RESUMO
Bile acids (BA) are signaling molecules that activate nuclear factors and g-protein coupled receptors signaling to maintain metabolic homeostasis. However, accumulation of toxic BA promotes liver injury by initiating inflammation, inducing apoptosis and causing oxidative stress leading to cirrhosis and liver failure. Augmenter of Liver Regeneration (ALR) is a hepatotrophic growth factor with anti-apoptotic and anti-oxidative properties that has been shown to improve mitochondrial and hepatic functions in rats after bile duct ligation. In the current study we aimed to analyze the regulation of the pro-survival protein, ALR, under conditions of cytotoxic concentrations of BA. Promoter studies of ALR (-733/+527â¯bp) revealed potential binding sites for various transcription factors like Egr-1, HNF4α and two bile acid response elements (BARE). Using a full-length and several truncated promoter constructs for ALR we analyzed promoter activity and showed that BA reduce ALR promoter activity whereas Egr-1 transfection induces it. EMSA and supershift analysis confirmed the specific binding of Egr-1 to its response element within ALR promoter and this binding was reduced upon simultaneous stimulation with BA. We also showed that ALR promoter activity and protein expression are induced by HNF4α1 and repressed by SHP. In conclusion, these results indicate that BA negatively regulate ALR expression by SHP activation.
Assuntos
Ácidos e Sais Biliares/farmacologia , Redutases do Citocromo/biossíntese , Regulação da Expressão Gênica/fisiologia , Fator 4 Nuclear de Hepatócito/metabolismo , Hepatócitos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Regulação para Baixo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Células Hep G2 , Humanos , Oxirredutases atuantes sobre Doadores de Grupo EnxofreRESUMO
Human augmenter of liver regeneration (hALR) is a sulfhydryl oxidase that is highly expressed in spermatogonia and early spermatocytes. To investigate the physiological effects of hALR in spermatogenesis, we generated a hALR transgenic mouse model driven by the human TSPY (testis-specific protein, Y-encoded) promoter that allows the transgene to be specifically activated in the testes. hALR content was found to be increased in both germ cells. The histological and TUNEL analysis of transgenic testes revealed a number of spermatogenetic defects including primary spermatocyte overpopulation followed by depletion through apoptosis, degenerating and detached nucleated germ cells, haploid cell loss and intraepithelial vacuoles of varying sizes. In line with these features, adult transgenic male mice also displayed a reduction in fertility. Our data suggest that regulated spatial and temporal expression of hALR is required for normal testicular development and spermatogenesis, and overexpression of hALR results in influencing the sperm morphology and quantity and the eventual reduction in male fertility. Present findings in the mouse may be of interest to human male fertility.
Assuntos
Redutases do Citocromo/biossíntese , Infertilidade Masculina/genética , Espermatogênese/fisiologia , Animais , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Oligospermia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Espermatogênese/genética , Testículo/metabolismoRESUMO
To gain new insight into the biological function of the human augmenter of liver regeneration (hALR) in HCC, we studied its involvement in radiation-induced damage and recovery of HCC cells. We found that hALR expression was up-regulated in both HCC tissues and multiple hepatoma cell lines and correlated significantly with increased radiation clonogenic survival after radiation treatment. Exogenous expression of hALR increased radiation resistance in SMMC-7721 cells, and the increased survival was accompanied by a decrease in apoptosis and a prolonged G(2)-M arrest after irradiation. Overexpression of ALR significantly increased the mitochondrial membrane potential, inhibited cytochrome c release, and opposed the loss of intracellular ATP levels after radiation. Moreover, knockdown of ALR by siRNA resulted in inhibition of viability in the absence of exogenously added oxidative stress and radiation sensitization in HepG2 cells. Importantly, hALR expression was very low in normal hepatocyte L02 cells, and hALR silencing had a minimal effect on L02 viability and radiation sensitivity. These results suggest that human ALR is important for hepatoma cell viability and involved in the protection of hepatoma cells against irradiation-induced damage by its association with mitochondria. Targeting hALR may be a promising novel approach to enhance the radiosensitivity of hepatoma cancers.
