RESUMO
Cervical cancer is caused by infections with human papillomaviruses (HPV) and genetic alternations in the cervical epithelium. While the former is well studied, the latter remains unclear. We report here that CYB5D2/Neuferricin possesses tumor suppressing activity towards cervical tumorigenesis. Ectopic expression of CYB5D2 did not affect HeLa cell proliferation and the cell's ability to form xenograft tumors, but significantly inhibited HeLa cell invasion in vitro and the cell-produced lung metastasis in NOD/SCID mice. Knockdown of CYB5D2 enhanced HeLa cell invasion. Two mutations in CYB5D2, the substitutions of arginine (R) 7 with either proline (P) or glycine (G), were reported in colon cancer. Both CYB5D2(R7P) and CYB5D2(R7G) were incapable of inhibiting HeLa cell invasion. CYB5D2 binds heme, in which aspartate (D) 86 is required. While CYB5D2(D86G) is heme-binding defective, it inhibited HeLa cell invasion. On the other hand, CYB5D2(R7P) and CYB5D2(R7G) bound heme but did not inhibit HeLa cell invasion. Collectively, CYB5D2 inhibits HeLa cell invasion independently of its heme binding. Furthermore, immunohistochemistry examination of CYB5D2 expression in 20 normal cervical tissues and 40 cervical squamous cell carcinomas (SCC) revealed a CYB5D2 reduction in 87.5% (35/40) of SCC. Analysis of CYB5D2 gene expression and genomic alteration data available from Oncomeine™ detected significant reductions of CYB5D2 mRNA in 40 SCCs and CYB5D2 gene copy number in 107 SCCs. Collectively, we provide evidence that CYB5D2 is a candidate tumor suppressor of cervical tumorigenesis.
Assuntos
Citocromos b5/biossíntese , Regulação Neoplásica da Expressão Gênica , Proteínas Supressoras de Tumor/biossíntese , Neoplasias do Colo do Útero/enzimologia , Animais , Citocromos b5/genética , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mutação , Invasividade Neoplásica , Proteínas Supressoras de Tumor/genética , Neoplasias do Colo do Útero/genéticaRESUMO
Despite all advances of heterologous expression of recombinant proteins in Escherichia coli, expression of multidomain disulphide-rich proteins faces some problems due to the absence of the possibility to monitor the process in real-time. Here we provide a CYB5-fusion system - cytochrome b5 fusion system for periplasmic expression of multimeric proteins with the possibility of process monitoring. We validated this system by Fab and scFv antibody fragments expression in order to improve antibody-derived molecules characterization and to simplify their usage. The combination of redox dependent absorbance spectrum of red-colored cytochrome b5 with its high value molar extinction coefficient allows us to monitor antibody fusion proteins localization, redox state and quantify them in reliable way in turbid solutions. Moreover, it was revealed that due to outstanding stability and solubility, cytochrome b5 significantly enhances expression level of Fab/scFv antibody fragments in Escherichia coli periplasm.
Assuntos
Citocromos b5 , Escherichia coli/metabolismo , Expressão Gênica , Periplasma/metabolismo , Anticorpos de Cadeia Única , Animais , Citocromos b5/biossíntese , Citocromos b5/química , Citocromos b5/genética , Escherichia coli/genética , Hidrocortisona/antagonistas & inibidores , Hidrocortisona/química , Periplasma/genética , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genéticaRESUMO
CYP3A4 is the most abundant cytochrome P450 in the human liver. The expression level of CYP3A4 when coexpressed with cytochrome b(5) (cyt b(5)) in Escherichia coli was 20-60% higher than that when it was expressed alone over an extended period (48-72 h). This time-dependent elevation in coexpression with cyt b(5) was a result of an increase in CYP3A4 mRNA half-life; no significant change in CYP3A4 degradation was seen in the bacterial protease fraction. These results suggest that the higher CYP3A4 levels observed upon coexpression with cyt b(5) primarily resulted from CYP3A4 mRNA stabilization by cyt b(5).
