Influence of Heme on Hepatic Cytochrome P450 Turnover

血红素对肝细胞色素 P450 周转的影响

基本信息

批准号：
8826728
负责人：
Maria Almira Correia
金额：
$ 55.53万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
1980
资助国家：
美国
起止时间：
1980-07-01 至 2016-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8826728
关键词：
ATP phosphohydrolase Abdomen Ablation Accounting Acute Affect Amino Acids Apoptosis Autophagocytosis Binding Biochemical Bone Marrow Carcinogens Catabolism Cells Chemicals Clinical Coupled Cytochrome P450 Cytochromes Defect Degradation Pathway Diet Drug Prescriptions Enzymes Etiology Event Fasting Ferritin Genes Genetic Health Heme Hemeproteins Hemoglobin Hepatic Hepatic Porphyrias Hepatocyte Hereditary Disease Homeostasis Hour Human Immune Immunity Immunoblotting Inborn Genetic Diseases Individual Infection Inflammation Inflammatory Infusion procedures Inherited Iron Knock-out Knockout Mice Life Lipids Liver Malignant Neoplasms Mediating Mitochondria Molecular Molecular Chaperones Mono-S Mus Myoglobin Neurologic Neurons Neurotransmitters Nitric Oxide Synthase Nuclear Nutrient Oxidative Phosphorylation Participant Pathway interactions Patients Pharmaceutical Preparations Phosphorylation Phosphotransferases Physiological Play Polyubiquitination Post-Translational Protein Processing Precipitation Process Production Prosthesis Protein Biosynthesis Proteins Proteomics RNA Interference Rattus Reaction Relative (related person)Reporting Rest Role Signal Pathway Signal Transduction Site-Directed Mutagenesis Sorting - Cell Movement Specificity Stable Isotope Labeling Starvation Stress Symptoms Toxin Tryptophan Tryptophanase Ubiquitin Virus Diseases Wild Type Mouse Xenobiotics adapter protein catalase cell growth drug clearance endoplasmic reticulum stress heme a heme biosynthesis inhibitor/antagonist insight neurotransmission response sensor small hairpin RNA stem synthetic enzyme transcription factor

项目摘要

DESCRIPTION (provided by applicant): Acute hepatic heme deficiency is a biochemical hallmark of acute clinical attacks of hepatic porphyrias, genetic disorders stemming from inherited defects in heme synthesis. In genetically predisposed individuals, such acute attacks are commonly triggered by some prescription drugs, starvation, infections and/or inflammation. Inhibition of heme synthesis in rat or mouse hepatocytes also results in acute heme depletion and consequent autoactivation of hepatic heme-regulated inhibitor HRI eIF2α kinase, an exquisite heme-sensor. The ensuing increased eIF2α phosphorylation causes global translational arrest of de novo synthesis of a myriad of hepatic proteins including cytochromes P450 (P450s), hemoprotein enzymes engaged in the breakdown and clearance of endo- and xenobiotics such as drugs, carcinogens, toxins, natural and chemical products. Such hepatic heme depletion also results in the translationally arrested synthesis of hepatic IκBα (a NFκB-inhibitor) and consequent nuclear activation of NFκB, a proinflammatory factor involved in inflammation, cancer, autophagy apoptosis and immunity. Concurrently, autophagic-lysosomal degradation (ALD) of existing and otherwise longer-lived hepatic P450s is also rapidly accelerated, in a heme-reversible manner. Activation of the other three eIF2α kinases is associated with NFκB-mediated autophagy, thereby indicating that hepatic HRI activation could similarly induce P450 ALD. The specific role of hepatic HRI in NFκB activation and consequent P450 ALD remains to be established and will be examined in hepatocytes from mice with genetic HRI knockout (Hri-/-) relative to wild type (Hri+/+) controls. Relatively little is mechanistically known about P450 ALD other than its recognition as a slow bulk process. Studies are therefore proposed to characterize the molecular determinants and participants operating in normal P450 ALD pathway, and to elucidate those responsible for accelerating P450 degradation upon hepatic heme depletion. Such elucidation will rely on approaches such as site-directed mutagenesis, lentiviral shRNA interference, immunoaffinity capture and proteomic analyses. The liver is a major supplier of proteins, lipids and nutrients to the rest of the body, and thus translational arrest of de novo synthesis coupled with enhanced ALD of hepatic proteins is bound to have far-reaching physiological/ pathophysiological consequences. Our preliminary proteomic analyses reveal that within a few hours of hepatic heme depletion, several hepatic proteins including P450s are decreased >2-fold, while a few are actually increased >2-fold. Studies are proposed in Hri-/- and Hri+/+ hepatocytes using SILAC (stable isotope labeling of amino acids in culture) coupled with proteomics to identify and quantify the hepatic proteins whose synthesis and/or degradation are so dramatically altered. The specific hepatic proteins whose turnover is markedly altered upon acute heme depletion and HRI activation may provide insight into the symptomatology and manifestations of acute hepatic porphyria. Moreover, such global alterations of hepatic P450 turnover would also rationalize the impaired drug clearance clinically observed in porphyric patients.