Assuntos
Carcinoma Hepatocelular/metabolismo , Redutases do Citocromo/metabolismo , Neoplasias Hepáticas/metabolismo , Animais , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos da radiação , Redutases do Citocromo/biossíntese , Relação Dose-Resposta à Radiação , Feminino , Raios gama , Humanos , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/efeitos da radiação , Dados de Sequência Molecular , Estresse Oxidativo/efeitos da radiação , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Tolerância a Radiação , Estudos Retrospectivos , Regulação para Cima/efeitos da radiaçãoRESUMO
AIMS: To establish a highly effective prokaryotic recombinant expression system for human augmenter of liver regeneration (hALR) and to characterize the recombinant hALR both in vitro and in vivo. METHODS: ALR cDNA was synthesized and inserted into expression vector pET28a+, the recombinant plasmid was transformed into BL21, and expression of hALR was induced by IPTG. Recombinant hALR (rhALR) was purified by sequential detergent wash, enterokinase (EK) digestion, gel-filtration, and chelating chromatography. The rhALR was identified by SDS-PAGE, immunoblotting, MALDI-TOF-MS, and N-terminal sequencer. Cell proliferative effect of rhALR on human hepatocytes was analyzed by MTT. The protective effect of rhALR on liver function was observed on CCl(4)-induced intoxicated mice. RESULTS: Recombinant expression plasmid of ALR [pET28(a+)-hALR] was confirmed by restriction enzyme digestion and DNA sequencing. The expressed rhALR constituted 30% of total bacterial protein. Molecular weight was 15,029 for monomer and 30,136 for dimer by mass determination. N-terminal was M-R-T-Q-Q, exactly the same as anticipated for hALR. The purified protein migrating at about 15 KD showed excellent antigenicity in immunoblotting. The rhALR also showed a strong stimulative effect on hepatocyte proliferation. ALT and AST levels, liver histological structure, as well as the survival rate of CCl(4)-intoxicated mice were significantly improved when rALR was administrated at 40 microg/kg or 200 microg/kg. CONCLUSIONS: The rhALR is successfully expressed highly effectively with anticipated MW, N-terminal, and antigenicity. It could play an important role in relieving acute hepatic injury and hepatic failure by promoting hepatic cell proliferation and improving liver function in CCl(4)-intoxicated mice.
Assuntos
Redutases do Citocromo/biossíntese , Falência Hepática Aguda/tratamento farmacológico , Animais , Intoxicação por Tetracloreto de Carbono , Linhagem Celular , Proliferação de Células , Redutases do Citocromo/genética , Redutases do Citocromo/isolamento & purificação , Redutases do Citocromo/uso terapêutico , Escherichia coli/metabolismo , Engenharia Genética , Vetores Genéticos/metabolismo , Hepatócitos/fisiologia , Humanos , Falência Hepática Aguda/induzido quimicamente , Masculino , Camundongos , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Plasmídeos/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/uso terapêuticoRESUMO
Acute and chronic hepatic failure is a devastating illness of varied causes with considerable mortality. Human augmenter of liver regeneration (hALR) is a hepatotrophic protein and the unique cytokine which can specially stimulate hepatic origin cells to grow regardless of genus. It has been proven that ALR can promote regeneration and avoid all kinds of injury in rat and canine models. In this study, the recombinant protein hALR was expressed successfully with recombinant prokaryotic expression vector pET28a(+) in Escherichia coli BL21 (DE3). We constructed the recombinant expression vector pET28a(+)/hALR with a full-length cDNA encoding hALR protein from normal human liver tissue by one-step reverse transcription-polymerase chain reaction and his-tag recognition sequence encoding polyhistidine (6 x His). Under IPTG (isopropyl-beta-d-thiogalactopyranoside) induction for 2 h at 37 degrees C, recombinant protein hALR was expressed. The expression of recombinant polyhistidine-tagged hALR was increased under low temperature and was confirmed that the temperature of 23 degrees C was the most suitable IPTG induction condition. Under low temperature induction of IPTG, recombinant protein can be expressed as a soluble protein. Recombinant protein hALR was also purified with His Bind Kits and characterized with SDS-PAGE and Western blotting. The results showed that recombinant hALR could be expressed as a soluble protein under low temperature induction of IPTG. The successful expression of ALR in E. coli makes it possible to further study its biological function and purified recombinant hALR could be developed into a new anti-hepatic damage product.