Assuntos
Citocromo P-450 CYP3A/biossíntese , Citocromos b5/biossíntese , Fígado/enzimologia , Oxirredução , Citocromo P-450 CYP3A/genética , Citocromos b5/genética , Escherichia coli , Regulação Bacteriana da Expressão Gênica , Humanos , Cinética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
OBJECTIVE: To investigate the optimal conditions of tri-expression of CYP3A4, POR and cyt b5 in Sf 9 cells. METHODS: The Sf 9 cells expressing CYP3A4, POR and cyt b5 were cultured in shaker flasks. The optimized conditions, including the temperature and rotation speed, the culture volume, the amount of surfactant and the culture time were studied. The expressed products in microsomes were used to metabolize the testosterone and their metabolic activity was determined. RESULTS: When the temperature and rotation speed of the shaker were 27 degree and 90 r/min, the cell density and culture volume were 5X105 cells/ml and 80-120 ml per 250 ml shaker flasks, respectively. When Pluronic F-68 was 0.1% and the culture time was 72 h, the condition was most suitable for culture of Sf 9 cells and expression of targeted proteins. When the ratio of the volume of three added viruses was 1:1:1, the expression condition was optimal, under which the Km, Vmax, and CLint for testosterone metabolism were 119.6 µmol/L,0.52 µmol/(min*g protein) and 4.34 ml/(min*g protein), respectively. CONCLUSION: The conditions of tri-expressing of CYP3A4, POR and cyt b5 have been optimized in the study and the product CYP3A4 is obtained with higher metabolic activity.
Assuntos
Citocromo P-450 CYP3A/biossíntese , Citocromos b5/biossíntese , NADPH-Ferri-Hemoproteína Redutase/biossíntese , Animais , Humanos , Insetos , Células Sf9RESUMO
TA (tail-anchored) proteins utilize distinct biosynthetic pathways, including TRC40 (transmembrane domain recognition complex of 40 kDa)-mediated, chaperone-dependent and/or unassisted routes to the ER (endoplasmic reticulum) membrane. We have addressed the flexibility of cytosolic components participating in these pathways, and explored the thermodynamic constraints of their membrane insertion, by exploiting recombinant forms of Sec61ß and Cytb5 (cytochrome b5) bearing covalent modifications within their TA region. In both cases, efficient membrane insertion relied on cytosolic factors capable of accommodating a surprising range of covalent modifications to the TA region. For Sec61ß, we found that both SGTA (small glutamine-rich tetratricopeptide repeat-containing protein α) and TRC40 can bind this substrate with a singly PEGylated TA region. However, by introducing two PEG [poly(ethylene glycol)] moieties, TRC40 binding can be prevented, resulting in a block of subsequent membrane integration. Although TRC40 can bind Sec61ß polypeptides singly PEGylated at different locations, membrane insertion is more sensitive to the precise location of PEG attachment. Modelling and experimentation indicate that this post-TRC40 effect results from an increased energetic cost of inserting different PEGylated TA regions into the lipid bilayer. We therefore propose that the membrane integration of TA proteins delivered via TRC40 is strongly dependent upon underlying thermodynamics, and speculate that their insertion is via a phospholipid-mediated process.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/biossíntese , Animais , Proteínas de Transporte/metabolismo , Citocromos b5/biossíntese , Citosol/metabolismo , Proteínas de Drosophila/biossíntese , Humanos , Chaperonas Moleculares , Polietilenoglicóis/metabolismo , Ligação Proteica , Transporte Proteico , Proteínas Recombinantes/metabolismo , Canais de Translocação SECRESUMO
Catalytic and immunochemical activities of cytochrome P450 (CYP) isoforms were investigated in argemone alkaloid, sanguinarine (SAN) intoxicated rats, pre-treated with different CYP inducers. SAN treated control (CON) and ethanol (ET), 3- methylcholantherene (MC) or dexamethasone (DEX) pre-exposed rats, resulted in 48, 64, 47 and 33% decrease in CYP content. SAN exposure to CON, and DEX, MC or ET pre-treated animals caused a decrease (22-37%) in glutathione-S-transferase (GST) activity, however, quinone reductase (QR) activity decreased (26-45%) in the MC pre-exposed group. Similarly, western-blot analysis of hepatic CYP1A1 and CYP1A2 showed a decrease (27-37%) in MC pre-treated SAN exposed animals. Further, a decrease in mortality in the SAN+MC (25%) group compared to SAN treated animals was also observed. The results suggest that inhibition of CYP 1A1, 1A2, 2D1, 2E1, 3A1, and Phase II enzymes by SAN augments its toxicity, whereas attenuation of SAN toxicity by MC may be due to removal of parent compound/metabolites from the body.
