Molecular aspects of brain aromatase cytochrome P450

Aromatase cytochrome P450 (P450arom) enzyme activity catalyses the conversion of androgens to estrogens in specific brain areas. During central nervous system (CNS) development local estrogen formation influences sexual differentiation of neural structures, regulates neuroendocrine functions and sexual behavior. A proposed mechanism (and re-examination) of the sexual differentiation of the rodent brain is presented. The metabolic pathway of androgen metabolism by P450arom was characterized in the medial basal hypothalamic (MBH) tissue from male rats during various prenatal and postnatal developmental intervals. The P450arom enzyme activity was determined using a saturating concentration of [³H]testosterone as the substrate, and the rates were quantified by scintillation counting. The MBH P450arom activity was highest during prenatal development (i.e. 3–6 pmol/h/mg protein), declined to moderate levels in newborns and infantile animals (approximately 1 pmol/h/mg protein) and then continued to decline to low activity rates in adult animals (approximately 80 fmol/h/mg protein). Regulation of the P450arom gene was characterized by a series of molecular biology studies where the controlling mechanism for brain P450arom was determined in MBH and amygdaloid tissue sites. Evidence for brain P450arom-specific mRNA in perinatal rats is presented as well as comparisons with rat ovary, a rat Leydig tumor cell line (R2C) and human fetal brain P450arom. Specifically, P450arom gene expression is driven in perinatal rat brain tissue by a different promoter compared to rat ovarian tissue or a R2C cell line, whereas human fetal brain tissue utilizes an almost identical promoter segment to that observed in the rodent. These findings provide an insight into the regulation of brain P450arom gene expression and suggest that there is an additional level of control for the expression of this gene during perinatal development. However, further study is necessary to understand the molecular basis of this complex developmental pattern of brain P450arom expression.     

Ligand-dependent maintenance of ethanol-inducible cytochrome P-450 in primary rat hepatocyte cell cultures

Administration of ethanol, dimethylsulphoxide, 2-propanol or imidazole to rats caused 2-7-fold increases in the level of hepatic ethanol-inducible cytochrome P-450 (P-450j), without any concomitant enhancement of corresponding mRNA. All the compounds were able to stabilize P-450j in hepatocyte cultures for at least three days, whereas P-450j mRNA rapidly disappeared from the cultures. A correlation was reached between the concentration of Me2SO, ethanol and 2-propanol necessary to maintain P-450j in the cell cultures and their binding affinities to the enzyme. It is suggested that the ligand-bound form of P-450j in the hepatocytes is protected from degradation.     

Formation of acetaldehyde adducts with ethanol-inducible P450IIE1 in vivo

Hepatic microsomes, obtained from rats pair-fed liquid diets supplemented with either ethanol or an isocaloric amount of carbohydrates (for 4 weeks), were subjected to crossed immunoelectrophoresis. Anti-acetaldehyde adduct-specific immunoglobulin reacted on the protein blots with a single major 52,000 dalton polypeptide. This same protein was recognized by antibodies specific for P450IIE1, an ethanol-inducible P450 isozyme. Furthermore, a single protein, also reactive with anti-P450IIE1 IgG, was isolated from liver microsomes of ethanol-fed rats by immunoaffinity chromatography on Sepharose-conjugated anti-acetaldehyde adduct IgG. These results indicate that P450IIE1 is a target protein for acetaldehyde binding in liver microsomes in vivo.     

Differential regulation of hepatic cytochrome P450 monooxygenases in streptozotocin-induced diabetic rats

The present investigation was carried out to study the expression of major cytochrome P450 (CYP) isozymes in streptozotocin-induced diabetes with concomitant insulin therapy. Male Sprague-Dawley rats were randomly assigned to untreated control, streptozotocin-induced diabetic, insulin-treated groups and monitored for 4 weeks. Uncontrolled hyperglycemia in the early phase of diabetes resulted in differential regulation of cytochrome P450 isozymes. CYP1B1, CYP1A2, heme oxygenase (HO)-2 proteins and CYP1A2-dependent 7-ethoxyresorufin O-deethylase (EROD) activity were upregulated in the hepatic microsomes of diabetic rats. Insulin therapy ameliorated EROD activity and the expression of CYP1A2, CYP1B1 and HO-2 proteins. In addition, CYP2B1 and 2E1 proteins were markedly induced in the diabetic group. Insulin therapy resulted in complete amelioration of CYP2E1 whereas CYP2B1 protein was partially ameliorated. By contrast, CYP2C11 protein was decreased over 99% in the diabetic group and was partially ameliorated by insulin therapy. These results demonstrate widespread alterations in the expression of CYP isozymes in diabetic rats that are ameliorated by insulin therapy.     

