Regulation of CYP Proteins by Reactive Nitrogen Species

活性氮对 CYP 蛋白的调节

基本信息

批准号：
7730011
负责人：
Edward Thomas Morgan
金额：
$ 32.85万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-02-01 至 2013-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7730011
关键词：
Acute Address Adverse reactions Amino Acids Area Attention Biological Assay Biology CYP2B1 gene CYP2B6 gene CYP3A4 gene Cell Line Cells Clinical Co-Immunoprecipitations Communicable Diseases Cyclic GMP-Dependent Protein Kinases Cysteine Cytochrome P450 Data Dexamethasone Disease Dose Down-Regulation Drug Interactions Drug Metabolic Detoxication Drug effect disorder Enzymes Funding Gel Genetic Transcription Hand Hepatic Hepatocyte Hexobarbital Human In Vitro Infection Inflammation Inflammatory Injection of therapeutic agent Interleukins Knowledge Laboratories Liver Mass Spectrum Analysis Messenger RNA Metabolism Methodology Modification Molecular Mutate Mutation Nitric Oxide Nitric Oxide Donors Nitric Oxide Synthase Nitroglycerin Nitrosation Patients Pharmaceutical Preparations Phenobarbital Physiological Production Proteins Proteomics Rattus Reactive Nitrogen Species Recruitment Activity Regulation Research Reverse Transcriptase Polymerase Chain Reaction Rodent Sampling Screening procedure Sodium Nitrite Stimulus System Therapeutic Agents Time Tyrosine Ubiquitin Ubiquitination Western Blotting Yeasts cDNA Library cytokine design drug clearance environmental chemical human CYP2B6 protein human NOS2A protein hypnotic in vivo inhibitor/antagonist multicatalytic endopeptidase complex mutant nitration novel oxidation protein degradation protein function public health relevance response sedative treatment effect ubiquitin-protein ligase yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): Many cytochrome P450 (P450) enzymes and their associated drug metabolizing activities are down- regulated in inflammatory and infectious disease states, resulting in adverse reactions due to impaired drug clearance. In order to predict drug-disease interactions, it is important to understand the mechanisms involved. The mechanisms of this down-regulation are only partially understood, and research to date has focused on P450 gene transcription. Our laboratory has identified a novel mode of post-transcriptional down-regulation of drug-metabolizing P450s. Elevated production of NO in rat hepatocytes caused by inflammatory stimuli elicits the stimulated degradation of CYP2B enzymes via the ubiquitin-proteasome system, and there is evidence that a similar mechanism exists in human hepatocytes. Preliminary data indicates that at least two other P450 proteins are also regulated by this mechanism. This application has 4 specific aims: 1) To elucidate the mechanism by which NO regulates rat and human CYP2B. This will involve studies of wild type and mutated CYP2B enzymes in various cell lines. 2) To characterize the molecular machinery for NO-dependent degradation of CYP2B. A yeast two-hybrid strategy will be used to identify the ubiquitin E3 ligase and other proteins that interact with CYP2B enzymes in a NO-dependent manner. 3) To study the down-regulation of rat hepatic CYP2B enzymes in vivo, and its consequences for drug-drug interactions. Rats will be administered NO donors and we will study the down-regulation of CYP2B enzymes in association with changes in sedative-hypnotic drug action; 4) To identify other proteins in rat hepatocytes that are targeted for degradation by physiologically-generated nitric oxide, using iTRAQ and other proteomic methodologies; These studies have implications not only for the function of CYP2B in metabolism of diverse therapeutic agents and in detoxification and bioactivation of environmental chemicals, but also for the toxicological, pharmacological, and physiological functions of other P450s that are regulated in the same way. They also have important implications for the broader field of NO and cytokine biology, since knowledge in the area of NO- and cytokine-stimulated protein degradation is limited. PUBLIC HEALTH RELEVANCE: People with ongoing infections or inflammatory diseases are more susceptible to the undesirable effects of drugs, because their livers cannot break down the drugs efficiently. This project will help us to understand what causes this change, and so allow us to predict what patients will need to have their drug doses adjusted to avoid this problem.

描述（由申请人提供）：许多细胞色素P450（P450）酶及其相关的药物代谢活性在炎症和传染病状态下受到调节，导致由于药物清除受损而导致不良反应。为了预测药物疾病的相互作用，了解所涉及的机制很重要。这种下调的机制仅被部分理解，迄今为止的研究集中在P450基因转录上。我们的实验室已经确定了一种新型的药物代谢P450的转录后下调模式。炎症刺激引起的NO产生NO的升高引起了CYP2B酶通过泛素 - 蛋白酶体系统的刺激降解，并且有证据表明人类肝细胞中存在类似的机制。初步数据表明，至少另外两种P450蛋白也受该机制调节。该应用具有4个特定目的：1）阐明没有调节大鼠和人CYP2B的机制。这将涉及各种细胞系中野生型和突变的CYP2B酶的研究。 2）表征分子机械，以无依赖性CYP2B降解。酵母双杂交策略将用于鉴定以无依赖性方式与CYP2B酶相互作用的泛素E3连接酶和其他蛋白质。 3）研究体内大鼠肝CYP2B酶的下调及其对药物相互作用的后果。将不给大鼠施用大鼠，我们将研究CYP2B酶的下调，以及镇静催眠药作用变化的变化； 4）使用ITRAQ和其他蛋白质组学方法论，在大鼠肝细胞中鉴定针对生理生成的一氧化氮降解的其他蛋白质；这些研究不仅对CYP2B在多种治疗剂的代谢以及环境化学物质的排毒和生物活化方面的作用有影响，还对其他P450的毒理学，药理和生理功能也具有同一方式调节的其他P450的毒理学，药理和生理功能。它们对NO和细胞因子生物学的更广泛领域也具有重要意义，因为在NO和细胞因子刺激的蛋白质降解领域的知识受到限制。公共卫生相关性：持续感染或炎症性疾病的人更容易受到药物不良影响的影响，因为他们的肝脏无法有效地分解药物。该项目将帮助我们了解导致这种变化的原因，因此让我们预测患者需要调整药物剂量以避免此问题。