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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。肝微粒体血红蛋白细胞色素P450 （P450）包括多种组成和诱导酶。这些单体血红蛋白（MW @ 50kDa）含有一个假血红素（铁原卟啉IX）片段/摩尔酶。尽管它们的血红素部分相同，但p450在功能上不同，这是由个体血红素-细胞色素微环境赋予的特性。p450在各种生理相关的内源性和外源性的氧化/还原代谢中起着重要作用。然而，尽管所有这些反应都会产生易于排泄的产物，但并非所有的反应都是有益的。p450催化一些底物代谢为自由基和其他可引起毒性/病理损伤的活性物质。此外，在某些氧化还原反应过程中，参与的P450在一个被归类为基于机制或“自杀”失活的过程中被牺牲。迄今为止，有三种不同的底物介导的P450失活机制被表征：(a)通过n -烷基/芳基化[即烯丙基异丙酰胺（AIA），索巴比塔（SB）]破坏假体血红素；(b)由反应中间体（氯霉素，SB， 11-十一烯酸）制备的细胞色素烷基化；(c)假体血红素被破坏成与细胞色素不可逆结合的产物[CCl4，螺内酯（SPL）， 3,5-二乙氧基-2,6-二甲基-4-乙基-1,4-二氢吡啶（DDEP）]。根据定义，“自杀式”失活发生在活性部位。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 -烷基化的这一标准。然而，“自杀失活”的标准并没有严格应用于药物诱导的P450破坏模式b或c，因为高度疏水的apoP450活性位点区域的结构分析和它们对蛋白水解消化（与一系列蛋白酶）的抗性迄今为止在很大程度上排除了它们明确的机制分类。利用赖氨酸内肽酶C和/或胃蛋白酶消化以及CNBr裂解，分离了两种不同的由SB烷基化的P450肽，并利用ESMS和MSLDI-MS对其进行了表征。这种结构表征有助于失活过程的明确机制阐明。P450假体血红素被破坏成碎片，在体内不可逆地修饰其活性位点的蛋白质，引发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，他们集中在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。利用高效液相色谱-肽图谱和MS核心设施目前提供的大量质谱方法，研究正在与博士合作进行。Burlingame， Medzihradszky和Maltby对血红素修饰物种进行了表征，并确定了被修饰的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，这提出了一些有趣的问题。虽然泛素化和降解是相关的，但尚不清楚这些事件是否需要磷酸化。因此，另一个目标是确定磷酸化在P450降解中的作用，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鉴定激酶的选择性抑制剂作为探针。因此，结合生化/免疫学方法，质谱方法将用于特异性阐明P450蛋白磷酸化位点及其性质。拟议的研究集中在P450生物学的生理相关但被忽视的方面。由于p450是内质网膜蛋白，阐明其周转将为其他内质网居民提供生物学原型。此外，这些研究主要集中在P450 3A4，主要的人类肝脏和肠道酶及其大鼠同源物，它负责超过60%的临床处方药的代谢，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. 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.