REGULATION OF LIVER HEME METABOLISM AND CYTOCHROME P-450

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 肝微粒体血红素蛋白细胞色素P450（P450）包括多种组成型和诱导型酶，每摩尔酶含有一个血红素（铁-原卟啉IX）部分。P450在各种生理学相关的内源性和外源性物质的氧化/还原代谢中起作用。在某些氧化还原反应的过程中，参与的P450在分类为基于机制或“自杀”失活的过程中被牺牲。一个这样的过程涉及P450假体血红素破坏成片段，不可逆地修改其蛋白质在其活性位点在体内，从而触发P450蛋白质降解。我们研究的长期目标集中在P450蛋白的血红素修饰使其易于降解的假设上。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的机制。使用HPLC-肽图谱和MS核心设施目前提供的大量质谱方法，正在与Burlingame，Medzihradszky和Maltby博士合作进行研究，以表征血红素修饰物种并确定修饰的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 3A 4蛋白被磷酸化，泛素化和降解的胞质26 S蛋白酶体，但精确的蛋白质参与这一过程，如泛素E3连接酶和伴侣蛋白（p97，Hsps 90/70）的作用仍有待确定。 虽然泛素化和降解是相关的，但还不清楚磷酸化是否是这些事件所必需的。 因此，另一个目的是通过表征所涉及的细胞激酶、磷酸化的蛋白质位点和使用所鉴定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，计划将生物化学/免疫学和交联/蛋白质组学方法相结合来鉴定这些蛋白质。 类似地，质谱方法将用于具体阐明P450蛋白磷酸化的位点，从而阐明其性质。 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是内质网膜蛋白的组成部分，可作为其他内质网居民ERAD的模型。 此外，这些研究集中在P450 3A 4，主要的人类肝脏和肠道酶及其大鼠直系同源物，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。

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.