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URI permanente para esta colecciónhttps://hdl.handle.net/10953/111

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Age-related changes of the nitric oxide system in the rat brain
(Elsevier BV, 2002) Siles, Eva; Martínez-Lara, Esther; Cañuelo, Ana R.; Sánchez, Ana M.; Hernández, Raquel; López-Ramos, Juan C.; Del Moral, María L.; Esteban, Francisco J.; Blanco, Santos; Pedrosa, Juan A.; Rodrigo, José; Peinado, María A.
Characterization of interaction sites in the Saccharomyces cerevisiae ribosomal stalk components
(Wiley, 2002-10-31) Lalioti, Vasiliki S.; Pérez-Fernández, Jorge; Remacha, Miguel; García Ballesta, Juan Pedro
The interactions among the yeast stalk components(P0, P1⍺, P1ꞵ, P2⍺ and P2ꞵ) and with EF-2 have been explored using immunoprecipitation, affinity chromatography and the two-hybrid system. No stable association was detected between acidic proteins of the same type. In contrast, P1⍺ and P1ꞵ were found to interact with P2ꞵ and P2⍺ respectively. An interaction of P0 with P1 proteins, but not with P2 proteins, was also detected. This interaction is strongly increased with the P0 carboxyl end, which is able to form a pentameric complex with the four acidic proteins. The P1/P2 binding site has been located between residues 212 and 262 using different C-terminal P0 fragments. Immunoprecipitation shows the association of EF-2 with protein P0. However, the interaction is stronger with the P1/P2 proteins than with P0 in the two-hybrid assay. This interaction improves using the 100-amino-acid-long C-end of P0 and is even higher with the last 50 amino acids. The data indicate a specific association of P1⍺ with P2ꞵ and of P1ꞵ with P2⍺ rather than the dimerization of the acidic proteins found in prokaryotes. In addition, they suggest that stalk assembly begins by the interaction of the P1 proteins with P0. Moreover, as functional interactions of the complete P0 were found to increase using protein fragments, the data suggest that some active sites are exposed in the ribosome as a result of conformational changes that take place during stalk assembly and function.
Glutathione S-transferase isoenzymatic response to aging in rat cerebral cortex and cerebellum
(Elsevier Inc., 2003) Martínez-Lara, Esther; Siles, Eva; Hernández, Raquel; Cañuelo, Ana R.; Del Moral, María L.; Jiménez, Ana; Blanco, Santos; López-Ramos, Juan C.; Esteban, Francisco J.; Pedrosa, Juan A.; Peinado, María A.
Upregulation of endothelial nitric oxide synthase maintains nitric oxide production in the cerebellum of thioacetamide cirrhotic rats
(Pergamon Press, 2004) Hernández, Raquel; Martínez-Lara, Esther; Del Moral, María L.; Blanco, Santos; Cañuelo, Ana R.; Siles, Eva; Esteban, Francisco J.; Pedrosa, Juan A.; Peinado, María A.
Immunohistochemistry of neuronal nitric oxide synthase and protein nitration in the striatum of the aged rat
(John Wiley & Sons, Inc., 2004) Del Moral, María L.; Esteban, Francisco J.; Hernández, Raquel; Blanco, Santos; Molina, Francisco J.; Martínez-Lara, Esther; Siles, Eva; Viedma, Gloria; Ruiz, Alharilla; Pedrosa, Juan A.; Peinado, María A.
Constitutive nitric oxide synthases are responsible for the nitric oxide production in the ischemic aged cerebral cortex
(Elsevier BV, 2005) Martínez-Lara, Esther; Cañuelo, Ana R.; Siles, Eva; Hernández, Raquel; Del Moral, María L.; Blanco, Santos; Pedrosa, Juan A.; Rodrigo, José; Peinado, María A.
Steatosis recovery after treatment with a balanced sunflower or olive oil-based diet: involvement of perisinusoidal stellate cells
(Baishideng Publishing Group Co., Limited, 2005) Hernández, Raquel; Martínez-Lara, Esthe; Cañuelo, Ana R.; Del Moral, María L.; Blanco, Santos; Siles, Eva; Jiménez, Ana; Pedrosa, Juan A.; Peinado, María A.
