Person: Mancheño Gómez, José Miguel
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First Name
José Miguel
Last Name
Mancheño Gómez
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Ciencias Químicas
Department
Area
Bioquímica y Biología Molecular
Identifiers
7 results
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Item Estudio del mecanismo de acción de la citotoxina alfa-sarcina(2002) Mancheño Gómez, José Miguel; Oñaderra Sánchez, Mercedes; Gavilanes Franco, José GLa x-sarcina es una toxina proteica sintetizada por el hongo aspergillus giganteus que para ejercer su acción citotóxica ha de penetrar en el interior celular, en donde inhibe la síntesis de proteinas mediante la modificación especifica e irreversible de los ribosomas. Si bien el mecanismo molecular de inactivación de los ribosomas se conoce en gran detalle, el modo mediante el cual la proteína accede al interior celular no se ha caracterizado. Hasta hoy día, no se han encontrado receptores de membrana para la proteína, aunque si se ha demostrado la capacidad de la x-sarcina de interaccionar con membranas modelo compuestas por fosfolípidos ácidos. En este trabajo de investigación se han extendido los estudios de la interacción alfa-sarcina-membranas modelo, formadas por un nuevo tipo de fosfolipido: fosfatidilserinas. Asimismo, se ha caracterizado la capacidad que tiene esta proteína de acceder al interior vesicular en ausencia de agentes permeabilizantes, concluyéndose que, efectivamente, esta proteína es capaz de alcanzar el interior de vesículas modelo. Otro punto abordado en este trabajo ha sido la caracterización cinética de los procesos de agregación y desestabilizaron de membranas inducidos por la toxina. La escala de tiempo en la que transcurren ambos procesos, del orden de milisegundos, hizo necesario el empleo de técnicas de flujo detenido. Un aspecto que se ha analizado también ha sido las bases estructurales en las que reside la capacidad de la proteína de interaccionar con membranas. En primer lugar, se ha propuesto un modelo de la conformación de la alfa-sarcina considerando la similitud de secuencia que posee con otras ribonucleasas fungicas cuya estructura tridimensional ha sido resuelta a gran resolucion. Este estudio predice la existencia de un núcleo hidrofobico formado por cuatro hebras beta, núcleo similar al que aparece en el resto de la ribonucleasas anteriores. Asimismo, la alfa-sarcina desnaturalizada mediante reducción y carboxiamidometilacion, que posee elementos de estructura beta y giros beta, según se deriva de estudios de dicroismo circular, interacciona con membranas modelo de un modo análogo al de la alfa-sarcina nativa. La capacidad de la alfa-sarcina desnaturalizada de interaccionar hidrofobicamente con membranas podría deberse, entre otras posibilidades, a la conservación del núcleo beta, anteriormente predicho. Por otro lado, la hipótesis de que esta región interacciona con membranas, es reforzada por el hecho de que el péptido sintético que corresponde a la región 116-139 de la secuencia de la alfa-sarcina, que englobaría dos de las cuatro hebras constituyentes del núcleo 116-139 de la secuencia de la alfa-sarcina, que englobaría dos de las cuatro hebras con un elevado contenido en estructura beta, tras interaccionar con membranas. Por todo ello, esta región ha sido propuesta como responsable del componente hidrofóbico de la interacción entre alfa-sarcina y bicapas modeloItem 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 Biochemical and structural studies of two tetrameric nucleoside 2′-deoxyribosyltransferases from psychrophilic and mesophilic bacteria: Insights into cold-adaptation(International Journal of Biological Macromolecules, 2021) Fernández Lucas, Jesús; Acebrón Ávalos, Iván; Wu, Ruiying Y.; Alfaro, Yohana; Acosta, Javier; Kaminski, Pierre A.; Arroyo Sánchez, Miguel; Joachimiak, Andrzej; Nocek, Boguslaw P.; De La Mata Riesco, Mª Isabel; Mancheño Gómez, José MiguelNucleoside 2′-deoxyribosyltransferases (NDTs) catalyze the cleavage of glycosidic bonds of 2′-deoxynucleosides and the following transfer of the 2′-deoxyribose moiety to acceptor nucleobases. Here, we report the crystal structures and biochemical properties of the first tetrameric NDTs: the type I NDT from the mesophilic bacterium Enterococcus faecalis V583 (EfPDT) and the type II NDT from the bacterium Desulfotalea psychrophila (DpNDT), the first psychrophilic NDT. This novel structural and biochemical data permitted an exhaustive comparative analysis aimed to shed light into the basis of the high global stability of the psychrophilic DpNDT, which has a higher melting temperature than EfPDT (58.5 °C versus 54.4 °C) or other mesophilic NDTs. DpNDT possesses a combination of unusual structural motifs not present neither in EfPDT nor any other NDT that most probably contribute to its global stability, in particular, a large aliphatic isoleucine-leucine-valine (ILV) bundle accompanied by a vicinal disulfide bridge and also an intersubunit disulfide bridge, the first described for an NDT. The functional and structural features of DpNDT do not fit the standard features of psychrophilic enzymes, which lead us to consider the implication of (sub)cellular levels together with the protein level in the adaptation of enzymatic activity to low temperatures.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 The cytotoxin α‐sarcin behaves as a cyclizing ribonuclease(FEBS Letters, 1998) Lacadena García-Gallo, Francisco Javier; Martínez Del Pozo, Álvaro; Valle Lacadena; Martínez Ruiz, Antonio; Mancheño Gómez, José Miguel; Oñaderra Sánchez, Mercedes; Gavilanes Franco, José GregorioThe hydrolysis of adenylyl(3PC5P)adenosine (ApA) and guanylyl(3PC5P)adenosine (GpA) dinucleotides by the cytotoxic protein K-sarcin has been studied. Quantitative analysis of the reaction has been performed through reversephase chromatographic (HPLC) separation of the resulting products. The hydrolysis of the 3P-5P phosphodiester bond of these substrates yields the 2P-3P cyclic mononucleotide; this intermediate is converted into the corresponding 3P-monophosphate derivative as the final product of the reaction. The values of the apparent Michaelis constant (KM), kcat and kcat/ KM have also been calculated. The obtained results fit into a twostep mechanism for the enzymatic activity of K-sarcin and allow to consider this protein as a cyclizing RNase. z 1998 Federation of European Biochemical Societies.Item Sequence determination and molecular characterization of gigantin, a cytotoxic protein produced by the mouldAspergillus giganteusIFO 5818(Archives of Biochemestry and Biophysics, 1997) Jérémie Wirth; Martínez Del Pozo, Álvaro; Mancheño Gómez, José Miguel; Martínez Ruiz, Antonio; Lacadena García-Gallo, Francisco Javier; Oñaderra Sánchez, Mercedes; Gavilanes Franco, José GregorioGigantin is a 17-kDa ribonuclease secreted by Aspergillus giganteus IFO 5818. The sequence of the genomic DNA coding for this protein is reported. The deduced amino acid sequence reveals nine amino acid variations with respect to alpha-sarcin, a well-characterized ribosome-inactivating protein from A. giganteus MDH 18894. The peptides obtained after tryptic digestion of reduced and carboxyamidomethylated gigantin have been chromatographically separated. The analysis of these peptides in comparison to those originating from alpha-sarcin corroborates the above sequence differences. These do not sensibly modify the conformation of the protein, based on the coincidence of the circular dichroism and fluorescence emission spectra of the two proteins. The obtained results are discussed in terms of the involvement of the distinctive residues in the immunological and catalytic properties that distinguish gigantin from alpha-sarcin.