RESUMEN
c-di-AMP is an important second messenger molecule that plays a pivotal role in regulating fundamental cellular processes, including osmotic and cell wall homeostasis in many Gram-positive organisms. In the opportunistic human pathogen Staphylococcus aureus, c-di-AMP is produced by the membrane-anchored DacA enzyme. Inactivation of this enzyme leads to a growth arrest under standard laboratory growth conditions and a re-sensitization of methicillin-resistant S. aureus (MRSA) strains to ß-lactam antibiotics. The gene coding for DacA is part of the conserved three-gene dacA/ybbR/glmM operon that also encodes the proposed DacA regulator YbbR and the essential phosphoglucosamine mutase GlmM, which is required for the production of glucosamine-1-phosphate, an early intermediate of peptidoglycan synthesis. These three proteins are thought to form a complex in vivo and, in this manner, help to fine-tune the cellular c-di-AMP levels. To further characterize this important regulatory complex, we conducted a comprehensive structural and functional analysis of the S. aureus DacA and GlmM enzymes by determining the structures of the S. aureus GlmM enzyme and the catalytic domain of DacA. Both proteins were found to be dimers in solution as well as in the crystal structures. Further site-directed mutagenesis, structural and enzymatic studies showed that multiple DacA dimers need to interact for enzymatic activity. We also show that DacA and GlmM form a stable complex in vitro and that S. aureus GlmM, but not Escherichia coli or Pseudomonas aeruginosa GlmM, acts as a strong inhibitor of DacA function without the requirement of any additional cellular factor. Based on Small Angle X-ray Scattering (SAXS) data, a model of the complex revealed that GlmM likely inhibits DacA by masking the active site of the cyclase and preventing higher oligomer formation. Together these results provide an important mechanistic insight into how c-di-AMP production can be regulated in the cell.
Asunto(s)
Inhibidores de Adenilato Ciclasa/metabolismo , Adenilil Ciclasas/metabolismo , Adenilil Ciclasas/ultraestructura , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Pared Celular/metabolismo , Fosfatos de Dinucleósidos/antagonistas & inhibidores , Fosfatos de Dinucleósidos/metabolismo , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/metabolismo , Operón/genética , Fosfoglucomutasa/metabolismo , Liasas de Fósforo-Oxígeno/metabolismo , Dominios Proteicos , Dispersión del Ángulo Pequeño , Sistemas de Mensajero Secundario/genética , Infecciones Estafilocócicas/genética , Staphylococcus aureus/metabolismo , Staphylococcus aureus/fisiología , Difracción de Rayos X/métodosRESUMEN
We previously demonstrated that uridine adenosine tetraphosphate (Up4A) exerts a potent vasodilator effect in the healthy porcine coronary vasculature. Since the coronary microvascular effects of Up4A after myocardial infarction (MI) are unknown, the present study investigated the response to Up4A in coronary microvessels from post-MI remodeled porcine myocardium, and the involvement of purinergic receptor subtypes. Coronary small arteries (diameter â¼150 µm) were dissected from the apex of Sham-operated swine and swine in which MI had been produced 5 weeks earlier by transient (2h) occlusion of the left circumflex coronary artery, and mounted on Mulvany wire myographs. Up4A (10(-9)-10(-5)M) produced coronary vasodilation that was reduced in MI as compared to Sham-operated swine. Up4A-induced vasodilation was reduced by P1 blockade with 8-phenyltheophylline in Sham-operated swine and to a lesser extent in MI, while the attenuation by the A2A receptor blocker SCH58261 was similar in Sham-operated and MI swine. Up4A-induced vasodilation remained unaffected by non-selective P2 receptor antagonist PPADS, but was attenuated by selective P2X1 and P2Y1 receptor antagonists MRS2159 and MRS2179, albeit to a similar extent in Sham-operated and MI swine. These responses were paralleled by similar mRNA expression levels of A2A, P2X1 and P2Y1 receptors in MI compared to slaughterhouse control swine. Finally, attenuation of Up4A-induced coronary vasodilation by nitric oxide synthase inhibition was not attenuated in MI as compared to Sham-operated swine. In conclusion, blunted coronary vasodilation in response to Up4A in MI swine is most likely due to reduced activation of P1, rather than P2, receptors and does not involve a loss of NO bioavailability.