Assuntos
Redutases do Citocromo/biossíntese , Redutases do Citocromo/genética , Escherichia coli/genética , Sequência de Bases , Clonagem Molecular , Temperatura Baixa , DNA Complementar/genética , Expressão Gênica , Humanos , Técnicas In Vitro , Isopropiltiogalactosídeo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SolubilidadeAssuntos
Redutases do Citocromo/genética , Fragmentos de Peptídeos/genética , Regiões Promotoras Genéticas/genética , Transcrição Gênica/genética , Animais , Células COS , Chlorocebus aethiops , Redutases do Citocromo/biossíntese , Humanos , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Fragmentos de Peptídeos/biossíntese , TransfecçãoRESUMO
Structural genes encoding quinol-cytochrome c reductase (QcR) were cloned and sequenced from nocardia-form actinomycete Rhodococcus rhodochrous. QcrC and qcrA encode diheme cytochrome cc and the Rieske Fe-S protein, respectively, while the qcrB product is a diheme cytochrome b. These amino acid sequences are similar to those of Corynebacterium and Mycobacterium, the members of high G+C content firmicutes. The presence of diheme cytochrome cc subunit as a sole c-type cytochrome in these organisms suggests the direct elecron transfer to cytochrome c oxidase. The N-terminal half of the Rieske Fe-S proteins of these bacteria has a unique structure with three transmembrane helices, while the C-terminal half sequence is conserved. A phylogenetic tree using the latter region showed that high G+C firmicutes form a clear clade with Thermus, but not with low G+C firmicutes.
Assuntos
Redutases do Citocromo/genética , Genes Bacterianos , Rhodococcus/genética , Sequência de Aminoácidos , Clonagem Molecular , Redutases do Citocromo/biossíntese , Redutases do Citocromo/química , Grupo dos Citocromos b/química , Grupo dos Citocromos b/genética , Complexo Citocromos b6f , Grupo dos Citocromos c/química , Grupo dos Citocromos c/genética , Dados de Sequência Molecular , Óperon , Filogenia , Rhodococcus/química , Rhodococcus/enzimologia , Alinhamento de SequênciaRESUMO
NADH:cytochrome b(5) reductase (FpD) is an enzyme capable of converting the prodrug mitomycin C (MC) into a DNA alkylating agent via reduction of its quininone moiety. In this study, Chinese hamster ovary (CHO) cells were transfected with a cDNA encoding rat FpD. Despite the demonstrated ability of this enzyme to reduce MC in vitro, a modest 5-fold level of overexpression of FpD activity in CHO cells did not increase the cytotoxicity of the drug over that seen with the parental cell line under either aerobic or hypoxic conditions. When the enzyme, which is predominantly localized in the mitochondria, was instead directed to the nucleus of cells by the fusion of the SV40 large T antigen nuclear localization signal sequence to the amino terminus of an FpD gene that lacked the membrane anchor domain, drug sensitivity was significantly enhanced at all concentrations of MC examined (2-10 microm) under both aerobic and hypoxic conditions, with greater cell kill occurring under hypoxia. The marked increase in drug sensitivity under hypoxia at 10 microm MC corresponded to a measurable increase in total MC-DNA adducts at the same concentration. The results indicate that the cytotoxicity of MC is modulated by the subcellular location of FpD, with greater cell kill occurring when bioactivation occurs in the proximity of its target, nuclear DNA.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Redutases do Citocromo/genética , Adutos de DNA/genética , Mitomicina/farmacologia , Animais , Células CHO , Morte Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Cricetinae , Redutases do Citocromo/biossíntese , Citocromo-B(5) Redutase , Adutos de DNA/efeitos dos fármacos , Resistência a Medicamentos/genética , Regulação Enzimológica da Expressão Gênica , RatosRESUMO
