白修饰在HNF 4a结合位点的Med 25蛋白水平。 我们的研究结果表明，改变Med 25的表达修改乙酰化和甲基化的组蛋白3上的某些赖氨酸27。 当表达Med 25时，该赖氨酸被乙酰化。 然而，当用小发夹环RNAi沉默Med 25时，H3 K27被三甲基化，这是基因沉默的原型。 这些结果表明，Med 25在CYP 2C 9近端HNF 4a结合位点诱导容许染色质状态。 类似地，共聚焦显微镜显示Med 25与关键组蛋白修饰标记共定位。 我们还使用甲醛辅助分离调节元件（FAIRE）确定了激活条件下开放CYP 2C 9染色质的水平。 FAIRE数据表明，CYP 2C 9近端启动子的HNF 4a位点周围的染色质在激活核受体CAR和HNF 4a和Med 25存在下是开放的，但当Med 25沉默时是封闭的。这些研究将在全球范围内扩展到更多的HNF 4a诱导基因。一项新的研究表明，雌激素受体α（ER α）诱导CYP 2C 9启动子活性的配体和外源性Med 25的存在下。 免疫沉淀研究表明ER α和Med 25之间的相互作用，在配体的存在或不存在。染色质免疫沉淀研究表明，Med 25结合的ERE（雌激素反应元件在ER α的存在下。在原代肝细胞中，Med 25和ER a增加了CYP 2C 9催化活性，但siMed 25使其沉默。最近我们发现亲电试剂和氧化应激通过AP-1蛋白与两个AP-1位点相互作用诱导人原代肝细胞中的CYP 2C 9和CYP 2C 19。 当分别被cJun和JunD占据时，两个站点之间存在循环。 许多药物是亲电的或已知被激活为亲电体。 这是一种新的CYP 2C 9和CYP 2C 19激活机制，不涉及异种感知受体CAR或PXR。 在合作研究中，我们发现，使用重组CYP 2C 19 *10等位基因（在我们的实验室发现），Plavidine（氯吡格雷）（一种预防血小板聚集的药物）的体外代谢清除率比正常CYP 2C 19低75%。 因此，虽然它不是一个失活的等位基因，但其活性大大受损。 使用不同CYP 2C 19底物的研究显示了不同程度的损伤。 CYP 2C 19将氯吡格雷从前药活化为活性药物。 CYP 2C 19 *10等位基因还干扰了许多针对无效CYP 2C 19 *2等位基因的基因分型检测。 这表明临床研究应包括CYP 2C 19 *10等位基因的基因分型。

In a new project, yeast and mammalian two-hybrid screens showed that Med25, a variable member of the mediator complex, is an HNF4a-binding protein. We have shown that Med25 is important for the recruitment of RNA Polymerase II to select sets of HNF4a-activated promoters such as the important drug-metabolizing gene cytochrome P450 2C9 (CYP2C9) and CYP3A4. We hypothesized that this involves direct interaction between Med25 and HNF4 to alter chromatin conformation of the CYP2C9 gene to a transcriptionally active state. Conformational change requires the modification of histones by enzymes that are recruited to target genes. Histone modifications include methylation or acetylation of lysine and arginine amino acids on histone N-terminal tails. For example, histone 3 lysine 4 dimethylation (H3K4me2) is associated with gene activation, while histone 3 lysine 27 trimethylation (H3K27me3) is a marker of gene silencing. In this study, we used HepG2 cells to determine the role of Med25 in the epigenetic regulation of HNF4a-dependent CYP2C9 expression. We performed chromatin immunoprecipitation to identify histone modifications at the HNF4a binding site in relation to Med25 protein levels. Our results indicate that altering Med25 expression modified acetylation and methylation of certain lysine 27 on histone 3. When Med 25 was expressed, this lysine was acetylated. However, when Med25 was silenced with small-hairpin looped RNAi, H3K27 was trimethylated which is prototypical in gene-silencing. These results indicate that Med25 induces a permissive chromatin state at the CYP2C9 proximal HNF4a binding site. Similarly, confocal microscopy revealed that Med25 colocalized with key histone modification markers. We have also determined levels of open CYP2C9 chromatin under activating conditions using formaldehyde-assisted isolation of regulatory elements (FAIRE). FAIRE data indicated that the chromatin around the HNF4a sites of the CYP2C9 proximal promoter was open in the presence of activating nuclear receptors CAR and HNF4a and Med25 but closed when Med25 was silenced. These studies will be extended globally to more HNF4a induced genes. A new study showed that the estrogen receptor alpha (ERa) induced CYP2C9 promoter activity in the presence of ligand and exogenous Med25. Immunoprecipitation studies showed interaction between ERa and Med25 in the presence or absence of ligand. Chromatin immunoprecipitation studies showed that Med25 bound to the ERE (estrogen responsive element in the presence of ERa. CYP2C9 catalytic activity was increased in primary hepatocytes by Med25 and ERa but silenced with siMed25. Recently we found that electrophiles and oxidative stress induce CYP2C9 and CYP2C19 in human primary hepatocytes through AP-1 proteins which interact with two AP-1 sites. There is looping between the two sites when occupied by cJun and JunD respectively. Many drugs are electrophilic or known to be activated to electrophiles. This is a new mechanism of activation of CYP2C9 and CYP2C19 not involving the xenosensing receptors CAR or PXR. In collaborative studies we showed that Plavix (clopidogrel) a drug which prevents platelet aggregation is metabolized with a 75% lower clearance in vitro using recombinant CYP2C19*10 allele (discovered in our laboratory) than normal CYP2C19. Thus, although it is not an inactive allele, its activity is greatly impaired. Studies with different CYP2C19 substrates showed different degrees of impairment. CYP2C19 activates clopidogrel from a prodrug to the active drug. The CYP2C19*10 allele also interferes with a number of genotyping tests for the null CYP2C19*2 allele. This suggests clinical studies should include genotyping of the CYP2C19*10 allele.