Browsing by Author "Gutiérrez-Merino, Carlos"
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- Actomyosin modulation by peroxynitritePublication . Tiago, Teresa; Silva, D.; Santos, Ana; Aureliano, M.; Gutiérrez-Merino, CarlosIn the present work we address the oxidative modifications accounting for the structural and functional impairment of the actomyosin complex under the oxidative stress mediated by peroxynitrite (ONOO-). Experiments on purified myosin and actin have shown that submicromolar ONOO- concentrations produce strong inhibition of the F-actin stimulated myosin ATPase activity. The peroxynitrite-induced actomyosin impairment correlated with structural modifications that decrease the thermal stability of both actin and myosin leading to partially unfolded states. The results suggest a major role for the highly reactive cysteines on actin and on myosin and also for some critical methionines on G-actin. 3-nitrotyrosine does not contribute significantly to the observed functional alterations.
- Binding modes of decavanadate to myosin and inhibition of the actomyosin ATPase activityPublication . Tiago, Teresa; Martel, Paulo; Gutiérrez-Merino, Carlos; Aureliano, M.Decavanadate, a vanadate oligomer, is known to interact with myosin and to inhibit the ATPase activity, but the putative binding sites and the mechanism of inhibition are still to be clarified. We have previously proposed that the decavanadate (V10O28 6−) inhibition of the actin-stimulated myosin ATPase activity is non-competitive towards both actin and ATP. A likely explanation for these results is that V10 binds to the so-called back-door at the end of the Pi-tube opposite to the nucleotide-binding site. In order to further investigate this possibility, we have carried out molecular docking simulations of the V10 oligomer on three different structures of the myosin motor domain of Dictyostelium discoideum, representing distinct states of the ATPase cycle. The results indicate a clear preference of V10 to bind at the back-door, but only on the “open” structures where there is access to the phosphate binding-loop. It is suggested that V10 acts as a “back-door stop” blocking the closure of the 50- kDa cleft necessary to carry out ATP-γ-phosphate hydrolysis. This provides a simple explanation to the non-competitive behavior of V10 and spurs the use of the oligomer as a tool to elucidate myosin back-door conformational changes in the process of muscle contraction.
- Decavanadate binding to a high affinity site near the myosin catalytic centre inhibits F-actin-stimulated myosin ATPase activityPublication . Tiago, Teresa; Aureliano, M.; Gutiérrez-Merino, CarlosDecameric vanadate (V10) inhibits the actin-stimulated myosin ATPase activity, noncompetitively with actin or with ATP upon interaction with a high-affinity binding site (Ki ) 0.27 ( 0.05 íM) in myosin subfragment-1 (S1). The binding of V10 to S1 can be monitored from titration with V10 of the fluorescence of S1 labeled at Cys-707 and Cys-697 with N-iodo-acetyl-N¢-(5-sulfo-1-naphthyl)- ethylenediamine (IAEDANS) or 5-(iodoacetamido) fluorescein, which showed the presence of only one V10 binding site per monomer with a dissociation constant of 0.16-0.7 íM, indicating that S1 labeling with these dyes produced only a small distortion of the V10 binding site. The large quenching of AEDANSlabeled S1 fluorescence produced by V10 indicated that the V10 binding site is close to Cys-697 and 707. Fluorescence studies demonstrated the following: (i) the binding of V10 to S1 is not competitive either with actin or with ADPâV1 or ADPâAlF4; (ii) the affinity of V10 for the complex S1/ADPâV1 and S1/ ADPâAlF4 is 2- and 3-fold lower than for S1; and (iii) it is competitive with the S1 “back door” ligand P1P5-diadenosine pentaphosphate. A local conformational change in S1 upon binding of V10 is supported by (i) a decrease of the efficiency of fluorescence energy transfer between eosin-labeled F-actin and fluorescein-labeled S1, and (ii) slower reassociation between S1 and F-actin after ATP hydrolysis. The results are consistent with binding of V10 to the Walker A motif of ABC ATPases, which in S1 corresponds to conserved regions of the P-loop which form part of the phosphate tube.
