RESUMO
Inhibition of sterol-14 alpha-demethylase, a cytochrome P450 (CYP51, Erg11p), is the mode of action of azole antifungal drugs, and with high frequencies of fungal infections new agents are required. New drugs that target fungal CYP51 should not inhibit human CYP51, although selective inhibitors of the human target are also of interest as anticholesterol agents. A strain of Saccharomyces cerevisiae that was humanized with respect to the amino acids encoded at the CYP51 (ERG11) yeast locus (BY4741:huCYP51) was produced. The strain was validated with respect to gene expression, protein localization, growth characteristics, and sterol content. The MIC was determined and compared to that for the wild-type parental strain (BY4741), using clotrimazole, econazole, fluconazole, itraconazole, ketoconazole, miconazole, and voriconazole. The humanized strain showed up to >1,000-fold-reduced susceptibility to the orally active azole drugs, while the topical agents showed no difference. Data from growth kinetic measurements substantiated this finding but also revealed reduced effectiveness against the humanized strain for the topical drugs. Cellular sterol profiles reflected the decreased susceptibility of BY4741:huCYP51 and showed a smaller depletion of ergosterol and accumulation of 14 alpha-methyl-ergosta-8, 24(28)-dien-3beta-6 alpha-diol than the parental strain under the same treatment conditions. This strain provides a useful tool for initial specificity testing for new drugs targeting CYP51 and clearly differentiates azole antifungals in a side-by-side comparison.
Assuntos
Antifúngicos/farmacologia , Azóis/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Sequência de Bases , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/genética , DNA Fúngico/genética , Farmacorresistência Fúngica/genética , Farmacorresistência Fúngica/fisiologia , Genes Fúngicos , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade da Espécie , Esterol 14-DesmetilaseRESUMO
The first two steps of the steroidogenic pathway were reproduced in Saccharomyces cerevisiae. Engineering of sterol biosynthesis by disruption of the delta 22-desaturase gene and introduction of the Arabidopsis thaliana delta 7-reductase activity and coexpression of bovine side chain cleavage cytochrome P450, adrenodoxin, and adrenodoxin reductase, lead to pregnenolone biosynthesis from a simple carbon source. Following additional coexpression of human 3 beta-hydroxysteroid dehydrogenase/isomerase, pregnenolone is further metabolized to progesterone. Steroid formation appears to be coupled to yeast sterol biosynthesis.
Assuntos
Engenharia Genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Pregnenolona/biossíntese , Progesterona/biossíntese , Saccharomyces cerevisiae/genética , Adrenodoxina/genética , Adrenodoxina/metabolismo , Animais , Arabidopsis , Bovinos , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Cromatografia Gasosa , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Ferredoxina-NADP Redutase/genética , Ferredoxina-NADP Redutase/metabolismo , Galactose/metabolismo , Técnicas de Transferência de Genes , Humanos , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Progesterona Redutase/genética , Progesterona Redutase/metabolismo , Proteínas de Saccharomyces cerevisiae , Esteroide Isomerases/genética , Esteroide Isomerases/metabolismoRESUMO
The design of a family shuffling strategy (CLERY: Combinatorial Libraries Enhanced by Recombination in Yeast) associating PCR-based and in vivo recombination and expression in yeast is described. This strategy was tested using human cytochrome P450 CYP1A1 and CYP1A2 as templates, which share 74% nucleotide sequence identity. Construction of highly shuffled libraries of mosaic structures and reduction of parental gene contamination were two major goals. Library characterization involved multiprobe hybridization on DNA macro-arrays. The statistical analysis of randomly selected clones revealed a high proportion of chimeric genes (86%) and a homogeneous representation of the parental contribution among the sequences (55.8 +/- 2.5% for parental sequence 1A2). A microtiter plate screening system was designed to achieve colorimetric detection of polycyclic hydrocarbon hydroxylation by transformed yeast cells. Full sequences of five randomly picked and five functionally selected clones were analyzed. Results confirmed the shuffling efficiency and allowed calculation of the average length of sequence exchange and mutation rates. The efficient and statistically representative generation of mosaic structures by this type of family shuffling in a yeast expression system constitutes a novel and promising tool for structure-function studies and tuning enzymatic activities of multicomponent eucaryote complexes involving non-soluble enzymes.