The Acidic Protein Binding Site Is Partially Hidden in the Free Saccharomyces cereVisiae Ribosomal Stalk Protein P0
(American Chemical Society, 2005-03-16) Pérez-Fernández, Jorge; Remacha, Miguel; Ballesta, Juan P.G.
The ribosomal stalk is essential for translation; however, its overall structure is poorly understood. Characterization of the region involved in the interactions between protein P0 and the 12kDa acidic proteins P1 and P2 is fundamental to understand the assembly and function of this structure in the eukaryotic ribosome. The acidic protein content is important for the ribosome efficiency and affects the translation of specific mRNAs. By usage of a series of progressively truncated fragments of protein P0 in the two-hybrid test, a region between positions 213 and 250 was identified as the minimal protein part able to interact with the acidic proteins. Extensions at either end affect the binding capacity of the fragment either positively or negatively depending on the number of added amino acids. Deletions inside the binding region confirm its in vivo relevance since they drastically reduce the P0 interacting capacity with the 12 kDa acidic proteins, which are severely reduced in the ribosome when the truncated protein is expressed in the cell. Moreover, recombinant His-tagged P0 fragments containing the binding site and bound to Ni2+-NTA columns can form a complex with the P1 and P2 proteins, which is able to bind elongation factor EF2. The results indicate the existence of a region in P0 that specifically interacts with the acidic proteins. These interactions are, however, hindered by the presence of neighbor protein domains, suggesting the need for conformational changes in the complete P0 to allow the assembly of the ribosomal stalk.
The 90S Preribosome Is a Multimodular Structure That Is Assembled through a Hierarchical Mechanism
(Taylor & Francis, 2007-05-21) Pérez-Fernández, Jorge; Román, Ángel; de las Rivas, Javier; Bustelo, Xosé R.; Dosil, Mercedes
The 90S preribosomal particle is required for the production of the 18S rRNA from a pre-rRNA precursor. Despite the identification of the protein components of this particle, its mechanism of assembly and structural design remain unknown. In this work, we have combined biochemical studies, proteomic techniques, and bioinformatic analyses to shed light into the rules of assembly of the yeast 90S preribosome. Our results indicate that several protein subcomplexes work as discrete assembly subunits that bind in defined steps to the 35S pre-rRNA. The assembly of the t-UTP subunit is an essential step for the engagement of at least five additional subunits in two separate, and mutually independent, assembling routes. One of these routes leads to the formation of an assembly intermediate composed of the U3 snoRNP, the Pwp2p/UTP-B, subunit and the Mpp10p complex. The other assembly route involves the stepwise binding of Rrp5p and the UTP-C subunit. We also report the use of a bioinformatic approach that provides a model for the topological arrangement of protein components within the fully assembled particle. Together, our data identify the mechanism of assembly of the 90S preribosome and offer novel information about its internal architecture.
Metabolism of reactive nitrogen species in pea plants under abiotic stress conditions
(OXFORD UNIV PRESS, 2008-09-18) Corpas, Francisco; Chaki, Mounira; Fernández-Ocaña, Ana; Valderrama, Raquel; Palma, José M.; Carreras, Alfonso; Begara-Morales, Juan C.; Airaki, Morad; del Río, Luis A.; Barroso, Juan B.
Nitric 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.
Protein targets of tyrosine nitration in sunflower (Helianthus annuus L.) hypocotyls
(OXFORD UNIV PRESS, 2009-08-28) Chaki, M.; Valderrama, R.; Fernández-Ocaña, AM.; Carreras, A.; López-Jaramillo, J.; Luque, F.; Palma, JM; Pedrajas, JR.; Begara-Morales, JC.; Sánchez-Calvo, B.; Gómez-Rodríguez, MV.; Corpas, FJ.; Barroso, JB.
Tyrosine 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.
Study of the nitric oxide system in the rat cerebellum during aging
(BioMed Central Ltd, 2010) Blanco, Santos; Molina, Francisco J.; Castro, Lourdes; Del Moral, María L.; Hernández, Raquel; Jiménez, Ana; Rus, Alma; Martínez-Lara, Esther; Siles, Eva; Peinado, María A.