Assuntos
Benzofenantridinas/toxicidade , Sistema Enzimático do Citocromo P-450/metabolismo , Inativação Metabólica/genética , Isoquinolinas/toxicidade , Animais , Argemone/química , Benzofenantridinas/análise , Benzofenantridinas/isolamento & purificação , Sistema Enzimático do Citocromo P-450/biossíntese , Citocromos b5/biossíntese , Indução Enzimática , Glutationa Transferase/biossíntese , Isoenzimas , Isoquinolinas/análise , Isoquinolinas/isolamento & purificação , Masculino , Microssomos Hepáticos/enzimologia , NAD(P)H Desidrogenase (Quinona)/biossíntese , NADPH-Ferri-Hemoproteína Redutase/biossíntese , Óleos de Plantas/química , Ratos , Ratos WistarRESUMO
The human adrenal reticularis produces the so-called adrenal androgens, dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEA-S). As opposed to the cortisol and aldosterone little is known regarding the mechanisms that regulate the production of the adrenal androgens. Several recent studies have shown that type II 3beta-hydroxysteroid dehydrogenase (HSD3B2), cytochrome b5 (CYB5), and steroid sulfotransferase (SULT2A1) play an important role in the regulation of adrenal androgen production. Specifically, adrenal production of DHEA-S is correlated with reticularis expression of SULT2A1 and CYB5. In contrast, HSD3B2 has an inverse correlation with adrenal androgen production likely due to its unique ability to remove precursors from the pathway leading to DHEA. Therefore, its expression is limited to the adrenal glomerulosa/fasciculata but not in reticularis. The differential expression of these three proteins appears to be critical for reticularis function. In this review, we focus on studies that have begun to define the mechanisms regulating the transcription of these genes. Understanding the mechanisms controlling differential expression of these proteins should provide novel information about the human adrenal reticularis and its production of DHEA and DHEA-S.
Assuntos
Glândulas Suprarrenais/metabolismo , Androgênios/biossíntese , Citocromos b5/biossíntese , Desidroepiandrosterona/biossíntese , Sulfato de Desidroepiandrosterona/metabolismo , Fator de Transcrição GATA6/fisiologia , Humanos , Progesterona Redutase/biossíntese , Receptores de Estrogênio/fisiologia , Esteroide 17-alfa-Hidroxilase/fisiologia , Fator Esteroidogênico 1/fisiologia , Sulfotransferases/biossíntese , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
A specialized vector backbone from the Protein Structure Initiative was used to express full-length human cytochrome b5 as a C-terminal fusion to His8-maltose binding protein in Escherichia coli. The fusion protein could be completely cleaved by tobacco etch virus protease, and a yield of approximately 18 mg of purified full-length human cytochrome b5 per liter of culture medium was obtained (2.3mg per g of wet weight bacterial cells). In situ proteolysis of the fusion protein in the presence of chemically defined synthetic liposomes allowed facile spontaneous delivery of the functional peripheral membrane protein into a defined membrane environment without prior exposure to detergents or other lipids. The utility of this approach as a delivery method for production and incorporation of monotopic (peripheral) membrane proteins is discussed.
