The hepatic endoplasmic reticulum (ER)-anchored hemoproteins, cytochromes P450 (P450s), are instrumental in the metabolism of physiologically relevant endobiotics and xenobiotics (clinically relevant drugs, environmental toxins and carcinogens). In the course of this metabolism, however, the participating enzyme is sacrificed in a mechanism-based """"""""suicide-inactivation"""""""". One form of this inactivation entails destruction of P450 prosthetic heme into fragments that irreversibly modify its protein at its active site. Such abnormal modification in vivo, triggers P450 degradation. The long-term goals of this proposal are centered on the hypothesis that heme-modification of the P450 protein predisposes it for degradation. Thus, they have centered on structural characterization of the heme-modified protein, and elucidation of the mechanism of its proteolytic degradation. Findings to date reveal that the heme-modified P450 3A4 protein is phosphorylated, ubiquitinated and degraded by the cytosolic 26S proteasome, raising some intriguing fundamental questions which are the subject of the present proposal: (i) How are the ER-bound P450 proteins delivered to the cytosolic proteasome? (ii) What precise sites (usually Lysepsilon-NH2) are required to be ubiquitinated for such P450 targeting to the 26S proteasome? (iii) Which particular ubiquitin conjugating enzymes are involved in this P450 targeting? And more importantly, is physiologically the native enzyme similarly disposed of as the heme-modified protein? While the ubiquitination and degradation are related, it is unclear whether phosphorylation is necessary for these events. Thus, the final aim (iv) 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. Immunological, morphological, site-directed mutagenesis, biochemical, protein chemistry and mass spectrometric approaches will be used in elucidating these issues, using intact freshly isolated rat hepatocytes, COS-7 cells, and S. cerevisiae as models. 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 focused on P450 3A4, the major human liver and intestinal enzyme and its rat orthologs, which are responsible for the metabolism of over 60 percent of clinically prescribed drugs, and are particularly susceptible to this biological fate. Finally, because the 26S proteasomal subunits are responsible for the generation of antigenic peptides, these studies may provide insight into the generation of P450 autoantibodies detected in sera of patients with chronic active and drug-induced hepatitis and hypersensitivity reactions.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM044037-09
Application #
6180429
Study Section
Special Emphasis Panel (ZRG1-ALTX-1 (01))
Project Start
1990-04-01
Project End
2003-03-31
Budget Start
2000-04-01
Budget End
2001-03-31
Support Year
9
Fiscal Year
2000
Total Cost
$197,299
Indirect Cost
Name
University of California San Francisco
Department
Pharmacology
Type
Schools of Medicine
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Kim, Sung-Mi; Wang, YongQiang; Nabavi, Noushin et al. (2016) Hepatic cytochromes P450: structural degrons and barcodes, posttranslational modifications and cellular adapters in the ERAD-endgame. Drug Metab Rev 48:405-33
Kim, Sung-Mi; Grenert, James P; Patterson, Cam et al. (2016) CHIP(-/-)-Mouse Liver: Adiponectin-AMPK-FOXO-Activation Overrides CYP2E1-Elicited JNK1-Activation, Delaying Onset of NASH: Therapeutic Implications. Sci Rep 6:29423
Lewis-Ballester, Ariel; Forouhar, Farhad; Kim, Sung-Mi et al. (2016) Molecular basis for catalysis and substrate-mediated cellular stabilization of human tryptophan 2,3-dioxygenase. Sci Rep 6:35169
Wang, YongQiang; Kim, Sung-Mi; Trnka, Michael J et al. (2015) Human liver cytochrome P450 3A4 ubiquitination: molecular recognition by UBC7-gp78 autocrine motility factor receptor and UbcH5a-CHIP-Hsc70-Hsp40 E2-E3 ubiquitin ligase complexes. J Biol Chem 290:3308-32
Correia, Maria Almira; Wang, YongQiang; Kim, Sung-Mi et al. (2014) Hepatic cytochrome P450 ubiquitination: conformational phosphodegrons for E2/E3 recognition? IUBMB Life 66:78-88
Wang, YongQiang; Guan, Shenheng; Acharya, Poulomi et al. (2012) Multisite phosphorylation of human liver cytochrome P450 3A4 enhances Its gp78- and CHIP-mediated ubiquitination: a pivotal role of its Ser-478 residue in the gp78-catalyzed reaction. Mol Cell Proteomics 11:M111.010132
Acharya, Poulomi; Liao, Mingxiang; Engel, Juan C et al. (2011) Liver cytochrome P450 3A endoplasmic reticulum-associated degradation: a major role for the p97 AAA ATPase in cytochrome P450 3A extraction into the cytosol. J Biol Chem 286:3815-28
Wang, YongQiang; Guan, Shenheng; Acharya, Poulomi et al. (2011) Ubiquitin-dependent proteasomal degradation of human liver cytochrome P450 2E1: identification of sites targeted for phosphorylation and ubiquitination. J Biol Chem 286:9443-56
Correia, Maria Almira; Sinclair, Peter R; De Matteis, Francesco (2011) Cytochrome P450 regulation: the interplay between its heme and apoprotein moieties in synthesis, assembly, repair, and disposal. Drug Metab Rev 43:1-26
Kim, Sung-Mi; Acharya, Poulomi; Engel, Juan C et al. (2010) Liver cytochrome P450 3A ubiquitination in vivo by gp78/autocrine motility factor receptor and C terminus of Hsp70-interacting protein (CHIP) E3 ubiquitin ligases: physiological and pharmacological relevance. J Biol Chem 285:35866-77

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