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：
8650281
负责人：
Maria Almira Correia
金额：
$ 55.31万
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 2017-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8650281
关键词：
ATP phosphohydrolase Abdomen Ablation Accounting Acute Affect Amino Acids Apoptosis Autophagocytosis Binding Biochemical Bone Marrow Carcinogens Catabolism Cells Chemicals Clinical Complement Factor B Coupled Cytochrome P450 Cytochromes Defect Degradation Pathway Diet Drug Prescriptions Enzymes Etiology Event Fasting Ferritin Genes Genetic 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 public health relevance 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 eIF2? kinase, an exquisite heme-sensor. The ensuing increased eIF2? 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 I?B? (a NF?B-inhibitor) and consequent nuclear activation of NF?B, 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 eIF2? kinases is associated with NF?B-mediated autophagy, thereby indicating that hepatic HRI activation could similarly induce P450 ALD. The specific role of hepatic HRI in NF?B 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（由申请人提供）：急性肝血红素缺乏是肝卟啉症急性临床发作的生化标志，肝卟啉症是由血红素合成遗传缺陷引起的遗传性疾病。在有遗传倾向的个体中，这种急性发作通常是由某些处方药、饥饿、感染和/或炎症引发的。抑制大鼠或小鼠肝细胞中的血红素合成也会导致急性血红素耗竭，并随后导致肝血红素调节抑制剂 HRI eIF2 的自动激活？激酶，一种精致的血红素传感器。随后的 eIF2 增加？磷酸化导致大量肝蛋白从头合成的整体翻译停滞，包括细胞色素 P450 (P450s)、参与内源性和外源性物质（如药物、致癌物、毒素、天然和化学产品）分解和清除的血红素蛋白酶。这种肝血红素耗竭还会导致肝 I?B? 合成的翻译停滞。（一种 NF?B 抑制剂）以及随后的 NF?B 核激活，NF?B 是一种参与炎症、癌症、自噬凋亡和免疫的促炎因子。同时，现有的和寿命较长的肝脏 P450 的自噬溶酶体降解 (ALD) 也以血红素可逆的方式迅速加速。激活其他三个eIF2？激酶与 NFκB 介导的自噬相关，从而表明肝脏 HRI 激活可以类似地诱导 P450 ALD。肝脏 HRI 在 NFκB 激活和随后的 P450 ALD 中的具体作用仍有待确定，并将在基因 HRI 敲除 (Hri-/-) 相对于野生型 (Hri+/+) 对照小鼠的肝细胞中进行检查。除了将 P450 ALD 视为缓慢的批量过程之外，人们对它的机理知之甚少。因此，建议进行研究来表征正常 P450 ALD 途径中运作的分子决定因素和参与者，并阐明那些在肝血红素耗尽时加速 P450 降解的因素。此类阐明将依赖于定点诱变、慢病毒 shRNA 干扰、免疫亲和捕获和蛋白质组分析等方法。肝脏是身体其他部位蛋白质、脂质和营养物质的主要供应者，因此从头合成的翻译停滞加上肝蛋白质 ALD 的增强必然会产生深远的生理/病理生理后果。我们的初步蛋白质组学分析表明，在肝血红素耗尽的几个小时内，包括 P450 在内的几种肝蛋白减少了 2 倍以上，而少数实际上增加了 2 倍以上。提出了在 Hri-/- 和 Hri+/+ 肝细胞中使用 SILAC（培养物中氨基酸的稳定同位素标记）与蛋白质组学相结合的研究，以鉴定和量化其合成和/或降解发生显着改变的肝蛋白质。急性血红素耗尽和 HRI 激活时其周转率显着改变的特定肝蛋白可能有助于深入了解急性肝卟啉症的症状和表现。此外，肝脏 P450 周转率的这种整体变化也可以合理解释临床上在卟啉症患者中观察到的药物清除受损的情况。