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

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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.