Assuntos
Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A2/genética , DNA Recombinante/genética , Biblioteca Gênica , Reação em Cadeia da Polimerase , Recombinação Genética/genética , Saccharomyces cerevisiae/genética , Sequência de Bases , Catálise , Clonagem Molecular , Técnicas de Química Combinatória/métodos , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Sondas de DNA , Desoxirribonuclease I/metabolismo , Vetores Genéticos/genética , Humanos , Hidroxilação , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Família Multigênica/genética , Naftalenos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência do Ácido Nucleico , Estatística como Assunto , Moldes Genéticos , Transformação GenéticaRESUMO
A mammalian cell expression plasmid, pH beta-Aro, containing the human placenta aromatase complementary DNA was constructed. The prepared plasmid was used to transfect breast cancer cells (MCF-7), noncancerous breast cells (HBL-100), and Chinese hamster ovary cells by a stable expression method. While the maximum velocities for aromatase expressed in three types of cells were different (10-201 pmol of [3H2O] formed/h/mg) using [1 beta, 2 beta-3H]androst-4-ene-3,17-dione as the substrate, the apparent Michaelis-Menten constants were found to be similar (39.9-57.8 nM) and were within the range determined for the enzyme existing in human placenta. The expressed activities were inhibited by the known aromatase inhibitors, 4-hydroxyandrostenedione and aminoglutethimide, at concentrations that normally inhibit the human placental aromatase. However, it was found that the inhibition profiles were different for aromatase expressed in different types of cells, suggesting that other factors, such as the uptake of the inhibitor, may also play a role in determining the inhibition efficiency. These constructed aromatase expressing mammalian cell lines will be very useful tools for aromatase inhibitor screening.
Assuntos
Aromatase/genética , DNA/genética , Aminoglutetimida/farmacologia , Androstenodiona/análogos & derivados , Androstenodiona/farmacologia , Animais , Aromatase/metabolismo , Inibidores da Aromatase , Sequência de Bases , Southern Blotting , Cricetinae , Cricetulus , Feminino , Expressão Gênica , Humanos , Cinética , Dados de Sequência Molecular , Ovário/citologia , Placenta/enzimologia , Plasmídeos/fisiologia , Reação em Cadeia da Polimerase , Gravidez , Transfecção , Células Tumorais CultivadasRESUMO
We investigated the transcriptional regulation of cytochrome P450 1A1 (CYP1A1) gene in human lymphoblastoid B cells and report that a high inducibility of CYP1A1 gene transcription by 2,3,7,8-tetrachlorodibenzo-p-dioxin is associated with glutathione S-transferase M1 (GSTM1) null genotype, whereas the presence of at least one GSTM1 allele is correlated with induction of only low levels of CYP1A1 mRNA by 2,3,7,8-tetrachlorodibenzo-p-dioxin. These data underline the major importance of the CYP1A1 inducibility phenotype associated with the homozygous GSTM1 null genotype in chemically induced cancers.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Regulação Enzimológica da Expressão Gênica , Glutationa Transferase/genética , Dibenzodioxinas Policloradas/farmacologia , Sequência de Bases , Linhagem Celular Transformada , Sistema Enzimático do Citocromo P-450/genética , Primers do DNA/química , Indução Enzimática/efeitos dos fármacos , Genótipo , Humanos , Linfoma de Células B/enzimologia , Dados de Sequência Molecular , Células Tumorais CultivadasRESUMO
Highly controlled supramolecular assemblies combining a genetically engineered redox protein, cytochrome b5, and modified oligonucleotides are presented. Modified b5 and DNA are covalently assembled through a hetero bifunctional cross-linker to give a unique hybrid molecular species. Moreover, the assembly includes a histidine tag head able to bind to modified phospholipids which lead to a new generation of self-assembled dynamic DNA chips. The interaction of the construction with a complementary oligonucleotide sequence can be monitored in real time by surface plasmon resonance using Biacore technology. The biochip, presented herein, features unique properties including tunable surface density of probes, very low non-specific interactions and optimization of hybridization efficiency. In addition, we demonstrated that the phase transition of the lipidic layer can modulate the dynamic of the association of the complex to the supported membrane. Potential applications of this new device are multiple including high sensitivity and high selectivity biochips, especially for studies of the DNA-ligands interactions in a biomimetic environment.
