Maria Almira Correia

Degradation of rat liver cytochromes P450 3A after their inactivation by 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine: Characterization of the proteolytic system☆

The suicide substrate 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4- dihydropyridine (DDEP) inactivates rat liver cytochrome P450 (P450) 3A isozymes through prosthetic heme alkylation of the apoprotein in a mechanism-based fashion, which marks them for rapid proteolysis. In this article, through the use of epitope-specific monoclonal antibodies, we show that both 3A1 and 3A2 isozymes are targeted for proteolysis. Furthermore, using intact rats, isolated rat hepatocytes, and rat liver subcellular fractions supplemented with ATP and MgCl2, as well as various proteolytic inhibitors as probes, we now report that the hepatic cytosolic ubiquitin-dependent proteolytic system rather than hepatic lysosomes is involved in the rapid degradation of DDEP-induced heme alkylated P450s 3A.

Pathways of carbamazepine bioactivation in vitro. III. The role of human cytochrome P450 enzymes in the formation of 2,3-dihydroxycarbamazepine.

Conversion of the carbamazepine metabolite 3-hydroxycarbamazepine (3-OHCBZ) to the catechol 2,3-dihydroxycarbamazepine (2,3-diOHCBZ) followed by subsequent oxidation to a reactive o-quinone species has been proposed as a possible bioactivation pathway in the pathogenesis of carbamazepine-induced hypersensitivity. Initial in vitro phenotyping studies implicated CYP3A4 as a primary catalyst of 2,3-diOHCBZ formation: 2-hydroxylation of 3-OHCBZ correlated significantly (r2 ≥ 0.929, P < 0.001) with CYP3A4/5 activities in a panel of human liver microsomes (n = 14) and was markedly impaired by CYP3A inhibitors (>80%) but not by inhibitors of other cytochrome P450 enzymes (≤20%). However, in the presence of troleandomycin, the rate of 2,3-diOHCBZ formation correlated significantly with CYP2C19 activity (r2 = 0.893, P < 0.001) in the panel of human liver microsomes. Studies with a panel of cDNA-expressed enzymes revealed that CYP2C19 and CYP3A4 were high (S50 = 30 μM) and low (S50 = 203 μM) affinity enzymes, respectively, for 2,3-diOHCBZ formation and suggested that CYP3A4, but not CYP2C19, might be inactivated by a metabolite formed from 3-OHCBZ. Subsequent experiments demonstrated that preincubation of 3-OHCBZ with human liver microsomes or recombinant CYP3A4 led to decreased CYP3A4 activity, which was both preincubation time- and concentration-dependent, but not inhibited by inclusion of glutathione or N-acetylcysteine. CYP3A4, CYP3A5, CYP3A7, CYP2C19, and CYP1A2 converted [14C]3-OHCBZ into protein-reactive metabolites, but CYP3A4 was the most catalytically active enzyme. The results of this study suggest that CYP3A4-dependent secondary oxidation of 3-OHCBZ represents a potential carbamazepine bioactivation pathway via formation of reactive metabolites capable of inactivating CYP3A4, potentially generating a neoantigen that may play a role in the etiology of carbamazepine-induced idiosyncratic toxicity.

Cytochrome P450 turnover.

Publisher Summary This chapter discusses the cytochrome P450 turnover. The hepatic microsomal hemoproteins, collectively termed as cytochrome P450, consist of a family of multiple isozymes, all of which recruit heme, as their essential prosthetic moiety. These hemoproteins are monomeric, containing one heme moiety per mole of enzyme. The iron of the heme moiety is coordinated to the thiol of a cysteine residue located in an invariable and highly conserved pentadecapeptide region in the COOH terminus of the apocytochrome. Nonetheless, it is obvious that factors which influence the synthesis and degradation of each constitutive moiety would control the normal turnover of P450 hemoproteins. Empirical approaches to the rigorous determination of the turnover of each individual P450 thus require a fundamental understanding of these critical controlling factors, if the half-life (t 1/2 ) parameters derived for each constitutive P450 moiety are to be truly representative of their “real” biological turnover and therefore meaningful.

