RESUMEN
Expression of two recombinant hepcidin homologues from Atlantic salmon, Salmo salar, characterization of their antimicrobial activity, and partial structural determination of the peptides is described. Expression was attempted in baculovirus and bacterial expression systems and the various purification and refolding methods used to determine the optimal strategy for production of active, correctly refolded hepcidin are reviewed.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Animales , Péptidos Catiónicos Antimicrobianos/biosíntesis , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Baculoviridae/genética , Cromatografía de Afinidad , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Endopeptidasas/metabolismo , Escherichia coli/genética , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Hepcidinas , Cuerpos de Inclusión/metabolismo , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Pliegue de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Salmo salar , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The cysteine-rich peptide hepcidin is an antimicrobial peptide and iron transport regulator that has been found in vertebrates including birds, fish and mammals. To elucidate the structure and biological function of fish hepcidin, which is difficult to produce synthetically, we have cloned several plasmid constructs encoding hepcidin from Japanese flounder, Paralichthys olivaceus, and tested expression of recombinant peptides, each with an N-terminal hexahistidine (6xHis) tag, in inclusion bodies or the periplasmic space of Escherichia coli. Hepcidin expressed in inclusion bodies was reduced, and subsequently refolded using a dilution technique with a cysteine redox system. The oxidized His-hepcidin monomer was separated from protein multimers and mass spectrometry analysis showed that the peptide was of the predicted size and contained four disulfide bonds. Removal of the 6xHis tag was attempted using enzymatic cleavage by Factor Xa and tobacco etch virus (TEV) protease or chemical cleavage by hydroxylamine. The Factor Xa cleavage was unsuccessful and hydroxylamine cleavage resulted in aggregation of cleaved peptide. TEV protease cleavage was successful but immediately resulted in hexamer formation despite varying reaction conditions (redox, non-redox, pH, temperature, target protein concentration, type of buffer). However, the recombinant His-hepcidin fusion peptide monomer showed considerable antimicrobial activity. NMR-based studies showed that hepcidin contained a rare vicinal disulfide linkage at the top of a loop structure and a short beta-sheet structure encompassing residues 7-13 and 19-25 that is stabilized by three disulfide bonds.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/genética , Animales , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Cromatografía Liquida , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Factor Xa/metabolismo , Lenguado , Hepcidinas , Espectrometría de Masas , Resonancia Magnética Nuclear Biomolecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Yeast co-expressing human elongase and desaturase genes were used to investigate whether the same desaturase gene encodes an enzyme able to desaturate n-3 and n-6 fatty acids with the same or different carbon chain length. The results clearly demonstrated that a single human Delta5 desaturase is active on 20:3n-6 and 20:4n-3. Endogenous Delta6 desaturase substrates were generated by providing to the yeast radiolabelled 20:4n-6 or 20:5n-3 which, through two sequential elongations, produced 24:4n-6 and 24:5n-3, respectively. Overall, our data suggest that a single human Delta6 desaturase is active on 18:2n-6, 18:3n-3, 24:4n-6 and 24:5n-3.
Asunto(s)
Ácido Graso Desaturasas/metabolismo , Ácidos Grasos Insaturados/metabolismo , Cromatografía Líquida de Alta Presión , Clonación Molecular , ADN Complementario/metabolismo , delta-5 Desaturasa de Ácido Graso , Humanos , Cinética , Linoleoil-CoA Desaturasa , Metabolismo de los Lípidos , Modelos Químicos , Saccharomyces cerevisiae/metabolismo , Factores de TiempoRESUMEN
1-Aminobenzotriazole (ABT) and its N-benzyl-1-aminobenzotriazole (BBT) and N-alpha-methylbenzyl (alpha-MB) derivatives were compared as isozyme-selective, lung-selective (vs. liver) mechanism-based inhibitors of cytochrome P-450 (P450) in noninduced, beta-naphthoflavone-induced and phenobarbital-induced guinea pigs 4 hr after i.v. administration. Isozyme-selective monooxygenase activities for lung P450 1A1, 2B4 and 4B1 orthologues (7-ethoxyresorufin O-deethylation for guinea pig P450 1A1, 7-pentoxyresorufin O-depentylation for P450 2Bx and 4-aminobiphenyl N-hydroxylation for P450 4Bx, respectively) were determined in pulmonary and hepatic microsomes. BBT and alpha-MB inactivated pulmonary P450 in an isozyme-selective manner; in non- and phenobarbital-induced animals the order of inactivation was 2Bx > 1A1 >>> 4Bx. In beta-naphthoflavone-induced animals, alpha-MB specifically inhibited 2Bx in the lung (>90% inactivation at 0.075 mumol/kg, whereas a 100-fold higher dose did not inhibit 4Bx or 1A1). BBT and alpha-MB also were highly selective for the inactivation of pulmonary vs. hepatic P450. In each case at least one of the doses administered caused marked inactivation of pulmonary 2Bx (>80% with alpha-MB and 50-70% with BBT) without inhibiting the hepatic monooxygenase activities. In contrast, ABT displayed little isozyme-selectively and little tissue-selectivity. The differences in tissue-selectivity of the inhibitors are due to BBT and alpha-MB being much more potent (100- to 1000-fold) inactivators of pulmonary P450 2Bx than ABT consistent with BBT and alpha-MB, but not ABT, serving as substrates for the lipophilic aromatic amine uptake system in the lung. In summary, BBT and alpha-MB, at appropriate doses, are isozyme-selective/specific (P450 2Bx), lung-specific inhibitors of P450 in guinea pig in vivo.
