RESUMEN
Ligand-biased receptor signaling has been proposed for several G-protein coupled receptors including the niacin receptor GPR109A. Coupling to the G(i/o) pathway has been shown to be responsible for the well described triglyceride lowering effect of nicotinic acid in mice, while activation of the ß-arrestin pathway has been suggested to be responsible for its peripheral vasodilatory effect that causes cutaneous flushing. Several ligands have been described to selectively induce triglyceride lowering without inducing flushing. Cellular impedance has been demonstrated to determine G-protein coupled receptors activation in a G-protein specific manner. Agonists, which induce triglyceride lowering, but not flushing show a profile in cellular impedance that is distinct from the one induced by niacin and those compounds that induce triglyceride lowering as well as flushing. The strength of the signal correlates with the activation of ß-arrestin.
Asunto(s)
Impedancia Eléctrica , Receptores Acoplados a Proteínas G/agonistas , Animales , Línea Celular , Humanos , Ligandos , Ratones , Niacina/farmacología , Receptores Acoplados a Proteínas G/análisis , Receptores Nicotínicos/análisis , Transducción de Señal , Triglicéridos/metabolismoRESUMEN
An assay was developed to determine the activity of peptide deformylase (PDF) inhibitors under conditions as close as possible to the physiological situation. The assay principle is the detection of N-terminal [35S]methionine labeling of a protein that contains no internal methionine. If PDF is active, the deformylation of the methionine renders the peptide a substrate for methionine aminopeptidase, resulting in the removal of the N-terminal methionine label. In the presence of a PDF inhibitor, the deformylation is blocked so that the N-formylated peptide is not processed and the label is detected. Using this assay, it is possible to determine the PDF activity under near-physiological conditions in a cell-free transcription-translation system as well as in intact bacterial cells.
Asunto(s)
Amidohidrolasas , Aminopeptidasas/antagonistas & inhibidores , Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Escherichia coli/efectos de los fármacos , Pruebas de Sensibilidad Microbiana/métodos , Proteínas Bacterianas/efectos de los fármacos , Extractos Celulares/análisis , Escherichia coli/enzimología , Metionina/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
New inhibitors of peptide deformylase (PDF) which are very potent against the isolated enzyme and show a certain degree of antibacterial activity have recently been synthesized by our group. Several lines of experimental evidence indicate that these inhibitors indeed interfere with the target enzyme in the bacterial cell. (i) The inhibition of Escherichia coli growth could be counteracted by overexpression of PDF from different organisms, including E. coli, Streptococcus pneumoniae, and Haemophilus influenzae. Conversely, reduced expression of PDF in S. pneumoniae resulted in an increased susceptibility to the inhibitors. (ii) Proteome analysis on two-dimensional gels revealed a shift for many proteins towards lower pI in the presence of PDF inhibitors, as would be expected if the proteins still carry their N-formyl-Met terminus. (iii) PDF inhibitors show no antimicrobial activity against E. coli under conditions that make growth independent of formylation and deformylation. The antibacterial activity in E. coli was characterized as bacteriostatic. Furthermore, the development of resistance in E. coli was observed to occur with high frequency (10(-7)). Resistant mutants show a reduced growth rate, and DNA sequence analysis revealed mutations in their formyl transferase gene. Taking all these aspects into account, we conclude that PDF may not be an optimal target for broad-spectrum antibacterial agents.
