The alternative exons 1 of the mouse aromatase cytochrome P-450 gene

Aromatase cDNA clones were isolated from cDNA libraries of mouse hypothalamus, amygdala and ovary. Analysis of the nucleotide sequences of the 5′ regions of the obtained cDNAs suggested that the mouse aromatase gene is tissue-specifically regulated by alternative exons 1. There were obvious differences between the 5′ regions of the brain and ovary aromatase cDNAs, but no difference was found between the sequences of the hypothalamus and amygdala ones. We further isolated a mouse genomic DNA clone containing brain- and ovary-specific exons 1. The brain specific exons 1 and their promoters were highly homologous in the human and mouse aromatase genes. In contrast there were several differences in the sequences among the promoter regions of the ovary-specific exons 1 of the mouse, human and rat aromatase genes, significant homology between their sequences was also observed. The present results demonstrate that expression of the mouse aromatase gene is also tissue-specifically regulated through the use of alternative exons 1 and promoters, as reported for man.     

昆虫基因启动子及细胞色素P450基因启动子研究进展

细胞色素P450是一类重要的解毒酶系。昆虫在各种内源或外源性有毒物质的胁迫下,通过调控体内细胞色素P450的过表达,对有毒化合物进行解毒代谢,从而适应不利环境。在昆虫体内,P450基因表达的调控主要发生在转录水平上,启动子作为基因的一部分,能够与RNA聚合酶结合形成转录起始复合体,进而控制基因表达的起始时间和表达程度。基于此,就昆虫启动子的分析及功能验证的主要方法、昆虫启动子的结构特征及昆虫细胞色素P450基因启动予的一些研究进展进行概述,以期为昆虫细胞色素P450基因启动子的深入研究提供借鉴。     

A three-dimensional model of aromatase cytochrome P450.

P450 hemeproteins comprise a large gene superfamily that catalyzes monooxygenase reactions in the presence of a redox partner. Because the mammalian members are, without exception, membrane-bound proteins, they have resisted structure-function analysis by means of X-ray crystallographic methods. Among P450-catalyzed reactions, the aromatase reaction that catalyzes the conversion of C19 steroids to estrogens is one of the most complex and least understood. Thus, to better understand the reaction mechanism, we have constructed a three-dimensional model of P450arom not only to examine the active site and those residues potentially involved in catalysis, but to study other important structural features such as substrate recognition and redox-partner binding, which require examination of the entire molecule (excepting the putative membrane-spanning region). This model of P450arom was built based on a "core structure" identified from the structures of the soluble, bacterial P450s (P450cam, P450terp, and P450BM-P) rather than by molecular replacement, after which the less conserved elements and loops were added in a rational fashion. Minimization and dynamic simulations were used to optimize the model and the reasonableness of the structure was evaluated. From this model we have postulated a membrane-associated hydrophobic region of aliphatic and aromatic residues involved in substrate recognition, a redox-partner binding region that may be unique compared to other P450s, as well as residues involved in active site orientation of substrates and an inhibitor of P450arom, namely vorozole. We also have proposed a scheme for the reaction mechanism in which a "threonine switch" determines whether oxygen insertion into the substrate molecule involves an oxygen radical or a peroxide intermediate.     

Three-dimensional model of cytochrome P450 human aromatase

A three-dimensional (3-D) structure of human aromatase (CYP 19) was modeled on the basis of the crystal structure of rabbit CYP2C5, the first solved X-ray structure of an eukaryotic cytochrome P450 and was evaluated by docking S-fadrozole and the steroidal competitive inhibitor (19R)-10-thiiranylestr-4-ene-3,17-dione, into the enzyme active site. According to a previous pharmacophoric hypothesis described in the literature, the cyano group of S-fadrozole partially mimics the steroid backbone C(17) carbonyl group of (19R)-10-thiiranylestr-4-ene-3,17-dione, and was oriented in a favorable position for H-bonding with the newly identified positively charged residues Lys 119 and Arg435. In addition, this model is consistent with the recent combined mutagenesis/modeling studies already published concerning the roles ofAsp309 and His480 in the aromatization of the steroid A ring.     

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.     

Three-dimensional model of cytochrome P450 human aromatase

A three-dimensional (3-D) structure of human aromatase (CYP19) was modeled on the basis of the crystal structure of rabbit CYP2C5, the first solved X-ray structure of an eukaryotic cytochrome P450 and was evaluated by docking S-fadrozole and the steroidal competitive inhibitor (19R)-10-thiiranylestr-4-ene-3,17-dione, into the enzyme active site. According to a previous pharmacophoric hypothesis described in the literature, the cyano group of S-fadrozole partially mimics the steroid backbone C(17) carbonyl group of (19R)-10-thiiranylestr-4-ene-3,17-dione, and was oriented in a favorable position for H-bonding with the newly identified positively charged residues Lys119 and Arg435. In addition, this model is consistent with the recent combined mutagenesis/modeling studies already published concerning the roles of Asp309 and His480 in the aromatization of the steroid A ring.     