- Decavanadate induces mitochondrial membrane depolarization and inhibits oxygen consumptionPublication . Soares, Sandra S.; Gutiérrez-Merino, Carlos; Aureliano, M.Decavanadate induced rat liver mitochondrial depolarization at very low concentrations, half-depolarization with 39 nM decavanadate, while it was needed a 130-fold higher concentration of monomeric vanadate (5 lM) to induce the same effect. Decavanadate also inhibits mitochondrial repolarization induced by reduced glutathione in vitro, with an inhibition constant of 1 lM, whereas no effect was observed up to 100 lM of monomeric vanadate. The oxygen consumption by mitochondria is also inhibited by lower decavanadate than monomeric vanadate concentrations, i.e. 50% inhibition is attained with 99 nM decavanadate and 10 lM monomeric vanadate. Thus, decavanadate is stronger as mitochondrial depolarization agent than as inhibitor of mitochondrial oxygen consumption. Up to 5 lM, decavanadate does not alter mitochondrial NADH levels nor inhibit neither FOF1-ATPase nor cytochrome c oxidase activity, but it induces changes in the redox steady-state of mitochondrial b-type cytochromes (complex III). NMR spectra showed that decameric vanadate is the predominant vanadate species in decavanadate solutions. It is concluded that decavanadate is much more potent mitochondrial depolarization agent and a more potent inhibitor of mitochondrial oxygen consumption than monomeric vanadate, pointing out the importance to take into account the contribution of higher oligomeric species of vanadium for the biological effects of vanadate solutions.
- Decavanadate interactions with actin: inhibition of G-actin polymerization and stabilization of decameric vanadatePublication . Ramos, Susana; Manuel, Miguel; Tiago, Teresa; Duarte, Rui O.; Martins, Jorge; Gutiérrez-Merino, Carlos; Moura, José J. G.; Aureliano, M.Decameric vanadate species (V10) inhibit the rate and the extent of G-actin polymerization with an IC50 of 68 ± 22 lM and 17 ± 2 lM, respectively, whilst they induce F-actin depolymerization at a lower extent. On contrary, no effect on actin polymerization and depolymerization was detected for 2 mM concentration of ‘‘metavanadate’’ solution that contains ortho and metavanadate species, as observed by combining kinetic with 51V NMR spectroscopy studies. Although at 25 C, decameric vanadate (10 lM) is unstable in the assay medium, and decomposes following a first-order kinetic, in the presence of G-actin (up to 8 lM), the half-life increases 5-fold (from 5 to 27 h). However, the addition of ATP (0.2 mM) in the medium not only prevents the inhibition of G-actin polymerization by V10 but it also decreases the half-life of decomposition of decameric vanadate species from 27 to 10 h. Decameric vanadate is also stabilized by the sarcoplasmic reticulum vesicles, which raise the half-life time from 5 to 18 h whereas no effects were observed in the presence of phosphatidylcholine liposomes, myosin or G-actin alone. It is proposed that the ‘‘decavanadate’’ interaction with G-actin, favored by the G-actin polymerization, stabilizes decameric vanadate species and induces inhibition of G-actin polymerization. Decameric vanadate stabilization by cytoskeletal and transmembrane proteins can account, at least in part, for decavanadate toxicity reported in the evaluation of vanadium (V) effects in biological systems.
