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（由申请人提供）：急性肝血红素缺乏是肝卟啉症急性发作的生化标志，肝卟啉症是由血红素合成遗传缺陷引起的遗传性疾病，在遗传易感个体中，这种急性发作通常由某些处方药、饥饿引发。、感染和/或炎症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和随后的肝脏自身激活。血红素调节抑制剂 HRI eIF2α 激酶，一种精致的血红素传感器，随后 eIF2α 磷酸化的增加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)、参与内切酶分解和清除的血红素蛋白酶。 - 以及药物、致癌物、毒素、天然和化学产品等异生物质也会导致肝血红素耗竭。肝脏 IκBα（一种 NFκB 抑制剂）合成的翻译停滞以及随之而来的 NFκB 的核激活，NFκB 是一种参与炎症、癌症、自噬凋亡和免疫的促炎因子，同时，现有的和其他寿命较长的自噬溶酶体降解 (ALD)。肝脏 P450 也以血红素可逆的方式迅速加速，与其他三种 eIF2α 激酶的激活相关。 NFκB 介导的自噬，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 敲除小鼠 (Hri-/) 的肝细胞中进行检查。 -) 相对于野生型 (Hri+/+) 对照，除了将其识别为 P450 ALD 外，人们对 P450 ALD 的机制知之甚少。因此，建议进行研究来表征正常 P450 ALD 途径中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，这种解释将依赖于定点诱变、慢病毒等方法。 shRNA 干扰、免疫亲和捕获和蛋白质组学分析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，因此可以阻止 de 的翻译。新合成加上肝蛋白 ALD 的增强必然会产生深远的生理/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。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）进行研究，实际上很少有增加超过 2 倍的研究。利用蛋白质组学来识别和定量合成和/或降解发生显着改变的特定肝蛋白，其周转率在急性血红素耗尽和 HRI 激活时显着改变，可以深入了解急性肝卟啉症的症状和表现。肝脏 P450 周转率的这种整体变化也可以使临床上在卟啉症患者中观察到的药物清除受损合理化。