Expression of a cytochrome P450 gene family in maize

Maize seedlings, like seedlings of many other plants, are rich in cytochrome P450 (P450) enzyme activity. Four P450 genes (CYPzm1–4), isolated from a seedling-specific cDNA library, are characterised by a transient and seedling-specific expression pattern. The maximum steady state mRNA levels are reached at 3 days in root and at 7 days in shoot tissue, respectively. All four genes belong to one gene family and are closely related to the CYP71 family of plant P450 genes, which includes the enzymes of the ripening avocado fruit (CYP71A1) and eggplant hypocotyls (CYP71A2, A3, A4). The expression of these related P450 genes in monocot and dicot plants indicates that these enzymes play a significant role in plants; however, the in vivo enzyme functions are unknown. The divergence of the four members of the maize gene family is sufficiently high to account for different substrate and/or reaction specificity. Although the general expression pattern of the four genes is identical, the maximum steady-state mRNA levels vary in different maize lines. In situ hybridisation reveals the highest mRNA levels in the coleoptile, the first developed leaflets, the ground tissue of the nodular complex, and in the cortex and pith of the region of cell division in the root. The mapping of the maize CYPzm genes shows that, as in animals, P450 genes of the same family can be clustered. The presence of the CYPzm gene cluster in maize argues for generation of distinct plant P450 gene families by gene duplication.     

Isolation of a full-length cDNA encoding mouse aromatase P450

A full-length cDNA clone for aromatase P450 has been isolated from a pregnant mouse ovarian cDNA library. The insert of this clone (2394 bp) contains a 1509-bp open reading frame encoding 503 amino acid residues together with a 46-bp 5'-untranslated stretch and an 839-bp 3'-untranslated region to which a poly(A) tract is attached. Northern blot analysis of ovarian RNA from pregnant mice reveals a major mRNA band of 2.5 kb with a minor band of 2.1 kb. Comparison of mouse aromatase P450 with that of rat, human, and chicken shows 91, 81, and 69% identity in the nucleotide sequence and 92, 79, and 69% identity in the deduced amino acid sequence, respectively. The membrane-spanning domain of mouse aromatase P450 is estimated to be an extremely hydrophobic segment located within the N-terminal region of the molecule. Furthermore, a highly conserved heme-binding domain is noticed.     

Expression of a full-length cDNA encoding bovine adrenal cytochrome P450C21

Two full-length cDNA clones encoding bovine adrenocortical P450C21 have been constructed in a eukaryotic expression vector using partial-length cDNAs whose structures have been previously reported. Following expression of these cDNAs in COS 1 cells, the substrate specificity of P450C21 was determined. Of the 18 steroids tested, progesterone, 17 alpha-hydroxyprogesterone, and 11 beta,17 alpha-dihydroxyprogesterone were found to be the only steroids to serve as substrates for this adrenal enzyme, a much higher degree of substrate specificity than has been reported for a hepatic 21-hydroxylase. The Vmax for 17 alpha-hydroxyprogesterone was 2.5 times greater than that for progesterone, whereas delta 5-steroids were unable to serve as substrate for this enzyme. A difference between the two cDNAs is located at amino acid 401 where one resultant enzyme contains tyrosine while the other contains histidine. This amino acid difference appears to have no effect on the kinetic properties of adrenal P450C21.     

Inhibition of human cytochrome P450 aromatase activity by butyltins

Organotin compounds are widely used as antifouling agents and bioaccumulate in the food chain. Tributyltin chloride (TBT) has been shown to induce imposex in female gastropods. On the basis of this observation it has been suggested that TBT acts as an endocrine disrupter inhibiting the conversion of androgens to estrogens mediated by the aromatase cytochrome P450 enzyme. However, to date, the molecular basis of TBT-induced imposex and in particular its putative inhibitory effects on human aromatase cytochrome P450 activity have not been investigated. Therefore, we examined the effects of the organotin compounds tetrabutyltin (TTBT), TBT, dibutyltin dichloride (DBT) and monobutyltin trichloride (MBT) on human placental aromatase activity. TBT was found to be a partial competitive inhibitor of aromatase activity with an IC(50) value of 6.2 microM with 0.1 microM androstenedione as substrate. TBT impaired the affinity of the aromatase to androstenedione but did not affect electron transfer from NADPH to aromatase via inhibiting the NADPH reductase. DBT acted as a partial but less potent inhibitor of human aromatase activity (65% residual activity), whereas TTBT and MBT had no effect. The residual activity of TBT-saturated aromatase was 37%. In contrast, human 3beta-HSD type I activity was only moderately inhibited by TBT (80% residual activity). Moreover, neither TTBT or DBT nor MBT inhibited the 3beta-HSD type I activity. Together, these results suggest that the environmental pollutants TBT and DBT, both present in marine organisms, textile and plastic products, may have specific impacts on the metabolism of sex hormones in humans.     

