REGULATION OF ETHANOL INDUCIBLE CYTOCHROME P450 2E1 (CYP2E1)

我们已经证明了细胞色素P450-2的多种调控机制：诱导 通过转录、信使核糖核酸稳定、激活信使核糖核酸翻译和 蛋白质稳定、转录抑制、信使核糖核酸降解 和蛋白质的降解。我们最近报道了转录 外源化合物YH439对细胞色素P421基因的抑制作用这个 这种合成的CYP2E1抑制剂的潜在有益作用是 碳素治疗急性肝炎体内模型的研究 四氯化物。活体肝胆成像分析显示 YH439能有效保护四氯化碳所致的肝损伤。这些 结果得到了相应水平的变化的证实 血清转氨酶和组织学评价。保护者 YH439的作用似乎是由于有效地抑制了 催化四氯化碳新陈代谢的细胞色素P450_2E_1， 引发自由基介导的组织损伤。的水平 并对乙醛加合物进行了研究。我们的免疫印迹数据，使用 抗乙醛-蛋白质加合物的多克隆抗体表明， 免疫反应条带(十二烷基硫酸钠-聚丙烯酰胺凝胶表观MR 37 kDa) 在用对照饲料处理的动物中没有检测到，而这条带 在以酒精流质饮食配对喂养的大鼠身上明显检测到。然而， 经YH439处理后，免疫反应性条带基本消失。 免疫细胞学分析显示，免疫反应抗原是 主要发现于中心周围区域，其中主要是CYP2E1 本地化。这些数据表明，乙醛-蛋白质加合物(MR 37 kDa)以依赖于细胞色素P450-2的方式产生。自CYP2E1以来 已知介导的新陈代谢会引起氧化应激，从而导致DNA 和蛋白质损伤，大鼠组织中DNA加合物的水平 不同的处理，包括不存在和存在的乙醇 YH439，正在用高效液相色谱法进行测定。我们还在寻找潜在的 人细胞色素P4502基因突变的研究 具有低水平和高水平的CYP2E1活性的个体。我们的数据 提示不同水平的CYP2E1活性并不对应 与细胞色素P4502基因多态性有关。此外，细胞色素P450-2E1的过量生产 在杆状病毒表达系统中正在进行研究 四种暴露赖氨酸残基中每一种的生物学作用 在一个模型化的CYP2E1蛋白结构中。这些赖氨酸残留物很好 泛素结合的候选者，导致蛋白酶体 细胞色素P450-2的降解。因此，这些赖氨酸残基中的每一个， 用丙氨酸突变泛素结合率的研究 以及随后的CYP2E1降解，以及阐明确切的 乙醇稳定细胞色素P450-2E1的机理。

We have demonstrated multiple regulatory mechanisms for CYP2E1: induction via transcription, mRNA stabilization, activation of mRNA translation and protein stabilization, suppression via transcription, mRNA degradations and protein degradation. We have recently reported transcriptional suppression of CYP2E1 gene by an exogenous compound, YH439. The potential beneficial effect of this synthetic inhibitor of CYP2E1 were studied in an in vivo model of acute hepatitis by treatment with carbon tetrachloride. In vivo hepatobiliary imaging analyses revealed that YH439 efficiently protects liver injury from carbon tetrachloride. These results were confirmed by the corresponding changes in the levels of serum transaminases and by histological evaluations. The protective effect of YH439 appears to be the result of effective suppression of CYP2E1, which catalyzes the metabolism of carbon tetrachloride, leading to the initiation of free-radical mediated tissue damage. The level of acetaldehyde adduct was also studied. Our immunoblot data, using polyclonal antibody against acetaldehyde-protein adduct, showed that an immunoreactive band (apparent Mr 37 kDa on SDS-polyacrylamide gel) was not detected in animals treated with control diet while this band was clearly detected in rats pair-fed with alcohol liquid diet. However, the immunoreactive band was virtually absent upon treatment with YH439. Immunocytological analyses revealed that immunoreactive antigen is primarily found in the pericentral region where CYP2E1 is mainly localized. These data suggest that the acetaldehyde-protein adduct (Mr 37 kDa) was produced in a CYP2E1-dependent manner. Since CYP2E1 -mediated metabolism is known to cause oxidative stress resulting in DNA and protein damage, the levels of DNA-adducts in rat tissues from different treatments, including ethanol in the absence and presence of YH439, are being measured by HPLC. We have also searched for potential mutations in the human CYP2E1 gene by analyzing DNA samples obtained from individuals with low and high levels of CYP2E1 activity. Our data suggest that the different levels of CYP2E1 activity do not correspond with CYP2E1 gene polymorphism. In addition, over-production of CYP2E1 in baculovirus expression system is being performed to study the biological role of each of the four exposed lysine residues, as viewed in a modeled CYP2E1 protein structure. These lysine residues are good candidates for ubiquitin conjugation, leading to the proteasomal degradation of CYP2E1. Each of these lysine residues is, therefore, being mutated with alanine to study the rates of ubiquitin conjugation and subsequent CYP2E1 degradation as well as to elucidate the exact mechanism of CYP2E1 stabilization by ethanol.