描述（由适用提供）：急性血红素缺乏症是卟啉炎急性临床攻击的生化标志，是由于血红素合成中遗传缺陷而引起的遗传疾病。在遗传易感的个体中，这种急性发作通常是由某些处方药，饥饿，感染和/或感染引起的。大鼠或小鼠肝细胞中血红素合成的抑制作用还会导致急性血红素部署，从而自动激活肝炎血红素调节的抑制剂HRIEIF2α激酶，这是一种独特的血红素发射器。确保增加的EIF2α磷酸化会导致多种肝蛋白的从头综合的全球翻译停滞，包括细胞色素P450（P450），血op蛋白酶，参与衰老和清除内生物，诸如药物，癌细胞和化学产品，造成毒素，天然和化学产品，天然和化学产品。耗竭还导致肝IκBα（NFκB抑制剂）翻译的合成，并导致NFκB的核激活，NFκB是一种参与炎症，癌症，自噬凋亡和免疫组织化学的促炎因子。同时，现有且寿命更长的肝脏P450的自噬 - 溶血体降解（ALD）也以血红素可逆的方式迅速加速。其他三种EIF2α激酶的激活与NFκB介导的自噬相关，从而表明肝HRI激活可能会影响P450 ALD。肝HRI在NFκB激活和随之而来的p450 ALD中的具体作用仍有待确定，并将在具有遗传HRI敲除（HRI - / - ）的小鼠的肝细胞中进行检查，相对于野生型（HRI+/+）对照。除了识别缓慢的过程外，对P450 ALD的机械知之甚少。因此，提出了研究以表征在正常P450 ALD途径中运行的分子确定剂和参与者，并阐明那些在肝血红素消耗后负责加速P450降解的人。这种阐明将依赖于诸如位置定向诱变，慢病毒shRNA干扰，免疫亲和力捕获和蛋白质组学分析等方法。肝脏是蛋白质，脂质和营养物质的主要供应商，因此翻译成从头合成的停滞以及增强的肝蛋白ALD势必会带来深远的生理/病理生理学后果。我们的初步蛋白质组学分析表明，在肝脏血红素部署的几个小时内，包括P450在内的几种肝蛋白降低了> 2倍，而实际上有少数人的增加> 2倍。在HRI - / - 和HRI+/+肝细胞中提出了研究（培养物中氨基酸的稳定同位素标记）以及蛋白质组学，以识别和量化其合成和/或降解的肝蛋白。急性血红素部署和HRI激活后，其营业额明显改变的特定肝蛋白可能会洞悉急性肝卟啉症的症状和表现。此外，这种肝P450的全球变化也将使斑岩患者临床观察到的药物清除受损合理化。