RESUMEN
Cholesterol conversion to bile acids is subject to a feedback regulatory mechanism by which bile acids down-regulate their own synthesis. This regulation occurs at the level of transcription of several genes encoding enzymes in the bile acid biosynthetic pathway. One of these enzymes is sterol 12alpha-hydroxylase/CYP8B1 (12alpha-hydroxylase), the specific enzyme required for cholic acid synthesis. The levels of this enzyme determine the ratio of cholic acid to chenodeoxycholic acid and thus the hydrophobicity of the circulating bile acid pool. Previous studies from this laboratory showed that fetoprotein transcription factor (FTF) is required for 12alpha-hydroxylase promoter activity and bile acid-mediated regulation. Here, we report that the short heterodimer partner (SHP) suppresses 12alpha-hydroxylase promoter activity via an interaction with FTF. Hepatic nuclear factor-4 (HNF-4) binds and activates the 12alpha-hydroxylase promoter and is required for 12alpha-hydroxylase promoter activity. Although HNF-4 interacts with SHP, it is not involved in SHP-mediated suppression of 12alpha-hydroxylase promoter activity. FTF and not HNF-4 is the factor involved in regulation of 12alpha-hydroxylase promoter activity by bile acids through its interaction with SHP. Finally, interaction of SHP with FTF displaces FTF binding to its sites within the 12alpha-hydroxylase promoter. These results provide insights into the mechanism of action of bile acid-mediated regulation of sterol 12alpha-hydroxylase transcription.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Receptores Citoplasmáticos y Nucleares/fisiología , Esteroide Hidroxilasas/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Ácidos y Sales Biliares/fisiología , Sitios de Unión , Línea Celular , Proteínas de Unión al ADN/fisiología , Regulación hacia Abajo , Genes Reporteros , Factor Nuclear 4 del Hepatocito , Humanos , Fosfoproteínas/fisiología , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Receptores Citoplasmáticos y Nucleares/química , Proteínas Represoras/química , Proteínas Represoras/fisiología , Esteroide 12-alfa-Hidroxilasa , Factores de Transcripción/fisiología , Transcripción GenéticaRESUMEN
Bile acid biosynthesis is subjected to feedback regulation whereby bile acids down-regulate their own synthesis. The major point of this regulation is at the level of cholesterol 7alpha-hydroxylase (7alpha-hydroxylase), which controls bile acid output from the classic pathway. This regulation is at the level of transcription of the gene. Two bile acid response elements have been localized within the 5'-flanking region of the rat gene and these elements overlap three nuclear receptor binding sites for hepatocyte nuclear factor (HNF-4), liver X receptor (LXR) and alpha(1)-fetoprotein transcription factor (FTF). Recently it has been shown that bile acids are physiological ligands for the farnesyl X receptor (FXR), which suggested that FXR could function by binding to one of the three nuclear receptor sites to mediate regulation of 7alpha-hydroxylase transcription by bile acids. In this study we show that FXR is indeed a crucial factor for bile acid-mediated regulation, but that it functions without binding to DNA. Furthermore, we also demonstrate that neither the LXR nor the HNF-4 sites are involved in bile acid-mediated regulation of 7alpha-hydroxylase transcription. Most importantly, we show that the FTF site is essential for regulation of 7alpha-hydroxylase by bile acids, similar to what we have recently demonstrated for another gene of the bile acid biosynthetic pathway, the sterol 12alpha-hydroxylase gene. These studies demonstrate the crucial role of FTF in the expression and regulation of a critical gene in the bile acid biosynthetic pathways.
Asunto(s)
Ácidos y Sales Biliares/fisiología , Colesterol 7-alfa-Hidroxilasa/genética , Proteínas de Unión al ADN/fisiología , Regulación Enzimológica de la Expresión Génica , Factores de Transcripción/fisiología , Animales , Proteínas Portadoras/metabolismo , Técnicas In Vitro , Masculino , Ratas , Receptores Citoplasmáticos y Nucleares , Transcripción Genética/fisiología , Transfección , alfa-FetoproteínasRESUMEN
Because oxidative stress is one of the main sources of severe cellular damage, cells have different defense weapons against reactive oxygen species. Ubiquitous plasma membrane redox systems play a role in defense against oxidative stress damage. On the other hand, a tightly controlled and localized production of reactive oxygen species by a plasma membrane NADPH oxidase can be used as a potent microbicidal weapon. This dual, prooxidant and antioxidant role of plasma membrane electron transport systems in defense is studied and discussed.
