REGULATION OF LIVER HEME METABOLISM AND CYTOCHROME P-450 INACTIVATION/HEPATIC D

肝脏血红素代谢和细胞色素 P-450 失活/肝 D 的调节

• 批准号: 7369058
• 负责人: Maria Almira Correia
• 金额: $ 0.81万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7369058
• 关键词: REGULATION; LIVER; HEME; METABOLISM; CYTOCHROME

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. The hepatic microsomal hemoproteins cytochromes P450 (P450) include multiple constitutive and inducible enzymes. These monomeric hemoproteins (MW @ 50kDa) contain one prosthetic heme (iron-protoporphyrin IX) moiety/mole of enzyme. In spite of their identical heme moieties, P450s differ functionally, a property conferred by individual heme-apocytochrome microenvironments. P450s are instrumental in the oxidative/reductive metabolism of various physiologically relevant endobiotics and xenobiotics. However, although all these reactions result in the formation of readily excretable products, not all are beneficial. P450s catalyze the metabolism of some substrates to radicals and other reactive species that can induce toxicity/pathological damage. Furthermore, in the course of certain redox reactions, the participating P450 is sacrificed in a process classified as a mechanism-based or "suicide" inactivation. To date, three distinct mechanisms of substrate-mediated P450 inactivation have been characterized: (a) prosthetic heme destruction via N-alkyl/arylation [i.e., allylisopropylacetamide (AIA), secobarbita (SB)]; (b) apocytochrome alkylatin by a reactive intermediate (chloramphenicol, SB, 11-undecynoic acid); and (c) destruction of the prosthetic heme to products that irreversibly bind to the apocytochrome [CCl4,spironolactone (SPL), 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP)]. By definition, "suicide" inactivations occur at the active site. Isolation and structural characterization of the N-alkylated heme has unequivocally established this criterion for N-alkylation of P450 heme. However, the criterion for "suicide inactivation" has not been rigorously applied to modes b or c of drug-induced P450 destruction because the inaccessibility to structural analyses of the highly hydrophobic apoP450 active site regions and their resistance to proteolytic digestion (with an array of proteases) have until now largely precluded their definitive mechanistic classification. Using lysyl endopeptidase C and/or pepsin digestion as well as CNBr cleavage, two different P450 peptides alkylated by SB have been isolated and characterized, using ESMS and MSLDI-MS. Such structural characterization has contributed to the definitive mechanistic elucidation of inactivation process. Destruction of P450 prosthetic heme into fragments that irreversibly modify its protein at its active site in vivo, triggers P450 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, raising some intriguing questions. 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, in combination with biochemical/immunological approaches, 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. Because P450s are integral ER-membrane proteins, elucidation of its turnover will provide a biological prototype for 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NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。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。这些单体血红素蛋白（Mff@50kDa）含有一个人工血红素（铁-原卟啉IX）部分/摩尔酶。尽管它们具有相同的血红素部分，但P450在功能上不同，这是由个体血红素-脱辅基细胞色素微环境赋予的特性。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。然而，尽管所有这些反应导致形成易于排泄的产物，但并非所有反应都是有益的。P450催化某些底物代谢为自由基和其他可诱导毒性/病理损伤的活性物质。此外，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。迄今为止，已经表征了底物介导的P450失活的三种不同机制：（a）通过N-烷基/芳基化的血红素修复破坏[即，烯丙基异丙基乙酰胺（AIA），secobarbita（SB）];（B）通过反应性中间体（氯霉素，SB，11-十一碳炔酸）使脱辅基细胞色素烷基化;和（c）将辅基血红素破坏成与脱辅基细胞色素不可逆结合的产物[CCl 4，螺内酯（SPL），3，5-二乙氧羰基-2，6-二甲基-4-乙基-1，4-二氢吡啶（DDEP）]。根据定义，"自杀"失活发生在活性位点。N-烷基化血红素的分离和结构表征明确地建立了P450血红素N-烷基化的标准。然而，"自杀失活"的标准还没有严格地应用于药物诱导的P450破坏的模式B或C，因为高度疏水性apoP450活性位点区域的结构分析的不可接近性和它们对蛋白水解消化（用一系列蛋白酶）的抗性迄今为止在很大程度上排除了它们的确定的机制分类。使用赖氨酰内肽酶C和/或胃蛋白酶消化以及溴化氰裂解，两种不同的P450肽烷基化SB已被分离和表征，使用ESMS和MSLDI-MS。这种结构表征有助于确定的机制阐明的失活过程。 P450假体血红素破坏成片段，在其体内的活性位点不可逆地修饰其蛋白质，触发P450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 3A4蛋白被磷酸化，泛素化和降解的胞质26 S蛋白酶体，提出了一些有趣的问题。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，结合生物化学/免疫学方法，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。由于P450是整合ER膜蛋白，阐明其营业额将提供其他ER居民的生物原型。此外，这些研究集中在P450 3A4，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。