Examinando por Autor "Carreras, Alfonso"
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Ítem Dual regulation of cytosolic ascorbate peroxidase (APX) by tyrosine nitration and S-nitrosylation(OXFORD UNIV PRESS, 2014-02) Begara-Morales, Juan Carlos; Sanchez-Calvo, Beatriz; Chaki, Mounira; Valderrama, Raquel; Mata-Pérez, Capilla; López-Jaramillo, Jaime; Padilla-Serrano, María Nieves; Carreras, Alfonso; Corpas, Francisco Javier; Barroso-Albarracín, Juan BautistaPost-translational modifications (PTMs) mediated by nitric oxide (NO)-derived molecules have become a new area of research, as they can modulate the function of target proteins. Proteomic data have shown that ascorbate peroxidase (APX) is one of the potential targets of PTMs mediated by NO-derived molecules. Using recombinant pea cytosolic APX, the impact of peroxynitrite (ONOO–) and S-nitrosoglutathione (GSNO), which are known to mediate protein nitration and S-nitrosylation processes, respectively, was analysed. While peroxynitrite inhibits APX activity, GSNO enhances its enzymatic activity. Mass spectrometric analysis of the nitrated APX enabled the determination that Tyr5 and Tyr235 were exclusively nitrated to 3-nitrotyrosine by peroxynitrite. Residue Cys32 was identified by the biotin switch method as S-nitrosylated. The location of these residues on the structure of pea APX reveals that Tyr235 is found at the bottom of the pocket where the haem group is enclosed, whereas Cys32 is at the ascorbate binding site. Pea plants grown under saline (150 mM NaCl) stress showed an enhancement of both APX activity and S-nitrosylated APX, as well as an increase of H₂O₂, NO, and S-nitrosothiol (SNO) content that can justify the induction of the APX activity. The results provide new insight into the molecular mechanism of the regulation of APX which can be both inactivated by irreversible nitration and activated by reversible S-nitrosylation.Ítem Función de las especies de Nitrógeno reactivo en la modulación de respuesta de plántulas de girasol (Helianthus annuus L.) frente a procesos de estrés biótico y abiótico(Jaén : Universidad de Jaén, 2012) Chaki, Mounira; Barroso-Albarracín, Juan Bautista; Carreras, Alfonso; Universidad de Jaén. Departamento de Biología ExperimentalEl óxido nítrico (NO) es la especies de nitrógeno reactivo más relevante, tiene un papel importamte en la regulación de funciones de planta. Así, se mostró su participación en los mecanismos de respuesta de planta frente al estrés biótico y abiótico. Sin embargo, poco es conocido sobre la participación de otras moléculas derivadas de NO como S-nitrosotioles, S-nitrosoglutation, peroxinitrito y 3-nitrotirosina. Por lo tanto, este trabajo se ha enfocado en el estudio del metabolismo de NO y moléculas derivadas de NO en hipocótilos de plantas de girasol (Helianthus annuus L.) sometidas a estrés biótico y abiótico. Para el estrés biótico, se ha estudiado la inoculación por el patógeno Plasmopara halstedii usando dos líneas de girasol HA89 susceptible donde el patógeno desarrolla su virulencia (interacción compatible) y la línea X55 donde el patógeno no puede desarrollarse (interacción incompatible). Por otro lado, se ha estudiado el estrés abiótico usando las mismas plantas que fueron sometidas a diferentes condiciones de estrés incluyendo alta y baja temperatura, daño mecánico, alta intensidad luminosa, luz continua y oscuridad.