Molecular identity and gene expression of aldosterone synthase cytochrome P450

11Beta-hydroxylase (CYP11B1) of bovine adrenal cortex produced corticosterone as well as aldosterone from 11-deoxycorticosterone in the presence of the mitochondrial P450 electron transport system. CYP11B1s of pig, sheep, and bullfrog, when expressed in COS-7 cells, also performed corticosterone and aldosterone production. Since these CYP11B1s are present in the zonae fasciculata and reticularis as well as in the zona glomerulosa, the zonal differentiation of steroid production may occur by the action of still-unidentified factor(s) on the enzyme-catalyzed successive oxygenations at C11- and C18-positions of steroid. In contrast, two cDNAs, one encoding 11beta-hydroxylase and the other encoding aldosterone synthase (CYP11B2), were isolated from rat, mouse, hamster, guinea pig, and human adrenals. The expression of CYP11B1 gene was regulated by cyclic AMP (cAMP)-dependent signaling, whereas that of CYP11B2 gene by calcium ion-signaling as well as cAMP-signaling. Salt-inducible protein kinase, a cAMP-induced novel protein kinase, was one of the regulators of CYP11B2 gene expression.     

Molecular characterization and expression of equine testicular cytochrome P450 aromatase

We characterized testicular equine aromatase and its expression. A 2707 bp cDNA was isolated, it encoded a polypeptide of 503 residues with a deduced molecular mass of 57.8 kDa. The sequence features were those of a cytochrome P450 aromatase, with a 78% polypeptide identity with the human counterpart. The gene has a minimal length of 74 kb comprising at least 9 exons and expresses a 2.8 kb mRNA in the testis. Transient cDNA transfections in E293 cells and in vitro translations in a reticulocyte lysate system allowed aromatase protein and activity detections. The activity increased with androstenedione as substrate in a dose-dependent manner. The isolation of testicular aromatase by a new immunoaffinity method demonstrated that the protein could exist either glycosylated or not with a 2 kDa difference. All these results taken together allow new structural studies to progress in the understanding of this cytochrome P450.     

Alpha-tocopherol regulation of hepatic cytochrome P450s and ABC transporters in rats

To test the hypothesis that supra-elevated hepatic alpha-tocopherol concentrations would up-regulate mechanisms that result in increased hepatic alpha-tocopherol metabolism and excretion, rats received daily subcutaneous alpha-tocopherol injections (10 mg/100 g body wt) and then were sacrificed on Day 0 or 12 h following their previous injection on Days 3, 6, 9, 12, 15, and 18. Liver alpha-tocopherol concentrations increased from 12 +/- 1 nmol/g (mean +/- SE) to 819 +/- 74 (Day 3), decreased at Day 9 (486 +/- 67), and continued to decrease through Day 18 (338 +/- 37). alpha-Tocopherol metabolites and their intermediates increased and decreased similarly to alpha-tocopherol albeit at lower concentrations. There were no changes in known vitamin E regulatory proteins, i.e., hepatic alpha-tocopherol transfer protein or cytochrome P450 (CYP) 4F. In contrast, both CYP3A and CYP2B, key xenobiotic metabolizing enzymes, doubled by Day 6 and remained elevated, while P450 reductase increased more slowly. Consistent with the decrease in liver alpha-tocopherol concentrations, a protein involved in biliary xenobiotic excretion, p-glycoprotein, increased at Day 9, doubling by Day 15. Thus hepatic alpha-tocopherol concentrations altered hepatic proteins involved in metabolism and disposition of xenobiotic agents.     

