Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Cocaine N-demethylation by microsomal cytochrome P450s is the principal pathway in cocaine bioactivation and hepatotoxicity. P450 isozymes involved in N-demethylation of cocaine have not been elucidated yet and they differ from species to species. In humans and mice, P4503A contributes to cocaine N-demethylase activity, whereas in rats, both P4503A and P4502B participate. In the present study, contribution of different P450 isozymes to cocaine N-demethylase activity was studied in vitro with fish liver microsomes. The specific cocaine N-demethylase activity was found to be 0.672 +/- 0.22 nmol formaldehyde formed/min/mg protein (mean +/- SD, n = 6). Cocaine N-demethylase exhibited biphasic kinetics, and from the Lineweaver-Burk plot, two K(m) values were calculated as 0.085 and 0.205 mM for the high- and low-affinity enzyme. These results indicate that N-demethylation of cocaine in mullet liver microsomes is catalyzed by at least two cytochrome P450 isozymes. Inhibitory effects of cytochrome P450 isozyme-selective chemical inhibitors, ketoconazole, cimetidine, SKF-525A, and quinidine, on cocaine N-demethylase activity were studied at 50, 100, and 500 micro M concentrations of these inhibitors. At 100 micro M final concentrations, ketoconazole (P4503A inhibitor), SKF-525A (inhibitor of both P4502B and P4503A), and cimetidine (P4503A inhibitor) inhibited N-demethylation activity by 73, 69, and 63%, respectively. Quinidine, P4502D-specific inhibitor, at 100 micro M final concentration, reduced N-demethylation activity down to 64%. Aniline, a model substrate for P4502E1, did not alter N-demethylase activity in the final concentration of 100 micro M. IC(50) values were calculated to be 20 micro M for ketoconazole, 48 micro M for cimetidine (both specific P4503A inhibitors), 164 micro M for quinidine (P4502D inhibitor), and 59 micro M for SKF-525A (inhibitor of both P4503A and P4502B). The contribution of P4502B to cocaine N-demethylase activity in mullet liver microsomes was further explored by the use of purified mullet cytochrome P4502B in the reconstituted system containing purified mullet P450 reductase and lipid. The turnover number was calculated as 4.2 nmol HCOH/(min nmol P450). Overall, these results show that P4503A and P4502B are the major P450s responsible for N-demethylation of cocaine, whereas contribution of P4502D is a minor one, and P4502E1 is not involved in the N-demethylation of cocaine in mullet liver microsomes.
J Biochem Mol Toxicol 2003
PMID:Catalyzation of cocaine N-demethylation by cytochromes P4502B, P4503A, and P4502D in fish liver. 1281 13

The PDA1 gene of the filamentous fungus Nectria haematococca MPVI (anamorph: Fusarium solani) encodes pisatin demethylase, a cytochrome P450. Pisatin is a fungistatic isoflavonoid produced by garden pea (Pisum sativum), a host for this fungus. Pisatin demethylase detoxifies pisatin and functions as a virulence factor for this fungus. Pisatin induces PDA1 expression both in cultured mycelia as well as during pathogenesis on pea. The regulatory element within PDA1 that provides pisatin-responsive expression was identified using a combination of in vivo functional analysis and in vitro binding analysis. The 40 bp pisatin-responsive element is located 635 bp upstream of the PDA1 transcription start site. This element was sufficient to provide strong pisatin-induced expression to a minimal promoter in vivo and was required for pisatin regulation of the PDA1 promoter. A gene encoding a DNA-binding protein specific to this 40 bp element was isolated from a N. haematococca cDNA library using the yeast one-hybrid screen. The cloned gene possesses sequence motifs found in the binuclear zinc (Cys 6-Zn 2) family of transcription factors unique to fungi. The results suggest that it is a regulator of this fungal cytochrome P450 gene and may provide pisatin-responsive regulation.
Mol Microbiol 2003 Jul
PMID:A binuclear zinc transcription factor binds the host isoflavonoid-responsive element in a fungal cytochrome p450 gene responsible for detoxification. 1282 15

The downregulation of the Reelin gene (RELN) that occurs in schizophrenic brains, which are characterized by pyramidal neurons with shortened dendrites and by reduced expression densities of dendritic spines, may well result from hypermethylation of the RELN promoter. In the adult mammalian brain, gamma-aminoburytic acid-secreting (GABAergic) interneurons release RELN into the extracellular matrix, where it binds with high affinity to the integrin receptors present at dendritic spine postsynaptic densities and likely plays a role, elaborated in this article, in synaptic plasticity. In heterozygous reeler mice, which are haploinsufficient in RELN, inhibitors of histone deacetylase increase DNA demethylase activity and restore RELN expression. Such inhibitors could thus be of therapeutic value in mitigating vulnerability to schizophrenia among high-risk individuals.
Mol Interv 2002 Feb
PMID:REELIN and schizophrenia: a disease at the interface of the genome and the epigenome. 1499 61

