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

• 批准号: 7957375
• 负责人: Maria Almira Correia
• 金额: $ 1.35万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7957375
• 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; Drug Prescriptions; Endoplasmic Reticulum Degradation Pathway; Enzymes; Event; Funding; Goals; Grant; Heme; Heme Iron; Hemeproteins; Hepatic; Human; Institution; Intestines; Liver; Mass Spectrum Analysis; 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

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.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 肝微体血球蛋白细胞色素P450(P450)包括多种构成酶和诱导酶，含有一种人工血红素(铁原卟啉IX)部分/摩尔酶。P450在各种生理相关的内生和外生物质的氧化/还原代谢中起重要作用。在某些氧化还原反应的过程中，参与的P450在被归类为基于机制的或“自杀”失活的过程中被牺牲。其中一个过程涉及将P450假体血红素破坏成片段，在体内不可逆转地修改其活性部位的蛋白质，从而引发P450蛋白质降解。我们研究的长期目标集中在这样一个假设上，即P450蛋白的血红素修饰使其易于降解。因此，他们集中在血红素修饰的蛋白质的结构特征，以及阐明其蛋白质降解的机制。利用高效液相-多肽图谱和微软核心设备目前提供的大量质谱学方法，正在与Burlingame、Medzihradszky和Maltby博士合作进行研究，以确定血红素修饰物种的特征并识别被修饰的P450蛋白的精确氨基酸残基。此外，到目前为止的研究结果表明，血红素修饰的P450 3A4蛋白被胞浆26S蛋白酶体磷酸化、泛素化和降解，但参与这一过程的确切蛋白质，如泛素E3连接酶和伴侣蛋白(p97，Hsps90/70)的作用仍未确定。虽然泛素化和降解是相关的，但目前还不清楚这些事件是否需要磷酸化。因此，另一个目标是通过表征所涉及的细胞激酶、被磷酸化的蛋白位点以及使用已识别的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，计划采用生化/免疫学和交联/蛋白质组学相结合的方法来鉴定这些蛋白质。同样，质谱学方法将被用来专门阐明P450蛋白磷酸化的位置，从而阐明其性质。拟议的研究集中在生理上相关但被忽视的P450生物学方面。P450是完整的内质网膜蛋白，可作为其他内质网居住者ERAD的模型。此外，这些研究集中在P450 3A4，这是人类主要的肝脏和肠道酶及其大鼠的同源物，负责超过60%的临床处方药物的代谢，特别容易受到这种生物命运的影响。