Asunto(s)
Vasos Coronarios/efectos de los fármacos , Fosfatos de Dinucleósidos/farmacología , Infarto del Miocardio/metabolismo , Miocardio/metabolismo , Agonistas del Receptor Purinérgico P1/farmacología , Vasodilatadores/farmacología , Remodelación Ventricular/efectos de los fármacos , Antagonistas del Receptor de Adenosina A2/farmacología , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/farmacología , Animales , Vasos Coronarios/fisiología , Fosfatos de Dinucleósidos/antagonistas & inhibidores , Corazón/efectos de los fármacos , Antagonistas de Receptores Purinérgicos P1/farmacología , Antagonistas del Receptor Purinérgico P2Y/farmacología , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacología , Pirimidinas/farmacología , Receptores Purinérgicos/biosíntesis , Receptores Purinérgicos P1/metabolismo , Teofilina/análogos & derivados , Teofilina/farmacología , Triazoles/farmacología , Vasodilatadores/antagonistas & inhibidores , Remodelación Ventricular/fisiologíaRESUMEN
(1) Diadenosine polyphosphates have been described to be present in the myocardium and exert purinergic- and nonreceptor-mediated effects. Since the electrophysiological properties of atrial myocardium are effectively regulated by A(1) receptors, we investigated the effect of diadenosine pentaphosphate (Ap(5)A) in rabbit myocardium. (2) Parameters of supraventricular electrophysiology and atrial vulnerability were measured in Langendorff-perfused rabbit hearts. Muscarinic potassium current (I(K(ACh/Ado))) and ATP-sensitive potassium current (I(K(ATP))) were measured by using the whole-cell voltage clamp method. (3) Ap(5)A prolonged the cycle length of spontaneously beating Langendorff perfused hearts from 225+/-14 (control) to 1823+/-400 ms (Ap(5)A 50 micro M; n=6; P<0.05). This effect was paralleled by higher degree of atrio-ventricular block. Atrial effective refractory period (AERP) in control hearts was 84+/-14 ms (n=6). Ap(5)A>/=1 micro M reduced AERP (100 micro M, 58+/-11 ms; n=6). (4) Extrastimuli delivered to hearts perfused with Ap(5)A- or adenosine (>/= micro M)-induced atrial fibrillation, the incidence of which correlated to the concentration added to the perfusate. The selective A(1)-receptor antagonist CPX (20 micro M) inhibited the Ap(5)A- and adenosine-induced decrease of AERP. Atrial fibrillation was no longer observed in the presence of CPX. (5) The described Ap(5)A-induced effects in the multicellular preparation were enhanced by dipyridamole (10 micro M), which is a cellular adenosine uptake inhibitor. Dipyridamole-induced enhancement was inhibited by CPX. (6) Ap(5)A (=1 mM) did neither induce I(K(Ado)) nor I(K(ATP)). No effect on activated I(K(Ado/ATP)) was observed in myocytes superfused with Ap(5)A. However, effluents from Langendorff hearts perfused with Ap(5)A 100 micro M activated I(K(Ado)) by using A(1) receptors. (7) Ap(5)A did not activate A(1) receptors in rabbit atrial myocytes. The Ap(5)A induced A(1)-receptor-mediated effects on supraventricular electrophysiology and vulnerability suggest that in the multicellular preparation Ap(5)A is hydrolyzed to yield adenosine, which acts via A(1) receptors. An influence on atrial electrophysiology or a facilitation of atrial fibrillation under conditions resulting in increased interstitial Ap(5)A concentrations might be of physiological/pathophysiological relevance.