The gene coding for expression of an endogenous soluble fusion protein comprising a b-type cytochrome-containing domain and a FAD-containing domain has been cloned from rat liver mRNA. The 1461-bp hemoflavoprotein gene corresponded to a protein of 493 residues with the heme- and FAD-containing domains comprising the amino and carboxy termini of the protein, respectively. Sequence analysis indicated the heme and flavin domains were directly analogous to the corresponding domains in microsomal cytochrome b(5) (cb5) and cytochrome b(5) reductase (cb5r), respectively. The full-length fusion protein was purified to homogeneity and demonstrated to contain both heme and FAD prosthetic groups by spectroscopic analyses and MALDI-TOF mass spectrometry. The cb5/cb5r fusion protein was able to utilize both NADPH and NADH as reductants and exhibited both NADPH:ferricyanide (k(cat) = 21.7 s(-1), K(NADPH)(m) = 1 microM. K(FeCN6)(m) = 8 microM) and NADPH:cytochrome c (k(cat) = 8.3 s(-1), K(NADPH)(m) = 1 microM. K(cyt c)(m) = 7 microM) reductase activities with a preference for NADPH as the reduced pyridine nucleotide substrate. NADPH-reduction was stereospecific for transfer of the 4R-proton and involved a hydride transfer mechanism with a kinetic isotope effect of 3.1 for NADPH/NADPD. Site-directed mutagenesis was used to examine the role of two conserved histidine residues, H62 and H85, in the heme domain segment. Substitution of either residue by alanine or methionine resulted in the production of simple flavoproteins that were effectively devoid of both heme and NAD(P)H:cytochrome c reductase activity while retaining NAD(P)H:ferricyanide activity, confirming that the former activity required a functional heme domain. These results have demonstrated that the rat cb5/cb5r fusion protein is homologous to the human variant and has identified the heme and FAD as the sites of interaction with cytochrome c and ferricyanide, respectively. Mutagenesis has confirmed the identity of both axial heme ligands which are equivalent to the corresponding residues in microsomal cytochrome b(5).
Assuntos
Redutases do Citocromo/biossíntese , Redutases do Citocromo/química , Citocromos b5/biossíntese , Citocromos b5/química , Histidina/química , Alanina/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Dicroísmo Circular , Clonagem Molecular , Grupo dos Citocromos c/metabolismo , Citocromo-B(5) Redutase , DNA Complementar/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Ferricianetos/metabolismo , Flavina-Adenina Dinucleotídeo/química , Cinética , Ligantes , Fígado/enzimologia , Magnetismo , Espectrometria de Massas , Metionina/química , Microssomos Hepáticos/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Prótons , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
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 cytochrome b reductase fragment of Neurospora crassa NADPH:nitrate reductase (EC 1.6.6.3) was overexpressed in Escherichia coli with a His-tag for purification after mutation of the NADPH binding site. The recombinant enzyme fragment was altered by site-directed mutagenesis guided by the three-dimensional structure of cytochrome b reductase fragment of corn NADH:nitrate reductase (EC 1.6.6.1). Substitution of Asp for Ser920 (using residue numbering for holo-NADPH:nitrate reductase of N. crassa) greatly increased preference for NADH. This mutant had nearly the same NADH:ferricyanide reductase kcat as wild-type with NADPH. Substitutions for Arg921 had little influence on coenzyme specificity, while substitution of Ser or Gln for Arg932 did. The cytochrome b reductase mutant with greatest preference for NADH over NADPH was the doubly substituted form, Asp for Ser920/Ser for Arg932, but it had low activity and low affinity for coenzymes, which indicated a general loss of specificity in the binding site. Steady-state kinetic constants were determined for wild type and mutants with NADPH and NADH. Wild type had a specificity ratio of 1100, which was defined as the catalytic efficiency (kcat/Km) for NADPH divided by catalytic efficiency for NADH, while Asp for Ser920 mutant had a ratio of 0.17. Thus, the specificity ratio was reversed by over 6000-fold by a single mutation. Preference for NADPH versus NADH is strongly influenced by presence/absence of a negatively charged amino acid side chain in the binding site for the 2' phosphate of NADPH in nitrate reductase, which may partially account for existence of bispecific NAD(P)H:nitrate reductases (EC 1.6.6.2).
