Person: García Ortega, Lucía
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First Name
Lucía
Last Name
García Ortega
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Ciencias Químicas
Department
Bioquímica y Biología Molecular
Area
Bioquímica y Biología Molecular
Identifiers
15 results
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Now showing 1 - 10 of 15
Item Papel de la horquilla amino-terminal como elemento clave en la funcionalidad de las ribotoxinas de "aspergillus"(2005) García Ortega, Lucía; Martínez del Pozo, Alvaro; Gavilanes Franco, José GLas ribotoxinas son una familia de ribonucleasas, con una característica citotoxicidad y alergenicidad. En esta Tesis Doctoral queda demostrado el papel esencial que un elemento de su estructura, la horquilla amino-terminal, juega en estas funciones. Esta región es responsable del obligatorio reconocimiento del ribosoma, necesario para la posterior inhibición de la biosíntesis de proteínas en las células diana mediante su actividad ribonucleasa específica sobre dicho sustrato. Y además, contiene epítopos que participan en la inducción de la respuesta alérgica en pacientes sensibilizados a Aspergillus. La caracterización de este elemento de las ribotoxinas ha significado un paso cualitativo a la hora de comprender el mecanismo por el cual actúan estas proteínas, y abre la puerta a su posible aplicación en el ámbito clínico.Item Characterization of a new toxin from the entomopathogenic fungus Metarhizium anisopliae: the ribotoxin anisoplin(Biological chemistry, 2017) Olombrada, Miriam; Medina, Pilar; Budia, Flor; Gavilanes, José; Martínez Del Pozo, Álvaro; García Ortega, LucíaMetarhizium anisopliae is an entomopathogenic fungus relevant in biotechnology with applications like malaria vector control. Studies of its virulence factors are therefore of great interest. Fungal ribotoxins are toxic ribonucleases with extraordinary efficiency against target ribosomes and suggested as potential insecticides. Here, we describe this ribotoxin characteristic activity in M. anisopliae cultures. Anisoplin has been obtained as a recombinant protein and further characterized. It is structurally similar to hirsutellin A, the ribotoxin from the entomopathogen Hirsutella thompsonii. Moreover, anisoplin shows the ribonucleolytic activity typical of ribotoxins and cytotoxicity against insect cells. How Metarhizium uses this toxin and possible applications are on perspective.Item The ribonucleolytic activity of the ribotoxin α-sarcin is not essential for in vitro protein biosynthesis inhibition(BBA-Proteins and Proteomics, 2011) Alvarez García, Elisa; Diago Navarro, Elizabeth; Herrero Galán, Elías; García Ortega, Lucía; López Villarejo, Juan; Olmo López, Nieves; Díaz Orejas, Ramón; Gavilanes, José G.; Martínez Del Pozo, ÁlvaroFungal ribotoxins are toxic secreted ribonucleases that cleave a conserved single phosphodiester bond located at the sarcin/ricin loop of the larger rRNA. This cleavage inactivates ribosomes leading to protein biosynthesis inhibition and cell death. It has been proposed that interactions other than those found at the active site of ribotoxins are needed to explain their exquisite specific activity. The study presented shows the ability of a catalytically inactive α-sarcin mutant (H137Q) to bind eukaryotic ribosomes and interfere with in vitro protein biosynthesis. The results obtained are compatible with previous observations that α-sarcin can promote cell death by a mechanism that is independent of rRNA cleavage, expanding the potential set of activities performed by this family of toxins.Item Influence of key residues on the heterologous extracellular production of fungal ribonuclease U2 in the yeast Pichia pastoris(Protein Expression and Purification, 2009) Álvarez García, Elisa; García Ortega, Lucía; De los Ríos, Vivian; Gavilanes, José G.; Martínez Del Pozo, ÁlvaroRibonuclease U2, secreted by the smut fungus Ustilago sphaerogena, is a cyclizing ribonuclease that displays a rather unusual specificity within the group of microbial extracellular RNases, best represented by RNase T1. Superposition of the three-dimensional structures of RNases T1 and U2 