Hepatic degradation of cytochrome P450 enzymes

描述（由申请方提供）：肝细胞色素P450（P450 s）是内质网膜锚定酶，参与内源性和外源性物质（即药物、致癌物、毒素、天然和化学产品）的分解。暴露于这些试剂可由于形成增加而增加肝脏P450含量，或由于失活/破坏和/或蛋白水解降解而减少肝脏P450含量。这种药物介导的P450含量调节可显著影响临床药物-药物相互作用（DDI）。因此，临床相关DDI通常来自药物介导的P450稳定（即乙醇（EtOH））以及增强的药物介导的P450降解（即葡萄柚汁呋喃香豆素）引起的P450转换改变。我们最近已经表明，P450如CYP 3A 4（主要的人类肝脏和肠道P450）和EtOH代谢CYP 2 E1的降解引起gp 78和CHIP E3泛素（Ub）连接酶和26 S蛋白酶体（UPD）的泛素化，这导致它们加速细胞处置。我们的体外研究表明，这种gp 78和CHIP介导的P450泛素化被它们的多位点蛋白磷酸化显著增强。泛素化的P450赖氨酸残基存在于带负电荷的酸性（Asp/Glu）和磷酸化（Ser/Thr）簇中。这使我们假设，蛋白磷酸化通过增强这些簇的带负电荷的特性增强了P450分子被这些E3 Ub连接酶识别，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的相关D/E和S/T残基进行定点诱变来测试这一假设沿着采用最先进的蛋白质组学方法来确定这些E3与P450泛素化的相关性。此外，我们还发现通过RNA干扰在大鼠肝细胞中敲低gp 78和CHIP导致功能活性肝P450水平增加。虽然这一发现与肝脏药物代谢和DDI具有临床相关性，但我们认为它也可能具有病理生理学意义：在不存在相关药物底物的情况下，这些P450通过无效氧化循环可能产生蛋白毒性反应性O2-物质（ROS），因此部分导致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升高。因此，我们的第二个具体目标是探索CHIP敲低后肝脏P450升高是否有助于CHIP-/-小鼠的这种病理学。还已知酗酒者中CYP 2 E1依赖性EtOH代谢增强类似地产生ROS、羟乙基自由基（HER）和脂质过氧化产物，这些产物损害细胞蛋白质，包括参与CYP 2 E1周转的UPD和自噬-溶酶体降解途径的蛋白质。我们的第三个具体目标是探索EtOH是否通过破坏正常的细胞内CYP 2 E1运输而损害CYP 2 E1周转，导致其向外质膜的迁移增加，从而被免疫监视系统识别，产生临床上与酒精性肝病、药物性肝炎和超敏反应综合征相关的致病性自身抗体。

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.