Improved cloning and expression of cytochrome P450s and cytochrome P450 reductase in yeast

Combination of the pYeDP60 yeast expression system with a modified version of the improved uracil-excision (USER) cloning technique provides a new powerful tool for high-throughput expression of eukaryotic cytochrome P450s. The vector presented is designed to obtain an optimal 5' untranslated sequence region for yeast (Kozak consensus sequence), and has been tested to produce active P450s and NADPH-cytochrome P450 oxidoreductase (CPR) after 5' end silent codon optimization of the cDNA sequences. Expression of two plant cytochrome P450s, Sorghum bicolor CYP79A1 and CYP71E1, and S. bicolor CPR2 using the modified pYeDP60 vector in all three cases produced high amounts of active protein. High-throughput functional expression of cytochrome P450s have long been a troublesome task due to the workload involved in cloning of each individual P450 into a suitable expression vector. The redesigned yeast P450 expression vector (pYeDP60u) offers major improvements in cloning efficiency, speed, fidelity, and simplicity. The modified version of the USER cloning system provides great potential for further development of other yeast vectors, transforming these into powerful high-throughput expression vectors.     

Molecular dynamics simulations of phenylimidazole inhibitor complexes of cytochrome P450 cam

Molecular dynamics simulations have been performed on three phenylimidazole inhibitor complexes ofP450 _cam, utilizing the X-ray structures and the AMBER suite of programs. Compared to their corresponding optimized X-ray structures, very similar features were observed for the 1-phenylimidazole (1-PI) and 2-phenylimidazole (2-PI) complexes during a 100 ps MD simulation. The 1-PI inhibitor binds as a Type II complex with the imidazole nitrogen as a ligand of the heme iron. Analysis of the inhibitor-enzyme interctions during the MD simulations reveals that electrostatic interactions of the imidazole with the heme and van der Waals interactions of the phenyl ring with nearby hydrophobic residues are dominant. By contrast, 2-PI binds as a Type I inhibitor in the substrate binding pocket, but not as a ligand of the iron. The interactions of this inhibitor are qualitatively different from that of the Type II 1-PI, being mainly electrostatic/H-bonding interactions with a bound water and polar residues. Although the third compound, 4-PI, in common with 1-PI, also binds as a Type II inhibitor, with one nitrogen of the imidazole as a ligand to the iron, the MD average binding orientation deviates significantly from the X-ray structure. The most important changes observed include: (1) the rotation of the imidazole ring of this inhibitor by about 90° to enhance electrostatic interactions of the imidazole NH group with the carbonyl group of LEU244, and (2) the rotation of the carbonyl group of ASP251 to form a H-bond with VAL254. An analysis of the H-bonding network surrounding this substrate in the optimized crystal structure revealed that there is no H-bonding partner either for the free polar NH group in the imidazole ring of 4-phenylimidazole or for the polar carbonyl group of the nearby ASP251 residue. The deviation of the dynamically averaged inhibitor-enzyme structure of the 4-PI complex from the optimized crystal structure can therefore be rationalized as a consequence of the optimization of the electrostatic interactions among the polar groups.     

Molecular cloning and expression analysis of a cytochrome P450 gene in tomato

A full-length cytochrome P450 cDNA, CYP71A2, was cloned from tomato (Lycopersicon esculentum Mill.) by RT-PCR and RACE. CYP71A2 (GenBank accession no. GQ370622) encoded a single polypeptide of 495 amino acid residues and shared 46–68% of identity with CYP71A1 which associated with avocado fruit ripening. The polypeptide, which held the conserved domains in all P450s, was classified as CYP71. CYP71A2-GFP fusion protein localised in the endoplasmic reticulum. The expression of CYP71A2 was detected in all the tissues (root, leaf, stem, bud, flower, immature green fruit, mature green fruit, breaker fruit, ripe fruit); however, the CYP71A2 expression was utmost in immature green fruit. During development of fruit, the expression of CYP71A2 reduced rapidly at mature green stage, then gradually increased at breaker and ripening stages. CYP71A2 was regulated by wounding, methyl jasmonate and ethylene. Promoter analysis indicated that CYP71A2 regulatory region had all the specific responding elements to these stresses. This suggested that the role of CYP71A2 is pleiotropic in tomato development and its adaptability to the environment.     

Thermophilic cytochrome P450 enzymes

Thermophilic cytochrome P450 enzymes are of potential interest from structural, mechanistic, and biotechnological points of view. The structures and properties of two such enzymes, CYP119 and CYP175A1, have been investigated and provide the foundation for future work on thermophilic P450 enzymes.     

