Person: Lacadena García-Gallo, Francisco Javier
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First Name
Francisco Javier
Last Name
Lacadena García-Gallo
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Ciencias Químicas
Department
Bioquímica y Biología Molecular
Area
Bioquímica y Biología Molecular
Identifiers
9 results
Search Results
Now showing 1 - 9 of 9
- Project number: 350
Item EChemTest: sistema de evaluación de la Calidad en Química(2022) Sánchez Benítez, Francisco Javier; Díaz Blanco, Cristina; Guerrero Martínez, Andrés; Gutiérrez Alonso, Ángel; Lacadena García-Gallo, Francisco Javier; Lainez Ferrando, Alfredo; Pilo Santos, Miguel; Villalba Díaz, MaríaTeresa; García Linares, SaraEste proyecto plantea la herramienta EChemTest como mecanismo de evaluación de la Calidad de un Grado relacionado con la Química. También presenta la oportunidad de evaluar cómo ha influido la docencia online en la adquisición de conocimientos, comparando con cursos anteriores. 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 Nanobody-based EGFR-targeting immunotoxins for colorectal cancer treatment(Biomolecules, 2023) Narbona Corral, Javier; Hernández Baraza, Luisa; García Gordo, Rubén; Sanz, Laura; Lacadena García-Gallo, Francisco JavierImmunotoxins (ITXs) are chimeric molecules that combine the specificity of a targeting domain, usually derived from an antibody, and the cytotoxic potency of a toxin, leading to the selective death of tumor cells. However, several issues must be addressed and optimized in order to use ITXs as therapeutic tools, such as the selection of a suitable tumor-associated antigen (TAA), high tumor penetration and retention, low kidney elimination, or low immunogenicity of foreign proteins. To this end, we produced and characterized several ITX designs, using a nanobody against EGFR (VHH 7D12) as the targeting domain. First, we generated a nanoITX, combining VHH 7D12 and the fungal ribotoxin α-sarcin (αS) as the toxic moiety (VHHEGFRαS). Then, we incorporated a trimerization domain (TIEXVIII) into the construct, obtaining a trimeric nanoITX (TriVHHEGFRαS). Finally, we designed and characterized a bispecific ITX, combining the VHH 7D12 and the scFv against GPA33 as targeting domains, and a deimmunized (DI) variant of α-sarcin (BsITXαSDI). The results confirm the therapeutic potential of α-sarcin-based nanoITXs. The incorporation of nanobodies as target domains improves their therapeutic use due to their lower molecular size and binding features. The enhanced avidity and toxic load in the trimeric nanoITX and the combination of two different target domains in the bispecific nanoITX allow for increased antitumor effectiveness.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 A New Optimized Version of a Colorectal Cancer-Targeted Immunotoxin Based on a Non-Immunogenic Variant of the Ribotoxin α-Sarcin(Cancers, 2023) Narbona Corral, Javier; García Gordo, Rubén; Tomé Amat, Jaime; Lacadena García-Gallo, Francisco JavierDue to its incidence and mortality, cancer remains one of the main risks to human health and lifespans. In order to overcome this worldwide disease, immunotherapy and the therapeutic use of immunotoxins have arisen as promising approaches. However, the immunogenicity of foreign proteins limits the dose of immunotoxins administered, thereby leading to a decrease in its therapeutic benefit. In this study, we designed two different variants of non-immunogenic immunotoxins (IMTXA33αSDI and IMTXA33furαSDI) based on a deimmunized variant of the ribotoxin α-sarcin. The inclusion of a furin cleavage site in IMTXA33furαSDI would allow a more efficient release of the toxic domain to the cytosol. Both immunotoxins were produced and purified in the yeast Pichia pastoris and later functionally characterized (both in vitro and in vivo), and immunogenicity assays were carried out. The results showed that both immunotoxins were functionally active and less immunogenic than the wild-type immunotoxin. In addition, IMTXA33furαSDI showed a more efficient antitumor effect (both in vitro and in vivo) due to the inclusion of the furin linker. These results constituted a step forward in the optimization of immunotoxins with low immunogenicity and enhanced antitumor activity, which can lead to potential better outcomes in cancer treatment.- Project number: 150
Item EChemTest como herramienta de evaluación en Química(2020) Sánchez Benítez, Francisco Javier; Villalba Díaz, Mayte; Lainez Ferrando, Alfredo; Lacadena García-Gallo, Francisco Javier; Gutiérrez Alonso, Ángel; Pilo Santos, MiguelEl proyecto pretende ser un modelo piloto para su futura implementación como mecanismo de evaluación de Calidad de un Grado relacionado con la Química. La metodología de trabajo pasa por la realización de diferentes pruebas de evaluación (EChemTest) compuestas por preguntas que abarcan todos los temas de la Química General. Estas pruebas se llevan cabo en diferentes momentos del curso académico, incluyendo el inicio y el final del mismo, y pretenden evaluar la adquisición de conocimientos y competencias por parte del alumnado. 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.Item Der p 1‑based immunotoxin as potential tool for the treatment of dust mite respiratory allergy(Scientific Reports, 2020) Lázaro‑Gorines, Rodrigo; López Rodríguez, Juan Carlos; Benedé Pérez, Sara; González, Miguel; Mayorga, Cristobalina; Vogel, Lothar; Martínez Del Pozo, Álvaro; Lacadena García-Gallo, Francisco Javier; Villalba Díaz, María TeresaImmunotoxins appear as promising therapeutic molecules, alternative to allergen-specifcimmunotherapy. In this work, we achieved the development of a protein chimera able to promote specifc cell death on efector cells involved in the allergic reaction. Der p 1 allergen was chosen as cell-targeting domain and the powerful ribotoxin α-sarcin as the toxic moiety. The resultant construction, named proDerp1αS, was produced and purifed from the yeast Pichia pastoris. Der p 1-protease activity and α-sarcin ribonucleolytic action were efectively conserved in proDerp1αS. Immunotoxin impact was assayed by using efector cells sensitized with house dust mite-allergic sera. Cell degranulation and death, triggered by proDerp1αS, was exclusively observed on Der p 1 sera sensitized-humRBL-2H3 cells, but not when treated with non-allergic sera. Most notably, equivalent IgE-binding and degranulation were observed with both proDerp1αS construct and native Der p 1 when using purifed basophils from sensitized patients. However, proDerp1αS did not cause any cytotoxic efect on these cells, apparently due to its lack of internalization after their surface IgEbinding, showing the complex in vivo panorama governing allergic reactions. In conclusion, herein we present proDerp1αS as a proof of concept for a potential and alternative new designs of therapeutic tools for allergies. Development of new, and more specifc, second-generation of immunotoxins following proDerp1αS, is further discussed