Assuntos
Citocromos b5/análise , Citocromos b5/química , DNA/análise , DNA/química , Bicamadas Lipídicas/química , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Ressonância de Plasmônio de Superfície/métodos , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/química , Substâncias Macromoleculares/análise , Substâncias Macromoleculares/química , Oxirredução , Fosfolipídeos/química , Ligação Proteica , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
A full-length human placental aromatase cDNA clone, Aro 2, was isolated upon screening a human placental cDNA library with an aromatase cDNA probe and an oligonucleotide probe whose sequence was derived from a human aromatase genomic clone. Nucleotide sequence microheterogeneity was found in the 3'-untranslated region among Aro 2 and in two previously described human aromatase cDNA clones. Both the minor sequence differences and the expression of a single protein species in placental tissue suggest the presence of different alleles for aromatase. Northern blot analyses using one cDNA and two oligonucleotide probes are consistent with the two mRNA messages of 2.9 and 2.5 kilobases arising in human placenta as a consequence of differential processing. Several yeast expression plasmids containing the aromatase cDNA we cloned were constructed. The enzyme was expressed in Saccharomyces cerevisiae. The expressed activity was inhibited by the known aromatase inhibitor, 4-hydroxyandrostenedione. A level of 2 micrograms aromatase/mg partially purified yeast microsomes was estimated by analyses of carbon monoxide difference spectra on microsomal fractions from yeast carrying plasmid pHARK/VGAL. Using [1 beta, 2 beta-3H]androst-4-ene-3,17-dione as the substrate, an apparent Michaels-Menken constant (Km) of 34 nM and a maximum velocity (Vmax) of 23 pmol [3H]water formed per min/mg protein were obtained for the yeast synthesized aromatase by transformation with plasmid pHARK/VGAL. The kinetic results are similar to those determined for human placental aromatase, and suggest that the yeast synthesized aromatase will be useful for further structure-function studies.
Assuntos
Aromatase/genética , Expressão Gênica , Placenta/enzimologia , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/genética , Redutases do Citocromo/fisiologia , Sondas de DNA , Vetores Genéticos , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Plasmídeos , Homologia de Sequência do Ácido Nucleico , TransfecçãoRESUMO
Cell engineering technology using recombinant microorganisms has created new opportunities in the development of innovative drugs. This article presents the use of living genetically engineered microorganisms, such as bacteria or yeasts, as a new delivery vehicle to the gastrointestinal tract. This 'biodrug' concept was demonstrated using recombinant Saccharomyces cerevisiae expressing the plant cytochrome P450 73A1. This enzyme provides a relevant model for potential therapeutic applications, such as 'biodetoxication' in the digestive environment. An artificial gastrointestinal tract simulating human digestion was chosen as a powerful tool to validate the biodrug concept. This approach offers a novel strategy for drug discovery and testing.
Assuntos
Terapia Biológica/tendências , Terapia Genética/tendências , Órgãos Artificiais , Sistema Digestório , Humanos , Lactobacillaceae , Saccharomyces cerevisiaeRESUMO
P450 S12, an engineered human P450 1A2 containing the 88-first amino-acids of the P450 1A1, demonstrates particularly high expression level in yeast while exhibiting catalytic properties very similar to the moderately expressed natural human P450 1A2. To facilitate P450 purification by nickel chelate chromatography, C-terminal extensions including histidine tags were tested. The -G(H)4 extension was found to be particularly efficient for permitting high expression levels without any catalytic alteration. This engineered P450 was purified to electrophoretic homogeneity (18 nmol/mg of protein) at a very high yield (87%) without any detectable formation of P420. P450 S12 activities were reconstituted in the presence of yeast and Arabidopsis thaliana (ATR1) NADPH-P450 reductases. The plant reductase supported better ethoxyresorufin-, methoxyresorufin- and phenacetin-O-dealkylase activities than the yeast reductase in reconstituted systems. Interestingly, polyclonal antibodies raised against purified P450 S12 selectively recognized in Western blot and fully immuno-inhibited the natural or recombinant P450 1A2 with very limited or no cross-reaction with P450 1A1 and other isoenzymes.