- Decavanadate toxicity effects following in vivo administrationPublication . S. Soares, Sandra; Gutiérrez-Merino, Carlos; Aureliano, M.Very few in vivo animal studies involving vanadium consider the contribution of decavanadate (V10) to vanadium biological effects. Recently, it is been suggested that decameric vanadate may not completely fall apart into other vanadate oligomers before induces changes in cell homeostasis, namely in several stress markers. An acute exposure of different fish species (Halobactrachus didactilus, Lusitanian toadfish, and Sparus aurata, gilthead seabream) to decavanadate, but not to other vanadate oligomers, induced different effects than vanadate in catalase activity, glutathione content, lipid peroxidation, mitochondrial superoxide anion production and vanadium accumulation, whereas both solutions seem to equally depress reactive oxygen species (ROS) production as well as total intracellular reducing power. Vanadium is accumulated in Sparus aurata mitochondria in particular when decavanadate is administrated. Moreover, exposure to different vanadate oligomers induced morphological changes in fish cardiac, hepatic and renal tissues causing tissues lesions in the liver and kidney, but not cardiac tissue. Nevertheless, the results highlight that different vanadate oligomers seem to follow, not only in vitro but also in vivo, different pathways, with different targets and effects. These recent findings, that are now summarized, point out the decameric vanadate species contributions to in vivo effects induced by vanadium in biological systems.
- Early disruption of the actin cytoskeleton in cultured cerebellar granule neurons exposed to 3-morpholinosydnonimine-oxidative stress is linked to alterations of the cytosolic calcium concentrationPublication . Tiago, Teresa; Marques-da-Silva, Dorinda; Samhan-Arias, A. K.; Aureliano, M.; Gutiérrez-Merino, CarlosCytoskeleton damage is a frequent feature in neuronal cell death and one of the early events in oxidantinduced cell injury. This work addresses whether actin cytoskeleton reorganization is an early event of SIN-1-induced extracellular nitrosative/oxidative stress in cultured cerebellar granule neurons (CGN). The actin polymerization state, i.e. the relative levels of G-/F-actin, was quantitatively assessed by the ratio of the fluorescence intensities of microscopy images obtained from CGN double-labelled with Alexa594- DNase-I (for actin monomers) and Bodipy-FL-phallacidin (for actin filaments). Exposure ofCGNto a flux of peroxynitrite as low as 0.5–1 M/min during 30 min (achieved with 0.1mMSIN-1) was found to promote alterations of the actin cytoskeleton dynamics as it increases the G-actin/F-actin ratio. Because L-type voltage-operated Ca2+ channels (L-VOCC) are primary targets in CGN exposed to SIN-1, the possible role of Ca2+ dynamics on the perturbation of the actin cytoskeleton was also assessed from the cytosolic Ca2+ concentration response to the L-VOCC’s agonist FPL-64176 and to the L-VOCC’s blocker nifedipine. The results showed that SIN-1 induced changes in the actin polymerization state correlated with its ability to decrease Ca2+ influx through L-VOCC. Combined analysis of cytosolic Ca2+ concentration and G-actin/Factin ratio alterations by SIN-1, cytochalasin D, latrunculin B and jasplakinolide support that disruption of the actin cytoskeleton is linked to cytosolic calcium concentration changes.
- Effects of reactive oxygen and nitrogen species on actomyosin and their implications for muscle contractilityPublication . Tiago, Teresa; Aureliano, M.; Gutiérrez-Merino, CarlosExperimental evidence accumulated during recent years is pointing out that numerous pathological conditions in skeletal and cardiac muscle are associated with an oxidative stress-induced muscle injury. Additionally, it has been postulated that several oxidants can directly alter contractile function by oxidative modification of the myofibril proteins – actin and myosin. Peroxynitrite (ONOO-), a potent biological oxidizing agent formed in the nearly instantaneous reaction of nitric oxide with superoxide anion, is increasingly recognized as playing a major role in the skeletal and cardiac muscle dysfunction. This is supported by detection of 3-nitrotyrosine, a protein modification produced by the reaction of peroxynitrite with tyrosine, on skeletal and cardiac muscle proteins during aging or in diseases associated with myocardial inflammation or ischemia/reperfusion insults. Although some studies point to a correlation of protein nitration with functional and structural modifications, the mechanism by which peroxynitrite may impair muscle contractility remains far from being elucidated. In the present review we address the role of reactive oxygen and nitrogen species on the structural and functional impairment of actomyosin ATPase activity and their implications for muscle contraction with particular emphasis on the oxidative modifications promoted by peroxynitrite on actin and myosin.