Hepatic Cytochrome P450 Degradation: Mechanistic Diversity of the Cellular Sanitation Brigade

Hepatic cytochromes P450 (P450s) are monotopic endoplasmic reticulum (ER)-anchored hemoproteins that exhibit heterogenous physiological protein turnover. The molecular/cellular basis for such heterogeneity is not well understood. Although both autophagic-lysosomal and nonlysosomal pathways are available for their cellular degradation, native P450s such as CYP2B1 are preferentially degraded by the former route, whereas others such as CYPs 3A are degraded largely by the proteasomal pathway, and yet others such as CYP2E1 may be degraded by both. The molecular/structural determinants that dictate this differential proteolytic targeting of the native P450 proteins remain to be unraveled. In contrast, the bulk of the evidence indicates that inactivated and/or otherwise posttranslationally modified P450 proteins undergo adenosine triphosphate-dependent proteolytic degradation in the cytosol. Whether this process specifically involves the ubiquitin (Ub)-/26S proteasome-dependent, the Ub-independent 20S proteasome-dependent, or even a recently characterized Ub- and proteasome-independent pathway may depend on the particular P450 species targeted for degradation. Nevertheless, the collective evidence on P450 degradation attests to a remarkably versatile cellular sanitation brigade available for their disposal. Given that the P450s are integral ER proteins, this mechanistic diversity in their cellular disposal should further expand the repertoire of proteolytic processes available for ER proteins, thereby extending the currently held general notion of ER-associated degradation.

CYP3A4-Mediated Carbamazepine (CBZ) Metabolism: Formation of a Covalent CBZ-CYP3A4 Adduct and Alteration of the Enzyme Kinetic Profile

Carbamazepine (CBZ) is a widely prescribed anticonvulsant whose use is often associated with idiosyncratic hypersensitivity. Sera of CBZ-hypersensitive patients often contain anti-CYP3A antibodies, including those to a CYP3A23 K-helix peptide that is also modified during peroxidative CYP3A4 heme-fragmentation. We explored the possibility that cytochromes P450 (P450s) such as CYP3A4 bioactivate CBZ to reactive metabolite(s) that irreversibly modify the P450 protein. Such CBZ-P450 adducts, if stable in vivo, could engender corresponding serum P450 autoantibodies. Incubation with CBZ not only failed to inactivate functionally reconstituted, purified recombinant CYP3A4 or CYP3A4 Supersomes in a time-dependent manner, but the inclusion of CBZ (0–1 mM) also afforded a concentration-dependent protection to CYP3A4 from inactivation by NADPH-induced oxidative uncoupling. Incubation of CYP3A4 Supersomes with 3H-CBZ resulted in its irreversible binding to CYP3A4 protein with a stoichiometry of 1.58 ± 0.15 pmol 3H-CBZ bound/pmol CYP3A4. Inclusion of glutathione (1.5 mM) in the incubation reduced this level to 1.09. Similar binding (1.0 ± 0.4 pmol 3H-CBZ bound/pmol CYP3A4) was observed after 3H-CBZ incubation with functionally reconstituted, purified recombinant CYP3A4(His)6. The CBZ-modified CYP3A4 retained its functional activity albeit at a reduced level, but its testosterone 6β-hydroxylase kinetics were altered from sigmoidal (a characteristic profile of substrate cooperativity) to near-hyperbolic (Michaelis-Menten) type, suggesting that CBZ may have modified CYP3A4 within its active site.

Effect of cobalt on microsomal cytochrome P-450: Differences between liver and intestinal mucosa

Summary Parenteral administration of cobalt to rats stimulated hepatic microsomal heme oxygenase (MHO) activity, depressed cytochrome P-450 content and reduced benzpyrene hydroxylase (BPH) activity, confirming previous reports. By contrast, in the intestinal mucosa, cobalt treatment not only stimulated MHO but in addition increased both cytochrome P-450 and BPH. This differential effect between liver and intestine suggests that the mechanism of the cobalt-mediated stimulation of MHO in the two tissues either differs and/or is unrelated to the turnover of cytochrome P-450.

