Influence of Heme on Hepatic Cytochrome P450 Synthesis and Degradation

描述(申请人提供)：肝脏血色素P450(P450)是内质网(ER)锚定的酶，用于分解内源和外源生物，如药物、致癌物、毒素、天然和化学产品。在暴露于这些制剂时，肝脏P450的含量可能由于其血红素和蛋白质部分的合成增加而增加，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之间的相互作用。由于P450的合成需要血红素，在基因遗传的急性血红素缺乏状态下的血红素合成缺陷会降低P450水平，从而损害摄入药物的新陈代谢。我们的发现表明，严重的肝脏血红素枯竭也可以通过切断P450等肝脏蛋白质的翻译来深刻地抑制它们的合成。这是由于真核细胞翻译起始因子eIF2的？亚基被推测的肝脏血红素敏感eIF2？当肝脏中的血红素严重耗尽时，其功能就会被释放。尽管该酶的特性长期以来一直悬而未决，但我们已经从大鼠肝脏和培养的大鼠肝细胞中克隆了该酶的cDNA，并对其进行了表达、纯化和功能鉴定，证实其为eIF2？激活剂。我们的第一个主要目标是(I)通过eIF2相互作用，通过各种最先进的技术，如哺乳动物双杂交、化学交联/蛋白质组分析、免疫共沉淀以及其组织/肝细胞内定位，建立其体内操作；以及(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。我们的目标是阐明翻译抑制可能损害关键的生理过程，从而不仅导致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，临床相关的药物相互作用也可以由药物介导的P450稳定以及药物促进的药物介导的P450降解引起的改变的P450代谢引起，例如由葡萄柚呋喃香豆素引起的。P450的这种内质网相关的降解需要它们的泛素化，从内质网中提取，然后被胞浆中的26S蛋白酶体降解。血红素导致泛素化P450的内质网积累，很可能是通过阻止它们的内质网提取和随后的降解。内质网提取需要ATP水解，因此可能涉及p97AAA ATPase或蛋白酶体19S AAA ATPase。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 ATPase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，从而调节摄入的药物和环境制剂在人类体内的影响和消除。与公众健康相关：肝脏细胞色素P450(P450)是参与分解药物、致癌物质、毒素、天然和化学制剂为水溶性产品的酶。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。我们的研究旨在阐明血红素在控制P450形成和周转的细胞过程中的作用。和血液中的血红蛋白一样，P450也是含有血红素的蛋白质，它们在肝脏中的形成需要血红素。然而，急性肝铁血红素耗竭，这是急性威胁生命的肝门脉症(临床上由血红素形成中的遗传缺陷定义)的特征，也通过功能性地激活一种正常的血红素控制的抑制物(eIF2？蛋白质合成的一个过程，我们希望进一步描述这个过程。此外，血红素还可以阻止P450的转换，我们建议从机械上剖析和进一步描述这一过程。被选为原型的P450是人体主要肝脏和肠道酶CYP3A4的同源物，以及另外两个肝脏P450，它们共同负责大约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.