Person: Martínez Ruiz, Antonio
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First Name
Antonio
Last Name
Martínez Ruiz
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Farmacia
Department
Bioquímica y Biología Molecular
Area
Bioquímica y Biología Molecular
Identifiers
31 results
Search Results
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Item Role of histidine-50, glutamic acid-96 and histidine-137 in the ribonucleolytic mechanism of the ribotoxin alpha-sarcin(Proteins: Structure, Function and Genetics, 1999) Lacadena García-Gallo, Francisco Javier; Martínez Del Pozo, Álvaro; Martínez Ruiz, Antonio; Pérez-Cañadillas, José Manuel; Bruix, Marta; Mancheño Gómez, José Miguel; Gavilanes Franco, José Gregorio; Oñaderra Sánchez, Mercedesalpha-Sarcin is a ribotoxin secreted by the mold Aspergillus giganteus that degrades the ribosomal RNA by acting as a cyclizing ribonuclease. Three residues potentially involved in the mechanism of catalysis--histidine-50, glutamic acid-96, and histidine-137--were changed to glutamine. Three different single mutation variants (H50Q, E96Q, H137Q) as well as a double variant (H50/137Q) and a triple variant (H50/137Q/E96Q) were prepared and isolated to homogeneity. These variants were spectroscopically (circular dichroism, fluorescence emission, and proton nuclear magnetic resonance) characterized. According to these results, the three-dimensional structure of these variants of alpha-sarcin was preserved; only very minor local changes were detected. All the variants were inactive when assayed against either intact ribosomes or poly(A). The effect of pH on the ribonucleolytic activity of alpha-sarcin was evaluated against the ApA dinucleotide. This assay revealed that only the H50Q variant still retained its ability to cleave a phosphodiester bond, but it did so to a lesser extent than did wild-type alpha-sarcin. The results obtained are interpreted in terms of His137 and Glu96 as essential residues for the catalytic activity of alpha-sarcin (His137 as the general acid and Glu96 as the general base) and His50 stabilizing the transition state of the reaction catalyzed by alpha-sarcin.Item Secretion of Recombinant Pro- and Mature Fungal α-Sarcin Ribotoxin by the Methylotrophic YeastPichia pastoris:The Lys–Arg Motif Is Required for Maturation(Protein Expression and Purification, 1998) Martínez Ruiz, Antonio; Martínez Del Pozo, Álvaro; Lacadena García-Gallo, Francisco Javier; Mancheño Gómez, José Miguel; Oñaderra Sánchez, Mercedes; Carlos López-Otı́n; Gavilanes Franco, José Gregorioα-Sarcin is a ribosome-inactivating protein from the moldAspergillus giganteus.The methylotrophic yeastPichia pastorishas been transformed with two plasmids (pHILD2preαS and pHILS1preαS), which contain the complete α-sarcin cDNA, including its original fungal leader peptide, under the control of yeast alcohol oxidase promoter. The second one is indeed fused to the signal sequence ofP. pastorisacid phosphatase. The transformed yeasts secreted both mature and pro-α-sarcin. The presence of this pro-α-sarcin in the yeast extracellular medium is due to an inefficient recognition of the pro-sequence by a putative Kex2p-like endopeptidase. A third plasmid accounting for a single mutation of the α-sarcin leader peptide was designed to produce a more efficient Kex2p recognition motif. This approach resulted in the extracellular production of only the mature protein, suggesting the existence of a two-step mechanism for processing its leader peptide. This recombinant α-sarcin is identical to the original fungal protein, according to activity and spectroscopic criteria. In addition, pro-α-sarcin, which has been characterized for the first time, also exhibits ribonucleolytic activity as the mature protein does. Therefore, protection of the producing cells against this kind of ribotoxins may depend on an efficient recognition of the signal sequence followed by translocation of the nascent polypeptide to the endoplasmic reticulum.Item Characterization of a natural larger form of the antifungal protein (AFP) from Aspergillus giganteus(Biochim Biophys Acta, 1997) Martínez Ruiz, Antonio; Martínez Del Pozo, Álvaro; Lacadena García-Gallo, Francisco Javier; Mancheño Gómez, José Miguel; Oñaderra Sánchez, Mercedes; Gavilanes Franco, José GregorioTwo major proteins, a-sarcin and an antifungal polypeptide AFP , are secreted by the mould Ž . Aspergillus giganteus MDH 18894 when it is cultured for 70–80 h. A third major protein is also found in the extracellular medium at 48–60 h, but it disappears as the culture proceeds. This protein has been isolated and characterized in terms of apparent molecular mass, electrophoretic and chromatographic behaviour, NH -terminal primary structure, amino acid content, spectroscopical 2 features, reactivity against anti-AFP antibodies, and antifungal activity. Based on the obtained results it would be an extracellular inactive precursor form of AFP, designated as the large form of AFP lf-AFP . Its amino acid composition is Ž . identical to that of AFP but containing six extra residues. NH -terminal sequence analysis of the first eight amino acid 2 residues of this polypeptide revealed that the extra residues can