Influence of Heme on Hepatic Cytochrome P450 Synthesis and Degradation

描述（由申请人提供）：肝血红蛋白细胞色素P450 （P450）是内质网（ER）锚定酶，参与分解内源性和外源性生物，如药物、致癌物质、毒素、天然和化学产品。暴露于这些物质后，肝脏P450含量可能因其血红素和蛋白质部分合成增加而增加，或因其失活/破坏和/或蛋白质水解降解而减少。已知这种药物介导的P450含量调节可显著影响临床药物-药物相互作用。由于P450的合成需要血红素，血红素合成缺陷，如遗传，急性血红素缺乏状态，临床称为肝卟啉症，可降低P450水平，从而损害摄入药物的代谢。我们的发现表明，严重的肝血红素消耗也可以通过关闭肝脏蛋白质的翻译，从而严重抑制肝脏蛋白质（如p450）的合成。这是由于？-真核翻译起始因子eIF2亚基被推测为肝血红素敏感的eIF2？激酶，当肝血红素严重耗尽时，其功能被释放。虽然这种肝激酶的身份一直难以确定，但我们从大鼠肝脏和培养的大鼠肝细胞中克隆了它的cDNA，表达、纯化和功能表征了这种酶，并证实了它的身份是eIF2？激酶。我们的第一个主要目标是(i)通过eIF2？-通过各种最先进的技术进行相互作用，如哺乳动物双杂交，化学交联/蛋白质组学分析，共免疫沉淀，以及其组织/肝细胞内定位；（ii）在小鼠中使用RNA干扰和靶向基因敲除来确定其在体内的生理和病理相关性。我们的目标是阐明可能损害关键生理过程的翻译抑制，从而不仅有助于急性肝性卟啉症的临床症状，而且还影响人类p450依赖的药物-药物相互作用。此外，现在越来越明显的是，临床相关的药物-药物相互作用也可以由药物介导的P450稳定和药物介导的P450降解（如葡萄柚呋喃香豆素）引起的P450转换改变引起。这种与内质网相关的p450降解包括它们的泛素化，从内质网中提取，随后由细胞质26S蛋白酶体进行蛋白水解。血红素导致泛素化p450的内质网积累，很可能是通过阻断它们的内质网提取和随后的降解。内质网的提取需要ATP水解，因此可能涉及p97 AAA ATP酶或蛋白酶体19S AAA ATP酶。因此，我们的第二个主要目标是以血红素为探针，表征p97和19S AAA atp酶在P450降解中的相对作用。这些研究有望阐明血红素如何影响这些P450蛋白的出生和死亡，从而调节人体摄入的药物和环境因子的作用和消除。公共卫生相关性：肝细胞色素P450 （P450）是参与将药物、致癌物、毒素、天然和化学制剂分解为水溶性产物的酶。暴露于这些药物可通过增强蛋白质降解来增加或减少肝脏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。我们的研究旨在阐明血红素在控制P450形成及其周转的细胞过程中的作用。p450和血红蛋白一样，是含血红素的蛋白质，它们在肝脏中的形成需要血红素。然而，急性肝血红素耗损，急性危及生命的肝卟啉症发作的标志（临床定义为血红素形成的遗传缺陷），也通过功能性激活正常血红素控制抑制剂(eIF2？激酶)的蛋白质合成，一个过程，我们希望进一步表征。此外，血红素还可以阻断P450的周转，我们建议对这一过程进行机械解剖和进一步表征。选择作为原型的p450是CYP3A4的同源物，CYP3A4是人类肝脏和肠道的主要酶，以及另外2种肝脏p450，它们共同负责约75%的临床相关药物、毒素和致癌物的代谢，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 engaged in the breakdown of endo- and xenobiotics such as drugs, carcinogens, toxins, natural and chemical products. On exposure to these agents, liver P450 content may be increased due to increased syntheses of its heme and protein moieties, 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. Because P450 synthesis requires heme, defective heme synthesis as in the genetically inherited, acute heme-deficient states clinically known as hepatic porphyrias, can lower P450 levels and thereby impair the metabolism of ingested drugs. Our finding indicates that severe hepatic heme depletion can also profoundly suppress the synthesis of hepatic proteins such as P450s, by shutting off their translation. This stems from increased phosphorylation of the ?-subunit of eukaryotic translational initiation factor eIF2 by a putative hepatic heme-sensitive eIF2? kinase, which is functionally unleashed when hepatic heme is severely depleted. Although the identity of this liver kinase had long remained elusive, we have cloned its cDNA from rat liver and cultured rat hepatocytes, expressed, purified, functionally characterized this enzyme and confirmed its identity as an eIF2? kinase. Our first major aim is to (i) establish its in vivo operation via eIF2?-interactions by various state-of-the-art techniques such as mammalian two-hybrid, chemical crosslinking/proteomic analyses, coimmunoprecipitation, as well as its tissue/intrahepatocellular localization; and (ii) use RNA interference and targeted gene knock out in mice to determine its in vivo physiological and pathological relevance. Our goal is to elucidate the translational suppression that may impair key physiological processes and thus contribute not only to the clinical symptoms of acute hepatic porphyrias, but also influence P450-dependent drug-drug interactions in man. Further, it is now increasingly evident that clinically relevant drug-drug interactions can also result from altered P450 turnover, elicited by drug-mediated P450 stabilization as well as enhanced drug-mediated P450 degradation, such as by the grapefruit furanocoumarins. Such ER-associated degradation of P450s entails their ubiquitylation, extraction from the ER and subsequent proteolysis by the cytosolic 26S proteasome. Heme results in an ER accumulation of ubiquitylated P450s, most likely by blocking their ER extraction and subsequent degradation. ER extraction requires ATP hydrolysis and thus could involve either the p97 AAA ATPase or the proteasomal 19S AAA ATPases. Thus, our second major goal is to characterize the relative roles of p97 and 19S AAA ATPases in this P450 degradation with heme as a probe. These studies are expected to elucidate how heme can affect the birth and death of these P450 proteins and thus modulate the effects and elimination of ingested drugs and environmental agents in man. PUBLIC HEALTH RELEVANCE: Liver 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. Our studies are aimed at elucidating the role of heme in the cellular processes that control P450 formation as well as their turnover. P450s, like blood hemoglobin, are heme-containing proteins, and their formation in the liver requires heme. However, acute liver heme depletion, a hallmark of acute life-threatening attacks of hepatic porphyrias (clinically defined by genetic defects in heme formation), also blocks P450 formation by functionally activating a normally heme-controlled inhibitor (eIF2? kinase) of protein synthesis, a process we wish to further characterize. Furthermore, heme can also block P450 turnover, a process we propose to mechanistically dissect and further characterize. The P450s chosen as prototypes are orthologs of CYP3A4, the major human liver and intestinal enzyme, and 2 other liver P450s that together are responsible for the metabolism of approximately 75% of clinically relevant drugs, toxins, and carcinogens, with consequently significant potential for drug-drug interactions and toxicity.