Sterol 14 alpha-demethylase (CYP51) is the single cytochrome P450 (CYP) required for sterol biosynthesis in different phyla, and it is the most widely distributed P450 gene family being found in all biological kingdoms. It catalyzes the first step following cyclization in sterol biosynthesis such as removal of the 14 alpha-methyl group from lanosterol in the cholesterol biosynthetic pathway, leading to formation of the initial substrate in steroid hormone biosynthesis. CYP51 from different phyla have low sequence similarity across kingdoms and contain only about 40 conserved amino acid residues in the whole family. An attempt to predict the possible role of these conserved residues is being made by a combination of the results of site-directed mutagenesis and information from the known crystal structure of sterol 14 alpha-demethylase from Mycobacterium tuberculosis.
Mol Cell Endocrinol 2004 Feb 27
PMID:CYP51--the omnipotent P450. 1502 90

Sterol 14alpha-demethylase (CYP51) is a member of the cytochrome P450 superfamily, widely found in animals, fungi, and plants but present in few prokaryotic groups. CYP51 is currently believed to be the ancestral cytochrome P450 that has been transferred from prokaryotes to eukaryotic kingdoms. We propose an alternate view of CYP51 evolution that has an impact on understanding the evolution of the entire CYP superfamily. Two hundred forty-nine bacterial and four archaeal CYP sequences have been aligned and a bacterial CYP tree designed, showing a separation of two branches. Prokaryotic CYP51s cluster to the minor branch, together with other eukaryote-like CYPs. Mycobacterial and methylococcal CYP51s cluster together (100% bootstrap probability), while Streptomyces CYP51 remains on a distant branch. A CYP51 phylogenetic tree has been constructed from 44 sequences resulting in a ((plant, bacteria),(animal, fungi)) topology (100% bootstrap probability). This is in accordance with the lanosterol/cycloartenol diversification of sterol biosynthesis. The lanosterol branch (nonphotosynthetic lineage) follows the previously proposed topology of animal and fungal orthologues (100% bootstrap probability), while plant and D. discoideum CYP51s belong to the cycloartenol branch (photosynthetic lineage), all in accordance with biochemical data. Bacterial CYP51s cluster within the cycloartenol branch (69% bootstrap probability), which is indicative of a lateral gene transfer of a plant CYP51 to the methylococcal/mycobacterial progenitor, suggesting further that bacterial CYP51s are not the oldest CYP genes. Lateral gene transfer is likely far more important than hitherto thought in the development of the diversified CYP superfamily. Consequently, bacterial CYPs may represent a mixture of genes with prokaryotic and eukaryotic origin.
J Mol Evol 2004 Jul
PMID:New aspects on lanosterol 14alpha-demethylase and cytochrome P450 evolution: lanosterol/cycloartenol diversification and lateral transfer. 1538 7

The antimutagenic activity of spearmint (Mentha spicata), a popular food flavoring agent, was studied in the Salmonella assay. Spearmint leaves were brewed in hot water for 5 min at concentrations up to 5% (w/v), and the water extracts were tested against the direct-acting mutagens 4-nitro-1,2-phenylenediamine (NPD) and 2-hydroxyamino-3-methyl-3H-imidazo[4,5-f]quinoline (N-OH-IQ) using Salmonella typhimurium strain TA98. Nontoxic concentrations of spearmint extract inhibited the mutagenic activity of N-OH-IQ in a concentration-dependent fashion, but had no effect against NPD. These experiments by design focused on the water extract consumed commonly as an herbal tea, but chloroform and methanol extracts of spearmint also possessed antimutagenic activity against N-OH-IQ. Water extract of spearmint inhibited the mutagenic activity of the parent compound, 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ), in the presence of rat liver S9; however, the concentration for 50% inhibition (IC50) against IQ was approximately 10-fold higher than in assays with N-OH-IQ minus S9. At concentrations similar to those used in the Salmonella assays, spearmint extract inhibited two of the major enzymes that play a role in the metabolic activation of IQ, namely, cytochromes P4501A1 and 1A2, based on ethoxyresorufin O-deethylase and methoxyresorufin O-demethylase assays in vitro. In vivo, rats were given spearmint water extract (2%; w/v) as the sole source of drinking fluid before, during, and after 2-week treatment with IQ; colonic aberrant crypt foci were inhibited significantly at 8 weeks (P < 0.05, compared with rats given IQ alone). Collectively, these findings suggest that spearmint tea protects against IQ and possibly other heterocyclic amines through inhibition of carcinogen activation and via direct effects on the activated metabolite(s).
Environ Mol Mutagen 2004
PMID:Antimutagenic activity of spearmint. 1552 23