Evaluation of the mechanism of aromatase cytochrome P450. A site-directed mutagenesis study.

Aromatase (CYP19) catalyzes three consecutive hydroxylation reactions converting C19 androgens to aromatic C18 estrogenic steroids. In this study, five human aromatase mutants (E302D, S478A, S478T, H480K, and H480Q) were prepared using a mammalian cell expression system. These mutants were evaluated by enzyme kinetic analysis, inhibitory profile studies, and reaction intermediate measurements. Three steroidal inhibitors [4-hydroxyandrostenedione (4-OHA), 7alpha-(4'-amino)phenylthio-1,4-androstandiene-3,17-dione (7alpha-APTADD), and bridge (2,19-methyleneoxy) androstene-3,17-dione (MDL 101003)], and four nonsteroidal inhibitors [aminoglutethimide (AG), CGS 20267, ICI D1033, and vorozole (R83842)] were used in the inhibitory profile studies. Our computer model of aromatase suggests that Glu302 is situated in the conserved I-helix region and located near the C-19 position of the steroid substrate. The model was supported by significant changes in kinetic parameters and a sevenfold increase in the Ki value of MDL 101,003 for the mutant E302D. As S478A was found to have kinetic properties similar to the wild-type enzyme and a much higher activity than S478T, Ser478 is thought to be situated in a rather restricted environment. There was a 10-fold increase in the Ki value of 7alpha-APTADD for S478T over that for the wild-type enzyme, suggesting that Ser478 might be near the C-7 position of the substrate. The reaction intermediate analysis revealed that significantly more 19-ol intermediate was generated by both S478A and S478T than the wild-type enzyme. These results would support a hypothesis that Ser478 plays a role in the first and second hydroxylation reactions. A positive charged amino acid is preferred at position 480 as shown by the fact that H480K has a significantly higher activity than H480Q. The Ki value of 4-OHA for H480Q was found to be three times that of the wild-type enzyme. In addition, significantly more 19-ol and 19-al intermediates were detected for both mutants H480K and H480Q than for the wild-type enzyme. Evaluation of the two mutations at His480 allows us to propose that this residue may participate in the aromatization reaction (the third step) by acting as a hydrogen bond donor for the C-3 keto group of the substrate. Furthermore, new products were generated when the enzyme was mutated at Ser478 and His480. Thus, these two residues must play an important role in the catalysis and are likely closer to the substrate binding site than previously predicted.     

A novel human cytochrome P450 gene (P450IIB): chromosomal localization and evidence for alternative splicing.

We have isolated from a single human liver cDNA library two clones which are highly homologous (78% over the coding region) to the major phenobarbital-inducible P450 from rat (P450IIB1). This is the first direct demonstration of the presence of the P450IIB gene subfamily in humans. This subfamily is much less extensive than the rodent homologues, but does appear to contain at least two genes. Of the cDNA clones isolated one is apparently normally spliced, whereas the other lacks exon 8 and retains all or part of intron 5. Both clones contain transcribed Alu sequences. The human P450IIB gene has been located to chromosome 19q12----19q13.2 using a probe derived from intron 5, and is close to the CYP 2A locus encoding cytochrome P450IIA2. Restriction fragment length polymorphisms have been found with the enzymes BamHI and MspI which will enable linkage to be determined between these two loci.     

Differential induction and tissue-specific expression of closely related members of the phenobarbital-inducible rabbit cytochrome P-450 gene family

We have examined the tissue-specific expression of three rabbit genes that are closely related members of a subfamily of the phenobarbital-inducible cytochrome P-450 gene family. Analysis of the levels of mRNA in liver revealed that (a) cytochrome P-450PBc1 mRNA was not detectable in livers from control animals but was present in livers from animals treated with phenobarbital, (b) cytochrome P-450PBc2 was present in control tissue and was increased by about 3-fold 24 h after phenobarbital treatment, and (c) the levels of cytochrome P-450PBc3 mRNA was the same in livers from control and treated animals. In the kidney, only P-450PBc2 mRNA was detected at a level 15% of that in the liver, and the levels increased about 3-fold after phenobarbital treatment. None of the mRNAs was detected in lung tissue. Multiple species of RNA were observed that hybridized to probes for cytochrome P-450PBc1 and P-450PBc2 cDNAs by Northern blot analysis ranging in size from 2300 to 4000 nucleotides. Differential sites for polyadenylation probably cause the heterogeneity in size. A single species of RNA of 2200 nucleotides that hybridized to cytochrome P-450PBc3 cDNA probes was observed. These data demonstrate that three closely related cytochrome P-450 genes are differentially responsive to phenobarbital treatment and that they exhibit different tissue-specific patterns of expression.