Asunto(s)
Inhibidores Enzimáticos del Citocromo P-450 , Isoenzimas/antagonistas & inhibidores , Pulmón/efectos de los fármacos , Pulmón/enzimología , Triazoles/farmacología , Animales , Benzoflavonas/farmacología , Activación Enzimática , Cobayas , Hígado/enzimología , Masculino , Fenobarbital/farmacología , Sensibilidad y Especificidad , beta-naftoflavonaRESUMEN
Guinea pig lung microsomes converted arachidonic acid (AA) to two classes of cytochrome P450 (P450)-dependent metabolites, 16- through 20-hydroxyeicosatetraenoic acids [(16-20)-OH-AA] and epoxyeicosatrienoic acids (EETs). The rate of formation of (16-20)-OH-AA was approximately 3-fold higher in microsomes from beta-naphthoflavone-induced versus untreated animals. In microsomes from untreated or induced animals EETs, the major class of P450 metabolites in guinea pig lung, were formed in a regioselective manner, with 8,9-, 11,12-, and 14,15-regioisomers accounting for > or = 90% of the total EETs. With isozyme-selective inhibitors and inhibitory antibodies the role of individual pulmonary P450 isozymes in AA metabolism was examined. Metyrapone and SKF-525A (P450 2B selective) inhibited EET formation by > or = 85% with little effect on (16-20)-OH-AA formation. 1-Aminobenzotriazole (1 mM), a mechanism-based inhibitor with low isozyme selectivity, inhibited the formation of both classes of metabolites by > 95%, whereas N-alpha-methylbenzyl-1-aminobenzotriazole (1 microM), a P450 2B-selective mechanism-based inhibitor, abolished EET formation with little effect on (16-20)-OH-AA formation. Antibodies to rabbit P450 2B4 also abolished EET formation without inhibiting the formation of (16-20)-OH-AA, whereas antibodies to rabbit P450 4B1 did not inhibit the formation of either class of metabolites. alpha-Naphthoflavone (P450 1A1 selective in lung) did not inhibit the formation of either class of metabolites. These data demonstrate that the guinea pig orthologue of P450 2B4 is solely responsible for the bioactivation of AA to EETs in guinea pig lung and that a form of P450 other than a 2B, 4B, or 1A isozyme, which is inducible by beta-naphthoflavone, is responsible for (16-20)-OH-AA formation.