Assuntos
Redutases do Citocromo/genética , Mutagênese Sítio-Dirigida , Neurospora crassa/enzimologia , Nitrato Redutases/genética , Fragmentos de Peptídeos/genética , Nucleotídeos de Pirimidina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Redutases do Citocromo/biossíntese , Citocromo-B(5) Redutase , Escherichia coli/enzimologia , Escherichia coli/genética , Modelos Moleculares , Dados de Sequência Molecular , Nitrato Redutase (NADPH) , Nitrato Redutases/metabolismo , Fragmentos de Peptídeos/química , Engenharia de Proteínas/métodos , Proteínas Recombinantes/química , Especificidade por Substrato/genéticaRESUMO
Two forms of NADH-cytochrome b5 reductase (b5R), an erythrocyte-restricted soluble form, active in methemoglobin reduction, and a ubiquitous membrane-associated form involved in lipid metabolism, are produced from one gene. In the rat, the two forms are generated from alternative transcripts differing in the first exon, however, biogenesis of human b5R was less understood. Recently, two different transcripts (M and S), differing in the first exon were also described in humans. Here, we have investigated the tissue-specificity and the role of the S-transcript in the generation of soluble b5R. By RNase protection assays designed to simultaneously detect alternative b5R transcripts in the same sample, the S transcript was undetectable in nonerythroid and in erythroleukemic K562 cells induced to differentiate, but was present in terminal erythroblast cultures, and represented a major b5R transcript in reticulocytes. Analysis of the translation products of the M- and S-transcripts in HeLa cells transfected with the corresponding cDNAs demonstrated that the S-transcript generates soluble b5R, presumably from an internal initiation codon. Our results indicate that the S-transcript is expressed at late stages of erythroid maturation to generate soluble b5R.
Assuntos
Redutases do Citocromo/biossíntese , Redutases do Citocromo/genética , Eritrócitos/enzimologia , Transcrição Gênica , Sequência de Aminoácidos , Animais , Diferenciação Celular , Citocromo-B(5) Redutase , DNA Complementar/genética , Eritrócitos/citologia , Células HeLa , Humanos , Dados de Sequência Molecular , Ratos , TransfecçãoRESUMO
NADH:cytochrome b5 reductase activates the mitomycins to alkylating intermediates in vitro. To investigate the intracellular role of this enzyme in mitomycin bioactivation, Chinese hamster ovary cell transfectants overexpressing rat NADH:cytochrome b5 reductase were generated. An NADH:cytochrome b5 reductase-transfected clone expressed 9-fold more enzyme than did parental cells; the levels of other mitomycin-activating oxidoreductases were unchanged. Although this enzyme activates the mitomycins in vitro, its overexpression in living cells caused decreases in sensitivity to mitomycin C in air and decreases in sensitivity to porfiromycin under both air and hypoxia. Mitomycin C cytotoxicity under hypoxia was similar to parental cells. Because NADH:cytochrome b5 reductase resides predominantly in the mitochondria of these cells, this enzyme may sequester these drugs in this compartment, thereby decreasing nuclear DNA alkylations and reducing cytotoxicity. A cytosolic form of NADH:cytochrome b5 reductase was generated. Transfectants expressing the cytosolic enzyme were restored to parental line sensitivity to both mitomycin C and porfiromycin in air with marked increases in drug sensitivity under hypoxia. The results implicate NADH:cytochrome b5 reductase in the differential bioactivation of the mitomycins and indicate that the subcellular site of drug activation can have complex effects on drug cytotoxicity.