Asunto(s)
Amidohidrolasas , Aminopeptidasas/antagonistas & inhibidores , Antibacterianos/farmacología , Farmacorresistencia Microbiana , Inhibidores Enzimáticos/farmacología , Escherichia coli/efectos de los fármacos , Pruebas de Sensibilidad Microbiana/métodos , Aminopeptidasas/genética , Aminopeptidasas/metabolismo , Antiinfecciosos Urinarios/farmacología , Proteínas Bacterianas/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Antagonismo de Drogas , Escherichia coli/enzimología , Escherichia coli/genética , Genes Bacterianos , Haemophilus influenzae/efectos de los fármacos , Haemophilus influenzae/metabolismo , Mutagénesis Insercional , Mutación , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo , Transformación Bacteriana , Trimetoprim/farmacologíaRESUMEN
The prenyltransferase undecaprenyl pyrophosphate synthetase (di-trans,poly-cis-decaprenylcistransferase; EC 2.5.1.31) was purified from the soluble fraction of Escherichia coli by TSK-DEAE, ceramic hydroxyapatite, TSK-ether, Superdex 200, and heparin-Actigel chromatography. The protein was labeled with the photolabile analogue of the farnesyl pyrophosphate analogue (E, E)-[1-3H]-(2-diazo-3-trifluoropropionyloxy)geranyl diphosphate and was detected on a sodium dodecyl sulfate-polyacrylamide gel as a protein with an apparent molecular mass of 29 kDa. This protein band was cut out from the gel, trypsin digested, and subjected to matrix-assisted laser desorption ionization mass spectrometric analysis. Comparison of the experimental data with computer-simulated trypsin digest data for all E. coli proteins yielded a single match with a protein of unassigned function (SWISS-PROT Q47675; YAES_ECOLI). Sequences with strong similarity indicative of homology to this protein were identified in 25 bacterial species, in Saccharomyces cerevisiae, and in Caenorhabditis elegans. The homologous genes (uppS) were cloned from E. coli, Haemophilus influenzae, and Streptococcus pneumoniae, expressed in E. coli as amino-terminal His-tagged fusion proteins, and purified over a Ni2+ affinity column. An untagged version of the E. coli uppS gene was also cloned and expressed, and the protein purified in two chromatographic steps. We were able to detect Upp synthetase activity for all purified enzymes. Further, biochemical characterization revealed no differences between the recombinant untagged E. coli Upp synthetase and the three His-tagged fusion proteins. All enzymes were absolutely Triton X-100 and MgCl2 dependent. With the use of a regulatable gene disruption system, we demonstrated that uppS is essential for growth in S. pneumoniae R6.
Asunto(s)
Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Transferasas Alquil y Aril/química , Secuencia de Aminoácidos , Animales , Bacterias/enzimología , Caenorhabditis elegans/enzimología , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Mapeo Cromosómico , Cromosomas Bacterianos , Clonación Molecular , Cinética , Datos de Secuencia Molecular , Peso Molecular , Mutagénesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimología , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The purpose of this study was to investigate the effects of retinoid analogues with different retinoid receptor specificity on the growth of human D10 and Cloudman S91 mouse melanoma cells. We compared the growth inhibitory effects with the ability of retinoids to downregulate cell surface expression of the melanocortin receptor (MC1-R). Retinoic acid receptor (RAR)-gamma-selective retinoids exerted the most prominent growth effects, with up to 68% and 69% inhibition in D10 and S91 cells, respectively. A retinoid X receptor (RXR)-selective compound inhibited cell growth by only 14% and 23% in D10 and S91 cells, respectively. Growth inhibition by RARalpha- and RARbeta-selective compounds was below 10% in both cells. In D10 cells, MC1-R downregulation was also induced most effectively by an RARgamma-selective retinoid (84% relative to controls). RARalpha-, RARbeta-and RXR-selective agonists induced only 16-24% MC1-R downregulation in these cells. The pattern for MC1-R downregulation was completely different in S91 cells. The RXR-selective compound was the most active (85%), followed by the RARalpha-selective agonist (58%), the RARgamma-selective compound (47%), and finally by the RARbeta-selective agonist (29%). We conclude that RARgamma-selective retinoids may have potential as therapeutic agents in melanoma. Different selectivity profiles for growth inhibition and MC1-R downregulation in S91 cells suggest that these two retinoid effects are not directly dependent on each other.