Ítem High temperature triggers the metabolism of S-nitrosothiols in sunflower mediating a process of nitrosative stress which provokes the inhibition of ferredoxin-NADP reductase by tyrosine nitration(WILEY, 2011-06) Chaki, Mounira; Valderrama, Raquel; Fernández-Ocaña, Ana; Carreras, Alfonso; Gómez-Rodríguez, María Victoria; López-Jaramillo, Jaime; Begara-Morales, Juan Carlos; Sánchez-Calvo, Beatriz; Luque-Vázquez, Francisco; Leterrier, Marina; Corpas, Francisco Javier; Barroso-Albarracín, Juan BautistaHigh temperature (HT) is considered a major abiotic stress that negatively affects both vegetative and reproductive growth. Whereas the metabolism of reactive oxygen species (ROS) is well established under HT, less is known about the metabolism of reactive nitrogen species (RNS). In sunflower (Helianthus annuus L.) seedlings exposed to HT, NO content as well as S-nitrosoglutathione reductase (GSNOR) activity and expression were down-regulated with the simultaneous accumulation of total S-nitrosothiols (SNOs) including S-nitrosoglutathione (GSNO). However, the content of tyrosine nitration (NO2-Tyr) studied by highperformance liquid chromatography with tandem mass spectrometry (LC–MS/MS) and by confocal laser scanning microscope was induced. Nitroproteome analysis under HT showed that this stress induced the protein expression of 13 tyrosine-nitrated proteins. Among the induced proteins, ferredoxin–NADP reductase (FNR) was selected to evaluate the effect of nitration on its activity after heat stress and in vitro conditions using 3-morpholinosydnonimine (SIN-1) (peroxynitrite donor) as the nitrating agent, the FNR activity being inhibited. Taken together, these results suggest that HT augments SNOs, which appear to mediate protein tyrosine nitration, inhibiting FNR, which is involved in the photosynthesis process.Ítem Mechanical wounding induces a nitrosative stress by down-regulation of GSNO reductase and an increase in S-nitrosothiols in sunflower (Helianthus annuus) seedlings(OXFORD UNIVERSITY PRESS, 2011-03) Chaki, Mounira; Valderrama, Raquel; Fernández-Ocaña, Ana; Carreras, Alfonso; Gómez-Rodríguez, María Victoria; Pedrajas, José Rafael; Begara-Morales, Juan Carlos; Sánchez-Calvo, Beatriz; Luque-Vázquez, Francisco; Leterrier, Marina; Corpas, Francisco Javier; Barroso-Albarracín, Juan BautistaNitric oxide (NO) and related molecules such as peroxynitrite, S-nitrosoglutathione (GSNO), and nitrotyrosine, among others, are involved in physiological processes as well in the mechanisms of response to stress conditions. In sunflower seedlings exposed to five different adverse environmental conditions (low temperature, mechanical wounding, high light intensity, continuous light, and continuous darkness), key components of the metabolism of reactive nitrogen species (RNS) and reactive oxygen species (ROS), including the enzyme activities L-argininedependent nitric oxide synthase (NOS), S-nitrosogluthathione reductase (GSNOR), nitrate reductase (NR), catalase, and superoxide dismutase, the content of lipid hydroperoxide, hydrogen peroxide, S-nitrosothiols (SNOs), the cellular level of NO, GSNO, and GSNOR, and protein tyrosine nitration [nitrotyrosine (NO2-Tyr)] were analysed. Among the stress conditions studied, mechanical wounding was the only one that caused a down-regulation of NOS and GSNOR activities, which in turn provoked an accumulation of SNOs. The analyses of the cellular content of NO, GSNO, GSNOR, and NO2-Tyr by confocal laser scanning microscopy confirmed these biochemical data. Therefore, it is proposed that mechanical wounding triggers the accumulation of SNOs, specifically GSNO, due to a downregulation of GSNOR activity, while NO2-Tyr increases. Consequently a process of nitrosative stress is induced in sunflower seedlings and SNOs constitute a new wound signal in plants.