Hepatic degradation of cytochrome P450 enzymes

描述（由申请方提供）：肝血红素蛋白细胞色素P450（P450 s）是内质网（ER）锚定酶，可分解内源性和外源性物质，即药物、致癌物、毒素、天然和化学产品。在暴露于这些试剂时，肝脏P450含量可能由于形成增加而增加，或由于其失活/破坏和/或蛋白水解降解而减少。已知这种药物介导的P450含量调节显著影响临床药物-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）引起的P450转换改变以及灭活后药物介导的P450降解增强（即葡萄柚汁呋喃香豆素）。失活的、结构受损的P450在称为ER相关降解（ERAD）的过程中引起泛素化和随后的26 S蛋白酶体降解（UPD），这导致它们加速细胞处置。尽管我们初步的体外研究已经鉴定gp 78泛素连接酶是泛素化CYP 3A 4（主要的人类肝脏和肠道P450）的酶，但其体内相关性仍然未知。因此，我们的第一个具体目标是使用最先进的蛋白质组学方法来确定其生理相关性。鉴于CYP 3A 4 ERAD还需要蛋白磷酸化以及与胞质伴侣的相互作用，我们的第二个具体目标是定义这些过程中的每一个的具体作用，并阐明它们是否在功能上相关或相互依赖。在生理上，一些P450以不同的半衰期（6-37小时）和通过不同的途径异步转换。因此，乙醇代谢CYP 2 E1在与乙醇结合时引起自噬-溶酶体降解（ALD），但在无底物时引起ERAD/UPD。建议进行研究，以阐明这种正常的CYP 2 E1营业额的中断是否会导致P450转运到外质膜的增加，从而被免疫监视系统识别，产生临床上与酒精性肝病，药物诱导的肝炎和过敏综合征相关的致病性P450自身抗体。由于UPD和ALD在酵母酿酒酵母中高度保守，并且这些途径中存在遗传缺陷/缺失的突变株，因此我们建议在培养的大鼠和人肝细胞之外使用酵母。我们的研究主要集中在人类肝脏CYP 3A 4和CYP 2 E1及其大鼠肝脏对应物。这些P450共同构成约40%的总人类肝脏P450含量，但负责约65%的临床处方药物、毒素和致癌物的代谢，因此具有显著的潜在DDI和毒性。更重要的是，由于UPD功能对许多重要生物过程的调节至关重要，而ALD具有重要的稳态和生物合成功能，因此正常UPD或ALD途径的破坏可导致各种人类疾病。我们提出的P450作为其原型ER蛋白底物的研究将大大促进我们对这些重要过程中所涉及的分子机制和卫生巡逻的理解。人类健康相关性：肝细胞色素P450（P450）是参与药物，致癌物，毒素，天然和化学试剂分解为水溶性产物的酶。暴露于这些药物可以增加肝脏P450含量或通过增强蛋白质降解降低肝脏P450含量，并且这种药物介导的P450含量调节可以显著影响临床药物-药物相互作用。事实上，临床相关的药物-药物相互作用是由于药物介导的P450稳定（即酒精/乙醇）或增强的药物介导的P450降解（即葡萄柚汁呋喃香豆素）引起的P450转换改变而发生的。我们的研究建议使用哺乳动物系统和酵母作为模型来阐明CYP 3A 4（主要的人类肝脏和肠道酶）和人类CYP 2 E1（与酒精性肝病有关的P450酶）的降解机制。这些P450共同负责约65%的临床相关药物，毒素和致癌物的代谢，因此具有药物相互作用和毒性的显著潜力。

DESCRIPTION (provided by applicant): The hepatic hemoproteins cytochromes P450 (P450s) are endoplasmic-reticulum (ER)-anchored enzymes that break down endo- and xenobiotics i.e. drugs, carcinogens, toxins, natural and chemical products. On exposure to these agents, liver P450 content may be increased due to increased formation, or reduced due to its inactivation/destruction and/or proteolytic degradation. Such drug-mediated modulation of P450 content is known to 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) as well as enhanced drug-mediated P450 degradation after their inactivation (i.e. grapefruit juice furanocoumarins). The inactivated, structurally damaged P450s incur ubiquitination and subsequent 26S proteasomal degradation (UPD) in a process known as ER-associated degradation (ERAD), which leads to their accelerated cellular disposal. Although our preliminary in vitro studies have identified gp78 ubiquitin- ligase as the enzyme which ubiquitinates CYP3A4, the major human liver and intestinal P450, its in vivo relevance remains unknown. Thus our first specific aim is to determine its physiological relevance using a state-of-the-art proteomic approach. Given that CYP3A4 ERAD also entails protein phosphorylation as well as interactions with cytosolic chaperones, our second specific aim is to define the specific role of each of these processes, and to elucidate if they are functionally associated or interdependent. Physiologically, some P450s turn over asynchronously with variable half-lives (6-37 h) and via different pathways. Thus, the ethanol-metabolizing CYP2E1 incurs autophagic-lysosomal degradation (ALD) when it is ethanol- bound, but ERAD/UPD when substrate-free. Studies are proposed to elucidate whether disruption of this normal CYP2E1 turnover results in increased P450 transport to the outer plasma membrane, whereupon it is recognized by the immune surveillance system, engendering pathogenic P450 autoantibodies clinically associated with alcoholic liver disease, drug-induced hepatitis, and hypersensitivity syndromes. Because both UPD and ALD are highly conserved in the yeast Saccharomyces cerevisiae and mutant strains with genetic defects/deletions in these pathways are available, we propose to use yeast in addition to cultured rat and human hepatocytes. Our studies are focused on human liver CYP3A4 and CYP2E1 and their rat liver counterparts. Together these P450s comprise ~40% of total human hepatic P450 content but are responsible for the metabolism of ~65% of clinically prescribed drugs, toxins, and carcinogens, with consequently significant potential for DDIs and toxicity. More importantly, because UPD function is essential to the regulation of many vital biological processes, and ALD has essential homeostatic and biosynthetic functions, disruption of normal UPD or ALD pathways can lead to various human diseases. Our proposed studies on P450s as their prototype ER protein substrates will considerably advance our understanding of the molecular mechanisms and the sanitation patrols involved in these vital processes. HUMAN HEALTH RELEVANCE: Hepatic cytochromes P450 (P450s) are enzymes engaged in the breakdown of drugs, carcinogens, toxins, natural and chemical agents to water-soluble products. Exposure to these agents can increase liver P450 content or reduce it by enhancing protein degradation and this drug-mediated modulation of P450 content can significantly influence clinical drug-drug interactions. Indeed, clinically relevant drug-drug interactions occur due to altered P450 turnover elicited by drug-mediated P450 stabilization (i.e. alcohol/ethanol) or enhanced drug-mediated P450 degradation (i.e. grapefruit juice furanocoumarins). Our studies propose to use mammalian systems and yeast as models for elucidating the mechanisms of degradation of CYP3A4, the major human liver and intestinal enzyme, and human CYP2E1, the P450 enzyme implicated in alcoholic liver disease. Together these P450s are responsible for the metabolism of ~65% of clinically relevant drugs, toxins, and carcinogens, with consequently significant potential for drug-drug interactions and toxicity.