Asunto(s)
Ácido 8,11,14-Eicosatrienoico/metabolismo , Ácido Araquidónico/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Pulmón/enzimología , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Animales , Benzoflavonas/farmacología , Cobayas , Pulmón/efectos de los fármacos , Masculino , Metirapona/farmacología , Microsomas/efectos de los fármacos , Microsomas/enzimología , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , NADP/metabolismo , Fenobarbital/farmacología , Proadifeno/farmacología , Triazoles/química , Triazoles/farmacología , beta-naftoflavonaRESUMEN
Guinea pig renal microsomes convert arachidonic acid to two classes of P450-dependent metabolites, epoxyeicosatrienoic acids (EET), and 16- through 20-hydroxyeicosatetraenoic acids [(16-20)-OH-AA)]. The rate of formation of these metabolites was not altered by beta-naphthoflavone induction, which increased P450 1A1-dependent 7-ethoxyresorufin O-deethylation activity approximately 100-fold. alpha-Naphthoflavone, which inhibits renal P450 1A1 in vitro, did not inhibit the formation of these metabolites in microsomes from induced animals. In induced animals, N-benzyl-1-aminobenzotriazole and N-alpha-methylbenzyl-1-aminobenzotriazole, administered i.v., inhibited microsomal 7-ethoxyresorufin O-deethylation activity by approximately 50% without inhibiting the formation of either class of arachidonic acid metabolites. In vitro these mechanism-based inhibitors inactivated 1A1 by > 90% without inhibiting EET or (16-20)-OH-AA formation. These data show that P450 1A1 does not bioactivate arachidonic acid to either (16-20)-OH-AA or EET in guinea pig kidney, and that N-benzyl-1-aminobenzotriazole and N-alpha-methylbenzyl-1-aminobenzotriazole selectively inactivate P450 1A1 in comparison to the P450 isozyme(s) that metabolize arachidonic acid in the kidney. In guinea pig liver beta-naphthoflavone treatment, which induces P450 1A1 and 1A2, increased the rate of the formation of (16-20)-OH-AA and EET and in vitro alpha-naphthoflavone inhibited the formation of these metabolites in induced hepatic microsomes by approximately 50 and approximately 35%, respectively. These data demonstrate that a beta-naphthoflavone-inducible isozyme, most likely 1A2, converts arachidonic acid to both (16-20)-OH-AA and EET in guinea pig liver.
Asunto(s)
Ácido Araquidónico/metabolismo , Sistema Enzimático del Citocromo P-450/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/metabolismo , Riñón/enzimología , Oxigenasas/efectos de los fármacos , Oxigenasas/metabolismo , Triazoles/farmacología , Animales , Benzoflavonas/farmacología , Radioisótopos de Carbono , Cromatografía Líquida de Alta Presión , Citocromo P-450 CYP1A1 , Inhibidores Enzimáticos del Citocromo P-450 , Activación Enzimática , Cobayas , Riñón/efectos de los fármacos , Masculino , Microsomas/efectos de los fármacos , Microsomas/metabolismo , Microsomas Hepáticos/enzimología , Oxidorreductasas/antagonistas & inhibidores , beta-naftoflavonaRESUMEN
The mechanism-based inactivation of the cytochrome P450 (P450) dependent monooxygenase system was studied in vivo in liver, lung, and kidney of untreated, phenobarbital-treated, and beta-naphthoflavone-treated guinea pigs 24 h after administration of 1-aminobenzotriazole (1-100 mg/kg, i.p.). Microsomal isozyme-selective or -specific monooxygenase activities were inhibited in a dose-dependent manner in all three organs. In the liver of untreated and phenobarbital-treated animals, 7-pentoxyresorufin O-depentylation (catalyzed primarily by P450 2Bx, an orthologue of rabbit P450 2B4/rat 2B1) was inhibited more than 7-ethoxyresorufin O-deethylation (P450 1A1), 4-aminobiphenyl N-hydroxylation (P450 1A2), erythromycin N-demethylation (P450 3A), or benzphetamine N-demethylation; in beta-naphthoflavone-treated animals, 4-amino-biphenyl N-hydroxylation activity was preferentially inhibited. In lung, the order of inactivation of monooxygenase activities was 4-aminobiphenyl N-hydroxylation (4Bx, the orthologue of rabbit 4B1) > 7-pentoxyresorufin O-depentylation activity (2Bx) > 7-ethoxyresorufin O-deethylation (1A1; for example 72, 53, and 29% inactivation, respectively, in phenobarbital-treated animals at 100 mg/kg). In all three tissues the loss in spectrally assayed P450 content corresponds quite well to the inhibition of monooxygenase activities. Thus, these studies show that 1-aminobenzotriazole is an effective inactivator of the pulmonary, hepatic, and renal P450 systems in guinea pigs following i.p. administration, and that P450 1A2 (liver) and P450 4Bx (lung), isozymes efficient for the oxidation of primary aromatic amines, are preferentially inactivated.