Ítem Metabolism of reactive nitrogen species in pea plants under abiotic stress conditions(OXFORD UNIV PRESS, 2008-09-18) Corpas, Francisco Javier; Chaki, Mounira; Fernández-Ocaña, Ana; Valderrama, Raquel; Palma, José Manuel; Carreras, Alfonso; Begara-Morales, Juan Carlos; Airaki, Morad; del-Río, Luis Alfonso; Barroso-Albarracín, Juan BautistaNitric oxide (NO) is a key signaling molecule in different physiological processes of animals and plants. However, little is known about the metabolism of endogenous NO and other reactive nitrogen species (RNS) in plants under abiotic stress conditions. Using pea plants exposed to six different abiotic stress conditions (high light intensity, low and high temperature, continuous light, continuous dark and mechanical wounding), several key components of the metabolism of RNS including the content of NO, S-nitrosothiols (RSNOs) and nitrite plus nitrate, the enzyme activities of L-arginine dependent nitric oxide synthase (NOS) and S-nitrosogluthathione reductase (GSNOR), and the profile of protein tyrosine nitration (NO2-Tyr) were analyzed in leaves. Low temperature was the stress that produced the highest increase of NOS and GSNOR activities, and this was accompanied by an increase in the content of total _NO and S-nitrosothiols, and an intensification of the immunoreactivity with an antibody against NO2-Tyr. Mechanical wounding, high temperature and light also had a clear activating effect on the different indicators of RNS metabolism in pea plants. However, the total content of nitrite and nitrate in leaves was not affected by any of these stresses. Considering that protein tyrosine nitration is a potential marker of nitrosative stress, the results obtained suggest that low and high temperature, continuous light and high light intensity are abiotic stress conditions that can induce nitrosative stress in pea plants.Ítem Protein targets of tyrosine nitration in sunflower (Helianthus annuus L.) hypocotyls(OXFORD UNIV PRESS, 2009-08-28) Chaki, Mounira; Valderrama, Raquel; Fernández-Ocaña, Ana; Carreras, Alfonso; López-Jaramillo, Jaime; Luque-Vázquez, Francisco; Palma, José Manuel; Pedrajas, José Rafael; Begara-Morales, Juan Carlos; Sánchez-Calvo, Beatriz; Gómez-Rodríguez, María Victoria; Corpas, Francisco Javier; Barroso-Albarracín, Juan BautistaTyrosine nitration is recognized as an important post-translational protein modification in animal cells that can be used as an indicator of a nitrosative process. However, in plant systems, there is scant information on proteins that undergo this process. In sunflower hypocotyls, the content of tyrosine nitration (NO₂-Tyr) and the identification of nitrated proteins were studied by high-performance liquid chromatography with tandem mass spectrometry (LC-MS/ MS) and proteomic approaches, respectively. In addition, the cell localization of nitrotyrosine proteins and peroxynitrite were analysed by confocal laser-scanning microscopy (CLSM) using antibodies against 3-nitrotyrosine and 3'-(p-aminophenyl) fluorescein (APF) as the fluorescent probe, in that order. The concentration of Tyr and NO₂- Tyr in hypocotyls was 0.56 mmol mg-¹ protein and 0.19 pmol mg-¹ protein, respectively. By proteomic analysis, a total of 21 nitrotyrosine-immunopositive proteins were identified. These targets include proteins involved in photosynthesis, and in antioxidant, ATP, carbohydrate, and nitrogen metabolism. Among the proteins identified, S- adenosyl homocysteine hydrolase (SAHH) was selected as a model to evaluate the effect of nitration on SAHH activity using SIN-1 (a peroxynitrite donor) as the nitrating agent. When the hypocotyl extracts were exposed to 0.5 mM, 1 mM, and 5 mM SIN-1, the SAHH activity was inhibited by some 49%, 89%, and 94%, respectively. In silico analysis of the barley SAHH sequence, characterized Tyr448 as the most likely potential target for nitration. In summary, the present data are the first in plants concerning the content of nitrotyrosine and the identification of candidates of protein nitration. Taken together, the results suggest that Tyr nitration occurs in plant tissues under physiological conditions that could constitute an important process of protein regulation in such a way that, when it is overproduced in adverse circumstances, it can be used as a marker of nitrosative stress.