The hepatic cytochromes P450 (P450s) are endoplasmic-reticulum membrane-anchored enzymes engaged in the breakdown of endo- and xenobiotics i.e. drugs, carcinogens, toxins, natural and chemical products. Exposure to these agents can increase liver P450 content due to increased formation, or reduce it due to inactivation/destruction and/or proteolytic degradation. Such drug-mediated modulation of P450 content can significantly influence clinical drug-drug interactions (DDIs). Thus, clinically relevant DDIs often emerge from altered P450 turnover elicited by drug-mediated P450 stabilization i.e. ethanol (EtOH), as well as enhanced drug-mediated P450 degradation (i.e. grapefruit juice furanocoumarins). We have recently shown that the degradation of P450s such as CYP3A4, the major human liver and intestinal P450, and the EtOH-metabolizing CYP2E1 incurs ubiquitination by gp78 and CHIP E3 ubiquitin (Ub)-ligases and the 26S proteasome (UPD) which leads to their accelerated cellular disposal. Our in vitro studies reveal that such gp78- and CHIP- mediated P450 ubiquitination is considerably enhanced by their multisite protein phosphorylation. The ubiquitinated P450 Lys-residues reside in negatively charged acidic (Asp/Glu) and phosphorylated (Ser/Thr) clusters. This leads us to hypothesize that protein phosphorylation by enhancing the negatively charged character of these clusters enhances P450 molecular recognition by these E3 Ub-ligases and thus would control its UPD. Thus our first specific aim is to test this hypothesis by site-directed mutagenesis of the relevant D/E and S/T residues in each cluster along with a state-of-the-art proteomic approach to determine their relevance to P450 ubiquitination by these E3s. In addition, we have also found that gp78- and CHIP- knockdown via RNA-interference in rat hepatocytes results in increased levels of functionally active hepatic P450s. While this finding is clinically relevant for hepatic drug metabolism and DDIs, we propose that it may also be pathophysiologically significant: In the absence of relevant drug substrates, these P450s through futile oxidative cycling could generate proteotoxic reactive O2-species (ROS), and thus partly contribute to age- dependent oxidative damage and elevated lipid peroxidation observed in the liver and other tissues of CHIP-/- mice. Thus our second specific aim is to explore whether elevated hepatic P450s upon CHIP knockdown contribute to this pathology in CHIP-/- mice. Enhanced CYP2E1-dependent EtOH metabolism in alcoholics is also known to similarly generate ROS, hydroxyethyl radicals (HER) and lipid peroxidation products that damage cellular proteins including those of UPD and autophagic-lysosomal degradation pathways involved in CYP2E1 turnover. Our third specific aim is to explore whether EtOH-impaired CYP2E1 turnover by disrupting the normal intracellular CYP2E1 trafficking results in its increased migration to the outer plasma membrane, whereupon it is recognized by the immune surveillance system, engendering pathogenic autoantibodies clinically associated with alcoholic liver disease, drug-induced hepatitis, and hypersensitivity syndromes.

Public Health Relevance

Hepatic cytochromes P450 (P450s) are enzymes engaged in the breakdown of drugs, carcinogens, toxins, natural and chemical agents to water-soluble products. Exposure to these agents can increase liver P450 content or reduce it by enhancing protein degradation and this drug-mediated modulation of P450 content can significantly influence clinical drug-drug interactions. Indeed, clinically relevant drug-drug interactions occur due to altered P450 turnover elicited by drug-mediated P450 stabilization (i.e. alcohol/ethanol) or enhanced drug-mediated P450 degradation (i.e. grapefruit juice furanocoumarins). Our studies propose to use mammalian systems as models for elucidating the mechanisms of degradation of CYP3A4, the major human liver and intestinal enzyme, and human CYP2E1, the P450 enzyme implicated in alcoholic liver disease. Together these P450s are responsible for the metabolism of ~ 65% of clinically relevant drugs, toxins, and carcinogens, with consequently significant potential for drug-drug interactions and toxicity. We also propose to determine whether impaired P450 degradation is pathophysiologically relevant to aggravation of alcoholic liver disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM044037-19
Application #
8302240
Study Section
Xenobiotic and Nutrient Disposition and Action Study Section (XNDA)
Program Officer
Okita, Richard T
Project Start
1990-04-01
Project End
2015-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
19
Fiscal Year
2012
Total Cost
$423,094
Indirect Cost
$149,247
Name
University of California San Francisco
Department
Pharmacology
Type
Schools of Medicine
DUNS #
094878337
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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