Moonlighting cytochrome P450 monooxygenases

Recently, cytochrome P450 170A1 (CYP170A1) has been found to be a bifunctional protein, which catalyzes both monooxygenase activity and terpene synthase activity by two distinct active sites in the well-established P450 protein structure. Therefore, CYP170A1 is identified clearly as a moonlighting protein. The known activities of a small number of the 13,000 members of the P450 superfamily fall into two general classes: promiscuous enzymes that are not considered as moonlighting and forms that participate in biosynthesis of endogenous compounds, such as steroids, vitamins and play different roles in different tissues, sometimes being moonlighting enzymes. Here, we review examples of moonlighting P450, which add to our understanding of the large CYP superfamily.     

Differential regulation of cytochrome P450 3A1 and P450 3A2 in rat liver following dexamethasone treatment

Induction of P450 3A1 and P450 3A2 was studied in adult rat liver following treatment with a single high dose of dexamethasone (DEX). The increase of both P450 3A1 and 3A2 occurred at the level of mRNA as well as protein. P450 3A isozymes thus induced were catalytically active. No constitutive expression of P450 3A1 mRNA or protein was observed in males or females. Constitutive expression of P450 3A2 mRNA and protein was observed in males but not in females. Additionally, in females, P450 3A2 was almost nondetectable compared to that in males, at any dose of DEX. A time course study following DEX treatment showed that P450 3A1 mRNA and protein were detectable in both sexes at 12 hours, increased until 48 hours, and then declined. The decline was more rapid in males. P450 3A2 mRNA and protein increased as early as 3 hours, increased further up to 48 hours, and slowly declined thereafter. A dose-response study indicated that P450 3A1 mRNA and protein progressively increased in both sexes from a dose of 30 mg/kg. In contrast, P450 3A2 mRNA and protein in males did not increase up to a dose of 30 mg/kg but increased at higher doses. Total P450 content and P450 3A catalytic activity were also found to increase with time and dose. © 1996 John Wiley & Sons, Inc.     

Expression and regulation of cytochrome P450 enzymes in primary cultures of human hepatocytes

The aim of this study was to test suitable culture conditions for maintaining normal cellular cytoarchitecture and inducibility of P450 enzymes in primary cultures of human hepatocytes by prototypical inducers. The selectivity and sensitivity of a sandwich culture system were determined by treating cultures with a number of clinically relevant drugs that are known to be inducers of either rodent and/or human P450 enzymes. The results showed that considerable induction of CYP3A4 activity is observed at DMSO concentrations greater than 0.1% (v/v). No differences in P450 induction response were observed between cultures maintained under different matrix conditions. However, the matrix condition considered to be optimal for maintaining cellular integrity, protein yields, and P450 enzyme induction was a sandwich configuration in combination with modified Chee's medium containing insulin (6.25 microg/mL) and dexamethasone (< or =0.1 microM). Under these conditions, induction of CYP3A4 occurred at clinically relevant drug concentrations, and maximal activities were achieved after 3 days of exposure. Overall, the response of human hepatocyte cultures to treatment with both positive and negative modulators was found to reflect that observed in vivo with respect to both enzyme specificity and potency of enzyme induction, although considerable sample-to-sample variability was observed in the magnitude of induction.     

The role of cytochrome P450 lysine residues in the interaction between cytochrome P450IA1 and NADPH-cytochrome P450 reductase.

Cytochrome P450IA1 (purified from hepatic microsomes of beta-naphthoflavone-treated rats) has been covalently modified with the lysine-modifying reagent acetic anhydride. Different levels of lysine residue modification in cytochrome P450IA1 can be achieved by varying the concentration of acetic anhydride. Modification of lysine residues in P450IA1 greatly inhibits the interaction of P450IA1 with NADPH-cytochrome P450 reductase. Modification of 1.0 and 3.3 mol lysine residues per mole P450IA1 resulted in 30 and 95% decreases, respectively, in 7-ethoxycoumarin hydroxylation by a reconstituted P450IA1/reductase complex. However, modification of 3.3 mol lysine residues per mole P450IA1 decreased only cumene hydroperoxide-supported P450-dependent 7-ethoxycoumarin hydroxylation by 30%. Spectral and fluorescence studies showed no indication of global conformational change of P450IA1 even with up to 8.8 mol lysine residues modified per mole P450IA1. These data suggest that at least three lysine residues in P450IA1 may be involved in the interaction with reductase. Identification of lysine residues in P450IA1 possibly involved in this interaction was carried out by [14C]acetic anhydride modification, trypsin digestion, HPLC separation, and amino acid sequencing. The lysine residue candidates identified in this manner were K97, K271, K279, and K407.