Assuntos
Anticorpos/imunologia , Citocromo P-450 CYP1A2/isolamento & purificação , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Citocromo P-450 CYP1A2/genética , Inibidores do Citocromo P-450 CYP1A2 , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Saccharomyces cerevisiae/genéticaRESUMO
The metabolism of benzo[a]pyrene (B[a]P) and its proximate mutagen B[a]P-7,8-dihydrodiol (7,8-diol) was investigated in the presence of human microsomal epoxide hydrolase and P450 1A1, 1A2, 2C8, 2C9, 2C18, 2C19, 2D6 and 3A4 expressed in the yeast Saccharomyces cerevisiae. P450 1A1 had the highest turnover numbers for the formation of all B[a]P metabolites, including phenols and dihydrodiols. P450 1A2, 2C8, 2C9, 2C18, 2C19 and 3A4, which are well represented in the liver, gave rise to the formation of appreciable amounts of 3-hydroxy-B[a]P and of some dihydrodiols from B[a]P. When 7,8-diol was used as substrate, P450 1A1 also exhibited the highest turnover numbers for the formation of tetrols, the hydrolysis products of the diolepoxides, whereas P450 1A2, 2C8, 2C19 and 3A4 showed moderate activities. In order to test the validity of the yeast system, the contribution of each P450 isoform to B[a]P and 7,8-diol metabolism was evaluated as the product of the turnover numbers of recombinant P450s by specific contents of each P450 in human liver microsomes. Calculated formation rates for each B[a]P and 7,8-diol metabolite globally matched experimental values. There is evidence that P450 3A4 and 2C9 play a major role in the formation of 3-hydroxy-B[a]P from B[a]P. Accumulation of the proximate mutagen 7,8-diol was predicted to be mainly driven by P450 1A2, 2C9 and 2C19, while formation of the genotoxic diolepoxides from 7,8-diol appeared to be dependent on P450 1A2 and 3A4 in the liver.
Assuntos
Benzo(a)pireno/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Di-Hidroxi-Di-Hidrobenzopirenos/metabolismo , Isoenzimas/metabolismo , Microssomos Hepáticos/enzimologia , Saccharomyces cerevisiae/metabolismo , Clonagem Molecular , Primers do DNA , Epóxido Hidrolases/metabolismo , Humanos , Cinética , Reação em Cadeia da Polimerase , Proteínas Recombinantes/metabolismoRESUMO
Mouse liver cytochrome P-450 P1 was produced in the yeast Saccharomyces cerevisiae transformed by various expression vectors. The relative efficiency of the phosphoglycerate kinase and GAL10-CYC1 promoters to direct the P-450 P1 mRNA synthesis was determined. The level of protein synthesis was found to be dependent on the amount of the 5'-noncoding sequence of the original cDNA removed during the construction. Yeast-synthesised P-450 P1 was found to be integrated into the microsomal membrane in a fully functional form, as judged by Western blotting, optical spectra and enzymatic activities. The amount of P-450 reached up to 0.6% of the microsomal protein level. A nucleotide sequence coding for a chimeric enzyme in which 40 N-terminal codons of P-450 P1 were replaced by 36 N-terminal codons of P-450 P3 was constructed and expressed in yeast. The resulting protein retained full P-450 P1 activity and was produced with a similar efficiency suggesting that the P-450 N-terminal sequence is not involved in structures critical for the substrate specificities of the P1 isoenzyme.
