Regulation of Heme Metabolism in the Liver

描述：（改编自申请人的摘要）：血红素起着关键作用， 作为重要细胞血红素蛋白的辅基，包括肝血红素蛋白， 细胞色素P450（CYP）和色氨酸2，3-双加氧酶（TDO），研究 这个应用程序的重点。这些肝血红素蛋白发挥独特的功能 作用：CYP致力于内源性物质的合成/代谢/解毒 和外源性物质（药物和毒素），而TDO是关键的限速酶 在不可逆的氧化L-色氨酸（L-Trp）降解中， L-Trp水平及其流入多巴胺能途径。因为CYP是 肝血红素的主要消耗者，血红素破坏导致急性血红素 消耗，在遗传易感的个体中，这可能会引发 被称为急性肝卟啉症的临床病症。在这些 个体，这种血红素耗竭通过剥夺 血红素，因此降低了异生素代谢潜力， 增加L-Trp转化为神经递质5-羟色胺， 导致这些血红素缺乏状态的神经症状。因此，在本发明中， 这些酶的正常生理功能严重依赖于 肝血红素状态以保证其结构完整性。然而，我们的研究， 数据显示，血红素也在转录上调节这些酶， 从理论上和/或后验上，从而揭示了血红素 在它们的从头合成和/或降解中。因此，研究 建议通过检查其血红素调节的机制来表征这种血红素调节。 通过确定它是否在（i）TDO和（ii）CYP 2B转录中发挥精确作用 通过其基因启动子区域中的特定“血红素反应元件”起作用 和/或血红素依赖性转录因子（TF）;（iii）翻译后 通过表征的作用调节自杀失活的CYP 2C 1 1 在其增强的蛋白水解降解中的血红素敏感性磷酸化;以及 (iv)以确定血红素是否也作为CYP 3A蛋白水解的触发剂， 通过不可逆的翻译后血红素修饰， 蛋白所提出的研究需要各种最先进的方法（DNA酶 I足迹、启动子缺失、凝胶迁移率和甲基化干扰 分析、定点诱变和TF共转染分析）以检查 转录激活机制，以及E.大肠杆菌酶 表达和纯化、免疫沉淀、血红素分离和/或 通过HPLC-肽图谱、氨基酸测序进行结构表征 和质谱分析来检测翻译后的修饰 （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力，尽管 血红素在这些重要的调节中的多种作用尚不清楚， 肝血红蛋白

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