Asunto(s)
Fibrilación Atrial/inducido químicamente , Fosfatos de Dinucleósidos/farmacología , Miocitos Cardíacos/efectos de los fármacos , Receptor de Adenosina A1/efectos de los fármacos , Acetilcolina/farmacología , Adenosina/antagonistas & inhibidores , Adenosina/biosíntesis , Adenosina/farmacología , Antagonistas del Receptor de Adenosina A1 , Animales , Fibrilación Atrial/fisiopatología , Función Atrial/efectos de los fármacos , Fosfatos de Dinucleósidos/antagonistas & inhibidores , Fosfatos de Dinucleósidos/metabolismo , Dipiridamol/farmacología , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Electrofisiología , Atrios Cardíacos/citología , Atrios Cardíacos/efectos de los fármacos , Atrios Cardíacos/fisiopatología , Frecuencia Cardíaca/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Perfusión/métodos , Conejos , Receptor de Adenosina A1/fisiología , Periodo Refractario Electrofisiológico/efectos de los fármacos , Transducción de Señal , Xantinas/farmacologíaRESUMEN
FHIT is a tumor suppressor gene that is frequently inactivated in human cancer. Although the Fhit protein is known to hydrolyze diadenosine triphosphate (Ap(3)A), this hydrolase activity is not required for Fhit-mediated oncosuppression. Indeed, the molecular mechanisms and the regulatory elements of Fhit oncosuppression are largely unknown. Here, we review physiological and pathological aspects of Fhit in the context of the Ap(n)A family of signaling molecules, as well as the involvement of Fhit in apoptosis and the cell cycle in cancer models. We also discuss recent findings of novel Fhit interactions that may lead to new hypotheses about biochemical mechanisms underlying the oncosuppressor activity of this gene.
Asunto(s)
Fosfatos de Dinucleósidos/antagonistas & inhibidores , Fosfatos de Dinucleósidos/genética , Genes Supresores de Tumor , Proteínas de Neoplasias/antagonistas & inhibidores , Animales , Humanos , Modelos Biológicos , Proteínas de Neoplasias/genéticaRESUMEN
Whereas the role of immune complexes in mediating renal cell and immune cell activation is well established, the contribution of sequence-specific immunomodulatory actions of the chromatin part remains unclear. Toll-like receptor-9 (TLR-9) mediates immunostimulatory effects of unmethylated microbial CpG-DNA. It was hypothesized that hypomethylated CpG-DNA in vertebrates may have similar effects and may contribute to disease progression in lupus nephritis. A synthetic G-rich DNA, known to block CpG-DNA effects, was used in this study. In macrophages, G-rich DNA suppressed CpG-DNA-but not LPS-induced production of CCL5 in a dose-dependent manner. Injections of G-rich DNA suppressed lymphoproliferation induced by CpG-DNA injections in mice. In MRL(lpr/lpr) mice with lupus nephritis, labeled G-rich DNA co-localized to glomerular immune complexes and was taken up into endosomes of TLR-9-positive infiltrating macrophages. Eleven-week-old MRL(lpr/lpr) mice that received injections of either saline or G-rich DNA for 13 wk revealed decreased lymphoproliferation and less autoimmune tissue injury in lungs and kidneys as compared with saline-treated controls. G-rich DNA reduced the levels of serum dsDNA-specific IgG2a as well as the renal immune complex deposits. This was consistent with the blocking effect of G-rich DNA on CpG-DNA-induced proliferation of B cells that were isolated from MRL(lpr/lpr) mice. As oligodeoxyribonucleotide 2114-treated MRL(lpr/lpr) mice were not exposed to exogenous CpG-DNA, these effects should relate to a blockade of CpG motifs in endogenous DNA. It is concluded that adjuvant activity of self-DNA contributes to the pathogenesis of lupus nephritis. Modulating the CpG-DNA-TLR-9 pathway may offer new opportunities for the understanding and treatment of lupus.
