Distinction between Human Cytochrome P450 (CYP) Isoforms and Identification of New Phosphorylation Sites by Mass Spectrometry

Redlich, Gorden; Zanger, Ulrich M.; Riedmaier, Stephan; Bache, Nicolai; Giessing, Anders; Eisenacher, Martin; Stephan, Christian; Meyer, Helmut E.; Jensen, Ole N.; Marcus, Katrin

doi:10.1021/pr800231w

Cited by 59 publications

(45 citation statements)

References 60 publications

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“…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: 80%

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

Wang

Guan²,

Acharya

et al. 2011

Journal of Biological Chemistry

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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 ...

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confidence: 80%

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

Wang

Guan²,

Acharya

et al. 2011

Journal of Biological Chemistry

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“…15 and references therein; Ref. 16). These studies have invariably implicated protein kinase (PK) A and PKC as protagonists in this process.…”

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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

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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...

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“…Human P450 3A4 is found mainly in the liver and accounts for the metabolism of about 50% of all xenobiotics [70]. Highly promiscuous by nature due to its large and flexible active site, substrate prediction with this enzyme has been very challenging.…”

Section: Mammalian P450smentioning

confidence: 99%

Use of Chemical Auxiliaries to Control P450 Enzymes for Predictable Oxidations at Unactivated C-H Bonds of Substrates

Auclair

Polic

2015

Advances in Experimental Medicine and Biology

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Cytochrome P450 enzymes (P450s) have the ability to oxidize unactivated C-H bonds of substrates with remarkable regio-and stereoselectivity. Comparable selectivity for chemical oxidizing agents is typically difficult to achieve. Hence, there is an interest in exploiting P450s as potential biocatalysts. Despite their impressive attributes, the current use of P450s as biocatalysts is limited. While bacterial P450 enzymes typically show higher activity, they tend to be highly selective for one or a few substrates. On the other hand, mammalian P450s, especially the drugmetabolizing enzymes, display astonishing substrate promiscuity. However, product prediction continues to be challenging. This review discusses the use of small molecules for controlling P450 substrate specificity and product selectivity. The focus will be on two approaches in the area: (1) the use of decoy molecules, and (2) the application of substrate engineering to control oxidation by the enzyme.

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Distinction between Human Cytochrome P450 (CYP) Isoforms and Identification of New Phosphorylation Sites by Mass Spectrometry

Cited by 59 publications

References 60 publications

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

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

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

Use of Chemical Auxiliaries to Control P450 Enzymes for Predictable Oxidations at Unactivated C-H Bonds of Substrates

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