REGULATION OF LIVER HEME METABOLISM AND CYTOCHROME P-450

肝脏血红素代谢和细胞色素 P-450 的调节

基本信息

批准号：
7724178
负责人：
Maria Almira Correia
金额：
$ 0.78万
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：
2008
资助国家：
美国
起止时间：
2008-06-01 至 2009-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7724178
关键词：
26S proteasome Active Sites Amino Acids Biochemical Biological Biology Collaborations Computer Retrieval of Information on Scientific Projects Database Core Facility Cytochrome P450 Cytochromes Drug Prescriptions Endoplasmic Reticulum Degradation Pathway Enzymes Event Funding Goals Grant Heme Heme Iron Hemeproteins Hepatic Human Institution Intestines Liver Melanocytic nevus Membrane Proteins Metabolism Modeling Modification Mole the mammal Molecular Chaperones Nature Orthologous Gene Oxidation-Reduction Peptide Mapping Phosphorylation Phosphotransferases Process Prosthesis Proteins Proteomics Rattus Reaction Regulation Research Research Personnel Resources Role Site Source Ubiquitination United States National Institutes of Health Xenobiotics base crosslink in vivo inhibitor/antagonist iron protoporphyrin IX neglect protein degradation suicide inactivation ubiquitin-protein ligase

项目摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Hepatic microsomal hemoproteins cytochromes P450 (P450s) include multiple constitutive and inducible enzymes, containing one prosthetic heme (iron-protoporphyrin IX) moiety/mole of enzyme. P450s are instrumental in the oxidative/reductive metabolism of various physiologically relevant endobiotics and xenobiotics. In the course of certain redox reactions, the participating P450 is sacrificed in a process classified as a mechanism-based or "suicide" inactivation. One such process involves destruction of P450 prosthetic heme into fragments that irreversibly modify its protein at its active site in vivo, thereby triggering P450 protein degradation. The long-term goals of our research are centered on the hypothesis that heme-modification of the P450 protein predisposes it for degradation. Thus, they have focussed on structural characterization of the heme-modified protein, and elucidation of the mechanism of its proteolytic degradation. Using HPLC-peptide mapping and the vast array of mass spectrometric approaches currently made available by the MS Core Facility, studies are in progress in collaboration with Drs. Burlingame, Medzihradszky and Maltby to characterize the heme-modifying species and identify the precise amino acid residue of the P450 protein that is modified. Furthermore, findings to date reveal that the heme-modified P450 3A4 protein is phosphorylated, ubiquitinated and degraded by the cytosolic 26S proteasome, but the precise proteins involved in this process such as the ubiquitin E3 ligase and the role of chaperones (p97, Hsps90/70) remain to be identified. While the ubiquitination and degradation are related, it is unclear whether phosphorylation is necessary for these events. Thus, another objective is to determine the role of phosphorylation in P450 degradation by characterizing the cellular kinases involved, the protein sites phosphorylated and the use of selective inhibitors of the identified kinases as probes. Accordingly, a combination of biochemical/immunological and cross-linking/proteomic approaches are planned to identify these proteins. Similarly, mass spectrometric approaches will be used to specifically elucidate the sites of P450 protein phosphorylation and consequently, its nature. The proposed studies center on a physiologically relevant but neglected aspect of P450 biology. P450s are integral ER-membrane proteins and serve as models for ERAD of other ER-residents. Furthermore, these studies are focussed on P450 3A4, the major human liver and intestinal enzyme and its rat orthologs, which are responsible for the metabolism of over 60% of clinically prescribed drugs, and are particularly susceptible to this biological fate.

该子项目是利用该技术的众多研究子项目之一资源由 NIH/NCRR 资助的中心拨款提供。子项目及研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金，因此可以在其他 CRISP 条目中表示。列出的机构是对于中心来说，它不一定是研究者的机构。肝微粒体血红素蛋白细胞色素 P450 (P450s) 包括多种组成型和诱导型酶，含有一个假体血红素（铁原卟啉 IX）部分/摩尔酶。 P450 在各种生理相关内生素和异生素的氧化/还原代谢中发挥着重要作用。在某些氧化还原反应过程中，参与的 P450 在基于机制或“自杀”失活的过程中被牺牲。其中一种过程涉及将 P450 假体血红素破坏成片段，从而在体内的活性位点不可逆地修饰其蛋白质，从而触发 P450 蛋白质降解。我们研究的长期目标集中在这样的假设上：P450 蛋白的血红素修饰使其易于降解。因此，他们专注于血红素修饰蛋白的结构表征，并阐明其蛋白水解降解机制。使用 HPLC 肽图谱和 MS 核心设施当前提供的大量质谱方法，与 Drs. 合作正在进行研究。 Burlingame、Medzihradszky 和 Maltby 描述了血红素修饰物种的特征，并鉴定了修饰的 P450 蛋白的精确氨基酸残基。此外，迄今为止的研究结果表明，血红素修饰的 P450 3A4 蛋白被胞质 26S 蛋白酶体磷酸化、泛素化和降解，但参与此过程的精确蛋白如泛素 E3 连接酶和伴侣蛋白（p97、Hsps90/ 70）仍有待确定。虽然泛素化和降解是相关的，但尚不清楚磷酸化是否是这些事件所必需的。因此，另一个目标是通过表征所涉及的细胞激酶、磷酸化的蛋白质位点以及使用已识别激酶的选择性抑制剂作为探针来确定磷酸化在 P450 降解中的作用。因此，计划结合生化/免疫学和交联/蛋白质组学方法来鉴定这些蛋白质。同样，质谱方法将用于专门阐明 P450 蛋白磷酸化的位点及其性质。拟议的研究集中在 P450 生物学的生理相关但被忽视的方面。 P450 是完整的 ER 膜蛋白，可作为其他 ER 驻留蛋白的 ERAD 模型。此外，这些研究主要集中在 P450 3A4，这是一种主要的人类肝脏和肠道酶及其大鼠直向同源物，它们负责超过 60% 的临床处方药物的代谢，并且特别容易受到这种生物学命运的影响。