Characterization of the Physiological Turnover of Native and Inactivated Cytochromes P450 3A in Cultured Rat Hepatocytes:  A Role for the Cytosolic AAA ATPase p97?

Molecular & Cellular Proteomics

Guan²,

Acharya

et al. 2012

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CYP3A4, an integral endoplasmic reticulum (ER)-anchored protein, is the major human liver cytochrome P450 enzyme responsible for the disposition of over 50% of clinically relevant drugs. Alterations of its protein turnover can influence drug metabolism, drug-drug interactions, and the bioavailability of chemotherapeutic drugs. Such CYP3A4 turnover occurs via a classical ER-associated degradation (ERAD) process involving ubiquitination by both UBC7/gp78 and UbcH5a/CHIP E2-E3 complexes for 26 S proteasomal targeting. These E3 ligases act sequentially and cooperatively in CYP3A4 ERAD because RNA interference knockdown of each in cultured hepatocytes results in the stabilization of a functionally active enzyme. We have documented that UBC7/gp78-mediated CYP3A4 ubiquitination requires protein phosphorylation by protein kinase (PK) A and PKC and identified three residues (Ser-478, Thr-264, and Ser-420) whose phosphorylation is required for intracellular CYP3A4 ERAD. We document herein that of these, Ser-478 plays a pivotal role in UBC7/ gp78-mediated CYP3A4 ubiquitination, which is accelerated and enhanced on its mutation to the phosphomimetic Asp residue but attenuated on its Ala mutation. Intriguingly, CYP3A5, a polymorphically expressed human liver CYP3A4 isoform (containing Asp-478) is ubiquitinated but not degraded to a greater extent than CYP3A4 in HepG2 cells. This suggests that although Ser-478 phosphorylation is essential for UBC7/gp78-mediated CYP3A4 ubiquitination, it is not sufficient for its ERAD. Additionally, we now report that CYP3A4 protein phosphorylation by PKA and/or PKC at sites other than Ser-478, Thr-264, and Ser-420 also enhances UbcH5a/CHIP-mediated ubiquitination. Through proteomic analyses, we identify (i) 12 additional phosphorylation sites that may be involved in CHIP-CYP3A4 interactions and (ii) 8 previously unidentified CYP3A4 ubiquitination sites within spatially associated clusters of Asp/Glu and phosphorylatable Ser/Thr residues that may serve to engage each E2-E3 complex. Collectively, our findings underscore the interplay between protein phosphorylation and ubiquitination in ERAD and, to our knowledge, provide the very first example of gp78 substrate recognition via protein phosphorylation. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.010132, 1-17, 2012. Hepatic cytochromes P450 (P450s)1 are endoplasmic reticulum (ER)-anchored hemoproteins involved in the metabolism of numerous endo-and xenobiotics. Of these, CYP3A4 is particularly noteworthy because it comprises 30% of the human liver microsomal P450 complement and is responsible for the metabolism of Ͼ50% clinically relevant drugs, as well as hepatotoxins such as aflatoxin B1 (1). In common with other ER-integral P450s, it is a monotopic protein, N-terminally anchored to the ER membrane, with the bulk of its catalytic domain in the cytosol. We have documented that CYP3A4, in common with its CYP3A orthologs, incurs ubiquitin (Ub)-dependent proteasomal degradation (UPD) in a classical ER-associated degradation (ERAD) process...

Section: Discussionsupporting

confidence: 89%

mentioning

confidence: 99%

Multisite Phosphorylation of Human Liver Cytochrome P450 3A4 Enhances Its gp78- and CHIP-mediated Ubiquitination

Molecular & Cellular Proteomics

Guan²,

Acharya

et al. 2012

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“…Cells were treated with vehicle (ethanol or DMSO) and proteasomal inhibitor MG-262 (20 M), MG-132 (20 M), or epoxomycin (10 M), with or without DDEP (100 M) for 0 -6 h. Hepatocytes from another human donor were also similarly cultured and treated with rifampin. On the 5th day they were treated with a combination of lysosomal inhibitors 3-methyladenine (3MA; 5 mM) and NH 4 Cl (50 mM) for 20 h, along with a DMSO vehicle control (16). Total cell lysate protein (20 g) was assayed by Western immunoblotting for CYP2E1 content as described above.…”

Section: ␥-S-[mentioning

confidence: 99%

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

Journal of Biological Chemistry

Guan²,

Acharya

et al. 2011

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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 ...

“…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.…”

mentioning

confidence: 99%

Human Liver Cytochrome P450 3A4 Ubiquitination

Journal of Biological Chemistry

Kim

Trnka³

et al. 2015

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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.