Asunto(s)
Inhibidores Enzimáticos del Citocromo P-450 , Riñón/enzimología , Hígado/enzimología , Pulmón/enzimología , Oxigenasas de Función Mixta/antagonistas & inhibidores , Triazoles/farmacología , Animales , Benzoflavonas/farmacología , Relación Dosis-Respuesta a Droga , Cobayas , Isoenzimas/antagonistas & inhibidores , Riñón/efectos de los fármacos , Hígado/efectos de los fármacos , Pulmón/efectos de los fármacos , Masculino , Microsomas/efectos de los fármacos , Microsomas/enzimología , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Fenobarbital/farmacología , beta-naftoflavonaRESUMEN
Interferon and interferon inducers are well known to depress the cytochrome P450-dependent hepatic mixed-function oxidase system and cause a decrease in the capacity of the liver to metabolize drugs and xenobiotics. In this study we have shown that the interferon-mediated changes in an induced form of hepatic cytochrome P450 (CYP4A) are mediated via a depression in the levels of mRNA as assessed by Northern blot and slot blot analyses using a 20-base synthetic oligodeoxyribonucleotide hybridization probe. Rats were pretreated with clofibrate to maximize CYP4A mRNA levels prior to the administration of polyinosinic acid.polycytidylic acid (poly IC), an alpha/beta interferon inducer. Hepatic CYP4A mRNA levels were decreased by 49 and 30% at 6 and 24 hr, respectively, following poly IC administration. In hepatic microsomes cytochrome P450 and functional CYP4A as measured by lauric acid hydroxylation, were not affected at 6 hr, but were depressed by 39 and 27%, respectively, 24 hr following poly IC administration. These results suggest that interferon depresses induced levels of hepatic drug metabolism by lowering the level of cytochrome P450 mRNAs and subsequent synthesis of cytochrome P450 apoproteins.
Asunto(s)
Sistema Enzimático del Citocromo P-450/análisis , Microsomas Hepáticos/enzimología , Poli I-C/farmacología , ARN Mensajero/análisis , Animales , Secuencia de Bases , Clofibrato , Sistema Enzimático del Citocromo P-450/biosíntesis , Inducción Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Interferones/biosíntesis , Ácidos Láuricos/metabolismo , Masculino , Microsomas Hepáticos/efectos de los fármacos , Datos de Secuencia Molecular , Ratas , Ratas EndogámicasRESUMEN
The potency and cytochrome P-450 (P-450) isozyme selectivity of 1-aminobenzotriazole (ABT) and three of its N-aralkylated analogues, N-benzyl-1-aminobenzotriazole (BBT), N-alpha-methylbenzyl-1-aminobenzotriazole (alpha MB), and the newly synthesized N-alpha-ethylbenzyl-1-aminobenzotriazole (alpha EB), as mechanism-based inhibitors were compared in pulmonary microsomes of untreated and beta-naphthoflavone (beta-NF)-induced guinea pigs. All four compounds were suicide substrates for pulmonary P-450, resulting in the loss of spectrally assayed hemoprotein (up to 50%). Monooxygenase activities were measured with isozyme-selective/specific substrates; the O-dealkylation of 7-pentoxyresorufin (PRF) for the guinea pig ortholog of rabbit P-450IIB4, the O-deethylation of 7-ethoxyresorufin for P-450IA1, and the N-hydroxylation of the aromatic amine 4-aminobiphenyl for P-450IVB1, BBT, alpha MB, and alpha EB were selective for the suicidal inhibition of P-450IIB4; for example, 1 microM alpha MB inactivated 95% of P-450IIB4-, and approximately 10% of P-450IA1- and IVB1-catalyzed, activity in microsomes from beta-NF-induced lungs. Isozyme selectivity was approximately the same for alpha EB and slightly lower for BBT, which inactivated relatively more P-450IA1. At low concentrations, 1 and 10 microM, respectively, ABT preferentially inactivated P-450IVB1, consistent with the efficient N-hydroxylation of aromatic amines by this form of P-450. alpha EB also was shown to efficiently inactivate P-450IIB4-catalyzed PRF activity in microsomes prepared from liver of phenobarbital-induced guinea pigs.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Inhibidores Enzimáticos del Citocromo P-450 , Pulmón/enzimología , Triazoles/farmacología , Animales , Femenino , Cobayas , Técnicas In Vitro , Isoenzimas/antagonistas & inhibidores , Hígado/enzimología , Pulmón/efectos de los fármacos , Masculino , Oxigenasas de Función Mixta/antagonistas & inhibidoresRESUMEN
During episodes of infectious disease the mixed function oxidase system is depressed and the capacity of the liver to metabolize drugs can be compromised in both animals and humans. The depression that occurs during viral infections is mediated via the production of interferon. This action of interferon requires the synthesis of an intermediate protein(s) yet to be identified. Using an oligonucleotide probe for a unique sequence in cytochrome P-450LA omega we have now shown that the mRNA for this isozyme is depressed following the administration of interferon inducers. The magnitude in the loss of mRNA corresponds to the magnitude of the loss in the levels of this isozyme. This depression is observed within 6 h of interferon exposure. It is concluded that the decrease in drug metabolism during viral infections is caused by an interferon-mediated loss in mRNA and subsequent cytochrome P-450 synthesis in the liver.