suggests that the RNase U2 His 101 would be the residue equivalent to the RNase T1 catalytically essential His 92. RNase U2 contains three disulfide bridges but only two of them are conserved among the family of fungal extracellular RNases. The non-conserved disulfide bond is established between Cys residues 1 and 54. Mispairing of the disulfide network due to the presence of two consecutive Cys residues (54 and 55) has been invoked to explain the presence of wrongly folded RNase U2 species when produced in P. pastoris. In order to study both hypotheses, the RNase U2 H101Q and C1/54S variants have been produced, purified, and characterized. The results obtained support the major conclusion that His 101 is required for proper protein folding when secreted by the yeast P. pastoris. On the other hand, substitution of the first Cys residue for Ser results in a mutant version which is more efficiently processed in terms of a more complete removal of the yeast α-factor signal peptide. In addition, it has been shown that elimination of the Cys 1-Cys 54 disulfide bridge does not interfere with RNase U2 proper folding, generating a natively folded but much less stable protein.Item The behaviour of sea anemone actinoporins at the water-membrane interface(Biochimica et Biophysica Acta - Biomembranes, 2011) García Ortega, Lucía; Alegre Cebollada, Jorge; García Linares, Sara; Bruix, Marta; Martínez Del Pozo, Álvaro; Gavilanes, José G.Actinoporins constitute a group of small and basic α-pore forming toxins produced by sea anemones. They display high sequence identity and appear as multigene families. They show a singular behaviour at the water-membrane interface: In aqueous solution, actinoporins remain stably folded but, upon interaction with lipid bilayers, become integral membrane structures. These membranes contain sphingomyelin, display phase coexistence, or both. The water soluble structures of the actinoporins equinatoxin II (EqtII) and sticholysin II (StnII) are known in detail. The crystalline structure of a fragaceatoxin C (FraC) nonamer has been also determined. The three proteins fold as a β-sandwich motif flanked by two α-helices, one of them at the N-terminal end. Four regions seem to be especially important: A cluster of aromatic residues, a phosphocholine binding site, an array of basic amino acids, and the N-terminal α-helix. Initial binding of the soluble monomers to the membrane is accomplished by the cluster of aromatic amino acids, the array of basic residues, and the phosphocholine binding site. Then, the N-terminal α-helix detaches from the β-sandwich, extends, and lies parallel to the membrane. Simultaneously, oligomerization occurs. Finally, the extended N-terminal α-helix penetrates the membrane to build a toroidal pore. This model has been however recently challenged by the cryo-EM reconstruction of FraC bound to phospholipid vesicles. Actinoporins structural fold appears across all eukaryotic kingdoms in other functionally unrelated proteins. Many of these proteins neither bind to lipid membranes nor induce cell lysis. Finally, studies focusing on the therapeutic potential of actinoporins also abound.Item Characterization of a new toxin from the entomopathogenic fungus Metarhizium anisopliae: the ribotoxin anisoplin(Biological Chemistry, 2016) Olombrada, Miriam; Medina, Pilar; Budia, Flor; Gavilanes, José G.; Martínez Del Pozo, Álvaro; García Ortega, LucíaMetarhizium anisopliae is an entomopathogenic fungus relevant in biotechnology with applications like malaria vector control. Studies of its virulence factors are therefore of great interest. Fungal ribotoxins are toxic ribonucleases with extraordinary efficiency against target ribosomes and suggested as potential insecticides. Here, we describe this ribotoxin characteristic activity in M. anisopliae cultures. Anisoplin has been obtained as a recombinant protein and further characterized. It is structurally similar to hirsutellin A, the ribotoxin from the entomopathogen Hirsutella thompsonii. Moreover, anisoplin shows the ribonucleolytic activity typical of ribotoxins and cytotoxicity against insect cells. How Metarhizium uses this toxin and