Degradation of Human CYP2E1 by the Proteasome

描述：(改编自申请人的摘要)：有很多人对 氧化应激在乙醇代谢机制中的作用 对肝脏有毒性。乙醇诱导细胞色素P450-2E_1的主要途径是通过 乙醇会产生一种氧化应激状态。除了乙醇，CYP2E1 氧化许多其他重要化合物，并在各种不同的 病理生理条件。CYP2E1的一个主要调控水平是 作为包括乙醇在内的几种CYP2E1的配体的转录后阶段 通过未鉴定的胞内保护酶免受降解 蛋白分解途径。此应用程序的目标是特定的目标1将 评价蛋白酶体复合体在人体降解细胞色素P450酶EI中的作用 肝细胞。我们将研究细胞色素P450 2E1在培养的人肝细胞中的周转。 主要的蛋白分解途径，以及是否需要泛素化 将确定CYP2E1的降解情况，以及乙醇对此的影响。 进程。特定目标2将评估活性氧物种在 通过蛋白酶体复合体触发CYP2E1的降解。一名员工的能力 多种抗氧化剂改变完整细胞和 将对重组的系统进行评估，并提供CYP2E1氧化的证据 将提供修改。具体目标3将评估 分子伴侣对细胞色素P450_2E_1稳态水平和转化率的影响。实验 将涉及研究热休克蛋白抑制剂或 免疫耗竭的抗体或免疫耗竭后的调节子在 重组系统，或添加抑制剂或添加质粒 将假定的调节子表达到完整的细胞模型中。乙醇和其他 细胞色素P450 2E 1配体阻断调节剂的作用？这些实验将 提供有关蛋白酶体和泛素化在蛋白质合成中作用的新信息 人肝细胞中细胞色素P450_2 E_1的转化，乙醇如何保护细胞色素P_2 E_1 降级，并找出可能引发该产品快速周转的因素 酵素。这些新发现的步骤中的任何一个的调节器都可能具有治疗作用 对高水平的CYP2E1的个体的影响。

DESCRIPTION: (Adapted from the Applicant's Abstract): There is much interest in the role of oxidative stress as contributing to mechanisms by which ethanol is hepatotoxic. Induction of CYP2E1 by ethanol appears to be a major pathway by which ethanol produces a state of oxidative stress. Besides ethanol, CYP2E1 oxidizes many other important compounds and is induced under a variety of pathophysiological conditions. A major level of regulation of CYP2E1 is at the posttranscriptional stage as several ligands for CYP2E1, including ethanol protect the enzyme against degradation by uncharacterized intracellular proteolytic pathways. The goal of this application is Specific Aim 1 will evaluate the role of the proteasome complex in CYP2EI degradation in human hepatocytes. Turnover of CYP2E1 in cultured human hepatocytes will be studied. The major proteolytic pathways, and whether ubiquitination is required for CYP2E1 degradation will be determined, as will the effects of ethanol on this process. Specific Aim 2 will evaluate the role of reactive oxygen species in triggering CYP2E1 degradation by the proteasome complex. The ability of a variety of antioxidants to alter CYP2E1 levels and turnover in intact cells and reconstituted systems will be evaluated, and evidence for CYP2E1 oxidative modification will be provided. Specific Aim 3 will evaluate the effect of molecular chaperones on steady state levels and turnover of CYP2E1. Experiments will involve studying the effects of inhibitors of heat shock proteins or antibodies or of irnmunodepletion followed by readdition of the modulator in the reconstituted system, or adding inhibitors or addition of plasmids expressing the putative modulator to intact cell models. Does ethanol and other CYP2E1 ligands block the effect of the modulator? These experiments will provide new information on the role of the proteasome and ubiquitination in CYP2E 1 turnover in human hepatocytes, how ethanol protects CYP2E1 against degradation, and identify factors which may trigger rapid turnover of this enzyme. Modulators of any of these newly identified steps may have therapeutic implications for individuals with high levels of CYP2E1.