Person: Gavilanes Franco, José Gregorio
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First Name
José Gregorio
Last Name
Gavilanes Franco
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Ciencias Biológicas
Department
Bioquímica y Biología Molecular
Area
Bioquímica y Biología Molecular
Identifiers
5 results
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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 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 Hirsutellin A: A Paradigmatic Example of the Insecticidal Function of Fungal Ribotoxins(Insects, 2013) Herrero Galán, Elías; García Ortega, Lucía; Olombrada Sacristán, Miriam; Lacadena García-Gallo, Javier; Martínez del Pozo, Álvaro; Gavilanes Franco, José Gregorio; Oñaderra Sánchez, MercedesThe fungal pathogen Hirsutella thompsonii produces an insecticidal protein named hirsutellin A (HtA), which has been described to be toxic to several species of mites, insect larvae, and cells. On the other hand, on the basis of an extensive biochemical and structural characterization, HtA has been considered to be a member of the ribotoxins family. Ribotoxins are fungal extracellular ribonucleases, which inactivate ribosomes by specifically cleaving a single phosphodiester bond located at the large rRNA. Although ribotoxins were brought to light in the 1960s as antitumor agents, their biological function has remained elusive. Thus, the consideration of hirsutellin A, an insecticidal protein, as a singular ribotoxin recalled the idea of the biological activity of these toxins as insecticidal agents. Further studies have demonstrated that the most representative member of the ribotoxin family, α-sarcin, also shows strong toxic action against insect cells. The determination of high resolution structures, the characterization of a large number of mutants, and the toxicity assays against different cell lines have been the tools used for the study of the mechanism of action of ribotoxins at the molecular level. The aim of this review is to serve as a compilation of the facts that allow identification of HtA as a paradigmatic example of the insecticidal function of fungal ribotoxins.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.