Pitx2c Modulates Cardiac-Specific Transcription Factors Networks in Differentiating Cardiomyocytes from Murine Embryonic Stem Cells
(Karger Publishers, 2010-03-09) Lozano-Velasco, Estefanía; Chinchilla, Ana; Martínez-Fernández, Sergio; Hernández-Torres, Francisco; Navarro-Gómez, Francisco; Lyons, Gary E.; Franco, Diego; Aránega, Amelia Eva
Aim: The knowledge of the molecular signals that control cell differentiation into cardiomyocytes is critical to apply cell-based therapies and repair an injured heart. The transcription factor Pitx2 has essential roles in the development of different organs including the heart. Although a direct role of Pitx2 in the developing myocardium has recently been reported, the molecular pathways driven by Pitx2 as well as its cardiac target genes remain largely unexplored. The aim of this study was to unravel the molecular mechanisms driven by Pitx2 during the process of cardiomyocyte differentiation in vitro in mouse embryonic stem cell-derived cardiomyocytes. Methods and Results: Pitx2c was overexpressed in the R1-embryonic stem cell line. mRNA levels and protein distribution of several specific cardiac genes were analyzed by real-time PCR and immunohistochemistry experiments in R1-embryonic stem cell-derived beating areas at different stages of in vitro differentiation. Our results show that overexpression of Pitx2c in embryonic stem cell-derived cardiomyocytes is able to dynamically upregulate several cardiac-enriched transcription factors such as Isl1, Mef2c and Gata4. Additionally, Pitx2c induces the expression of chamber-specific cardiac genes such as Tbx5, Nppa and Cx40. These data were validated in an in vivo model of Pitx2 loss of function. Conclusion: Taken together, these results demonstrate that Pitx2 plays a major role reinforcing the transcriptional program of cardiac differentiation.
MicroRNA profiling during mouse ventricular maturation: a role for miR-27 modulating Mef2c expression
(Oxford, 2010-08-24) Chinchilla, Ana; Lozano-Velasco, Estefanía; Daimi, Houria; Esteban-Ruiz, Francisco José; Crist, Colin; Aránega, Amelia Eva; Franco, Diego
Aims: Non-coding RNA has been recently demonstrated to be a novel mechanism for modulation of gene expression at the post-transcriptional level. The importance of microRNAs in the cardiovascular system is now apparent. Mutations of distinct microRNAs have provided evidence for fundamental roles of microRNAs during cardiovascular development. However, there is limited information about global microRNA profiles during mouse heart development. In this study, we have gained insight from the expression profiles of microRNAs during mouse ventricular development by microarray and qRT-PCR analysis. Methods and results: Our microarray analysis reveals that relatively few microRNAs display either increasing or decreasing expression profiles during ventricular chamber formation. Interestingly, most of the differentially expressed microRNAs display a rather discrete peak of expression at particular developmental stages. Furthermore, we demonstrate that micro-RNA-27b (miR-27b) displays an overt myocardial expression during heart development and that the transcription factor-encoding gene Mef2c is an miR-27b target. Conclusion: Our data present a comprehensive profile of microRNA expression during ventricular maturation, providing an entry point for investigation of the functional roles of the most abundantly and differentially expressed microRNAs during cardiogenesis
Is endothelial-nitric-oxide-synthase-derived nitric oxide involved in cardiac hypoxia/reoxygenation-related damage?
(Indian Academy of Sciences, 2011) Rus, Alma; Peinado, María A.; Blanco, Santos; Del Moral, María L.
RNA polymerase I–specific subunits promotepolymerase clustering to enhance the rRNA genetranscription cycle
(Rockefeller University Press, 2011-01-24) Albert, Benjamin; Léger-Silvestre, Isabelle; Normand, Christophe; Ostermaier, Martin K.; Pérez-Fernández, Jorge; Panov, Kostya I.; Zomerdijk, Joost C.B.M.; Schultz, Patrick; Gadal, Olivier
RNA polymerase I (Pol I) produces large ribosomal RNAs (rRNAs). In this study, we show that the Rpa49 and Rpa34 Pol I subunits, which do not have counterparts in Pol II and Pol III complexes, are functionally conserved using heterospecific complementation of the human and Schizosaccharomyces pombe orthologues in Saccharomyces cerevisiae. Deletion of RPA49 leads to the disappearance of nucleolar structure, but nucleolar assembly can be restored by decreasing ribosomal gene copy number from 190 to 25. Statistical analysis of Miller spreads in the absence of Rpa49 demonstrates a fourfold decrease in Pol I loading rate per gene and decreased contact between adjacent Pol I complexes. Therefore, the Rpa34 and Rpa49 Pol I–specific subunits are essential for nucleolar assembly and for the high polymerase loading rate associated with frequent contact between adjacent enzymes. Together, our data suggest that localized rRNA production results in spatially constrained rRNA production, which is instrumental for nucleolar assembly.