A role for protein phosphorylation in cytochrome P450 3A4 ubiquitin-dependent proteasomal degradation

Cytochromes P450 (P450s) incur phosphorylation. Although the precise role of this post-translational modification is unclear, marking P450s for degradation is plausible. Indeed, we have found that after structural inactivation, CYP3A4, the major human liver P450, and its rat orthologs are phosphorylated during their ubiquitin-dependent proteasomal degradation. Peptide mapping coupled with mass spectrometric analyses of CYP3A4 phosphorylated in vitro by protein kinase C (PKC) previously identified two target sites, Thr264 and Ser420. We now document that liver cytosolic kinases additionally target Ser478 as a major site. To determine whether such phosphorylation is relevant to in vivo CYP3A4 degradation, wild type and CYP3A4 with single, double, or triple Ala mutations of these residues were heterologously expressed in Saccharomyces cerevisiae pep4Δ strains. We found that relative to CYP3A4wt, its S478A mutant was significantly stabilized in these yeast, and this was greatly to markedly enhanced for its S478A/T264A, S478A/S420A, and S478A/T264A/S420A double and triple mutants. Similar relative S478A/T264A/S420A mutant stabilization was also observed in HEK293T cells. To determine whether phosphorylation enhances CYP3A4 degradation by enhancing its ubiquitination, CYP3A4 ubiquitination was examined in an in vitro UBC7/gp78-reconstituted system with and without cAMP-dependent protein kinase A and PKC, two liver cytosolic kinases involved in CYP3A4 phosphorylation. cAMP-dependent protein kinase A/PKC-mediated phosphorylation of CYP3A4wt but not its S478A/T264A/S420A mutant enhanced its ubiquitination in this system. Together, these findings indicate that phosphorylation of CYP3A4 Ser478, Thr264, and Ser420 residues by cytosolic kinases is important both for its ubiquitination and proteasomal degradation and suggest a direct link between P450 phosphorylation, ubiquitination, and degradation.

CYP3A4 ubiquitination by gp78 (the tumor autocrine motility factor receptor, AMFR) and CHIP E3 ligases

Human liver CYP3A4 is an endoplasmic reticulum (ER)-anchored hemoprotein responsible for the metabolism of >50% of clinically prescribed drugs. After heterologous expression in Saccharomyces cerevisiae, it is degraded via the ubiquitin (Ub)-dependent 26S proteasomal pathway that utilizes Ubc7p/Cue1p, but none of the canonical Ub-ligases (E3s) Hrd1p/Hrd3p, Doa10p, and Rsp5p involved in ER-associated degradation (ERAD). To identify an Ub-ligase capable of ubiquitinating CYP3A4, we examined various in vitro reconstituted mammalian E3 systems, using purified and functionally characterized recombinant components. Of these, the cytosolic domain of the ER-protein gp78, also known as the tumor autocrine motility factor receptor (AMFR), an UBC7-dependent polytopic RING-finger E3, effectively ubiquitinated CYP3A4 in vitro, as did the UbcH5a-dependent cytosolic E3 CHIP. CYP3A4 immunoprecipitation coupled with anti-Ub immunoblotting analyses confirmed its ubiquitination in these reconstituted systems. Thus, both UBC7/gp78 and UbcH5a/CHIP may be involved in CYP3A4 ERAD, although their relative physiological contribution remains to be established.

Morphine metabolism revisited. II. Isolation and chemical characterization of a glutathionylmorphine adduct from rat liver microsomal preparations

Incubation of tritium-labeled morphine and cold glutathione (GSH) or cold morphine and tritiated GSH with liver microsomal preparations obtained from phenobarbital-treated rats led to the identification by high performance liquid chromatography (HPLC) of a glutathionylmorphine adduct. Liquid secondary ion mass spectral analysis established the molecular weight of the metabolite to be 590 which corresponds to the mass of a mono-GSH-morphine adduct. High resolution (360 and 500 MHz) 1H-NMR experiments have led to the tentative assignment of the structure of this metabolite as 10-alpha-S-glutathionylmorphine. Based on both in vivo and in vitro data, the formation of this product appears to be mediated by cytochrome P-450 and to involve a reactive intermediate that may be responsible for the observed covalent binding of radiolabeled morphine to proteins and, at least in part, for the morphine-induced depletion of GSH in the rat.

Morphine metabolism revisited: I. metabolic activation of morphine to a reactive species in rats

Treatment of fasted rats with relatively high doses of morphine rapidly results in depletion of hepatic glutathione (GSH) content and marked elevation of serum transaminase activity. Such morphine-induced response has been generally attributed to central nervous system mediated effects of the drug. We now report that this response might be due to a direct effect of the drug in the liver. That is, its metabolic activation to reactive electrophilic metabolite(s), by the hepatic cytochrome P-450-dependent mixed function oxidase system. Structure-activity relationships of morphine and its congeners indicate that the (-)-3-hydroxy-N- methylmorphinan moiety is linked with the potential of these opioids to deplete hepatic GSH and to raise serum transaminases in rats.