Assuntos
Citocromos b5/biossíntese , Lipossomos/metabolismo , Proteínas de Transporte/genética , Clonagem Molecular/métodos , Endopeptidases/metabolismo , Vetores Genéticos , Heme/metabolismo , Humanos , Proteínas Ligantes de Maltose , Proteínas Recombinantes de Fusão/biossínteseRESUMO
We investigated the direct constitution of membrane proteins into giant liposomes in cell-free (in vitro) protein synthesis. Giant liposomes were present in a translation reaction cocktail of a wheat germ cell-free protein translation system. Apo cytochrome b(5) (b5) and its fusion proteins were synthesized and directly localized in the liposomes. After the translation reaction, the proteo-liposomes were isolated by simplified discontinuous density-gradient centrifugation. Apo cytochrome b(5) conjugated dihydrofolate reductase (DHFR) was synthesized in the same procedure and the protein was directly displayed on the liposome surface. b5 acts as a "hydrophobic tag" for recruitment to the liposome surface.
Assuntos
Citocromos b5/biossíntese , Lipossomos/metabolismo , Biossíntese de Proteínas , Proteínas/metabolismo , Eletroforese em Gel de Poliacrilamida , Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência , Tetra-Hidrofolato Desidrogenase/metabolismo , Fatores de TempoRESUMO
Sex steroid synthesis requires the 17,20 lyase activity of P450c17, which is enhanced by cytochrome b5, acting as an allosteric factor to promote association of P450c17 with its electron donor, P450 oxidoreductase. Cytochrome b5 is preferentially expressed in the fetal adrenal and postadrenarchal adrenal zona reticularis; the basis of this tissue-specific, developmentally regulated transcription of the b5 gene is unknown. We found b5 expression in all cell lines tested, including human adrenal NCI-H295A cells, where its mRNA is reduced by cAMP and phorbol ester. Multiple sites, between -83 and -122 bp upstream from the first ATG, initiate transcription. Deletional mutagenesis localized all detectable promoter activity within -327/+15, and deoxyribonuclease I footprinting identified protein binding at -72/-107 and -157/-197. DNA segments -65/-40, -114/-70 and -270/-245 fused to TK32/Luc yielded significant activity, and mutations in their Sp sites abolished that activity; electrophoretic mobility shift assay (EMSA) showed that Sp3, but not Sp1, binds to these Sp sites. Nuclear factor 1 (NF-1) and GATA-6, but not GATA-4 bind to the NF-1 and GATA sites in -157/-197. In Drosophila S2 cells, Sp3 increased -327/Luc activity 58-fold, but Sp1 and NF-1 isoforms were inactive. Mutating the three Sp sites ablated activity without or with cotransfection of Sp1/Sp3. In NCI-H295A cells, mutating the three Sp sites reduced activity to 39%; mutating the Sp, GATA, and NF-1 sites abolished activity. In JEG-3 cells, GATA-4 was inactive, GATA-6 augmented -327/Luc activity to 231% over the control, and steroidogenic factor 1 augmented activity to 655% over the control; these activities required the Sp and NF-1 sites. Transcription of cytochrome b5 shares many features with the regulation of P450c17, whose activity it enhances.