Asunto(s)
Melanoma Experimental/metabolismo , Melanoma/metabolismo , Receptores de Corticotropina/biosíntesis , Retinoides/farmacología , Tretinoina/farmacología , Animales , División Celular/efectos de los fármacos , AMP Cíclico/metabolismo , Regulación hacia Abajo , Humanos , Melanoma/patología , Melanoma Experimental/patología , Ratones , Receptores de Melanocortina , Receptores de Ácido Retinoico/fisiología , Receptor alfa de Ácido Retinoico , Receptores X Retinoide , Relación Estructura-Actividad , Factores de Transcripción/fisiología , Tretinoina/análogos & derivados , Células Tumorales Cultivadas , Receptor de Ácido Retinoico gammaRESUMEN
Retinoids exert their pleiotropic effects on cell differentiation and proliferation through specific nuclear receptors, the retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Two biologically highly active natural retinoids have been identified, all-trans-retinoic acid (t-RA) and 9-cis-retinoic acid (9-cis-RA). The RXRs exclusively bind 9-cis-RA, whereas the RARs bind both isomers of RA with comparable affinity. Recently published results suggest that RARs have the same binding site for t-RA and 9-cis-RA but with different determinants (1-3). Antagonist binding on RARalpha has been suggested to induce distinct conformational changes in comparison with agonist binding. To elucidate the region minimally required for efficient binding of agonist (t-RA and 9-cis-RA) and antagonist Ro 41-5253 to the RARalpha, we generated N- and C-terminally truncated mutants of the receptor. Characterization of these deletion mutant proteins using protease mapping and ligand binding experiments revealed that different parts of the ligand-binding domain are necessary for t-RA, 9-cis-RA, and antagonist binding. Three distinct regions of the ligand-binding domain of the human retinoic acid receptor-alpha are required for binding of t-RA (RARalpha187-402), 9-cis-RA (RARalpha188-409), and the antagonist Ro 41-5253 (RARalpha226-414).
Asunto(s)
Benzoatos/metabolismo , Cromanos/metabolismo , Receptores de Ácido Retinoico/química , Receptores de Ácido Retinoico/metabolismo , Tretinoina/metabolismo , Alitretinoína , Sitios de Unión , Análisis Mutacional de ADN , Cartilla de ADN , Humanos , Cinética , Mutagénesis Insercional , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Receptor alfa de Ácido Retinoico , Retinoides/metabolismo , Eliminación de SecuenciaRESUMEN
Three subtypes of retinoic acid receptors (RAR), termed RAR alpha, RAR beta, and RAR gamma, have been described. They are composed of different structural domains, including distinct domains for DNA and ligand binding. RARs specifically bind all-trans-retinoic acid (RA), 9-cis-RA, and retinoid analogs. In this study, we examined the functional role of cysteine and arginine residues in the ligand-binding domain of hRAR alpha (hRAR alpha-LBD, amino acids 154 to 462). All conserved cysteine and arginine residues in this domain were mutated by site-directed mutagenesis, and the mutant proteins were characterized by blocking reactions, ligand-binding experiments, transactivation assays, and protease mapping. Changes of any cysteine residue of the hRAR alpha-LBD had no significant influence on the binding of all-trans RA or 9-cis RA. Interestingly, residue C-235 is specifically important in antagonist binding. With respect to arginine residues, only the two single mutations of R-276 and R-394 to alanine showed a dramatic decrease of agonist and antagonist binding whereas the R272A mutation showed only a slight effect. For all other arginine mutations, no differences in affinity were detectable. The two mutations R217A and R294A caused an increased binding efficiency for antagonists but no change in agonist binding. From these results, we can conclude that electrostatic interactions of retinoids with the RAR alpha-LBD play a significant role in ligand binding. In addition, antagonists show distinctly different requirements for efficient binding, which may contribute to their interference in the ligand-inducible transactivation function of RAR alpha.