be perfectly accommodated within the DNA-deduced sequence of the precursor form of AFP. Its alignment with precursor sequences of different proteins, secreted by a variety of Aspergillus spp., reveals the existence of a common tetrapeptide at the carboxy-terminal end of their leader peptides. This sequence would be IlerLeu-Xaa-Yaa-Arg, being mostly Xaa and Yaa an acid residue Asp Ž . rGlu and alanine, respectively. The presence of lf-AFP as an extracellular protein would be in perfect agreement with the existence of this tetrapeptide motif, that can be involved in the protein secretion mechanisms of filamentous fungi.Item Regulation of Ras Signaling by S-Nitrosylation(Antioxidants, 2023) Simão, Sónia; Agostinho, Rafaela Ribeiro; Martínez Ruiz, Antonio; Araújo, Inês MariaRas are a family of small GTPases that function as signal transduction mediators and are involved in cell proliferation, migration, differentiation and survival. The significance of Ras is further evidenced by the fact that Ras genes are among the most mutated oncogenes in different types of cancers. After translation, Ras proteins can be targets of post-translational modifications (PTM), which can alter the intracellular dynamics of the protein. In this review, we will focus on how S-nitrosylation of Ras affects the way these proteins interact with membranes, its cellular localization, and its activity. S-Nitrosylation occurs when a nitrosyl moiety of nitric oxide (NO) is covalently attached to a thiol group of a cysteine residue in a target protein. In Ras, the conserved Cys118 is the most surface-exposed Cys and the preferable residue for NO action, leading to the initiation of transduction events. Ras transduces the mitogen-activated protein kinases (MAPK), the phosphoinositide-3 kinase (PI3K) and the RalGEF cellular pathways. S-Nitrosylation of elements of the RalGEF cascade remains to be identified. On the contrary, it is well established that several components of the MAPK and PI3K pathways, as well as different proteins associated with these cascades, can be modified by S-nitrosylation. Overall, this review presents a better understanding of Ras S-nitrosylation, increasing the knowledge on the dynamics of these proteins in the presence of NO and the underlying implications in cellular signalingItem S-Nitrosylation of Ras Mediates Nitric Oxide-Dependent Post-Injury Neurogenesis in a Seizure Model(Antioxidants & Redox Signaling, 2018) Santos, Ana Isabel; Pereira Carreira, Bruno; Izquierdo-Álvarez, Alicia; Ramos, Elena; Lourenço, Ana Sofia; Santos, Daniela Filipa; Morte, Maria Inês; Ribeiro, Luís Filipe; Marreiros, Ana; Sánchez-López, Nuria; Marina, Anabel; Monteiro Carvalho, Caetana; Martínez Ruiz, Antonio; Araújo, Inês MariaAims: Nitric oxide (NO) is involved in the upregulation of endogenous neurogenesis in the subventricular zone and in the hippocampus after injury. One of the main neurogenic pathways activated by NO is the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway, downstream of the epidermal growth factor receptor. However, the mechanism by which NO stimulates cell proliferation through activation of the ERK/MAPK pathway remains unknown, although p21Ras seems to be one of the earliest targets of NO. Here, we aimed at studying the possible neurogenic action of NO by posttranslational modification of p21Ras as a relevant target for early neurogenic events promoted by NO in neural stem cells (NSCs). Results: We show that NO caused S-nitrosylation (SNO) of p21Ras in Cys118, which triggered downstream activation of the ERK/MAPK pathway and proliferation of NSC. Moreover, in cells overexpressing a mutant Ras in which Cys118 was replaced by a serine–C118S–, cells were insensitive to NO, and no increase in SNO, in ERK phosphorylation, or in cell proliferation was observed. We also show that, after seizures, in the presence of NO derived from inducible nitric oxide synthase, there was an increase in p21Ras cysteine modification that was concomitant with the previously described stimulation of proliferation in the dentate gyrus. Innovation: Our work identifies p21Ras and its SNO as an early target of NO during signaling events that lead to NSC proliferation and neurogenesis. Conclusion: Our data highlight Ras SNO as an early event leading to NSC proliferation, and they may provide a target for NO-induced stimulation of neurogenesis with implications for brain repairItem Mitochondrial complex I deactivation is related to superoxide production in acute hypoxia(Redox Biology, 2017) Hernansanz-Agustín, Pablo; Ramos, Elena; Navarro, Elisa; Parada, Esther; Sánchez-López, Nuria; Peláez-Aguado, Laura; Cabrera-García, J. Daniel; Tello, Daniel; Buendia, Izaskun; Marina, Anabel; Egea, Javier; López, Manuela G.; Bogdanova, Anna; Martínez Ruiz, AntonioMitochondria use oxygen as the final acceptor of the respiratory chain, but its incomplete reduction can also produce reactive oxygen species (ROS), especially superoxide. Acute hypoxia produces a superoxide burst in different cell types, but the triggering mechanism is still unknown. Herein, we show that complex I is involved in this superoxide burst under acute hypoxia in endothelial cells. We have also studied the possible mechanisms by which complex I could be involved in this burst, discarding reverse electron transport in complex I and the implication of PTEN-induced putative kinase 1 (PINK1). We show that complex I transition from the active to ‘deactive’ form is enhanced by acute hypoxia in endothelial cells and brain tissue, and we suggest that it can trigger ROS production through its Na+/H+ antiporter activity. These results highlight the role of complex I as a key actor in redox signalling in acute hypoxia.- Project number: 237