possible applications are on perspective.Item Fungal Ribotoxins(ELS, 2018) García Ortega, Lucía; Palacios Ortega, Juan; Martínez Del Pozo, ÁlvaroFungal ribotoxins constitute a family of extracellular ribonucleases with exquisite specificity against rRNA (ribonucleic acid). They induce apoptotic death of cells after inhibiting protein translation. Ribosomes become functionally incompetent because ribotoxins cleave one single phosphodiester bond, located at a unique and universally conserved loop, needed for elongation factors function. As secreted proteins, ribotoxins need to cross the membrane of their target cells in order to exert their catalytic activity, and they do it without receptor mediation. Using lipid model systems, it has been shown that they are able to enter cells with membranes enriched in acidic phospholipids. Both membrane-interacting and ribosomal-recognition activities are characterised by distinct structural features. Even though the natural function of ribotoxins is not known yet, their production by entomopathogenic fungi has suggested their insecticidal role. After decades of detailed study, the biotechnological potential of ribotoxins in pest control and as antitumour agents is becoming evident.Item Fungal extracellular ribotoxins as insecticidal agents(Insect Biochemistry and Molecular Biology, 2013) Olombrada Sacristán, Miriam; Herrero Galán, Elías; Tello, Daniel; Oñaderra Sánchez, Mercedes; Gavilanes, José G.; Martínez Del Pozo, Álvaro; García Ortega, LucíaFungal ribotoxins were discovered almost 50 years ago as extracellular ribonucleases (RNases) with antitumoral properties. However, the biological function of these toxic proteins has remained elusive. The discovery of the ribotoxin HtA, produced by the invertebrates pathogen H. thompsonii, revived the old proposal that insecticidal activity would be their long searched function. Unfortunately, HtA is rather singular among all ribotoxins known in terms of sequence and structure similarities. Thus, it was intriguing to answer the question of whether HtA is just an exception or, on the contrary, the paradigmatic example of the ribotoxins function. The work presented uses HtA and -sarcin, the most representative member of the ribotoxins family, to show their strong toxic action against insect larvae and cells.Item Hirsutellin A: A Paradigmatic Example of the Insecticidal Function of Fungal Ribotoxins(Insects, 2013) Herrero Galán, Elías; García Ortega, Lucía; Lacadena García-Gallo, Francisco Javier; Olombrada Sacristán, Miriam; 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 Involvement of loops 2 and 3 of alpha-sarcin on its ribotoxic activity(Toxicon, 2015) Castaño Rodríguez, Carlos; Olombrada Sacristán, Miriam; Partida Hanon, Angélica; Lacadena García-Gallo, Francisco Javier; Oñaderra Sánchez, Mercedes; Gavilanes, José G.; García Ortega, Lucía; Martínez Del Pozo, ÁlvaroRibotoxins are a family of fungal ribosome-inactivating proteins displaying highly specific ribonucleolytic activity against the sarcin/ricin loop (SRL) of the larger rRNA, with a-sarcin as its best-characterized member. Their toxicity arises from the combination of this activity with their ability to cross cell membranes. The involvement of a-sarcin's loops 2 and 3 in SRL and ribosomal proteins recognition, as well as in the ribotoxin-lipid interactions involving cell penetration, has been suggested some time ago. In the work presented now different mutants have been prepared in order to study the role of these loops in their ribonucleolytic and lipid-interacting properties. The results obtained confirm that loop 3 residues Lys 111, 112, and 114 are key actors of the specific recognition of the SRL. In addition, it is also shown that Lys 114 and Tyr 48 conform a network of interactions which is essential for the catalysis. Lipid-interaction studies show that this Lys-rich region is indeed involved in the phospholipids recognition needed to cross cell membranes. Loop 2 is shown to be responsible for the conformational change which exposes the region establishing hydrophobic interactions with the membrane inner leaflets and eases penetration of ribotoxins target cells.