Asunto(s)
Estrés Oxidativo/fisiología , Especies Reactivas de Oxígeno/fisiología , Animales , Infecciones Bacterianas/patología , Membrana Celular/metabolismo , Transporte de Electrón , Proteínas de Unión al GTP/metabolismo , Enfermedad Granulomatosa Crónica/metabolismo , Enfermedad Granulomatosa Crónica/patología , Humanos , NADPH Oxidasas/metabolismo , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Cholesterol conversion to bile acids occurs via the "classic" (neutral) or the "alternative" (acidic) bile acid biosynthesis pathways. Sterol 12alpha-hydroxylase/CYP8b1 is the specific enzyme required for cholic acid synthesis. The levels of this enzyme determine the ratio of cholic acid to chenodeoxycholic acid and thus the hydrophobicity of the circulating bile acid pool. Expression of the 12alpha-hydroxylase gene is tightly down-regulated by hydrophobic bile acids. In this study, we report the characterization of two DNA elements that are required for both the 12alpha-hydroxylase promoter activity and bile acid-mediated regulation. Mutation of these elements suppresses 12alpha-hydroxylase promoter activity. Mutations of any other part of the promoter do not alter substantially the promoter activity or alter regulation by bile acids relative to the wild type promoter. These two DNA elements bind alpha(1)-fetoprotein transcription factor (FTF), a member of the nuclear receptor family. We also show that overexpression of FTF in a non-liver cell line activates the sterol 12alpha-hydroxylase promoter. These studies demonstrate the crucial role of FTF for the expression and regulation of a critical gene in the bile acid biosynthetic pathways.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Proteínas de Unión al ADN/metabolismo , Regiones Promotoras Genéticas , Esteroide Hidroxilasas/genética , Factores de Transcripción/metabolismo , Transcripción Genética/fisiología , Animales , Secuencia de Bases , Línea Celular , Ácido Cólico/biosíntesis , Clonación Molecular , Secuencia de Consenso , Regulación Enzimológica de la Expresión Génica/fisiología , Biblioteca Genómica , Humanos , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Ratas , Proteínas Recombinantes/biosíntesis , Eliminación de Secuencia , Esteroide 12-alfa-Hidroxilasa , Transfección , Células Tumorales CultivadasRESUMEN
Phosphate-activated glutaminase (GA) is overexpressed in certain types of tumour but its exact role in tumour cell growth and proliferation is unknown. Here we describe the isolation of a full-length cDNA clone of human breast cancer ZR75 cells, by a combination of lambdagt10 cDNA library screening and the rapid amplification of cDNA ends ('RACE') technique. The cDNA of human GA is 2408 nt with a 1806-base open reading frame encoding a 602-residue protein with a predicted molecular mass of 66309 Da. The deduced amino acid sequence contains a putative mitochondrial import presequence of 14 residues at the N-terminal end. Heterologous expression and purification in Escherichia coli yielded a product of the expected molecular size that was recognized by using antibodies against the recombinant human GA. Sequence analyses showed that human GA was highly similar to the rat liver enzyme. Northern gel analysis revealed that the gene is present in human liver, brain and pancreas, in which a major transcript of 2.4 kb was demonstrated, but not in kidney, heart, skeletal muscle, lung or placenta. These results strongly suggest that the first human GA cloned, the GA from ZR-75 breast cancer cells, and presumably those from human liver and brain, are liver-type isoenzymes, in sharp contrast with the present view that considers the kidney type as the isoform expressed in all tissues with GA activity, with the exception of postnatal liver.