Assuntos
Glândulas Suprarrenais/citologia , Citocromos b5/biossíntese , Citocromos b5/genética , Fator de Transcrição GATA6/metabolismo , Proteínas de Homeodomínio/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Sítios de Ligação , Northern Blotting , Linhagem Celular , Linhagem Celular Tumoral , Citocromos b5/metabolismo , DNA/química , DNA/metabolismo , DNA Complementar/metabolismo , Desoxirribonuclease I/metabolismo , Drosophila , Deleção de Genes , Genes Reporter , Células HeLa , Humanos , Luciferases/metabolismo , Dados de Sequência Molecular , Mutagênese , Mutagênese Sítio-Dirigida , Mutação , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Oligonucleotídeos/química , Ésteres de Forbol/metabolismo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Ligação Proteica , Isoformas de Proteínas , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleases/metabolismo , Fator Esteroidogênico 1 , Ativação Transcricional , TransfecçãoRESUMO
The structure-function relationships of the 43-amino-acid membrane-binding domain of cytochrome b5 have been examined in two mutant forms of the protein. In one mutant, two tryptophans in the membrane-binding domain, at positions 108 and 112, were replaced by leucines, and in the second mutant, in addition, aspartic acid 103 was also replaced by leucine. The fluorescence emission spectra of the three proteins and their degree of quenching by brominated lipids indicate that the mutations are not producing major conformational changes or allowing a deeper degree of penetration of the domain into the bilayer. The hydrophobicities of the three proteins were compared, by determining strengths of self-association and membrane affinities, and it was found that the protein with two additional leucines was much less hydrophobic and the one with three additional leucines was much more hydrophobic than the native cytochrome. It appears that small changes in amino acid composition, which produce no gross changes in the structure of the membrane-binding domain, will nevertheless produce very large changes in the strengths of self- and membrane-association. These differences in self-association had profound effects on the times required for membrane-association to reach equilibrium.
Assuntos
Citocromos b5/química , Citocromos b5/metabolismo , Lipossomos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cromatografia em Gel , Citocromos b5/biossíntese , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Cinética , Fígado/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Coelhos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometria de FluorescênciaRESUMO
This chapter describes the design, practical construction, and characterization of P-DNA and their applications in building a new generation of DNA chips. P-DNAs are artificial covalent assemblies involving a histidine tag head able to bind to modified phospholipids, a core protein domain derived from cytochrome b5 by genetic engineering that features specific spectroscopic and electrochemical properties useful for detection, a synthetic linker acting as a spacer, and an oligonucleotide acting as a probe. P-DNA has the property of being able to efficiently self-associate to a supported bilayer including nickel-iminodiacetate-modified phospholipids. The construction of P-DNA and its interaction with a complementary oligonucleotide sequence can be monitored in real time by surface plasmon resonance using a Biacore system or equivalent. P-DNA chips feature unique properties including tunable surface density of probes; very low nonspecific interaction with external DNA; lateral mobility, minimizing-steric interaction; optimization of hybridization efficiency; and, potentially, recognition by multiple probes of a single target and perfectly defined and homogeneous structure, permitting high density up to a compact monolayer. Potential applications of this new device are multiple, including high-sensitivity and high-selectivity chips for DNA-DNA, DNA-RNA, or DNA-protein interactions.
Assuntos
Técnicas Biossensoriais/métodos , DNA , Proteínas , Sequência de Aminoácidos , Citocromos b5/biossíntese , Citocromos b5/genética , DNA/análise , Dados de Sequência Molecular , Biossíntese de Proteínas , Engenharia de Proteínas , Proteínas/análise , Proteínas/genética , Alinhamento de Sequência , Temperatura , Fatores de TempoRESUMO