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, M.; Valderrama, R.; Fernández-Ocaña, AM.; Carreras, A.; Gómez-Rodríguez, MV.; Pedrajas, JR.; Begara-Morales, jc.; Sánchez-Calvo, B.; Luque, F.; Leterrier, M.; Corpas, FJ.; Barroso, JB.
Nitric 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.
PITX2 Insufficiency Leads to Atrial Electrical and Structural Remodeling Linked to Arrhythmogenesis
(American Heart Association, 2011-04-21) Chinchilla, Ana; Daimi, Houria; Lozano-Velasco, Estefanía; Domínguez-Macías, Jorge Nicolás; Caballero-Collado, Ricardo; Delpón, Eva; Tamargo, Juan; Cinca, Juan; Hove-Madsen, Leif; Aránega, Amelia Eva; Franco, Diego
Background: Pitx2 is a homeobox transcription factor that plays a pivotal role in early left/right determination during embryonic development. Pitx2 loss-of-function mouse mutants display early embryonic lethality with severe cardiac malformations, demonstrating the importance of Pitx2 during cardiogenesis. Recently, independent genome-wide association studies have provided new evidence for a putative role of PITX2 in the adult heart. These studies have independently reported several risk variants close to the PITX2 locus on chromosome 4q25 that are strongly associated with atrial fibrillation in humans. Methods and Results: We show for the first time that PITX2C expression is significantly decreased in human patients with sustained atrial fibrillation, thus providing a molecular link between PITX2 loss of function and atrial fibrillation. In addition, morphological, molecular, and electrophysiological characterization of chamber-specific Pitx2 conditional mouse mutants reveals that atrial but not ventricular chamber-specific deletion of Pitx2 results in differences in the action potential amplitude and resting membrane potential in the adult heart as well as ECG characteristics of atrioventricular block. Lack of Pitx2 in atrial myocardium impairs sodium channel and potassium channel expression, mediated in part by miRNA misexpression. Conclusions: This study thus identifies Pitx2 as an upstream transcriptional regulator of atrial electric function, the insufficiency of which results in cellular and molecular changes leading to atrial electric and structural remodeling linked to arrhythmogenesis.
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, M.; Valderrama, R.; Fernández-Ocaña, AM.; Carreras, A.; Gómez-Rodríguez, MV.; López-Jaramillo, J.; Begara-Morales, JC.; Sánchez-Calvo, B.; Luque, F.; Leterrier, M.; Corpas, FJ.; Barroso, JB.
High 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.
Elucidation of the assembly events required for the recruitment of Utp20, Imp4 and Bms1 onto nascent pre-ribosomes
(Oxford University Press, 2011-06-30) Pérez-Fernández, Jorge; Martín-Marcos, Pilar; Dosil, Mercedes
The 90S pre-ribosome, also known as the small subunit (SSU) processome, is a large multisubunit particle required for the production of the 18S rRNA from a pre-rRNA precursor. Recently, it has been shown that the formation of this particle entails the initial association of the tUTP subunit with the nascent pre-RNA and, subsequently, the binding of Rrp5/UTP-C and U3 snoRNP/UTP-B subunits in two independent assembly branches. However, the mode of assembly of other 90S pre-ribosome components remains obscure as yet. In this study, we have investigated the assembly of three proteins (Utp20, Imp4 and Bms1) previously regarded as potential nucleating factors of the 90S particle. Here, we demonstrate that the loading of those three proteins onto the pre-rRNA takes place independently of Rrp5/UTP-C and, instead, occurs downstream of the tUTP and U3/UTP-B subcomplexes. We also demonstrate that Bms1 and Utp20 are required for the recruitment of a subset of proteins to nascent pre-ribosomes. Finally, we show that proteins associated through secondary steps condition the stability of the two assembly branches in partially assembled preribosomes. These results provide new information about the functional relationships among 90S particle components and the events that are required for their stepwise incorporation onto the primary pre-rRNA.

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