The hepatic microsomal hemoproteins cytochromes P450 (P450) are instrumental in the oxidative/reductive metabolism of various physiologically relevant endobiotics and xenobiotics (drugs, environmental toxins and carcinogens). Although all these reactions generate readily excretable products, not all are beneficial: Some substrates may be metabolized to radicals and other reactive species that can induce toxicity/pathological damage. In the course of certain redox reactions, the participating P450 is sacrificed in a process classified as a mechanism-based or """"""""suicide"""""""" inactivation. A novel form of this entails oxidative fragmentation of the prosthetic heme to yet uncharacterized products that alkylate/acylate the apoP450 and result in its rapid loss. The overall goals of this application thus center on our hypothesis that drug-mediated heme-alkylation of the P450s in the rat liver brands them for proteolytic disposal. They specifically build on our recent findings that the heme-alkylation occurs at the P450 active site, and that heme-alkylated P450s are rapidly proteolysed by a rat liver cytosolic ATP/ubiquitin (Ub)- dependent system. Using cumeme hydroperoxide (CuOOH) to suicidally inactivate P450s 3A1 and 2B1, two major heme-alkylated active-site apoP450 peptides have been isolated, sequenced and characterized by mass spectrometry (MS). Although the mass (19-49 units) of the alkylating heme fragments is known, their precise structural/chemical identification and determination of their attachment site have been hampered by the prohibitive mass and relative hydrophobicity of each peptide (45 and 33 amino acid residues, for 3A1 and 2B1, respectively). In continuation of these studies, proteolytic size-reduction of these alkylated peptides (and others isolated, but yet to be characterized) to fragments <2kDa that can be more readily analyzed by tandem MS is proposed. Furthermore, the studies on the ATP/Ub-dependent proteolytic degradation of the heme-alkylated apo450s 3A, will be extended by identifying the specific ubiquitination sites (epsilon-NH(2) of Lys- residues, or alpha-NH(2)?), and the mode of ubiquitination (multiubiquitination or polyubiquitination) of CuOOh-inactivated P450s 3A (and 2B1) as well as characterizing the rat liver enzymes (Ub protein ligase E3? multicatalytic protease complex? proteasome? or other hepatic ATP-dependent proteases) engaged in the ATP/Ub-dependent degradation of heme-alkylated P450s 3A. The proposed studies thus center on an important but relatively neglected aspect of P450 regulation and turnover. The P450s to be studied are the orthologs of the major P450 (3A4) in the human liver and intestine, known to metabolize an array of clinically useful drugs and carcinogens. At a molecular level heme-alkylated/ubiquitinated P450s provide an excellent prototype of ER-proteins that incur posttranslational modification before their degradation. Heme-alkylation thus apparently converts P450s to structurally aberrant proteins. Whether this is abnormal or a feature of their normal turnover is to be probed.
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