RESUMEN
Glioblastomas (GBMs), the most frequent brain tumours, are highly invasive and their prognosis is still poor despite the use of combination treatment. MG624 is a 4-oxystilbene derivative that is active on α7- and α9-containing neuronal nicotinic acetylcholine receptor (nAChR) subtypes. Hybridisation of MG624 with a non-nicotinic resveratrol-derived pro-oxidant mitocan has led to two novel compounds (StN-4 and StN-8) that are more potent than MG624 in reducing the viability of GBM cells, but less potent in reducing the viability of mouse astrocytes. Functional analysis of their activity on α7 receptors showed that StN-4 is a silent agonist, whereas StN-8 is a full antagonist, and neither alters intracellular [Ca2+] levels when acutely applied to U87MG cells. After 72 h of exposure, both compounds decreased U87MG cell proliferation, and pAKT and oxphos ATP levels, but only StN-4 led to a significant accumulation of cells in phase G1/G0 and increased apoptosis. One hour of exposure to either compound also decreased the mitochondrial and cytoplasmic ATP production of U87MG cells, and this was not paralleled by any increase in the production of reactive oxygen species. Knocking down the α9 subunit (which is expressed at relatively high levels in U87MG cells) decreased the potency of the effects of both compounds on cell viability, but cell proliferation, ATP production, pAKT levels were unaffected by the presence of the noncell-permeable α7/α9-selective antagonist αBungarotoxin. These last findings suggest that the anti-tumoral effects of StN-4 and StN-8 on GBM cells are not only due to their action on nAChRs, but also to other non-nicotinic mechanisms.
Asunto(s)
Compuestos de Amonio/farmacología , Antineoplásicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Estilbenos/farmacología , Adenosina Trifosfato/metabolismo , Animales , Astrocitos/efectos de los fármacos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Fenómenos Fisiológicos Celulares/efectos de los fármacos , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Ligandos , Ratones , Proteínas Proto-Oncogénicas c-akt/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores Nicotínicos/genética , Receptor Nicotínico de Acetilcolina alfa 7/genéticaRESUMEN
Arthrogryposis multiplex congenita (AMC) is a developmental condition characterized by multiple joint contractures resulting from reduced or absent fetal movements. Homozygosity mapping of disease loci combined with whole exome sequencing in a consanguineous family presenting with lethal AMC allowed the identification of a homozygous frameshift deletion in UNC50 gene (c.750_751del:p.Cys251Phefs*4) in the index case. To assess the effect of the mutation, an equivalent mutation in the Caenorhabditis elegans orthologous gene was created using CRISPR/Cas9. We demonstrated that unc-50(kr331) modification caused the loss of acetylcholine receptor (AChR) expression in C. elegans muscle. unc-50(kr331) animals were as resistant to the cholinergic agonist levamisole as unc-50 null mutants suggesting that AChRs were no longer expressed in this animal model. This was confirmed by using a knock-in strain in which a red fluorescent protein was inserted into the AChR locus: no signal was detected in unc-50(kr331) background, suggesting that UNC-50, a protein known to be involved in AChR trafficking, was no longer functional. These data indicate that biallelic mutation in the UNC50 gene underlies AMC through a probable loss of AChR expression at the neuromuscular junction which is essential for the cholinergic transmission during human muscle development.
Asunto(s)
Artrogriposis/genética , Artrogriposis/metabolismo , Mutación del Sistema de Lectura , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Receptores Colinérgicos/metabolismo , Alelos , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Unión Neuromuscular/genética , Unión Neuromuscular/metabolismo , Linaje , Transporte de Proteínas , Receptores Colinérgicos/genética , Mortinato/genéticaRESUMEN
The ATP synthase from Escherichia coli was isolated and reconstituted into liposomes. The ATP hydrolysis by these proteoliposomes was coupled to proton pumping, and the ensuing inner volume acidification was measured by the fluorescent probe 9-amino-6-chloro-2-methoxyacridine (ACMA). The ACMA response was calibrated by acid-base transitions, and converted into internal pH values. The rates of internal acidification and of ATP hydrolysis were measured in parallel, as a function of P(i) or ADP concentration. Increasing P(i) monotonically inhibited the hydrolysis rate with a half-maximal effect at 510µM, whereas it stimulated the acidification rate up to 100-200µM, inhibiting it only at higher concentrations. The ADP concentration in the assay, due both to contaminant ADP in ATP and to the hydrolysis reaction, was progressively decreased by means of increasing pyruvate kinase activities. Decreasing ADP stimulated the hydrolysis rate, whereas it inhibited the internal acidification rate. The quantitative analysis showed that the relative number of translocated protons per hydrolyzed ATP, i.e. the relative coupling ratio, depended on the concentrations of P(i) and ADP with apparent K(d) values of 220µM and 27nM respectively. At the smallest ADP concentrations reached, and in the absence of P(i), the coupling ratio dropped down to 15% relative to the value observed at the highest ADP and P(i) concentrations tested. In addition, the data indicate the presence of two ADP and P(i) binding sites, of which only the highest affinity one is related to changes in the coupling ratio.