Asunto(s)
Fosfatos de Dinucleósidos/farmacología , Nefritis Lúpica/inmunología , Oligodesoxirribonucleótidos/farmacología , Animales , Enfermedades Autoinmunes/prevención & control , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Línea Celular , Metilación de ADN , Fosfatos de Dinucleósidos/antagonistas & inhibidores , Nefritis Lúpica/prevención & control , Activación de Linfocitos/efectos de los fármacos , Trastornos Linfoproliferativos/inmunología , Trastornos Linfoproliferativos/prevención & control , Macrófagos/fisiología , Ratones , Ratones MutantesRESUMEN
T4 RNA ligase has been shown to synthesize nucleoside and dinucleoside 5'-polyphosphates by displacement of the AMP from the E-AMP complex with polyphosphates and nucleoside diphosphates and triphosphates. Displacement of the AMP by tripolyphosphate (P3) was concentration dependent, as measured by SDS/PAGE. When the enzyme was incubated in the presence of 0.02 mm [alpha-32P] ATP, synthesis of labeled Ap4A was observed: ATP was acting as both donor (Km, microm) and acceptor (Km, mm) of AMP from the enzyme. Whereas, as previously known, ATP or dATP (but not other nucleotides) were able to form the E-AMP complex, the specificity of a compound to be acceptor of AMP from the E-AMP complex was very broad, and with Km values between 1 and 2 mm. In the presence of a low concentration (0.02 mm) of [alpha-32P] ATP (enough to form the E-AMP complex, but only marginally enough to form Ap4A) and 4 mm of the indicated nucleotides or P3, the relative rate of synthesis of the following radioactive (di)nucleotides was observed: Ap4X (from XTP, 100); Ap4dG (from dGTP, 74); Ap4G (from GTP, 49); Ap4dC (from dCTP, 23); Ap4C (from CTP, 9); Ap3A (from ADP, 5); Ap4ddA, (from ddATP, 1); p4A (from P3, 200). The enzyme also synthesized efficiently Ap3A in the presence of 1 mm ATP and 2 mm ADP. The following T4 RNA ligase donors were inhibitors of the synthesis of Ap4G: pCp > pAp > pA2'p.
Asunto(s)
Bacteriófago T4/enzimología , Fosfatos de Dinucleósidos/biosíntesis , ARN Ligasa (ATP)/metabolismo , Adenosina Monofosfato/metabolismo , Catálisis , Fosfatos de Dinucleósidos/antagonistas & inhibidores , Concentración de Iones de Hidrógeno , Especificidad por SustratoRESUMEN
The diadenosine polyphosphates diadenosine tetraphosphate (Ap4A) and diadenosine pentaphosphate (Ap5A) are costored with ATP and released in a calcium-dependent manner from neural preparations in vitro. By means of a push-pull perfusion system, samples from conscious rat were collected from the caudate putamen area, and nucleotide compounds were analyzed by HPLC. The adenine dinucleotides were not detectable before systemic amphetamine injection. The maximal levels were reached 20 min after injection, independently of the dose. The EC50 values for amphetamine-induced release of dinucleotides were 2.04 +/- 0.15 and 2.43 +/- 0.36 mg/kg for Ap4A and Ap5A, respectively. Amphetamine doses higher than 5 mg/kg did not increase the dinucleotide release, the maximal values being 12.9 +/- 0.9 and 11.5 +/- 0.9 pmol/fraction for Ap4A and Ap5A, respectively, which corresponds with 64.5 and 57.5 nM in the samples. Adenosine and AMP were present in push-pull samples from rat brain under basal conditions. Their levels were 15 pmol/fraction (75 nM) and 50 pmol/fraction (250 nM) for adenosine and AMP, respectively. A significant increase was obtained for both compounds after amphetamine injection. The adenosine increase reached 45 pmol/sample (225 nM), which was 200% of the basal value 20 min after the stimulant administration. The increase at other times was not significant. The AMP levels increased significantly from 10 to 50 min. The maximal level was reached 20 min after amphetamine injection, with 150 pmol/fraction (750 nM), which represents a 200% increase with respect to the basal level. The adenine dinucleotide release was blocked by the dopamine receptor antagonist haloperidol, which returned the levels to the control basal values.