Hepatic degradation of cytochrome P450 enzymes

描述(申请人提供)：肝脏血球蛋白细胞色素P450(P450)是内质网(ER)锚定的酶，可分解内源和外源物质，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的含量可能由于形成增加而增加，或由于其失活/破坏和/或蛋白质降解而减少。这种药物介导的对P450含量的调节被认为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稳定化(即乙醇)引起的P450代谢的改变以及药物失活后的药物介导的P450的加速降解(例如西柚汁呋喃香豆素)。失活的、结构受损的P450在一个被称为内质网相关降解(ERAD)的过程中引起泛素化和随后的26S蛋白酶体降解(UPD)，导致它们加速细胞降解。虽然我们的初步体外研究已经确定gp78泛素连接酶是泛素化CYP3A4的酶，该酶是人类肝脏和肠道的主要P450，但它在体内的相关性尚不清楚。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CYP3A4 ERAD还涉及蛋白质磷酸化以及与胞浆伴侣蛋白的相互作用，我们的第二个特定目标是定义这些过程中每个过程的特定作用，并阐明它们在功能上是相关的还是相互依赖的。生理上，一些P450通过不同的途径以不同的半衰期(6-37h)进行异步化翻转。因此，乙醇代谢的CYP2E1在乙醇结合时会发生自噬-溶酶体降解(ALD)，而在无底物时会导致ERAD/UPD降解。有人建议进行研究，以阐明这种正常的CYP2E1转换是否会导致P450转运到外膜的增加，从而被免疫监测系统识别，从而产生临床上与酒精性肝病、药物性肝炎和超敏综合征相关的致病性P450自身抗体。由于UPD和ALD在酿酒酵母中都是高度保守的，并且在这些途径中存在遗传缺陷/缺失的突变株，我们建议除了使用培养的大鼠和人肝细胞外，还可以使用酵母。我们的研究主要集中在人肝细胞色素P3A4和细胞色素P450_2E_1及相应的大鼠肝细胞。这些P450总共占人类肝脏P450总含量的40%，但负责代谢~65%的临床处方药、毒素和致癌物，因此具有显著的DDIS和毒性。更重要的是，由于UPD功能对许多重要的生物过程的调节是必不可少的，而ALD具有必要的动态平衡和生物合成功能，正常的UPD或ALD途径的破坏可能导致各种人类疾病。我们提出的以P450作为其原型ER蛋白底物的研究将极大地促进我们对这些重要过程中涉及的分子机制和卫生巡逻的理解。与人类健康相关：肝脏细胞色素P450(P450)是一种酶，参与将药物、致癌物、毒素、天然和化学试剂分解为水溶性产品。暴露于这些药物可以增加或通过促进蛋白质降解来减少肝脏P450的含量，这种药物介导的P450含量的调节可以显著影响临床药物之间的相互作用。事实上，临床上相关的药物相互作用的发生是由于药物介导的P450稳定(即酒精/乙醇)或增强药物介导的P450降解(即西柚汁呋喃香豆素)引起的P450周转的改变。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人类肝脏和肠道的主要酶--细胞色素P3A4和与酒精性肝病有关的P450酶--人类细胞色素P450的降解机制。这些P450共同负责代谢~65%的临床相关药物、毒素和致癌物，因此具有显著的药物-药物相互作用和毒性潜力。

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.