Human Liver Cytochrome P450 3A4 Ubiquitination

Abstract

Background: CYP3A4, a major human liver drug-metabolizing enzyme, is degraded upon phosphorylation and ubiquitination. Results: CYP3A4 phosphorylation occurs within negatively charged surface clusters that are important for electrostatic interactions with positively charged patches of the ubiquitination enzymes. Conclusion: These phosphorylated clusters enhance CYP3A4 molecular recognition by the ubiquitination complexes. Significance: This is the first mechanistic example of UBC7-gp78 substrate recognition.

Hepatic cytochromes P450: structural degrons and barcodes, posttranslational modifications and cellular adapters in the ERAD-endgame

“…However, the advent of much more sensitive LC-MS/MS methodology has led to the recognition that Ser129 is but one of 16 different CYP2E1 S/T-residues that are phosphorylated. Because CYP2E1 Ser129 mutation to Ala or Gly had little effect on its turnover upon transfection into COS 7 cells (Freeman & Wolf, 1994), we believe that some of these other 16 phosphorylated residues must be involved, as phosphorylation does indeed significantly enhance both CYP2E1 and CYP3A4 ERAD/UPD (Wang et al, 2009, 2011, 2012, 2015; Correia et al, 2014). Indeed, similar analyses of in vitro phosphorylated CYP3A4 revealed that PKA phosphorylates 5 Ser residues (S116, S119, S134, S259 and S478), whereas PKC phosphorylates 14 Ser/Thr residues (T92, S100, T103, S116, S119, S131, S134, T136, S139, S259, T264, T284, S398, S420).…”

“…Such ERAD involves posttranslational phosphorylation of the P450 proteins by cytosolic kinases (Korsmeyer et al, 1999; Wang et al, 2001; Wang et al, 2009, 2011, 2012, 2015), their ubiquitination by the concerted action of the E1-Ub-activating enzyme, E2 Ub-conjugating enzyme/E3 Ub-ligase complexes (Morishima et al, 2005; Pabarcus et al, 2009; Wang et al, 2009, 2011, 2012, 2015; Kim et al, 2010), and subsequent degradation by the cytosolic 26S proteasome (Correia et al 1992b, 2005; Tierney et al, 1992; Roberts, 1997; Murray & Correia, 2001; Correia, 2003; Correia et al, 2005; Morishima et al, 2005; Liao et al, 2006; Correia & Liao, 2007; Faouzi et al, 2007; Fig. 1).…”

“…Interestingly, CYP3A5 that has a phosphomimetic D478 instead of S478 is constitutively ubiquitinated to a much greater extent than CYP3A4, but not degraded quite as rapidly (Wang et al, 2012). Although the relevance of the other 12 phosphorylated CYP3A residues has not been quite as extensively probed, yet we now know that S259 and T284, as constituents of two CYP3A4 phosphodegrons ( see below ) are also quite relevant to its ubiquitination by E2/E3-complexes (Wang et al, 2015). Collectively, these findings argue that phosphorylation is an important determinant of P450 ERAD/UPD, albeit not the only one.…”

“…Its cytosolic, functionally active C-terminal domain (309–643 residues) is composed of an intrinsic RING-finger Ub-ligase (residues 341–378), Cue1-like domain (residues 456–497) for binding Ub and elongating polyUb-chains, Ubc7/Ube2g2-binding (G2BR; residues 579–600), substrate recognition and putative p97/VCP-interacting motif (VIM; residues 626–643) (Fang et al, 2001; Chen et al, 2006; Kostova et al, 2007; Das et al, 2009, 2013; Metzger et al, 2012; Chen et al, 2012). Except for VIM, all the other gp78-domains are essential for Ubc7-dependent CYP3A4-ubiquitination (Wang et al, 2015). Lentiviral shRNAi-mediated gp78-knockdown in cultured rat hepatocytes verified its physiological role in CYP3A ERAD (Kim et al, 2010).…”

“…Chemical crosslinking coupled with LC-MS/MS analyses of Ubc7/gp78-ubiquitinated CYP3A4 peptide digests revealed that CYP3A4-K251, flanking its negatively charged (E258-pS259-E262-D263-pT264) DEST-cluster, interacts with gp78-K313 residing in a positively charged (R307-R308-R310-H312-K313) patch flanking the Ubc7-interacting gp78-RING finger domain, whereas CYP3A4-K257 is found crosslinked to gp78-K586 in another positively charged Q584-R585-K586 patch within the gp78-C-terminal Ubc7-interacting-G2BR domain (Wang et al, 2015). Simultaneous site-directed mutagenesis of both these positively charged gp78 patches were found to attenuate not only its CYP3A4 ubiquitination in vitro (Wang et al, 2015), but also that of human CYP2E1 (Fig.…”