Asunto(s)
Neoplasias de la Mama/enzimología , Glutaminasa/genética , Glutamina/metabolismo , Proteínas de Neoplasias/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Escherichia coli/genética , Femenino , Expresión Génica , Biblioteca de Genes , Glutaminasa/biosíntesis , Humanos , Datos de Secuencia Molecular , Proteínas de Neoplasias/biosíntesis , Proteínas Recombinantes/biosíntesis , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
Our laboratory described a ca. 34-kDa protein of the HeLa S cell surface that bound an antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl) urea (LY181984) with high affinity and that exhibited NADH oxidase and protein disulfide-thiol interchange activities also inhibited by LY181984. The quinone site inhibitor 8-methyl-N-vanillyl-6-noneamide (capsaicin) also blocked these same enzymatic activities. Using capsaicin inhibition as the criterion, the drug-responsive oxidase was released from the surface of HeLa S cells and purified. The activity of the released capsaicin-inhibited oxidase was resistant to heating at 50 degrees C and to protease digestion. After heating and proteinase K digestion, the activity was isolated in >90% yield by FPLC as an apparent 50- to 60-kDa multimer. Final purification by preparative SDS-PAGE yielded a capsaicin-inhibited NADH oxidase activity of a specific activity indicative of >500-fold purification relative to the plasma membrane. The final activity correlated with a ca. 34-kDa band on SDS-PAGE. Matrix-assisted laser desorption mass spectroscopy as well as reelectrophoresis of the 34-kDa band indicated that the ca. 34-kDa material was a stable mixture of 22-, 17-, and 9.5-kDa components which occasionally migrated as a ca. 52-kDa complex. The purified complex tended to multimerize and formed insoluble 10- to 20-nm-diameter amyloid rods. The components of the purified 34-kDa complex were blocked to N-terminal amino acid sequencing and were resistant to further protease digestion. After multimerization into amyloid rods, the protein remained resistant to proteases even under denaturing conditions and to cyanogen bromide either with or without prior alkylation.
Asunto(s)
Endopeptidasas/metabolismo , Células HeLa/enzimología , Proteínas de la Membrana/metabolismo , Complejos Multienzimáticos/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Ácidos , Aminoácidos/análisis , Amiloide/metabolismo , Capsaicina/farmacología , Cromatografía Líquida de Alta Presión , Bromuro de Cianógeno , Electroforesis en Gel de Poliacrilamida , Endopeptidasa K/metabolismo , Femenino , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Calor , Humanos , Hidrólisis , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/aislamiento & purificación , Peso Molecular , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/aislamiento & purificación , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , NADH NADPH Oxidorreductasas/aislamiento & purificación , Fragmentos de Péptidos/aislamiento & purificaciónRESUMEN
The pH dependence of the phosphate-activated glutaminase isolated from Ehrlich tumour cells suggests a functional role for two prototropic groups with apparent pKa of 9.3 and 7.7 at the active site of the protein; these pKa values are compatible with cysteine and histidine residues, respectively. This possibility was investigated by chemical modification studies of the purified enzyme. N-Ethylmaleimide fully inactivated the purified glutaminase; the reaction order was very close to 1.0, suggesting that N-ethylmaleimide modifies glutaminase at a single essential site. Spectrophotometric studies of the isolated protein treated with diethyl pyrocarbonate indicate that two histidine residues are modified. Since glutaminase is loosely associated to the inner mitochondrial membrane, modification experiments were also carried out using mitochondrial membrane fractions. N-Ethylmaleimide and diethyl pyrocarbonate gave similar results in mitochondria membrane-bound enzyme to those obtained with purified enzyme. Glutamate, which behaves as a competitive inhibitor of the enzyme, partially protected the inactivation caused by N-ethylmaleimide in membrane-bound experiments. The results suggest the existence of a critical histidine residue(s) in the tumour glutaminase, and strongly support the notion that a cysteine residue, which is located at (or near) the active site, is involved in the catalytic mechanism as well.
Asunto(s)
Cisteína/metabolismo , Glutaminasa/metabolismo , Histidina/metabolismo , Animales , Sitios de Unión , Carcinoma de Ehrlich/enzimología , Dietil Pirocarbonato/farmacología , Inhibidores Enzimáticos/farmacología , Etilmaleimida/farmacología , Glutaminasa/antagonistas & inhibidores , Glutaminasa/aislamiento & purificación , Concentración de Iones de Hidrógeno , Membranas Intracelulares/enzimologíaRESUMEN
All the biological membranes contain oxidoreduction systems actively involved in their bioenergetics. Plasma membrane redox systems seem to be ubiquitous and they have been related to several important functions, including not only their role in cell bioenergetics, but also in cell defense through the generation of reactive oxygen species, in iron uptake, in the control of cell growth and proliferation and in signal transduction. In the last few years, an increasing number of mechanistic and molecular studies have deeply widened our knowledge on the function of these plasma membrane redox systems. The aim of this review is to summarize what is currently known about the components and physiological roles of these systems.