Assuntos
Quimera , Sistema Enzimático do Citocromo P-450/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Animais , Sistema Enzimático do Citocromo P-450/biossíntese , Escherichia coli/genética , Regulação da Expressão Gênica , Vetores Genéticos , Camundongos , Microssomos Hepáticos/enzimologia , Dados de Sequência Molecular , Plasmídeos , Regiões Promotoras Genéticas , Biossíntese de Proteínas , RNA Mensageiro/genética , Saccharomyces cerevisiae/metabolismoRESUMO
We have constructed, in the yeast Saccharomyces cerevisiae, a mosaic assembly of genes by in vivo recombination of partially homologous sequences. The approach was tested on cDNAs encoding functionally distinct mammalian cytochromes P-450 (P-450). The selection for recombinant cDNAs used the transformation of yeast cells, which required the recircularization of a linearized plasmid by recombination of two partially homologous cDNAs. Libraries of mosaic genes with bipartite or tripartite structures were generated by intramolecular and intermolecular recombination events. The presence of yeast promoter and terminator sequences on the flanking sides of the recombined cDNAs has allowed the synthesis of encoded mosaic proteins. A library of yeast clones producing recombinant mouse P-450 P1 and rabbit P-450 LM4 was screened using functional criteria to identify chimeras with shuffled substrate specificity. Restriction mapping of mosaic genes, biochemical analysis of the synthesized proteins, comparison of chimeric enzymes, and the alignment of sequences with bacterial P-450 camphor hydroxylase of known three-dimensional structure, all suggest that the P-450 P1 amino acid residues 203-238 play a major role in the control of cytochrome activity toward carcinogenic polycyclic aromatic hydrocarbons. Similar approaches to structure-function analysis are believed to be applicable to other protein families.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , DNA Recombinante/análise , Engenharia de Proteínas/métodos , Saccharomyces cerevisiae/genética , Expressão Gênica , Biblioteca Gênica , Mosaicismo , Plasmídeos , Transformação GenéticaRESUMO
Cytochrome P-450 (Cyp) 51 or lanosterol-C14-demethylase is the main target for antifungal compounds of the triazole family like ketoconazole (Kz). Disruption of the associated NADPH-P-450 reductase-encoding gene (YRED) is not lethal, but decreases by about 20-fold the Kz resistance (KzR) of wild-type (wt) Saccharomyces cerevisiae. Transformation of a YRED-disrupted strain by a yeast genomic library based on a multicopy vector allowed us to identify a suppressor of Kz hypersensitivity. Deletion analysis of the 5-kb cloned fragment indicated that yeast cytochrome b5-encoding gene (CYB5), which encodes a 120-amino-acid (aa) protein, is required and sufficient for the suppressor effect. The encoded polypeptide shares about 30% aa identity with mammalian cytochromes b5 (Cyb5). CYB5 disruption and tetrad analysis demonstrate that yeast Cyb5 is not required for growth in a Yred+ strain. Determination of the microsomal content of b-type cytochromes by differential spectra indicated the presence of a strongly decreased or null Cyb5 level in the disrupted strain. This confirms that we have cloned the gene encoding the major microsomal form of Cyb5 which appears not to be essential. Minor Cyb5 isoforms could also be present in yeast or other redox proteins could substitute for the pleiotropic roles of Cyb5 in the sterol and lipid biosynthesis pathways.