Asunto(s)
Adenosina Difosfato/metabolismo , Escherichia coli/enzimología , Fosfatos/metabolismo , ATPasas de Translocación de Protón/metabolismo , Adenosina Trifosfato/metabolismo , Catálisis , Membrana Celular/enzimología , Cloroplastos/enzimología , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Fosforilación , Plásmidos , Piruvato Quinasa/metabolismoRESUMEN
In the ATP synthases of Escherichia coli ADP and phosphate exert an apparent regulatory role on the efficiency of proton transport coupled to the hydrolysis of ATP. Both molecules induce clearly biphasic effects on hydrolysis and proton transfer. At intermediate concentrations (approximately 0.5-1 microM and higher) ADP inhibits hydrolysis and proton transfer; a quantitative analysis of the fluxes however proves that the coupling efficiency remains constant in this concentration range. On the other hand at nanomolar concentrations of ADP (a level obtainable only using an enzymatic ATP regenerating system) the efficiency of proton transport drops progressively, while the rate of hydrolysis remains high. Phosphate, at concentrations>or=0.1 mM, inhibits hydrolysis only if ADP is present at sufficiently high concentrations, keeping the coupling efficiency constant. At lower ADP levels phosphate is, however, necessary for an efficiently coupled catalytic cycle. We present a model for a catalytic cycle of ATP hydrolysis uncoupled from the transport of protons. The model is based on the available structures of bovine and yeast F1 and on the known binding affinities for ADP and Pi of the catalytic sites in their different functional states. The binding site related to the inhibitory effects of Pi (in association with ADP) is identified as the alphaHCbetaHC site, the pre-release site for the hydrolysis products. We suggest, moreover, that the high affinity site, associated with the operation of an efficient proton transport, could coincide with a conformational state intermediate between the alphaTPbetaTP and the alphaDPbetaDP (similar to the transition state of the hydrolysis/synthesis reaction) that does not strongly bind the ligands and can exchange them rather freely with the external medium. The emptying of this site can lead to an unproductive hydrolysis cycle that occurs without a net rotation of the central stalk and, consequently, does not translocate protons.
Asunto(s)
Adenosina Trifosfato/metabolismo , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/metabolismo , Adenosina Difosfato/metabolismo , Animales , Dominio Catalítico , Bovinos , Hidrólisis , Técnicas In Vitro , Liposomas , Modelos Biológicos , Fosfatos/metabolismo , ProtonesRESUMEN
The ATP hydrolysis activity and proton pumping of the ATP synthase of Escherichia coli in isolated native membranes have been measured and compared as a function of ADP and Pi concentration. The ATP hydrolysis activity was inhibited by Pi with an half-maximal effect at 140 microM, which increased progressively up in the millimolar range when the ADP concentration was progressively decreased by increasing amounts of an ADP trap. In addition, the relative extent of this inhibition decreased with decreasing ADP. The half-maximal inhibition by ADP was found in the submicromolar range, and the extent of inhibition was enhanced by the presence of Pi. The parallel measurement of ATP hydrolysis activity and proton pumping indicated that, while the rate of ATP hydrolysis was decreased as a function of either ligand, the rate of proton pumping increased. The latter showed a biphasic response to the concentration of Pi, in which an inhibition followed the initial stimulation. Similarly as previously found for the ATP synthase from Rhodobacter caspulatus [P. Turina, D. Giovannini, F. Gubellini, B.A. Melandri, Physiological ligands ADP and Pi modulate the degree of intrinsic coupling in the ATP synthase of the photosynthetic bacterium Rhodobacter capsulatus, Biochemistry 43 (2004) 11126-11134], these data indicate that the E. coli ATP synthase can operate at different degrees of energetic coupling between hydrolysis and proton transport, which are modulated by ADP and Pi.
