REGULATION OF LIVER CYTOCHROME P450 TURNOVER/HEPATIC DEGRADATION OF P450 ENZYMES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The hepatic microsomal hemoproteins cytochromes P450 (P450) include multiple constitutive and inducible enzymes. that are instrumental in the oxidative/reductive metabolism of various physiologically relevant endobiotics and xenobiotics. These substrates can induce the content of various P450s via transcriptional/translational mechanisms but also via protein stabilization i.e. inhibition of its proteolytic degradation. This degradation proceeds via ubiquitin-dependent proteasomal degradation (UPD), autophagic-lysosomal degradation (ALD) or in some cases by both pathways. The long-term goals of our research are centered on the characterization of these processes at the molecular level: the degrons and posttranslational modifications that mark these proteins for cellular disposal and the molecular partners involved therein. Specifically, using mass spectrometric and/or proteomic analyses we wish to characterize the sites of P450 ubiquitination, the chaperones and factors involved in their degradation. Studies are in progress in collaboration with Drs. Burlingame, Medzihradszky and Maltby to identify the precise amino acid residue of the P450 protein that is ubiquitinated. In parallel our studies also focus on the role of heme in regulating the synthesis and turnover of hepatic P450s. 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.