Assuntos
Sobrevivência Celular/fisiologia , Redutases do Citocromo/metabolismo , Mitomicina/toxicidade , Animais , Células CHO , Núcleo Celular/enzimologia , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Redutases do Citocromo/biossíntese , Citocromo-B(5) Redutase , Citoplasma/enzimologia , Cinética , Microssomos/enzimologia , Mitocôndrias/enzimologia , Mitomicina/farmacocinética , Oxirredução , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Succinato Desidrogenase/metabolismo , TransfecçãoRESUMO
Two forms of NADH-cytochrome b5 reductase (b5R), soluble and membrane-bound, are known. A hypothesis that the human soluble form b5R is generated through post-translational processing of the membrane-bound form was previously proposed. In this study, the 5'-rapid amplification of cDNA ends for human reticulocyte, liver, brain, and HL-60 cell mRNAs revealed the ubiquitous presence of an alternative type of human b5R mRNA which can probably be translated into the soluble form b5R directly; however, the erythroid-specific transcript of the b5R gene was not found. This type of b5R mRNA initiating from at least two sites contains a non-coding new first exon located between the first two exons of the human b5R gene identified before. In addition, this new first exon shares 62% homology with the first exon and its 3'-flanking intron sequences of rat erythroid-specific b5R mRNA, whereas the 5'-flanking region of the new first exon possesses features of house-keeping gene. These results might be important to understand the regulation mechanism of human b5R biosynthesis and divergent evolution of the gene regulation.
Assuntos
Redutases do Citocromo/biossíntese , Redutases do Citocromo/genética , Éxons , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Encéfalo/enzimologia , Citocromo-B(5) Redutase , Primers do DNA , Células HL-60/enzimologia , Humanos , Fígado/enzimologia , Dados de Sequência Molecular , Especificidade de Órgãos , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Ratos , Reticulócitos/enzimologia , Transcrição GênicaRESUMO
Many eukaryote promoters, particularly those for so-called housekeeping genes, have multiple GC boxes which are the binding sites of the transcription factor, Sp1. It has been proposed that Sp1 binds to the multiple GC boxes, and then the GC box-bound Sp1 interact with each other to synergistically stimulate transcription. Here, we describe a Sp1-dependent promoter which does not necessarily fit the synergistic activation mechanism. The promoter of the human NADH-cytochrome b5 reductase-encoding gene (CYTB5R) possesses five potential GC box sequences. Deletion and mutagenesis studies coupled with CAT assays revealed that three out of five GC box-like sequences were functionally active and activated transcription additively (rather than synergistically). Our results suggested that Sp1-mediated activation of transcription occurs in a promoter context-dependent manner.
Assuntos
Redutases do Citocromo/biossíntese , Redutases do Citocromo/genética , Hominidae/genética , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/metabolismo , Animais , Composição de Bases , Sequência de Bases , Cloranfenicol O-Acetiltransferase/biossíntese , Citocromo-B(5) Redutase , Desoxirribonuclease I , Expressão Gênica , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/biossínteseRESUMO
Higher plant species, particularly tobacco, have been used as hosts for expression of both eucaryotic and procaryotic cytochromes P450. Strategies for subcellular localization and for providing access to a source of reducing equivalents have varied. While expression of the P450 gene can be confirmed by the appearance of mRNA or antigen, recovery of activity for in vitro studies is very difficult. Some plant-expressed heterologous cytochromes P450 dramatically alter the phenotype of the plant, so in vivo activity such as premature senescence or P450-specific alterations in herbicide sensitivity confirm that expression of a functional protein has occurred.
Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/genética , Plantas/enzimologia , Plantas/genética , Redutases do Citocromo/biossíntese , Células Eucarióticas/enzimologia , Organelas/enzimologiaRESUMO
We have isolated and characterised a novel yeast gene, CBR (cytochrome b reductase), encoding a 35-kDa yeast novobiocin-binding protein. The predicted protein sequence of CBR displays considerable similarity to both plant nitrate reductases and mammalian cytochrome b5 reductases indicating that it is a putative member of the flavoprotein pyridine-nucleotide-cytochrome-reductase family. Disruption of CBR is not lethal under various growth conditions, suggesting the presence of some functional overlap with other reductases, possibly with the cytochrome P-450 reductase.
