Influence of Heme on Hepatic Cytochrome P450 Synthesis and Degradation

肝血红素蛋白细胞色素P450（P450 s）是内质网（ER）锚定的酶 从事分解内源性和外源性物质，如药物，致癌物质，毒素，天然和 化学产品.在暴露于这些药剂时，肝脏P450含量可能会增加，这是由于 其血红素和蛋白质部分的合成，或由于其失活/破坏和/或蛋白水解而减少 降解已知这种药物介导的P450含量调节显著影响临床应用。 药物相互作用因为P450的合成需要血红素，所以在P450的合成中存在血红素合成缺陷。 一种遗传性急性血红素缺乏状态，临床上称为肝卟啉症，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如P450的合成， 翻译.这是真核生物翻译起始的a亚基磷酸化增加的结果 因子eIF 2通过假定的肝血红素敏感性eIF 2a激酶，当肝血红素敏感性eIF 2a激酶在功能上释放时， 血红素严重耗尽虽然这种肝激酶的身份长期以来一直是难以捉摸的，我们有 从大鼠肝脏和培养的大鼠肝细胞中克隆了该酶，表达并纯化了它。 是（i）进一步表征该酶结构和功能;（ii）确认其作为eIF 2a的身份 激酶;（iii）通过各种现有技术，如哺乳动物细胞培养，建立其体内eIF 2a-相互作用。 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;和（iv）在小鼠中使用RNA干扰和靶向基因敲除， 确定其与肝蛋白和P450合成的体内相关性。我们的目标是阐明 翻译抑制可能会损害关键的生理过程，因此不仅有助于 急性肝卟啉症的临床症状，但也影响P450依赖性药物-药物相互作用， 伙计此外，现在越来越明显的是，临床相关的药物-药物相互作用也可由以下因素引起： 通过药物介导的P450稳定以及增强药物介导的P450引起的P450转换改变 降解，如葡萄柚呋喃香豆素。这种ER相关的P450降解需要 它们的泛素化，从ER中提取，随后通过胞质26 S蛋白酶体进行蛋白水解。 血红素导致泛素化P450的ER积累，最有可能是通过阻断它们的ER提取， 随后的退化。ER提取需要ATP水解，因此可能涉及p97 AAA ATP酶或蛋白酶体19 S AAA ATP酶。因此，我们的第二个主要目标是描述相对 p97和19 S AAA ATP酶在P450降解中的作用。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，从而调节细胞的增殖。 人体摄入的药物和环境因素的影响和消除。

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 syntheses of hepatic proteins such as P450s, by shutting off their translation. This results from increased phosphorylation of the a-subunit of eukaryotic translational initiation factor elF2 by a putative hepatic heme-sensitive elF2a kinase, that is functionally unleashed when hepatic heme is severely depleted. Although the identity of this liver kinase had long remained elusive, we have cloned this enzyme from rat liver and cultured rat hepatocytes, expressed and purified it. Our first major aim is to (i) structurally and functionally characterize this enzyme further; (ii) confirm its identity as an elF2a kinase; (iii) establish its in vivo elF2a-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 (iv) use RNA interference and targeted gene knock out in mice to determine its in vivo relevance to hepatic protein and P450 syntheses. 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 ubiquitination, extraction from the ER and subsequent proteolysis by the cytosolic 26S proteasome. Heme results in an ER accumulation of ubiquitinated 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.