Ketamine is a common intravenous anesthetic and a frequent drug of abuse, alone or in combination with cocaine. However, the pharmacokinetic effects of ketamine have not been fully investigated. This study determined the effects of ketamine on cytochrome P-450 (P-450)-dependent catalytic activities, protein levels, and hepatotoxicity using male Wistar rats treated with 10, 20, 40, or 80 mg/kg ketamine intraperitoneally twice daily for 4 d. Treatment with ketamine produced a dose-dependent increase of pentoxyresorufin O-dealkylation activity of liver microsomes. Treatment with 80 mg/kg ketamine resulted in 14-, 3-, and 2-fold rise in O-dealkylation of pentoxyresorufin, ethoxyresorufin, and methoxyresorufin of rat liver microsomes, respectively. The treatment produced 31% and 86% increases in 7-ethoxycoumarin O-deethylation and erythromycin N-demethylation, respectively. In addition, aniline hydroxylation activity was elevated by 62%. Protein blot analysis of liver microsomal proteins revealed that 80 mg/kg ketamine induced P-450 1A, 2B, 2E1, and 3A proteins by 2-, 13-, 2-, and 2-fold, respectively. In reversibility study, ketamine-induced pentoxyresorufin O-dealkylation, 7-ethoxycoumarin O-deethylation, erythromycin N-demethylation, and methoxyresorufin O-demethylation activities of liver microsomes prepared from rats 4 d after ketamine treatment were 75%, 48%, 29%, and 38% lower than the respective activities of liver microsomes prepared from rats 1 d after treatment. Protein blot analysis showed that ketamine-induced P-450 2B1/2 proteins also decreased in a time-dependent manner in 4 d. In hepatotoxicity study, treatment of rats with 1 ml/kg CCl4 produced a 7-fold increase in serum alanine aminotransferase activity level and a 17-fold rise in rats pretreated with 80 mg/kg ketamine for 4 d. Treatment of ICR mice with 120 mg/kg cocaine produced a 17% mortality, whereas the same dose of cocaine produced a 50% mortality in mice pretreated with ketamine. Treatment of mice with 100 mg/kg cocaine produced a 76-fold increase in serum alanine aminotransferase activity level and a 260-fold rise in mice pretreated with 80 mg/kg ketamine for 4 d. The present study shows that ketamine induces the expression of multiple forms of P-450 in rat liver microsomes and increases CCl4-induced liver toxicity and cocaine-mediated acute toxicity. Other potential pharmacological or toxicological events related to ketamine use need to be further explored.
Assuntos
Anestésicos Dissociativos/toxicidade , Sistema Enzimático do Citocromo P-450/biossíntese , Ketamina/toxicidade , Fígado/efeitos dos fármacos , Animais , Tetracloreto de Carbono/administração & dosagem , Cocaína/administração & dosagem , Citocromos b5/biossíntese , Sinergismo Farmacológico , Ketamina/administração & dosagem , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Mortalidade , NADPH-Ferri-Hemoproteína Redutase/biossíntese , Ratos , Ratos Wistar , Redução de PesoRESUMO
The developmental expressions of cytochrome b5 (b5), 17 alpha-hydroxylase/17,20-lyase cytochrome P450 (P450c17), and 3 beta-hydroxysteroid dehydrogenase were examined in primate fetal adrenals by immunocytochemistry from 50-160 d gestation. The expression of b5 was evident at 50 d in the developing fetal zone (FZ), but decreased markedly through midgestation, then increased again from 150 d to term. Similar changes in the temporal expression was observed for P450c17. Whereas P450c17 was induced largely in the transitional zone (TZ; outer-most FZ), b5 expression was strongest in FZ cells further from the capsule, although overlap between these regions involved a narrow band of cells beneath the TZ that may represent the developing zona reticularis. Thus, the induction of b5 in the FZ and of P450c17 in the TZ of the fetal adrenal late in gestation coincided temporally with the prepartum rise in dehydroepiandrosterone previously reported. These data are consistent with the proposed role of b5 in supporting 17,20-lyase activity of P450c17. However, the lack of cytochrome b5 and P450c17 expression in the FZ of the developing macaque adrenal cortex for much of the second and third trimesters distinguishes it from the mature zona reticularis seen in adult animals.
Assuntos
Córtex Suprarrenal/enzimologia , Citocromos b5/biossíntese , Desenvolvimento Embrionário e Fetal/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação Enzimológica da Expressão Gênica/genética , Córtex Suprarrenal/embriologia , Androgênios/biossíntese , Animais , Citocromos b5/genética , Feminino , Idade Gestacional , Imuno-Histoquímica , Macaca mulatta , Gravidez , Esteroide 17-alfa-Hidroxilase/biossíntese , Esteroide 17-alfa-Hidroxilase/genética , Zona Reticular/crescimento & desenvolvimentoRESUMO
A cDNA clone isolated by differentially screening a cytokinin-induced haustorial cDNA library of Cuscuta reflexa was sequenced and identified as the gene coding for cytochrome b5, based on the similarity of the deduced amino-acid sequence with that of the cauliflower (60% identity) and tobacco (78% identity) proteins. The 5'-UTR is unusually long (720 bp) and contains 14 potential start codons (ATG) and 10 short ORFs.