- Inhibition of skeletal muscle S1-myosin ATPase by peroxynitritePublication . Tiago, Teresa; S, Simão; Aureliano, M.; Martín-Romero, Francisco Javier; Gutiérrez-Merino, CarlosExposure of myosin subfragment 1 (S1) to 3-morpholinosydnonimine (SIN-1) produced a time-dependent inhibition of the F-actin-stimulated S1 Mg2+-ATPase activity, reaching 50% inhibition with 46.7 ( 8.3 íM SIN-1 for 8.7 íM S1, that is, at a SIN-1/S1 molar ratio of approximately 5.5. The inhibition was due to the peroxynitrite produced by SIN-1 decomposition because (1) decomposed SIN-1 was found to have no effect on S1 ATPase activity, (2) addition of SIN-1 in the presence of superoxide dismutase and catalase fully prevented inhibition by SIN-1, and (3) micromolar pulses of chemically synthesized peroxynitrite produced inhibition of F-actin-stimulated S1 Mg2+-ATPase activity. In parallel, SIN-1 produced the inhibition of the nonphysiological Ca2+-dependent and K+/EDTA-dependent S1 ATPase activity of S1 and, therefore, suggested that the inhibition of F-actin-stimulated S1 Mg2+-ATPase activity is produced by the oxidation of highly reactive cysteines of S1 (Cys707 and Cys697), located close to the catalytic center. This point was further confirmed by the titration of S1 cysteines with 5,5¢-dithiobis(2- nitrobenzoic acid) and by the parallel decrease of Cys707 labeling by 5-(iodoacetamido)fluorescein, and it was reinforced by the fact that other common protein modifications produced by peroxynitrite, for example, protein carbonyl and nitrotyrosine formation, were barely detected at the concentrations of SIN-1 that produced more than 50% inhibition of the F-actin-stimulated S1 Mg2+-ATPase activity. Differential scanning calorimetry of S1 (untreated and treated with different SIN-1 concentrations) pointed out that SIN-1, at concentrations that generate micromolar peroxynitrite fluxes, impaired the ability of ADPâV1 to induce the intermediate catalytic transition state and also produced the partial unfolding of S1 that leads to an enhanced susceptibility of S1 to trypsin digestion, which can be fully protected by 2 mM GSH.
- Mitochondria as a target for decavanadate toxicity in Sparus aurata heartPublication . S. Soares, Sandra; Gutiérrez-Merino, Carlos; Aureliano, M.In a previous in vivo study we have reported that vanadium distribution, antioxidant enzymes activity and lipid peroxidation in Sparus aurata heart are strongly dependent on the oligomeric vanadate species being administered. Moreover, it was suggested that vanadium is accumulated in mitochondria, in particular when V10 was intravenously injected. In this work we have done a comparative study of the effects of V10 and monomeric vanadate (V1) on cardiac mitochondria from S. aurata. V10 inhibits mitochondrial oxygen consumption with an IC50 of 400 nM, while the IC50 for V1 is 23 M. V10 also induced mitochondrial depolarization at very low concentrations, with an IC50 of 196 nM, and 55 Mof V1was required to induce the same effect. Additionally, up to 5 M V10 did inhibit neither F0F1-ATPase activity nor NADH levels and it did not affect respiratory complexes I and II, but it induced changes in the redox steady-state of complex III. It is concluded that V10 inhibits mitochondrial oxygen consumption and induces membrane depolarization more strongly than V1, pointing out that mitochondria is a toxicological target for V10 and the importance to take into account the contribution of V10 to the vanadate toxic effects.