Asunto(s)
Receptores de Ácido Retinoico/química , Receptores de Ácido Retinoico/metabolismo , Tretinoina/metabolismo , Alanina , Secuencia de Aminoácidos , Animales , Arginina , Secuencia de Bases , Benzoatos/metabolismo , Sitios de Unión , Cromanos/metabolismo , Cisteína , Humanos , Cinética , Ligandos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Mutación Puntual , Biosíntesis de Proteínas , Conejos , Reticulocitos/metabolismo , Receptor alfa de Ácido Retinoico , Retinoides/metabolismo , Homología de Secuencia de Aminoácido , Transcripción Genética , Activación Transcripcional , Receptor de Ácido Retinoico gammaRESUMEN
The broad spectrum of physiological activities of retinoids is mediate d by two types of receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Though they have 9-cis retinoic acid as a common ligand, the amino acid sequence of their ligand binding domains is only distantly related (27%). This fact makes it probable that the ligand binding pockets of RARs and RXRs differ significantly with respect to their three dimensional structure. Therefore, one can expect that selective ligands for these receptor subclasses do exit. A clear example of a naturally existing RAR-selective retinoid is all-trans retinoic acid. Here we report on two synthetic retinoids which are very closely related to retinoic acid in structure yet show good receptor subclass selectivity. These compounds have a saturated double bond in the polyene side chain between either the 7, 8 or 9, 10 carbon atoms and are highly RAR or RXR selective, respectively (as shown by receptor binding, transactivation activity and the ability to induce RXR homodimer formation). In addition, we present compounds of the synthetic arotinoid class which are highly RAR selective. Interestingly the corresponding '9-cis analogs' are not able to bind or activate RXR alpha and show greatly reduced activity on the RARs.
Asunto(s)
Receptores de Ácido Retinoico/efectos de los fármacos , Retinoides/farmacología , Factores de Transcripción/efectos de los fármacos , Animales , Núcleo Celular/metabolismo , Ligandos , Ratones , Receptores X Retinoide , Relación Estructura-ActividadRESUMEN
Metabolic defects in phytanic acid catabolism have been shown to be connected with a number of human diseases which can lead to lethal defects of the nervous system and other organs. These effects are probably a result of the very high accumulation of phytanic acid in tissues throughout the body, due to defects in phytanic acid oxidation, the peroxisome being a major site for this process. The nuclear hormone receptors peroxisome proliferator-activated receptor and retinoid X receptor (RXR) have been shown to function as transcription factors in the control of the peroxisomal enzyme expression. Known activators of peroxisome proliferator-activated receptor include polyunsaturated fatty acids and, for RXR, the 9-cis isomer of retinoic acid. Here we report that phytanic acid is also a natural ligand for RXR alpha, being able to activate a RXR-responsive promoter. We present evidence that phytanic acid binds to RXR alpha, promotes formation of an RXR alpha/RXR response element complex (as detected by gel retardation), and induces a RXR alpha conformational change similar to that induced by 9-cis-retinoic acid (as detected by protease sensitivity). These results suggest an involvement of RXR alpha in the control of fatty acid metabolism and could imply that RXRs have a role in the disease effects resulting from defective phytanic acid catabolism.
Asunto(s)
Ácido Fitánico/metabolismo , Receptores de Ácido Retinoico/agonistas , Factores de Transcripción/agonistas , Secuencia de Bases , Relación Dosis-Respuesta a Droga , Escherichia coli/genética , Humanos , Ligandos , Datos de Secuencia Molecular , Receptores de Ácido Retinoico/genética , Proteínas Recombinantes/metabolismo , Enfermedad de Refsum/metabolismo , Receptores X Retinoide , Factores de Transcripción/genética , Activación Transcripcional/efectos de los fármacos , Tretinoina/metabolismoRESUMEN
Cellular responsiveness to retinoic acid and its metabolites is conferred through two distinct families of receptors: the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Herein, we report on the identification and characterization of several conformationally restricted retinoids, which selectively bind and activate RX receptors. Under the influence of retinoids, HL-60 myelocytic leukemia cells differentiate into granulocytes. This effect is mediated by RAR alpha, as has been demonstrated through the use of a selective RAR alpha antagonist (Apfel, C., Bauer, F., Crettaz, M., Forni, L., Kamber, M., Kaufmann, F., LeMotte, P., Pirson, W., and Klaus, M. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 7129-7133). Here, we show that conformationally restricted RXR-specific retinoids, at doses that are per se inactive, are able to potentiate by up to one order of magnitude the pro-differentiating effects of all-trans retinoic acid and an RAR alpha-selective synthetic retinoid. We also present evidence that these RXR-selective ligands are able to bind to a DNA RXR.RAR heterodimer complex. This finding demonstrates that agonists for RARs and RXRs can synergistically promote HL-60 differentiation, which could be mediated through a heterodimer of these receptors.