Assuntos
Citocromos b5/biossíntese , Citocininas/farmacologia , Plantas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Brassica/genética , Galinhas , Citocromos b5/genética , DNA Complementar/química , DNA Complementar/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta , Plantas/efeitos dos fármacos , Plantas/metabolismo , Plantas Tóxicas , Homologia de Sequência de Aminoácidos , Nicotiana/genéticaRESUMO
Two isoforms of mammalian cytochrome b5, which have homologous cytosolic amino-terminal catalytic domains, are located one on endoplasmic reticulum (ER b5) the other on mitochondrial outer membranes (OM b5). A cDNA coding for the previously unknown carboxyl-terminal domain of OM b5 was cloned and a chimera between the catalytic domain of ER b5 and the carboxyl-terminal region of OM b5 was expressed in cultured mammalian cells. The chimera localized to mitochondria, indicating that the carboxyl-terminal 43 amino acids of OM b5 contain sufficient information to target the catalytic domain of ER b5 to the mitochondrial outer membrane.
Assuntos
Citocromos b5/química , Citocromos b5/metabolismo , Membranas Intracelulares/metabolismo , Mitocôndrias/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Chlorocebus aethiops , Clonagem Molecular , Citocromos b5/biossíntese , Primers do DNA , Retículo Endoplasmático/metabolismo , Rim , Fígado/metabolismo , Mamíferos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
The widely accepted catalytic cycle of cytochromes P450 (CYP) involves the electron transfer from NADPH cytochrome P450 reductase (CPR), with a potential for second electron donation from the microsomal cytochrome b5/NADH cytochrome b5 reductase system. The latter system only supported CYP reactions inefficiently. Using purified proteins including Candida albicans CYP51 and yeast NADPH cytochrome P450 reductase, cytochrome b5 and NADH cytochrome b5 reductase, we show here that fungal CYP51 mediated sterol 14alpha-demethylation can be wholly and efficiently supported by the cytochrome b5/NADH cytochrome b5 reductase electron transport system. This alternative catalytic cycle, where both the first and second electrons were donated via the NADH cytochrome b5 electron transport system, can account for the continued ergosterol production seen in yeast strains containing a disruption of the gene encoding CPR.
Assuntos
Candida albicans/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/fisiologia , Redutases do Citocromo/metabolismo , Citocromos b5/metabolismo , NADH NADPH Oxirredutases/metabolismo , Oxirredutases/metabolismo , Catálise , Clonagem Molecular , Redutases do Citocromo/biossíntese , Redutases do Citocromo/genética , Citocromo-B(5) Redutase , Citocromos b5/biossíntese , Citocromos b5/genética , Transporte de Elétrons , Cinética , NADH NADPH Oxirredutases/genética , NADPH-Ferri-Hemoproteína Redutase , Proteínas Recombinantes/metabolismo , Esterol 14-Desmetilase , Fatores de TempoRESUMO
The present study evaluates the potential of smokeless tobacco to modify the chemopreventive efficacy of minor dietary constituents, including garlic, mace or black mustard, via modulating the competing pathways of hepatic detoxication system and antioxidant defense mechanism in murine system. Garlic (100 mg/kg b.w. per day) by gavage and mace (1% w/w) or black mustard (1% w/w) in diet induced a significant increase in the levels of glutathione-S-transferase (GST), acid-soluble sulfhydryl (-SH), cytochrome b5 (Cyt.b5) and cytochrome P-450 (Cyt.P-450) in murine liver. The hepatic levels of GST and -SH were significantly depressed whereas microsomal Cyt.b5, Cyt.P-450 and MDA levels were elevated in groups treated with smokeless tobacco (50 or 100 mg/kg b.w. per day). The data revealed the inhibitory potential of smokeless tobacco on garlic-induced hepatic GST/GSH system besides the significant augmentation by smokeless tobacco on garlic or mace or black mustard-induced microsomal cytochromes. The possible implications of modulation in competing bioactivation and detoxication pathways in the process of chemical carcinogenesis are discussed.