Item Mejora de la docencia en Bioquímica Aplicada y Clínica: desarrollo de una revista digital sobre investigación en Medicina Molecular elaborado por los alumnos y puesta en marcha de la metodología didáctica mediante clases invertidas(2020) Oset Gasque, María Jesús; Escrivá Pons, Fernando; Iniesta Serrano, María Pilar; Gómez Hernández, María De La Almudena; Escribano Illanes, Óscar; García Redondo, Alberto; Martínez Ruiz, Antonio; Roncero Romero, Cesáreo; Álvarez Escolá, Carmen; Juan Chocano, María Del Carmen De Item Acute hypoxia produces a superoxide burst in cells(Free Radical Biology and Medicine, 2014) Hernansanz-Agustín, Pablo; Izquierdo-Álvarez, Alicia; Sánchez-Gómez, Francisco J.; Ramos, Elena; Villa-Piña, Tamara; Lamas, Santiago; Bogdanova, Anna; Martínez Ruiz, AntonioOxygen is a key molecule for cell metabolism. Eukaryotic cells sense the reduction in oxygen availability (hypoxia) and trigger a series of cellular and systemic responses to adapt to hypoxia, including the optimization of oxygen consumption. Many of these responses are mediated by a genetic program induced by the hypoxia-inducible transcription factors (HIFs), regulated by a family of prolyl hydroxylases (PHD or EGLN) that use oxygen as a substrate producing HIF hydroxylation. In parallel to these oxygen sensors modulating gene expression within hours, acute modulation of protein function in response to hypoxia is known to occur within minutes. Free radicals acting as second messengers, and oxidative posttranslational modifications, have been implied in both groups of responses. Localization and speciation of the paradoxical increase in reactive oxygen sp+ecies production in hypoxia remain debatable. We have observed that several cell types respond to acute hypoxia with a transient increase in superoxide production for about 10 min, probably originating in the mitochondria. This may explain in part the apparently divergent results found by various groups that have not taken into account the time frame of hypoxic ROS production. We propose that this acute and transient hypoxia-induced superoxide burst may be translated into oxidative signals contributing to hypoxic adaptation and preconditioningItem Signalling by NO-induced protein S-nitrosylation and S-glutathionylation: Convergences and divergences(Cardiovascular research, 2007) Martínez Ruiz, Antonio; Lamas, SantiagoThe role of nitric oxide in several signalling routes has been clearly established. In recent years increasing attention has been paid to its ability to produce covalent protein post-translational modifications in conjunction with other reactive oxygen and nitrogen species. Among these, the modification of cysteine residues has been shown to be of particular importance due to the functional relevance of many of them. In this review, we focus on the modification of the cysteine thiol by incorporation of a NO moiety (S-nitrosylation) or of a glutathione moiety (S-glutathionylation). Both modifications are produced by different reactions induced by nitric oxide-related species. We discuss the differences and similarities of both modifications, and their relationships, in regard to the biochemical mechanisms that produce them, including the enzymatic activities that may catalyze some of them and their subcellular compartmentalization. Even when biochemical knowledge is one step ahead of the demonstration of their pathophysiological relevance, we also describe the potential role of both modifications in several processes in which both post-translational modifications are involved. © 2007 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.Item Ribotoxins are a more widespread group of proteins within the filamentous fungi than previously believed(Toxicon, 1999) Martínez Ruiz, Antonio; Richard Kao; Julian Davies; Martínez Del Pozo, Álvaroa-Sarcin, restrictocin and mitogillin are the best known members of the family of fungal ribotoxins. In recent years, new members of this family have been discovered and characterised. In this work, we study the occurrence of ribotoxins among di erent species of fungi. The presence of ribotoxins has been identi®ed in some new species by means of genetic studies, as well as expression and activity assays. The ribotoxin genes have been partially sequenced, and demonstrate a high degree of similarity. These studies demonstrate that these toxins are more widespread than previously considered. This is surprising, considering the ribotoxins are such speci®c and potent toxins, of unknown biological function. These studies con®rm the hypothesis that these proteins are naturally engineered toxins derived from ribonucleases of broad substrate speci®city.