Assuntos
Citocromos b5/genética , Cetoconazol/farmacologia , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Fúngico , Resistência Microbiana a Medicamentos/genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Fenótipo , Mapeamento por Restrição , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Homologia de Sequência de Aminoácidos , Transformação GenéticaRESUMO
We have engineered yeast genomic DNA to construct a set of strains producing various relative amounts of yeast NADPH-P450 reductase (Yred) and human cytochrome b5 (Hb5). Expression of cDNAs encoding human P450 1A1, 1A2, 3A4, 19A and mouse P450 1A1 in the different oxido-reduction backgrounds thus constituted were achieved after strain transformation by plasmid-based P450-encoding expression cassettes. The results indicate that the level of Yred strongly affects all activities tested. In contrast, the amount of Hb5 affects activities in a manner that is dependent both on the P450 isoform considered and the Yred level. In a strain containing optimized amounts of Hb5 and Yred, human P450 3A4-specific testosterone-6 beta-hydroxylase activity can be enhanced as much as 73-fold in comparison with the activity observed in a wild-type strain. Bioconversion of sterols or xenobiotics was easily achieved in vivo using this new co-expression system.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Citocromos b5/genética , Citocromos b5/fisiologia , Regulação Enzimológica da Expressão Gênica/genética , NADPH-Ferri-Hemoproteína Redutase/genética , NADPH-Ferri-Hemoproteína Redutase/fisiologia , Saccharomyces cerevisiae/enzimologia , Animais , Biotransformação , Regulação Fúngica da Expressão Gênica , Engenharia Genética/métodos , Humanos , Camundongos , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genéticaRESUMO
Cytochrome P450 IA2, a liver-specific member of the 3-methylcholanthrene-inducible family, is never detected in established cell lines. With the aim of isolating cells stably producing this protein, we have used rat and mouse hepatoma cells as recipients in transfection experiments involving rabbit cytochrome P450 IA2 cDNA. We report here the isolation of five hepatoma cell clones expressing functional P450 IA2. The level of expression is comparable to that found in COS cells transiently transformed by other P450 cDNAs. It ranges between 0.4 and 1.6 pmol P450 IA2/mg total cell protein.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Animais , Southern Blotting , Células Clonais , Regulação Enzimológica da Expressão Gênica , Fígado/enzimologia , Neoplasias Hepáticas Experimentais , Camundongos , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Coelhos , Ratos , Especificidade da Espécie , Transfecção , Células Tumorais CultivadasRESUMO
Human monoamine oxidases A and B were expressed under the control of a galactose inducible promoter in Saccharomyces cerevisiae. The two MAO isoenzymes were found located in the yeast mitochondrial outer membrane, probably in different orientations as suggested by controlled proteolysis experiments. A high level of both human MAO-A or -B activities is measured in intact mitochondria without the need for any detergent solubilisation step. The substrate and inhibitor selectivities of the membrane-bound MAOs are highly similar to those of purified human enzymes. The level of MAO-B activity, however, is selectively lowered when bound to the membrane.
Assuntos
Membranas Intracelulares/enzimologia , Mitocôndrias/enzimologia , Monoaminoxidase/metabolismo , Saccharomyces cerevisiae/genética , Sequência de Bases , Clonagem Molecular , Clorgilina/farmacologia , DNA , Humanos , Cinética , Dados de Sequência Molecular , Monoaminoxidase/genética , Pargilina/farmacologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/ultraestrutura , Especificidade por SubstratoRESUMO
Cytochrome P-450s constitute a superfamily of mono-oxygenases which require the association with specific redox enzymes bound to the endoplasmic reticulum membrane for their activity. Conditions for the functional expression of these mammalian enzymes in yeast cells and the respective merits and limitations of currently used P-450 expression systems, are considered. The dependence of the mouse P-450 IA1 specific activity on the cytochrome expression level in yeast microsomes is studied and results demonstrate that the low amounts of endogenous NADPH-cytochrome P-450 reductase and cytochrome b5 which are naturally present, are limiting for the heterologous monooxygenase activities. The sequences encoding human liver cytochrome b5, the native and a modified form of the yeast NADPH-cytochrome P-450 reductase were cloned by making use of PCR techniques, over-expressed in yeast as functional forms, and characterized. New vectors allowing a high level of mammalian P-450 expression upon induction were also constructed and tested. A strategy for the construction of a co-expression system allowing maximal activity of mammalian cytochrome P-450s is discussed.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Oxigenases/genética , Animais , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/metabolismo , Citocromos b5/genética , Citocromos b5/metabolismo , Expressão Gênica , Humanos , NADPH-Ferri-Hemoproteína Redutase/genética , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Oxigenases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Here we describe a DNA-chip-based method for high-throughput sequence mapping. This involves competitive hybridization between short and differentially labeled fluorescent oligonucleotide probes and glass-supported PCR products. Competition between an excess of oligonucleotide probes targeting the same sequence segment improves sequence discrimination and reduces sensitivity to experimental conditions such as probe concentrations, hybridization, and washing temperatures and durations. The method was found to be particularly adapted to sequence mapping of combinatorial libraries obtained by DNA shuffling between members of a gene family. We present an application of this technique for the characterization of recombination biases in combinatorial libraries used in directed evolution.