Sterol 14alpha-demethylase (CYP51) is one of the known major targets for azole antifungals. Therapeutic side effects of these antifungals are based on interactions of the azoles with the human analogue enzyme. This study describes for the first time a comparison of a human CYP51 (HU-CYP51) homology model with a homology model of the fungal CYP51 of Candida albicans (CA-CYP51). Both models are constructed by using the crystal structure of Mycobacterium tuberculosis MT-CYP51 (PDB code: 1EA1). The binding mode of the azole ketoconazole is investigated in molecular dynamics simulations with the GROMACS force field. The usage of special parameters for the iron azole complex binding is necessary to obtain the correct complex geometry in the active site of the enzyme models. Based on the dynamics simulations it is possible to explain the enantioselectivity of the human enzyme and also to predict the binding mode of the isomers of ketoconazole in the active site of the fungal model.
J Comput Aided Mol Des 2005 Mar
PMID:Molecular design of two sterol 14alpha-demethylase homology models and their interactions with the azole antifungals ketoconazole and bifonazole. 1605 69

Infection with the protozoan, Trypanosoma cruzi, is the cause of Chagas disease that occurs widely throughout Latin America. T. cruzi contains sterol biosynthesis enzymes, and produces sterol products similar to those found in fungi. Antifungal drugs that inhibit ergosterol biosynthesis have potent anti-T. cruzi activity in vitro and in animal models. In this report, we describe the effects of sterol biosynthesis inhibitors (simvistatin, zaragosic acid, terbinafine, a lanosterol synthase inhibitor, ketoconazole, and tridemorph) on the regulation of two sterol biosynthesis genes and their protein products. Culturing T. cruzi in the presence of the lanosterol synthase inhibitor, terbinafine, or ketoconazole increased mRNA levels of the sterol C14-demethylase gene approximately 7-12-fold. The sterol C14-demethylase protein levels were also elevated. The effects of the sterol biosynthesis inhibitors on hydroxymethylglutaryl-CoA reductase expression were minimal. Control of the upregulation of sterol C14-demethylase appears to be mediated through the 3'-untranslated region of the gene. The findings demonstrate that T. cruzi can specifically regulate gene expression in response to derangements in its cellular functions.
Mol Biochem Parasitol 2005 Nov
PMID:Upregulation of sterol C14-demethylase expression in Trypanosoma cruzi treated with sterol biosynthesis inhibitors. 1616 33

We have previously reported that carcinogenic nickel compounds decreased global histone H4 acetylation and silenced the gpt transgene in G12 Chinese hamster cells. However, the nature of this silencing is still not clear. Here, we report that nickel ion exposure increases global H3K9 mono- and dimethylation, both of which are critical marks for DNA methylation and long-term gene silencing. In contrast to the up-regulation of global H3K9 dimethylation, nickel ions decreased the expression and activity of histone H3K9 specific methyltransferase G9a. Further investigation demonstrated that nickel ions interfered with the removal of histone methylation in vivo and directly decreased the activity of a Fe(II)-2-oxoglutarate-dependent histone H3K9 demethylase in nuclear extract in vitro. These results are the first to show a histone H3K9 demethylase activity dependent on both iron and 2-oxoglutarate. Exposure to nickel ions also increased H3K9 dimethylation at the gpt locus in G12 cells and repressed the expression of the gpt transgene. An extended nickel ion exposure led to increased frequency of the gpt transgene silencing, which was readily reversed by treatment with DNA-demethylating agent 5-aza-2'-deoxycytidine. Collectively, our data strongly indicate that nickel ions induce transgene silencing by increasing histone H3K9 dimethylation, and this effect is mediated by the inhibition of H3K9 demethylation.
Mol Cell Biol 2006 May
PMID:Nickel ions increase histone H3 lysine 9 dimethylation and induce transgene silencing. 1664 69

Studies initiated to investigate the expression of cytochrome P450 2E1 (CYP2E1) in rat brain demonstrated low but detectable protein and mRNA expression in control rat brain. Though mRNA and protein expression of CYP2E1 in brain was several fold lower as compared to liver, relatively high activity of N-nitrosodimethylamine demethylase (NDMA-d) was observed in control rat brain microsomes. Like liver, pretreatment with CYP2E1 inducers such as ethanol or pyrazole or acetone significantly increased the activity of brain microsomal NDMA-d. Kinetic studies also showed an increase in the Vmax and affinity (Km) of the substrate towards the brain enzyme due to increased expression of CYP2E1 in microsomes of brain isolated from ethanol pretreated rats. In vitro studies using organic inhibitors, specific for CYP2E1 and anti-CYP2E1 significantly inhibited the brain NDMA-d activity indicating that like liver, NDMA-d activity in rat brain is catalyzed by CYP2E1. Olfactory lobes exhibited the highest CYP2E1 expression and catalytic activity in control rats. Furthermore, several fold increase in the mRNA expression and activity of CYP2E1 in cerebellum and hippocampus while a relatively small increase in the olfactory lobes and no significant change in other brain regions following ethanol pretreatment have indicated that CYP2E1 induction maybe involved in selective sensitivity of these brain areas to ethanol induced free radical damage and neuronal degeneration.
Mol Cell Biochem 2006 Jun
PMID:Expression of constitutive and inducible cytochrome P450 2E1 in rat brain. 1665 27


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