Assuntos
Proteínas de Transporte/genética , Redutases do Citocromo/genética , Genes Fúngicos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Animais , Northern Blotting , Western Blotting , Proteínas de Transporte/análise , Proteínas de Transporte/biossíntese , Cromatografia de Afinidade , Mapeamento Cromossômico , Cromossomos Fúngicos , Clonagem Molecular , Redutases do Citocromo/análise , Redutases do Citocromo/biossíntese , Citocromo-B(5) Redutase , DNA Fúngico/isolamento & purificação , DNA Fúngico/metabolismo , Escherichia coli , Biblioteca Gênica , Humanos , Dados de Sequência Molecular , Nitrato Redutases/genética , Novobiocina/metabolismo , Fases de Leitura Aberta , Plantas/enzimologia , RNA Fúngico/isolamento & purificação , RNA Fúngico/metabolismo , Ratos , Mapeamento por Restrição , Homologia de Sequência de AminoácidosRESUMO
Interleukin-1 has been shown to prolong the survival of rats exposed to lethal concentrations of oxygen. This oxygen tolerance has been attributed by some workers to an increase of manganese superoxide dismutase. We report here that the administration of interleukin-1 to male adult rats results in (i) significant decrease of pulmonary cytochrome P450 at 24 and 72 hours, (ii) decrease of P450 IIB1 mRNA at 24 and 72 hours and (iii) significant decrease of superoxide anion generation from pulmonary microsomes isolated from treated rats. To the best of our knowledge, this is the first report to demonstrate these effects of interleukin-1 on pulmonary P450 and its oxidase activity (O2- generation). On the basis of these results and several earlier reports in which various P450 depressants have been shown to depress superoxide production from microsomes and to prolong the lives of rodents in hyperoxia, we conclude that oxygen tolerance induced by interleukin-1 administration is likewise mediated, at least in part, by reduced generation of superoxide anion from cytochrome P450 monooxygenase system.
Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/genética , Interleucina-1/farmacologia , Pulmão/fisiologia , RNA Mensageiro/metabolismo , Superóxidos/metabolismo , Animais , Sequência de Bases , Northern Blotting , Citocromo P-450 CYP2B1 , Redutases do Citocromo/biossíntese , Citocromos b5/biossíntese , Cinética , Pulmão/efeitos dos fármacos , Pulmão/enzimologia , Masculino , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Oxirredutases/genética , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos , Proteínas Recombinantes/farmacologia , Valores de Referência , Fatores de TempoRESUMO
Inducibility of oxidative stress in rat liver in vivo by menadione-associated redox cycling activation under redox enzyme modulating conditions was examined by monitoring hepatocyte injury and 8-hydroxydeoxyguanosine (8-OHdG) levels of liver DNA. In addition, the treatment-associated liver tumor initiating activity was assessed in terms of development of gamma-glutamyl-transpeptidase (GGT)- and glutathione S-transferase placental form (GST-P)-positive foci and hyperplastic nodules. With or without following menadione treatment (50 mg/kg, i.g.), redox enzyme modulations of increased cytochrome P450 reductase activity induced by phenobarbital (PB)-Na (100 mg/kg, i.p. for 5 days), inhibition of DT-diaphorase by dicumarol (25 mg/kg, i.p.) and depletion of glutathione by phorone (200 mg/kg, i.p.), with or without further supplement of iron EDTA-Na-Fe(III) (70 mg/kg, i.p.), caused both substantial hepatocyte necrosis and 8-OHdG production in Fischer 344 male rats. Subsequent feeding with a 0.05% PB diet for 64 weeks resulted in slightly increased development of GGT-positive foci but not GST-P positive lesions or hyperplastic nodules, suggesting a lack of tumor-initiating activity of the oxidative DNA damage associated with redox enzyme modulations with or without menadione.