REGULATION OF ETHANOL INDUCIBLE CYTOCHROME P450 GENE

我们先前已经分离了编码该蛋白的cDNA和基因组克隆。 乙醇诱导的细胞色素P450 IIE 1，并已证明六个不同的 其表达的调控类型。 在过去的一年里，我们 继续研究一种实验性的抗疟疾药物 代理YH 439，并进一步表征了P450 IIE 1的新机制 由外源化合物YH 439调节。 P450 IIE 1活性和 YH 439在一定时间内有效地抑制了蛋白水平， 剂量依赖性 核转录检测证实 YH 439在2小时内即抑制P450 IIE 1基因的转录 YH 439给药后。 与此相反，P450 IA 1/2基因是 由YH 439诱导的转录。 为了阐明的机制， YH 439的转录调控，P4502 E1的启动子区和 P4501 A1/2基因正在积极研究阴性和阳性 元素。 我们已经证明YH 439可以激活P450 IA 1/2 人肝癌细胞系HepG 2. 在这个细胞系中， YH 439激活芳香烃受体（Ah受体）， 以前显示介导二恶英的毒性作用以及其他 环境污染物。 我们之前已经发布了P450 IIE 1 在怀孕期间受到抑制， 正常水平（1天内）。 我们还 研究了妊娠期间其他P450的表达， 基因表达的机制。 核连续转录分析 P450 IIE 1和其他P450基因主要在细胞膜上表达。 转录后水平。 与罗伯茨博士合作， Shoaf，肝，脑， 和其他组织进行了研究，在酒精消费和 酒精戒断期 酒精升高了P450 IIE 1 5- 在检查的所有组织中折叠。 戒酒后，P450 IIE 1 水平迅速（12小时内）恢复到正常水平， 我们先前关于P450 IIE 1相对较短的半衰期及其 通过乙醇和丙酮稳定蛋白质。 P450 IIE 1稳定性 乙醇通过干扰P450 IIE 1的结合介导 与泛素结合，使P450 IIE 1快速降解。

We have previously isolated both the cDNA and genomic clones encoding the ethanol-inducible cytochrome P450IIE1 and have demonstrated six distinct types of regulation of its expression. During the past year, we have continued to investigate the effect of an experimental anti-cirrhotic agent YH439 and have further characterized a novel mechanism of P450IIE1 regulation by an exogenous compound, YH439. P450IIE1 activity and protein level were effectively suppressed by YH439 in a time- and dose-dependent manner. Nuclear run-on transcription assays confirmed that YH439 inhibited transcription of P450IIE1 gene as early as two hours post-YH439 administration. In contrast, P450IA1/2 genes were transcriptionally induced by YH439. To elucidate the mechanisms of the transcriptional regulations by YH439, the promoter regions of P4502E1 and P4501A1/2 genes are being actively studied for negative and positive elements, respectively. We have shown that YH439 activates the P450IA1/2 genes in the human hepatocarcinoma cell line, HepG2. In this cell line, YH439 activates the aromatic hydrocarbon receptor (Ah receptor), previously shown to mediate the toxic effects of dioxine as well as other environmental contaminants. We have previously published that P450IIE1 was pretranslationally suppressed during pregnancy and that it returned to normal level (within 1 day) upon parturition. We have also investigated the expression of other P450s during pregnancy and their mechanism of gene expression. Nuclear run-on transcription analyses revealed that P450IIE1 and other P450 genes are mainly regulated at the post-transcriptional level. In collaboration with Drs. Roberts and Shoaf, the levels of P450IIE1 and other P450 isoenzymes in liver, brain, and other tissues were studied during alcohol consumption and after alcohol withdrawal period. Alcohol elevated the levels of P450IIE1 5- fold in all tissues examined. After withdrawal of alcohol, P450IIE1 level rapidly (within 12 hours) returned to the normal level, supporting our earlier report on the relatively short half-lives of P450IIE1 and its protein stabilization by ethanol and acetone. The P450IIE1 stabilization by ethanol was mediated by interference of the conjugation of P450IIE1 with ubiquitin, which renders rapid degradation of P450IIE1.