Asunto(s)
ATPasas de Translocación de Protón Bacterianas/metabolismo , Escherichia coli/enzimología , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Aminoacridinas/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Escherichia coli/efectos de los fármacos , Fluorescencia , Hidrólisis/efectos de los fármacos , Fosfatos/metabolismo , Bombas de Protones/metabolismo , Piruvato Quinasa/metabolismoRESUMEN
The assembly of neurotransmitter receptors in the endoplasmic reticulum limits the number of receptors delivered to the plasma membrane, ultimately controlling neurotransmitter sensitivity and synaptic transfer function. In a forward genetic screen conducted in the nematode C. elegans, we identified crld-1 as a gene required for the synaptic expression of ionotropic acetylcholine receptors (AChR). We demonstrated that the CRLD-1A isoform is a membrane-associated ER-resident protein disulfide isomerase (PDI). It physically interacts with AChRs and promotes the assembly of AChR subunits in the ER. Mutations of Creld1, the human ortholog of crld-1a, are responsible for developmental cardiac defects. We showed that Creld1 knockdown in mouse muscle cells decreased surface expression of AChRs and that expression of mouse Creld1 in C. elegans rescued crld-1a mutant phenotypes. Altogether these results identify a novel and evolutionarily-conserved maturational enhancer of AChR biogenesis, which controls the abundance of functional receptors at the cell surface.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Receptores Colinérgicos/metabolismo , Sinapsis/metabolismo , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Técnicas de Silenciamiento del Gen , Prueba de Complementación Genética , Cardiopatías Congénitas , Ratones , Células Musculares , Unión Proteica , Proteína Disulfuro Isomerasas/genética , Multimerización de ProteínaRESUMEN
The ATP synthase in chromatophores of Rhodobacter caspulatus can effectively generate a transmembrane pH difference coupled to the hydrolysis of ATP. The rate of hydrolysis was rather insensitive to the depletion of ADP in the assay medium by an ATP regenerating system (phospho-enol-pyruvate (PEP) and pyruvate kinase (PK)). The steady state values of DeltapH were however drastically reduced as a consequence of ADP depletion. The clamped concentrations of ADP obtained using different PK activities in the assay medium could be calculated and an apparent Kd approximately 0.5 microM was estimated. The extent of proton uptake was also strongly dependent on the addition of phosphate to the assay medium. The Kd for this effect was about 70 microM. Analogous experiments were performed in membrane fragment from Escherichia coli. In this case, however, the hydrolysis rate was strongly inhibited by Pi, added up to 3 mM. Inhibition by Pi was nearly completely suppressed following depletion of ADP. The Kd's for the ADP and Pi were in the micromolar range and submillimolar range, respectively, and were mutually dependent from the concentration of the other ligand. Contrary to hydrolysis, the pumping of protons was rather insensitive to changes in the concentrations of the two ligands. At intermediate concentrations, proton pumping was actually stimulated, while the hydrolysis was inhibited. It is concluded that, in these two bacterial organisms, ADP and phosphate induce a functional state of the ATP synthase competent for a tightly coupled proton pumping, while the depletion of either one of these two ligands favors an inefficient (slipping) functional state. The switch between these states can probably be related to a structural change in the C-terminal alpha-helical hairpin of the epsilon-subunit, from an extended conformation, in which ATP hydrolysis is tightly coupled to proton pumping, to a retracted one, in which ATP hydrolysis and proton pumping are loosely coupled.
Asunto(s)
ATPasas de Translocación de Protón Bacterianas/fisiología , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Cromatóforos Bacterianos/metabolismo , Sitios de Unión , Transporte Biológico , Membrana Celular/metabolismo , Escherichia coli/enzimología , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Hidrólisis , Fuerza Protón-Motriz , Protones , Piruvato Quinasa/metabolismo , Rhodobacter capsulatus/enzimología , Rhodobacter capsulatus/metabolismoRESUMEN
Nucleus positioning is key for intracellular organization, cell differentiation, and organ development and is affected in many diseases, including myopathies due to alteration in amphiphysin-2 (BIN1). The actin and microtubule cytoskeletons are essential for nucleus positioning, but their crosstalk in this process is sparsely characterized. Here, we report that impairment of amphiphysin/BIN1 in Caenorhabditis elegans, mammalian cells, or muscles from patients with centronuclear myopathy alters nuclear position and shape. We show that AMPH-1/BIN1 binds to nesprin and actin, as well as to the microtubule-binding protein CLIP170 in both species. Expression of the microtubule-anchoring CAP-GLY domain of CLIP170 fused to the nuclear-envelope-anchoring KASH domain of nesprin rescues nuclear positioning defects of amph-1 mutants. Amphiphysins thus play a central role in linking the nuclear envelope with the actin and microtubule cytoskeletons. We propose that BIN1 has a direct and evolutionarily conserved role in nuclear positioning, altered in myopathies.