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

Abstract

CYP3A4 is a dominant human liver cytochrome P450 enzyme engaged in the metabolism and disposition of >50% of clinically relevant drugs and held responsible for many adverse drugdrug interactions. CYP3A4 and its mammalian liver CYP3A orthologs are endoplasmic reticulum (ER)-anchored monotopic proteins that undergo ubiquitin (Ub)-dependent proteasomal degradation (UPD) in an ER-associated degradation (ERAD) process. These integral ER proteins are ubiquitinated in vivo, and in vitro studies have identified the ER-integral gp78 and the cytosolic co-chaperone, CHIP (C terminus of Hsp70-interacting protein), as the relevant E3 Ub-ligases, along with their cognate E2 Ub-conjugating enzymes UBC7 and UbcH5a, respectively. Using lentiviral shRNA templates targeted against each of these Ub-ligases, we now document that both E3s are indeed physiologically involved in CYP3A ERAD/UPD in cultured rat hepatocytes. Accordingly, specific RNAi resulted in ≈80% knockdown of each hepatic Ub-ligase, with a corresponding ≈2.5-fold CYP3A stabilization. Surprisingly, however, such stabilization resulted in increased levels of functionally active CYP3A, thereby challenging the previous notion that E3 recognition and subsequent ERAD of CYP3A proteins required ab initio their structural and/or functional inactivation. Furthermore, coexpression in HepG2 cells of both CYP3A4 and gp78, but not its functionally inactive RING-finger mutant, resulted in enhanced CYP3A4 loss greater than that in corresponding cells expressing only CYP3A4. Stabilization of a functionally active CYP3A after RNAi knockdown of either of the E3s, coupled with the increased CYP3A4 loss on gp78 or CHIP coexpression, suggests that ERAD-associated E3 Ub-ligases can influence clinically relevant drug metabolism by effectively regulating the physiological CYP3A content and consequently its function.The CYP3A subfamily of hepatic cytochrome P450 hemoproteins includes CYP3A4, the dominant human liver P450 enzyme responsible for the metabolism of more than 50% of clinically relevant drugs and other xenobiotics (1). The CYPs 3A, 2 in common with many hepatic P450s, are excellent examples of integral endoplasmic reticulum (ER) membrane-anchored monotopic proteins, with their N termini embedded in the ER and their catalytic domains exposed to the cytosol. Using various in vivo and in vitro reconstituted eukaryotic systems, we have shown that both native 3 and structurally inactivated CYPs 3A incur ubiquitin (Ub)-dependent proteasomal degradation (UPD), in a typical ER-associated degradation (ERAD) process involving phosphorylation, ubiquitination, ER membrane extraction into the cytosol, and subsequent degradation by the 26S proteasome (2-13). Indeed mechanism-based CYP3A inactivation often results in active site structural lesions within their cytosolic domain (2, 6, 7), thereby qualifying these proteins as bona fide ERAD-C substrates.The pathways of P450 degradation appear to be highly conserved in all eukaryotes from yeast to man (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22...

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

“…Cotreatment of rifampin-treated human hepatocytes with the ALD inhibitors 3MA and NH 4 Cl for 20 h also led to a significant CYP2E1 stabilization (Fig. 4B), with no appreciable A plasmid encoding human liver CYP2E1 was heterologously expressed in yeast strains defective in proteasomal degradation (hrd2-1) or deficient in vacuolar degradation (pep4⌬), and corresponding isogenic wild type (WT) yeast strains, and its degradation was followed by the stationary chase method as described previously (46,50). Yeast cells were harvested at various stages of logarithmic culture growth corresponding to an A 600 of 0.8 (E, early), 12 h after reaching this value (M, middle stage), or 20 h after reaching an A 600 of 0.8 (L, late), as required for monitoring ALD in yeast.…”

“…Analyses of in Vitro CYP2E1 Ubiquitination and the Effects of PKA and/or PKC on This Process-Two E2-E3 complexes have been shown to catalyze P450 ubiquitination in vitro: UbcH5a/ CHIP in the case of CYP2E1 (22), and both UbcH5a/CHIP and UBC7/gp78 in the case of CYP3A4 (49,50). To enhance the detection of the ubiquitinated species, P450s were first inactivated by CuOOH in vitro before incubation in each of the reconstituted E2/E3 systems.…”

“…Yeast cells were harvested at various stages of logarithmic culture growth corresponding to an A 600 of 0.8 (E, early), 12 h after reaching this value (M, middle stage), or 20 h after reaching an A 600 of 0.8 (L, late), as required for monitoring ALD in yeast. Microsomes were subjected to IB analyses with sheep anti-CYP2E1 IgG as the primary antibody and rabbit anti-sheep HRP-coupled secondary IgG, followed by ECL detection as described previously (50). Prototypical immunoblots are shown with a purified human liver CYP2E1 standard included as a standard (Std).…”

Human Liver Cytochrome P450 3A4 Ubiquitination

“…Human cytosolic gp78C (C-terminal domain 309 -643 residues; 63 kDa) (E3) and its truncated and site-directed mutants, murine UBC7 (E2) and human C terminus of Hsc70-Interacting protein (CHIP), were expressed in E. coli and purified as described previously (30,(37)(38)(39)(40). C-terminally His 6 -tagged human CYP3A4 wild type (CYP3A4WT) and all mutants were incorporated into the pCWori ϩ vector and expressed in DH5␣ cells grown in TB media at 37°C with isopropyl 1-thio-␤-Dgalactopyranoside induction and continuous monitoring of P450 content by the reduced CO-binding spectral assay (53).…”

“…We and others have documented that in this ERAD-C process, CYPs 3A and CYP2E1 are first phosphorylated by protein kinases (PKA and PKC) (30, 34 -38), ubiquitinated by the cytosolic UbcH5a-CHIP-Hsc70-Hsp40 and UBC7-dependent ERintegral polytopic gp78 E2-E3-Ub-ligase complexes (29,30,(37)(38)(39)(40), extracted from the ER membrane by the Npl4-Ufd1-p97/VCP-AAA ATPase chaperone complex (12,41), and then delivered to the 26 S proteasome for degradation. Herein, we document that although each of these E2-E3 systems can function quite independently in vitro, when present concurrently as in vivo, their roles in CYP3A4 ubiquitination are complementary rather than redundant.…”