Assuntos
Análise de Sequência com Séries de Oligonucleotídeos/métodos , Sequência de Bases , Biotecnologia , Mapeamento Cromossômico/métodos , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A2/genética , DNA Bacteriano/genética , Evolução Molecular Direcionada , Escherichia coli/genética , Biblioteca Gênica , Genes Bacterianos , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos/estatística & dados numéricos , Sondas de Oligonucleotídeos/genética , Plasmídeos/genética , Sensibilidade e EspecificidadeRESUMO
In order to investigate the expression of five different forms of microsomal cytochrome P-450 including P-450 2 (P450IIBI), 3b (P450IIC3), 3c (P450IIIA4), 4 and 6 (P450IA2 and A1), hepatocytes were isolated from untreated rabbit and maintained in primary monolayer cultures in serum free modified Waymouth medium in the absence and in the presence of various classes of inducers including phenobarbital (PB), rifampicin (RIF), dexamethasone (DEX) and B-naphthoflavone (BNF). In untreated cultures the level of the various forms of P-450, determined by immunoblot with the use of specific antibodies, generally declined with time but at markedly different rates. In cultures treated with the inducers decline of the various forms was either unaffected, reduced, or even reversed, so that 96 hr after plating some of these forms appeared to be induced several-fold with respect to the untreated cultures. The forms 2 and 3c were co-induced by PB, RIF or DEX; as in vivo, BNF induced forms 4 and 6. Induction of forms 2, 3c, 4 and 6 was accompanied by stimulation of related monooxygenase activities, benzphetamine demethylase, progesterone 6B hydroxylase and benzpyrene hydroxylase and ethoxyresorufin deethylase, respectively. In all cases, induction was accompanied by an increased rate of de novo synthesis of the protein, determined by radio-immunoprecipitation assay with the use of specific antibodies on [3H]-Leu labeled cell lysate. Both induction and increased de novo synthesis were time- and inducer concentration-dependent. In cultures treated with RIF or BNF de novo synthesis of P-450 3c or of P-450 4 and 6 was correlated with the level of their specific mRNA quantitated from northern blots probed with either pLM3c-4.1 or pLM6.1, two plasmids containing inserted cDNA coding for P-450 3c or P-450 6, respectively. We conclude from these experiments that rabbit hepatocytes in primary monolayer cultures represent suitable models for studying regulation induction and pharmacological implications of the microsomal cytochromes P-450.
Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Microssomos Hepáticos/enzimologia , Animais , Benzoflavonas/farmacologia , Células Cultivadas , Sistema Enzimático do Citocromo P-450/genética , Dexametasona/farmacologia , Indução Enzimática/efeitos dos fármacos , Feminino , Masculino , Fenobarbital/farmacologia , RNA Mensageiro/metabolismo , Coelhos , Rifampina/farmacologia , beta-NaftoflavonaRESUMO
In human and murine lymphoid organs, circulating 3 beta-hydroxysteroids, including pregnenolone (PREG), dehydroepiandrosterone (DHEA), and epiandrosterone (EPIA), are 7 alpha-hydroxylated by a cytochrome P450 identified in the hippocampus as P4507B1. Mouse and human lymphoid organs produced different patterns of 3 beta-hydroxysteroid 7 alpha-hydroxylation with the absence of pregnenolone and epiandrosterone hydroxylation in human and mouse, respectively. Both 7 alpha-hydroxy-DHEA and 7 alpha-hydroxy-EPIA triggered a significant increase of antitetanus toxoid and anti-Bordetella pertussis toxins IgGs production in cultures of activated B + T cells derived from human tonsils, whereas both 7 alpha-hydroxy-PREG and 7 alpha-hydroxy-DHEA increased the immune response in mouse. Paracrine action of 7 alpha-hydroxysteroids resulted from their production in cells of the lymphoid organs. Comparison of P4507B1 sequences in rat, human, and two mouse species showed that one amino acid change might explain important differences in KM for 7 alpha-hydroxylation, and suggested that such differences might contribute to the extent of immune response.