Liver Cytochrome P450 3A Endoplasmic Reticulum-associated Degradation

Abstract

The CYP3A subfamily of hepatic cytochromes P450, being engaged in the metabolism and clearance of >50% of clinically relevant drugs, can significantly influence therapeutics and drug-drug interactions. Our characterization of CYP3A degradation has indicated that CYPs 3A incur ubiquitin-dependent proteasomal degradation (UPD) in an endoplasmic reticulum (ER)-associated degradation (ERAD) process. Cytochromes P450 are monotopic hemoproteins N-terminally anchored to the ER membrane with their protein bulk readily accessible to the cytosolic proteasome. Given this topology, it was unclear whether they would require the AAA-ATPase p97 chaperone complex that retrotranslocates/dislocates ubiquitinated ER-integral and luminal proteins into the cytosol for proteasomal delivery. To assess the in vivo relevance of this p97-CYP3A association, we used lentiviral shRNAs to silence p97 (80% mRNA and 90% protein knockdown relative to controls) in sandwich-cultured rat hepatocytes. This extensive hepatic p97 knockdown remarkably had no effect on cellular morphology, ER stress, and/or apoptosis, despite the well recognized strategic p97 roles in multiple important cellular processes. However, such hepatic p97 knockdown almost completely abrogated CYP3A extraction into the cytosol, resulting in a significant accumulation of parent and ubiquitinated CYP3A species that were firmly ER-tethered. Little detectable CYP3A accumulated in the cytosol, even after concomitant inhibition of proteasomal degradation, thereby documenting a major role of p97 in CYP3A extraction and delivery to the 26 S proteasome during its UPD/ERAD. Intriguingly, the accumulated parent CYP3A was functionally active, indicating that p97 can regulate physiological CYP3A content and thus influence its clinically relevant function.Hepatic cytochrome P450 hemoproteins (P450s) 2 of the CYP3A subfamily include CYP3A4, the major human liver drug metabolizing P450 enzyme engaged in the metabolism of over 50% of clinically relevant drugs and other xenobiotics (1). In common with other microsomal P450s (2-10), CYPs 3A are integral endoplasmic reticulum (ER) membrane-anchored monotopic proteins, with their N terminus embedded in the ER and their catalytic domain exposed to the cytosol. Our findings in various in vivo and in vitro reconstituted eukaryotic systems have documented that both native 3 and structurally inactivated CYPs 3A incur ubiquitin (Ub)-dependent proteasomal degradation (UPD) (11)(12)(13)(14)(15)(16)(17)(18)(19)(20), in a typical ER-associated degradation (ERAD) process (21-28). Indeed, CYPs 3A qualify as bona fide ERAD-C substrates following mechanismbased inactivation by certain agents (29 -31) (see below).Thus, consistent with a typical ERAD process, we have found that CYP3A ERAD involves posttranslational phosphorylation by cytosolic kinases (15,32,33); ubiquitination by the ER-integral "glycoprotein" 78/autocrine motility factor receptor (gp78/AMFR) and the cytosolic C terminus of Hsp70-interacting protein (CHIP) E3 Ub ligases along with their respecti...

Ubiquitin-dependent Proteasomal Degradation of Human Liver Cytochrome P450 2E1

“…5C) as described previously (40). In control (non-DDEPtreated) cells, CYP2E1 was present largely as the parent 50-kDa species with traces of HMM species at time 0 h (Fig.…”

“…Rat hepatocytes cultured exactly as described above were pretreated with ethanol (200 mM)/acetone (20 mM) to induce CYP2E1 for 3 days, weaned off these CYP2E1-stabilizing inducers for another day, and then subjected to 35 S-pulse-chase analyses on the 5th day (40). Following chase, they were treated with DDEP (100 M), and 30 min later with vehicle (DMSO), KT-5720 (5 M), BisIII (5 M), or staurosporine (1 M).…”

“…S1) and donor 3 cells treated with MG-132 or 3-MA ϩ NH 4 Cl as described previously in B (see under "Experimental Procedures"). The immunoprecipitates from lysates were subjected to IB analyses with a rabbit anti-Ub antibody, as detailed (8,58). 1st lane (extreme left) corresponds to 0-h controls.…”

Liver Cytochrome P450 3A Ubiquitination in Vivo by gp78/Autocrine Motility Factor Receptor and C Terminus of Hsp70-interacting Protein (CHIP) E3 Ubiquitin Ligases

“…Lipofectamine 2000 was obtained from Invitrogen. Common cell culture media, supplements, culture plasticware, and commercial sources of protease inhibitors and dexamethasone (Dex) have been reported previously (52,53). Goat polyclonal IgGs were raised commercially against purified recombinant rat hepatic CYP3A23 and purified by Hi-Trap protein A-Sepharose affinity chromatography.…”

“…After 18 -20 h, the medium was changed, and cells were overlaid with 0.25 mg/ml Matrigel. Cells were cultured as described (4,(52)(53)(54) in Williams' E medium containing insulintransferrin-selenium G, 0.1 M Dex, 50 units/ml penicillin/ streptomycin, 2 mM L-glutamine, and 0.1% w/v BSA. Cells were maintained for 2 days with a daily change of medium to enable the recovery and restoration of hepatic function (55).…”