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Anfetamina/farmacología , Cuerpo Estriado/metabolismo , Fosfatos de Dinucleósidos/antagonistas & inhibidores , Antagonistas de Dopamina/farmacología , Adenosina/metabolismo , Adenosina Monofosfato/metabolismo , Animales , Fosfatos de Dinucleósidos/metabolismo , Haloperidol/farmacología , Masculino , Perfusión , Ratas , Ratas WistarRESUMEN
Extracellular P1,P4-diadenosine 5'-tetraphosphate (Ap4A) has been implicated as a modulator of cell stress. We have previously demonstrated specific receptors for Ap4A at the surface of cardiac myocytes (Walker et al., 1993a). In addition, we have isolated a monoclonal antibody (mAb TL4) that recognized the Ap4A receptor and inhibited binding of Ap4A to its receptor (Walker & Hilderman, 1993). As part of our effort to characterize the Ap4A receptor building domain, we screened a random phage peptide library with mAb TL4. After affinity purification of specifically bound phage, we isolated 38 individual phage clones. Twenty-eight of these clones bound mAb TL4 in ELISA and dot blot analyses. Twenty-two of the twenty-eight individual clones contained inserts with an RGS tripeptide sequence. Synthetic RGS peptide specifically inhibits the binding of mAb TL4 to its membrane receptor. Furthermore, the RGS peptide also inhibits [3H]Ap4A binding to its receptor. These data are consistent with the RGS peptide mimicking part of the mAb TL4 recognition site on the Ap4A receptor. The The RGS peptide may be used to help characterize the Ap4A receptor binding domain and to help determine the physiological significance of the interaction between Ap4A and its receptor.
Asunto(s)
Fosfatos de Dinucleósidos/antagonistas & inhibidores , Fosfatos de Dinucleósidos/metabolismo , Miocardio/metabolismo , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Receptores Purinérgicos P2/metabolismo , Secuencia de Aminoácidos , Animales , Bacteriófagos , Membrana Celular/metabolismo , Bases de Datos Factuales , Ensayo de Inmunoadsorción Enzimática , Epítopos , Ratones , Datos de Secuencia Molecular , Oligopéptidos/química , Antagonistas del Receptor Purinérgico P2 , Relación Estructura-ActividadRESUMEN
Stimulation of a Ca(2+)-induced Ca(2+)-release channel from skeletal muscle sarcoplasmic reticulum by various adenosine(5')oligophospho(5')adenosines (ApnA, n = 2-6) by a rapid quenching technique using radioactive calcium was studied. Ap4A, Ap5A and Ap6A, as well as adenosine 5'-[beta, gamma-methylene]triphosphate (AdoPP [CH2]P), a non-hydrolyzable ATP analogue, stimulated the Ca(2+)-release channel, whereas Ap2A and Ap3A had no effect. At a concentration of 0.5 mM, the order of stimulation was AdoPP[CH2]P less than Ap4A less than Ap5A much less than Ap6A. As well as having the highest affinity (0.44 mM for half-maximal stimulation), Ap6A showed an extraordinarily high Hill coefficient of 3.3 (1.9 for AdoPP[CH2]P, 2.1 for Ap5A). The stimulating effect of Ap6A was reversible, yet its dissociation proceeded very slowly. Stimulation of Ca2+ release by Ap6A was counteracted by Mg2+ and ruthenium red. A 2',3'-dialdehyde derivative of Ap6A, which is a chemical probe for amino groups, stimulated irreversibly the Ca(2+)-release channel and modified some high-molecular-mass sarcoplasmic reticulum proteins, possibly including the channel protein. Our data suggest that Ap6A stimulates the Ca2+ channel by binding to the activation site of the channel subunit and simultaneously preventing the spontaneous decay of the Ca2+ channel by keeping together two of the four channel subunits by bridging them with its two adenosine groups.