Asunto(s)
Membrana Celular/metabolismo , Metabolismo Energético/fisiología , Animales , División Celular/fisiología , Membrana Celular/enzimología , Transporte de Electrón/fisiología , Hierro/metabolismo , Modelos Biológicos , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
A procedure based on CHAPS-agarose-polyacrylamide electrophoresis and in situ staining of activity was used to detect a Zn(2+)-sensitive component of Ehrlich cell plasma membrane redox system. The procedure is so powerful that it allows to use crude plasma membrane fractions and can be easily adapted for use in an electrophoretic approach to the purification of this protein.
Asunto(s)
Membrana Celular/enzimología , Oxidorreductasas/análisis , Coloración y Etiquetado/métodos , Zinc , Animales , Carcinoma de Ehrlich , Ácidos Cólicos , Electroforesis en Gel de Poliacrilamida , Femenino , Ratones , Oxidación-Reducción , Células Tumorales CultivadasRESUMEN
Diacyl glycerols and phorbol esters, which activate protein kinases C, stimulated Ehrlich ascites tumor cell ferricyanide reductase activity. On the contrary, selective inhibition of active protein kinases C with bis-indolyl maleimide did not change the rate of ferricyanide reduction by Ehrlich cells. Selective inhibitors of phosphoprotein phosphatases, okadaic acid and cyclosporin A, also stimulated plasma membrane redox system. Taking all these data together, protein kinases or phosphoprotein phosphatases seemed to be involved in the multiple and complex regulation of Ehrlich cell plasma membrane redox system.
Asunto(s)
Membrana Celular/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteína Quinasa C/fisiología , Animales , Carcinoma de Ehrlich , Ciclosporina/farmacología , Inhibidores Enzimáticos/farmacología , Éteres Cíclicos/farmacología , NADH NADPH Oxidorreductasas/metabolismo , Ácido Ocadaico , Oxidación-Reducción , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Proteína Quinasa C/antagonistas & inhibidores , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
A ferricyanide-utilizing NADH dehydrogenase (NADH-ferricyanide oxidoreductase) from the plasma membrane of Ehrlich ascites tumour cells has been purified about 1500-fold to apparent homogeneity. The method comprises the isolation of an enriched plasma membrane fraction, solubilization with Triton X-100, ion-exchange chromatography, ammonium sulphate precipitation, Cibacron Blue chromatography and fast-protein liquid chromatography with a Superose-6 gel filtration column. The specific activity of the final pool was more than 61 units/mg protein. The pure enzyme examined by SDS/PAGE displayed only one type of subunit with an apparent molecular mass of 32.0 kDa. The molecular mass of the native protein (117.0 kDa) was estimated by gel filtration; these results suggest a protein composed of four subunits of identical molecular mass. The enzyme was stable in the pH interval between 6 and 9, with maximum activity at pH values from 7.5 to 8.5. The purified enzyme showed Michaelis-Menten kinetics for the substrates, with apparent K(m) values of 4.3 X 10(-5) M and 6.7 X 10(-5) M for NADH and ferricyanide respectively. The isolated protein was strongly inhibited by Zn2+ and the thio-specific reagents mersalyl and p-chloromercuribenzenesulphonic acid.
Asunto(s)
Carcinoma de Ehrlich/enzimología , Ferricianuros/metabolismo , NADH Deshidrogenasa/aislamiento & purificación , NADH Deshidrogenasa/metabolismo , Animales , Membrana Celular/enzimología , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Femenino , Concentración de Iones de Hidrógeno , Cinética , Ratones , Peso Molecular , NADH Deshidrogenasa/antagonistas & inhibidores , SolubilidadRESUMEN
Ehrlich cell plasma membrane ferricyanide reductase activity increased in the presence of mastoparan, a generic activator of G proteins, using either whole cells or isolated plasma membrane-fractions. Agents that increase intracellular cAMP also increased the rate of ferricyanide reduction by Ehrlich cells. For the first time, evidence is shown on a modulation of plasma membrane redox system by cGMP. In fact, permeant analogs of cGMP, dibutyryl cGMP, and 8-bromo-cGMP increased the rate of ferricyanide reduction by the Ehrlich cell plasma membrane redox system. Furthermore, specific inhibition of cGMP-phosphodiesterases by dipyridamole was also accompanied by an enhancement in the rate of ferricyanide reduction. On the other hand, treatments expected to increase cytoplasmic Ca2+ concentrations were accompanied by a remarkable stimulation of the reductase activity. Taking all these data together, it seems that the Ehrlich cell plasma membrane redox system is under a multiple and complex regulation by different signal transduction pathways involving G proteins, cyclic nucleotides, and Ca2+ ions.
