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

Abstract

Human liver CYP2E1 is a monotopic, endoplasmic reticulumanchored cytochrome P450 responsible for the biotransformation of clinically relevant drugs, low molecular weight xenobiotics, carcinogens, and endogenous ketones. CYP2E1 substrate complexation converts it into a stable slow-turnover species degraded largely via autophagic lysosomal degradation. Substrate decomplexation/withdrawal results in a fast turnover CYP2E1 species, putatively generated through its futile oxidative cycling, that incurs endoplasmic reticulum-associated ubiquitin-dependent proteasomal degradation (UPD). CYP2E1 thus exhibits biphasic turnover in the mammalian liver. We now show upon heterologous expression of human CYP2E1 in Saccharomyces cerevisiae that its autophagic lysosomal degradation and UPD pathways are evolutionarily conserved, even though its potential for futile catalytic cycling is low due to its sluggish catalytic activity in yeast. This suggested that other factors (i.e. post-translational modifications or "degrons") contribute to its UPD. Indeed, in cultured human hepatocytes, CYP2E1 is detectably ubiquitinated, and this is enhanced on its mechanismbased inactivation. Studies in Ubc7p and Ubc5p genetically deficient yeast strains versus corresponding isogenic wild types identified these ubiquitin-conjugating E2 enzymes as relevant to CYP2E1 UPD. Consistent with this, in vitro functional reconstitution analyses revealed that mammalian UBC7/gp78 and UbcH5a/CHIP E2-E3 ubiquitin ligases were capable of ubiquitinating CYP2E1, a process enhanced by protein kinase (PK) A and/or PKC inclusion. Inhibition of PKA or PKC blocked intracellular CYP2E1 ubiquitination and turnover. Here, through mass spectrometric analyses, we identify some CYP2E1 phosphorylation/ubiquitination sites in spatially associated clusters. We propose that these CYP2E1 phosphorylation clusters may serve to engage each E2-E3 ubiquitination complex in vitro and intracellularly.Hepatic cytochromes P450 (P450s) 2 are endoplasmic reticulum (ER)-anchored hemoproteins involved in the metabolism of numerous endo-and xenobiotics. These substrates can modulate P450 content, diversity, and/or function (see Refs. 1, 2 and references therein) through induction via either increased synthesis or protein stabilization, i.e. half-life prolongation (3-9). By contrast, "suicide" substrate/inactivators accelerate the degradation of certain P450s and dramatically curtail their halflives (10 -23). Such substrate-mediated P450 induction and/or enhanced turnover can influence the severity and the time course of certain pharmacokinetic/pharmacodynamic drugdrug interactions and is an important therapeutic consideration (24 -27).P450 turnover has been proposed to involve various proteolytic mechanisms (6 -9, 28 -38). However, it is now increasingly evident that in common with other type I monotopic ER proteins, P450s such as CYPs 3A (both native and structurally inactivated) undergo ER-associated degradation (ERAD) involving the ubiquitin (Ub)-dependent 26 S proteasomal system (UPS) (6 ...

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

“…Our own LC-MS/MS analyses of in vitro phosphorylated human liver CYP2E1 peptide digests further revealed that Ser129 is a major (>98%) PKA-phosphorylation site both in the native and CuOOH-inactivated proteins (Wang et al, 2011). This residue is also correspondingly phosphorylated by PKC to >23% and 32%, respectively (Wang et al, 2011). 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.…”

“…Thus, although all CYPs 3A as well as all structurally/functionally inactivated P450s are predominantly ERAD/UPD targets (Correia et al 1987, 1992a, 1992b, 2005; Correia, 2003; Tierney et al, 1992; Sohn et al, 1991; Dai & Cederbaum, 1995; Roberts, 1997; Schmiedlin-Ren, 1997; Korsmeyer et al, 1997; Wang et al, 1999; Murray & Correia, 2001; Morishima et al, 2005; Liao et al, 2006; Correia & Liao, 2007; Faouzi et al, 2007; Lee et al, 2008), native P450s such as rat liver CYPs 2B1 and 2C11 are predominantly ERAD-II substrates requiring ALD instead of UPD, henceforth referred to as the ERAD/ALD pathway (Masaki et al, 1987; Ronis & Ingelman-Sundberg, 1989; Ronis et al, 1991; Murray et al, 2002; Liao et al, 2005). On the other hand, human or rat liver CYP2E1 utilizes both ERAD/UPD and ERAD/ALD pathways, depending on whether it is suicidally inactivated or native/substrate-free, or native/substrate-bound, respectively (Song et al, 1989; Roberts et al, 1995; Bardag-Gorce, 2002; Morishima et al, 2005; Wang et al, 2011). Thus, although a preferential P450 routing into either pathway apparently exists, the pathway not chosen may simply represent a convenient alternative backup, in eventualities such as ER-stress when ERAD/UPD is overloaded and clogged with misfolded/aggregated proteins awaiting clearance, and competition for the ERAD/UPD cellular machinery is insurmountable.…”

“…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).…”

Coat Protein Regulation by CK2, CPIP, HSP70, and CHIP Is Required for Potato Virus A Replication and Coat Protein Accumulation

“…Protein phosphorylation has been linked both to activation (41) and to inhibition (42,43) of proteasome-mediated degradation of the target protein. One of the questions of the current study was to address if CK2-mediated phosphorylation affects degradation of CP via the CPIP pathway.…”

Effect of Oridonin on Cytochrome P450 Expression and Activities in HepaRG Cell

“…Protein ubiquitination is a pathway for a variety of ­proteins and plays a critical role in post-translational modification [35]. Previous studies have suggested that CYP2E1 protein contains 37 lysyl residues, which could be considered a candidate for ubiquitin conjugation, and there are likely 2 subtypes of ubiquitination systems involved in the degradation of the free CYP2E1 [36]. Therefore, the difference between mRNA and ­protein level may have been partly expanded, although only scarce information on ubiquitin-mediated CYP450 family degradation is currently available.…”