Assuntos
Anticarcinógenos , Sistema Enzimático do Citocromo P-450/biossíntese , Citocromos b5/biossíntese , Alho , Glutationa Transferase/biossíntese , Peroxidação de Lipídeos , Fígado/enzimologia , Mostardeira , Plantas Medicinais , Plantas Tóxicas , Tabaco sem Fumaça/toxicidade , Animais , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Malondialdeído/metabolismo , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Compostos de Sulfidrila/metabolismoRESUMO
We have demonstrated previously that musk xylene, a non-mutagenic carcinogen, is a novel and specific inducer of CYP1A2 in rats (Iwata et al., Biochem Biophys Res Commun 184: 149-153, 1992). In the present study, the effects of musk xylene (50, 100 or 200 mg/kg body weight, i.p., for 5 consecutive days) on both Phase I and Phase II metabolizing enzymes in rat liver were investigated further and more completely. Among the mixed-function oxidases monitored, 7-ethoxycoumarin deethylase and 7-pentoxyresorufin depentylase activities were increased at all dose levels from 1.6- to 1.7-fold and 2.6- to 3.1-fold, respectively. Benzo[a]pyrene hydroxylase activity was increased significantly at only the 200 mg/kg dose level of musk xylene (1.5-fold). Regarding Phase II enzymes, activities of both cytosolic DT-diaphorase and glutathione S-transferase (GST) were increased up to 2.0- to 2.4-fold by musk xylene in a dose-dependent manner. Western blot analysis revealed that the changes in these activities were caused by increases in the amounts of DT-diaphorase and GST Ya subunit. Microsomal UDP-glucoronyltransferase (UDPGT) activity assayed with p-nitrophenol as substrate was increased 1.6- to 2.0-fold. These results show that musk xylene induces both Phase I cytochrome P450 mixed-function oxidase (CYP1A2 specific) and Phase II metabolizing enzyme systems (DT-diaphorase, GST Ya subunit and UDPGT) in rat liver.
Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Fígado/efeitos dos fármacos , Xilenos/farmacologia , Animais , Peso Corporal , Citocromos b5/biossíntese , Di-Hidrolipoamida Desidrogenase/biossíntese , Indução Enzimática/efeitos dos fármacos , Fígado/enzimologia , Masculino , Ratos , Ratos WistarRESUMO
On the first day of lactation, material rats were treated with a single low dose of 5 mg/kg body weight of 3,3',4,4'-tetrachlorobiphenyl (TCB) or 2,2',4,4',5,5'-hexachlorobiphenyl (HCB) or with a combination of both congeners. Lactational transfer of these polychlorinated biphenyls (PCBs) was found in neonates and significant increases in microsomal cytochrome P450, cytochrome b5 and in glutathione-S-transferase activity were observed. Treatment with HCB did not increase neonatal ethoxyresorufin-O-de-ethylation (EROD) activities whereas a more than 26-fold increase in EROD activity was noted in response to exposure to TCB. However, EROD activities were increased more than 65-fold in response to the combined exposure to TCB and HCB. Exposure via milk to TCB caused a significant reduction in the N-demethylation of aminopyrine, but the combined exposure to TCB and HCB produced a significant reduction in the N-demethylation of dimethylnitrosamine. Lactational transfer of either TCB or HCB reduced marginally peroxisomal enzyme activities; however, exposure to a combination of TCB and HCB resulted in the highly significant reduction in KCN-insensitive palmitoyl-CoA oxidation and acetyl-CoA oxidation. Contrary to the reduction of these enzyme activities, the specific concentrations of CYP4A1 were significantly increased when neonates were exposed to either TCB or HCB. The largest induction, however, was observed in response to the combined exposure to both PCBs. Evidence is presented to suggest an induction of CYP4A1 which may be independent of the molecular substitution pattern of the two PCBs used in our studies but on a possible mode of synergistic interaction.