Hepatic degradation of cytochrome P450 enzymes

描述(申请人提供)：肝脏细胞色素P450(P450)是内质网膜锚定的酶，参与分解内源和外源生物，即药物、致癌物、毒素、天然和化学产品。暴露在这些制剂中会因形成增加而增加肝脏P450的含量，或因灭活/破坏和/或蛋白质降解而减少P450的含量。这种药物介导的对P450含量的调节可以显著影响临床药物相互作用(DDiS)。因此，临床上相关的DDiS通常出现在药物介导的P450稳定(即乙醇)以及药物促进的P450降解(即西柚汁呋喃香豆素)所引起的P450代谢的改变。我们最近发现，人体主要的肝脏和肠道中的P450，如细胞色素P450 3A4和乙醇代谢的细胞色素P450-1的降解，会引起gp78和芯片E3泛素(Ub)连接酶以及26S蛋白酶体(Upd)的泛素化，从而加速它们的细胞降解。我们的体外研究表明，这种由gp78和ChIP介导的P450泛素化通过其多位点蛋白磷酸化而显著增强。泛素化的P450赖氨酸残基位于带负电荷的酸性(Asp/Glu)和磷酸化(Ser/Thr)簇中。这导致我们假设蛋白质的磷酸化通过增强这些簇的负电荷特性来增强这些E3Ub连接酶对P450的分子识别，从而控制其UPD。因此，我们的第一个具体目标是通过对每个簇中相关的D/E和S/T残基进行定点突变来验证这一假设，并采用最先进的蛋白质组学方法来确定它们与这些E3的P450泛素化的相关性。此外，我们还发现，在大鼠肝细胞中，通过RNA干扰的gp78和ChIP基因敲除导致功能活跃的肝脏P450水平增加。虽然这一发现在临床上与肝脏药物代谢和DDIS相关，但我们认为它也可能具有病理生理学意义：在缺乏相关药物底物的情况下，这些P450通过无效的氧化循环可以产生蛋白毒性反应性O2-物种(ROS)，从而在一定程度上导致CHIP-/-小鼠肝脏和其他组织中观察到的年龄依赖性氧化损伤和脂质过氧化增加。因此，我们的第二个具体目标是探索CHIP基因敲除后肝脏P450的升高是否与CHIP-/-小鼠的这种病理有关。酒精依赖的乙醇代谢增强也同样会产生ROS、羟乙基自由基(HER)和脂质过氧化产物，从而破坏细胞蛋白，包括与CYP2E1代谢有关的UPD和自噬-溶酶体降解途径。我们的第三个特定目的是探索乙醇通过破坏细胞内正常的CYP2E1转运来破坏CYP2E1的周转是否导致其向外膜迁移增加，从而被免疫监视系统识别，从而产生与酒精性肝病、药物性肝炎和超敏综合征相关的致病自身抗体。

DESCRIPTION (provided by applicant): The hepatic cytochromes P450 (P450s) are endoplasmic-reticulum membrane-anchored enzymes engaged in the breakdown of endo- and xenobiotics i.e. drugs, carcinogens, toxins, natural and chemical products. Exposure to these agents can increase liver P450 content due to increased formation, or reduce it due to inactivation/destruction and/or proteolytic degradation. Such drug-mediated modulation of P450 content can significantly influence clinical drug-drug interactions (DDIs). Thus, clinically relevant DDIs often emerge from altered P450 turnover elicited by drug-mediated P450 stabilization i.e. ethanol (EtOH), as well as enhanced drug-mediated P450 degradation (i.e. grapefruit juice furanocoumarins). We have recently shown that the degradation of P450s such as CYP3A4, the major human liver and intestinal P450, and the EtOH-metabolizing CYP2E1 incurs ubiquitination by gp78 and CHIP E3 ubiquitin (Ub)-ligases and the 26S proteasome (UPD) which leads to their accelerated cellular disposal. Our in vitro studies reveal that such gp78- and CHIP- mediated P450 ubiquitination is considerably enhanced by their multisite protein phosphorylation. The ubiquitinated P450 Lys-residues reside in negatively charged acidic (Asp/Glu) and phosphorylated (Ser/Thr) clusters. This leads us to hypothesize that protein phosphorylation by enhancing the negatively charged character of these clusters enhances P450 molecular recognition by these E3 Ub-ligases and thus would control its UPD. Thus our first specific aim is to test this hypothesis by site-directed mutagenesis of the relevant D/E and S/T residues in each cluster along with a state-of-the-art proteomic approach to determine their relevance to P450 ubiquitination by these E3s. In addition, we have also found that gp78- and CHIP- knockdown via RNA-interference in rat hepatocytes results in increased levels of functionally active hepatic P450s. While this finding is clinically relevant for hepatic drug metabolism and DDIs, we propose that it may also be pathophysiologically significant: In the absence of relevant drug substrates, these P450s through futile oxidative cycling could generate proteotoxic reactive O2-species (ROS), and thus partly contribute to age- dependent oxidative damage and elevated lipid peroxidation observed in the liver and other tissues of CHIP-/- mice. Thus our second specific aim is to explore whether elevated hepatic P450s upon CHIP knockdown contribute to this pathology in CHIP-/- mice. Enhanced CYP2E1-dependent EtOH metabolism in alcoholics is also known to similarly generate ROS, hydroxyethyl radicals (HER) and lipid peroxidation products that damage cellular proteins including those of UPD and autophagic-lysosomal degradation pathways involved in CYP2E1 turnover. Our third specific aim is to explore whether EtOH-impaired CYP2E1 turnover by disrupting the normal intracellular CYP2E1 trafficking results in its increased migration to the outer plasma membrane, whereupon it is recognized by the immune surveillance system, engendering pathogenic autoantibodies clinically associated with alcoholic liver disease, drug-induced hepatitis, and hypersensitivity syndromes.