Degradation of Human CYP2E1 by the Proteasome

描述：（改编自申请人的摘要）： 氧化应激的作用是促进乙醇 肝毒性乙醇对CYP2E1的诱导作用似乎是一个主要途径， 乙醇会产生氧化应激状态。除了乙醇，CYP2E1 氧化许多其他重要的化合物，并在各种 病理生理条件。CYP2E1的主要调节水平是在 作为CYP2E1的几种配体，包括乙醇 保护酶免受未表征的细胞内降解 蛋白水解途径此应用程序的目标是具体目标1将 评价蛋白酶体复合物在人CYP2EI降解中的作用 肝细胞将研究人肝细胞培养物中CYP 2E1的转换。 主要的蛋白水解途径，以及是否需要泛素化 将测定CYP2E1降解以及乙醇对此的影响 过程具体目标2将评估活性氧在以下方面的作用： 通过蛋白酶体复合物触发CYP2E1降解。的能力 多种抗氧化剂改变完整细胞中的CYP2E1水平和周转率， 将对复溶系统进行评价，并提供CYP2E1氧化 将进行修改。具体目标3将评估 分子伴侣对稳态水平和CYP2E1转换的影响。实验 将涉及研究热休克蛋白抑制剂的作用， 抗体或免疫耗竭，然后再加入调节剂， 重组系统，或添加抑制剂或添加质粒 将推定的调节剂表达至完整细胞模型。乙醇和其他 CYP2E1配体阻断调节剂的作用？这些实验将 提供了新的信息，蛋白酶体和泛素化的作用， 人肝细胞中的CYP2E1周转，乙醇如何保护CYP2E1免受 退化，并确定可能引发这种快速周转的因素， 酵素任何这些新发现的步骤的调节剂可能具有治疗作用。 对CYP2E1高水平个体的影响。

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.