Substrate-, hormone-, and cAMP-regulated cytochrome P450 degradation.

Eliasson, Erik; Johansson, Inger; Ingelman‐Sundberg, Magnus

doi:10.1073/pnas.87.8.3225

Cited by 97 publications

(56 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…7 This PKC-mediated Ser 129 phosphorylation is further enhanced by CuOOH-mediated structural inactivation of the protein (Table 3). Although such Ser 129 phosphorylation has been postulated to trigger its rapid proteolytic degradation (28,29), other CYP2E1 phosphorylatable residues must exist, as mutation of Ser 129 to Ala or Gly, while retaining enzyme activity, failed to stabilize the enzyme (52 , and Thr 373 ) were also PKA targets. CYP2E1 structural inactivation not only further enhanced the extent of their phosphorylation but also uncovered two additional sites Ser 387 and Thr 431 phosphorylated to a significant extent (Table 3).…”

Section: Discussionmentioning

confidence: 99%

“…Thus, in the situation of low stoichiometry, the method may indeed estimate smaller values because the difference in the ionization efficiency between phosphorylated and nonphosphorylated peptides is not known. 5 In contrast to this native substrate-free CYP2E1 that is very highly phosphorylated by PKA, it is worth noting that substrate-complexed CYP2E1 is apparently more resistant to PKA-mediated phosphorylation (28,29), which may account for its greater proteolytic stability. respectively), and similar semi-quantitative analyses indicated that this extent was further increased to 1.44 Ϯ 0.07, 1.10 Ϯ 0.32, and 2.1 Ϯ 0.28%, respectively, upon CuOOH-mediated structural inactivation (Tables 1 and 2; supplemental Figs.…”

Section: Identification Of Human Liver Cyp2e1 Residues Phosphorylatedmentioning

confidence: 99%

“…Using LC-MS/MS analyses, we have identified several CYP2E1 residues, in addition to the previously identified Ser 129 (28,(51)(52)(53) specifically phosphorylated by PKA or PKC in the native enzyme. Furthermore, we document that this phosphorylation is further enhanced after CYP2E1 structural inactivation by cumene hydroperoxide (CuOOH).…”

mentioning

confidence: 99%

See 2 more Smart Citations

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

Wang

Guan²,

Acharya

et al. 2011

Journal of Biological Chemistry

View full text Add to dashboard Cite

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 ...

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Identification Of Human Liver Cyp2e1 Residues Phosphorylatedmentioning

confidence: 99%

See 1 more Smart Citation

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

Wang

Guan²,

Acharya

et al. 2011

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…All agents which increase Ah-receptor to the AhRE activates transcription of the CYP1A1 gene. The Ah receptor in its active form CYP2E1 degradation also cause phosphorylation of the enzyme on Ser-129, while substrates which prevent actually consists of a heterodimer comprising the ligand binding domain (ALBD) and the Ah-receptor nuclear this phosphorylation also prevent degradation [29 ]. Phosphorylation of Ser-129 triggers a rapid loss of transporter (ARNT).…”

Section: Mechanism Of Enzyme Inductionmentioning

confidence: 99%

Relevance of induction of human drug‐metabolizing enzymes: pharmacological and toxicological implications

Park

Kitteringham

Pirmohamed

et al. 1996

Brit J Clinical Pharma

View full text Add to dashboard Cite

1Human drug‐metabolizing systems can be induced, or activated, by a large number of exogenous agents including drugs, alcohol, components in the diet and cigarette smoke, as well as by endogenous factors. 2Such perturbation of enzyme activity undoubtedly contributes to both intra‐ and inter‐individual variation both with respect to the rate and route of metabolism for a particular drug. Induction may, in theory, either attenuate the pharmacological response or exacerbate the toxicity of a particular drug, or both. 3The clinical impact of enzyme induction will depend upon the number of different enzyme isoforms affected and the magnitude of the inductive response within an individual, and also on the therapeutic indices of the affected substrates. 4The toxicological implications will be determined either by any change in the route of metabolism, or by a disturbance of the balance between activation and detoxication processes, which may be isozyme selective.

show abstract

“…Moreover, we find that each of these kinases significantly phosphorylates additional, previously unidentified CYP3A4 Ser/Thr residues. Because PKA-dependent protein phosphorylation is generally believed to stem from signaling cascades, the physiological relevance of the observed in vitro CYP3A4 Ser-478 phosphorylation by the PKA catalytic subunit remains equivocal, despite the overwhelming evidence from our own laboratory and that of others that PKA is indeed involved in P450 phosphorylation (10,(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). In support of the in vivo relevance of this phosphorylation, we now also provide proteomic evidence that the corresponding residue Ser-479 in the orthologous rat liver CYP3A23 is indeed phosphorylated intracellularly in cultured hepatocytes upon acceleration of its ERAD through 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP)-elicited CYP3A suicide inactivation.…”

mentioning

confidence: 99%

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

Wang

Guan²,

Acharya

et al. 2012

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

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...

show abstract

Substrate-, hormone-, and cAMP-regulated cytochrome P450 degradation.

Cited by 97 publications

References 21 publications

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

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

Relevance of induction of human drug‐metabolizing enzymes: pharmacological and toxicological implications

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

Contact Info

Product

Resources

About