Asunto(s)
Calcio/metabolismo , Carcinoma de Ehrlich/metabolismo , GMP Cíclico/metabolismo , Sistemas de Mensajero Secundario , Transducción de Señal , Animales , Bucladesina/farmacología , Calcimicina/farmacología , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Membrana Celular/metabolismo , GMP Dibutiril Cíclico/farmacología , Inhibidores Enzimáticos/farmacología , Femenino , Cinética , Ratones , NADH NADPH Oxidorreductasas/metabolismo , Neomicina/farmacología , Oxidación-Reducción , Esfingosina/farmacología , Tapsigargina/farmacologíaRESUMEN
Ferricyanide reductase activity of plasma membranes isolated from Ehrlich ascites tumour cells was very sensitive to trypsin treatment. The decreases of activity observed after treatment with different glycosidases suggests that ferricyanide reductase is a glycoprotein. The opposite effects of phospholipase A2 and phospholipase C on the redox activity indicate that the phospholipidic environment plays an important role in the function of ferricyanide reductase. Sodium ions at millimolar concentrations, and some divalent cations at micromolar concentrations (Ca2+, Mg2+, Sr2+, and Mn2+) behaved as stimulators of ferricyanide reductase activity.
Asunto(s)
Carcinoma de Ehrlich/metabolismo , Membrana Celular/metabolismo , Animales , Cationes Bivalentes/farmacología , Glicósido Hidrolasas/farmacología , NADH NADPH Oxidorreductasas/efectos de los fármacos , NADH NADPH Oxidorreductasas/metabolismo , Oxidación-Reducción , Fosfolipasas/farmacología , Tripsina/farmacologíaRESUMEN
The existence of histidine residue(s) implicated in the active site of NADH-ferricyanide oxidoreductase in plasma membrane vesicles isolated from Ehrlich ascites tumour cells is investigated. The shape of the pH-dependence curve of the enzyme activity suggests that one or more histidine residues are located at (or near) the active site of the enzyme. This hypothesis is supported by the following experimental data: the loss of activity after treatment with diethyl pyrocarbonate (DEPC) or photooxidation by using Rose bengal, and the strong inhibition caused by Zn2+ ions at micromolar concentrations. The combined arguments support the statement that histidine plays an essential role in the catalytic activity of NADH-ferricyanide oxidoreductase from Ehrlich ascites tumour cells.
Asunto(s)
Carcinoma de Ehrlich/enzimología , Histidina/metabolismo , Animales , Sitios de Unión , Membrana Celular/enzimología , Cloruros , Dietil Pirocarbonato , Concentración de Iones de Hidrógeno , NADH NADPH Oxidorreductasas/metabolismo , Rosa Bengala , Células Tumorales Cultivadas , Compuestos de ZincRESUMEN
Na(+)-dependent amino isobutyric acid transport by two neuroblastoma cell lines with and without amplification of the oncogene N-myc is studied. Surprisingly, the contribution of system A is greater in the cell line showing no N-myc amplification. Preliminary data support a role for essential tyrosine and cysteine residues in the active center of the carriers, mainly in system A.
Asunto(s)
Ácidos Aminoisobutíricos/metabolismo , Neuroblastoma/metabolismo , Sodio/metabolismo , Aminoácidos/farmacología , Transporte Biológico/efectos de los fármacos , Amplificación de Genes , Genes myc , Células Tumorales CultivadasRESUMEN
Ehrlich cell plasma membrane vesicles have a ferricyanide reductase activity that shows two phases. These two phases were kinetically characterized. Evidence is presented for a differential effect of trypsin on both phases.
Asunto(s)
Carcinoma de Ehrlich/enzimología , Animales , Membrana Celular/enzimología , Cinética , Ratones , NADH NADPH Oxidorreductasas/metabolismo , Concentración Osmolar , Cloruro de Sodio/farmacología , Tripsina/farmacologíaRESUMEN
In different neuroblastoma cell lines and transfected clones, an increasing plasma membrane redox activity correlates with amplification and enhanced expression of the N-myc oncogene. Furthermore, plasma membrane redox activity is partially inhibited by retinoic acid in neuroblastoma cells with multiple copies of the N-myc oncogene but not in neuroblastoma cells with only one copy of this gene.