Regulation of Heme Metabolism in the Liver

描述：(改编自申请者摘要)：血红素起着举足轻重的作用 作为包括肝脏在内的重要细胞血红蛋白的修复体部分 细胞色素P450(Cyps)和色氨酸2，3-双加氧酶(Tdo)，研究 此应用程序的焦点。这些肝血球蛋白发挥着独特的功能 角色：Cyps致力于内生生物的合成/代谢/解毒 和外源生物(药物和毒素)，而TDO是关键的限速酶 控制的不可逆氧化L-色氨酸(L-色氨酸)的降解 L-色氨酸水平及其进入5-羟色胺能通路的研究。因为Cyps是 肝脏血红素的主要消耗者，CYP血红素破坏会导致急性血红素 枯竭，在遗传倾向的个体中，这会引发 临床症状称为急性肝性门静脉病。在这些 在个体中，这种血红素缺乏会通过剥夺细胞色素P450和细胞色素DO的功能来损害细胞色素P450和细胞色素DO 因此降低了异种生物的代谢潜力，并 增加L-色氨酸向神经递质5-羟色胺的转化可能是 造成这些血红素缺乏症的神经学症状。因此， 这些酶的正常生理功能严重依赖于 肝脏血红素的结构完整性状况。然而，我们的学习是为了 数据显示，血红素也在转录上调节这些酶， 翻译和/或翻译后，从而揭示了亚铁血红素的作用 它们的从头合成和/或降解。因此，研究是 建议通过研究其对血红素的调节来机械地描述这种调节 通过确定它在(I)TDO和(II)CYP2B转录中的确切作用 通过其基因启动子区域的特定“血红素反应元件”起作用 和/或依赖于血红素的转录因子；(Iii)翻译后 对自杀失活的细胞色素P450受体1的调控作用 促进蛋白质降解过程中对血红素敏感的磷酸化作用； (Iv)确定血红素是否也是CYP3A蛋白水解酶的触发物 ITS不可逆的翻译后血红素修饰的周转 蛋白。提出的研究需要各种最先进的方法(DNase I足迹、启动子缺失、凝胶迁移率和甲基化干扰 分析、定点突变和转铁蛋白共转染分析)以检查 转录激活的机制，以及大肠杆菌酶 表达和纯化、免疫沉淀、血红素分离和/或 用高效液相色谱-多肽图谱、氨基酸测序进行结构表征 和质谱学检测翻译后修饰 (磷酸化和血红素修饰)。据信， 总的来说，这些研究将提供对多功能的洞察，尽管 血红素在调节这些重要因素中的多重作用尚不清楚 肝脏血球蛋白

DESCRIPTION: (Adapted from the Applicant's Abstract): Heme plays a pivotal role as the prosthetic moiety of vital cellular hemoproteins that include hepatic cytochromes P450 (CYPs) and tryptophan 2,3-dioxygenase (TDO), the research focus of this application. These hepatic hemoproteins play distinct functional roles: CYPs are committed to synthesis/metabolism/detoxification of endobiotics and xenobiotics (drugs and toxins), whereas TDO is the key rate-limiting enzyme in the irreversible oxidative L-tryptophan (L-Trp) degradation that controls L-Trp levels and its flux into serotonergic pathways. Because CYPs are the major consumers of hepatic heme, CYP heme destruction results in acute heme depletion, and in genetically predisposed individuals this can trigger the clinical conditions known as the acute hepatic porphyrias. In these individuals, such heme depletion impairs both CYP and TDO function by depriving them of heme, with consequently reduced xenobiotic metabolizing potential and increased L-Trp conversion to the neurotransmitter serotonin that may be responsible for the neurologic symptoms of these heme-deficient states. Thus, normal physiologic function of these enzymes is critically dependent on the hepatic heme status for their structural integrity. However, our studies to date reveal that heme also regulates these enzymes transcriptionally, translationally and/or posttranslationally, thereby revealing a role for heme in their de novo synthesis and/or degradation. Accordingly, studies are proposed to mechanistically characterize this heme regulation by examining its precise role in (i) TDO and (ii) CYP2B transcription by determining whether it acts through specific "heme responsive elements" in their gene promoter regions and/or heme-dependent transcription factors (TFs); (iii) posttranslational regulation of suicidally inactivated CYP2C1 1 by characterizing the role of heme-sensitive phosphorylation in its enhanced proteolytic degradation; and (iv) to determine whether heme also serves as a trigger for CYP3A proteolytic turnover through irreversible posttranslational heme modification of its protein. The studies proposed entail various state-of-the-art approaches (DNAse I footprint, promoter deletion, gel-mobility, and methylation interference analyses, site-directed mutagenesis and TF cotransfection analyses) to examine the mechanics of transcriptional activation, as well as E. coli enzyme expression and purification, immunoprecipitation, heme isolation and/or structural characterization through HPLC-peptide mapping, amino acid sequencing and mass spectrometry for examination of the postranslational modification (phosphorylation and heme-modification) of CYPs. It is believed that collectively, these studies will provide insight into the versatile, albeit ill-understood multiple roles of heme in the regulation of these important liver hemoproteins