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Thomas M.C.,Institute Parasitologia y Biomedicina Lopez Neyra CSIC | Macias F.,Institute Parasitologia y Biomedicina Lopez Neyra CSIC | Lopez M.C.,Institute Parasitologia y Biomedicina Lopez Neyra CSIC
Trends in Parasitology | Year: 2010

Transposable elements (TEs) are dynamic elements that can reshape host genomes by generating rearrangements with the potential to create or disrupt genes, to shuffle existing genes, and to modulate their patterns of expression. In the genomes of parasites that infect mammals several TEs have been identified that probably have been maintained throughout evolution due to their contribution to gene function and regulation of gene expression. This review addresses how TEs are organized, how they colonize the genomes of mammalian parasites, the functional role these elements play in parasite biology, and the interactions between these elements and the parasite genome. © 2010 Elsevier Ltd.


Fernandez-Orgiler A.,CSIC - Biological Research Center | Alonso A.,CSIC - Biological Research Center | Alcolea P.J.,CSIC - Biological Research Center | Thomas M.C.,Institute Parasitologia y Biomedicina Lopez Neyra CSIC | Larraga V.,CSIC - Biological Research Center
Nucleic Acids Research | Year: 2016

Leishmania infantum is a protozoan parasite that is phagocytized by human macrophages. The host macrophages kill the parasite by generating oxidative compounds that induce DNA damage. We have identified, purified and biochemically characterized a DNA polymerase θ from L. infantum (LiPolθ), demonstrating that it is a DNA-dependent DNA polymerase involved in translesion synthesis of 8oxoG, abasic sites and thymine glycol lesions. Stably transfected L. infantum parasites expressing LiPolθ were significantly more resistant to oxidative and interstrand cross-linking agents, e.g. hydrogen peroxide, cisplatin and mitomycin C. Moreover, LiPolθ-overexpressing parasites showed an increased infectivity toward its natural macrophage host. Therefore, we propose that LiPolθ is a translesion synthesis polymerase involved in parasite DNA damage tolerance, to confer resistance against macrophage aggression. © 2016 The Author(s).


Sanchez-Luque F.J.,Institute Parasitologia y Biomedicina Lopez Neyra CSIC | Lopez M.C.,Institute Parasitologia y Biomedicina Lopez Neyra CSIC | MacIas F.,Institute Parasitologia y Biomedicina Lopez Neyra CSIC | Thomas M.C.,Institute Parasitologia y Biomedicina Lopez Neyra CSIC
Nucleic Acids Research | Year: 2011

L1Tc is a non-LTR LINE element from Trypanosoma cruzi that encodes its transposition machinery and bears an internal promoter. Herewith, we report the identification of an in vitro active hepatitis delta virus-like ribozyme located in the first 77nt at the 5′-end of the L1Tc mRNA (L1TcRz). The data presented show that L1TcRz has a co-transcriptional function. Using gel-purified uncleaved RNA transcripts, the data presented indicate that the kinetics of the self-cleaving, in a magnesium-dependent reaction, fits to a two-phase decay curve. The cleavage point identified by primer extension takes place at +1 position of the element. The hydroxyl nature of the 5′-end of the 3′-fragment generated by the cleavage activity of L1TcRz was confirmed. Since we have previously described that the 77-nt long fragment located at the 5′-end of L1Tc has promoter activity, the existence of a ribozyme in L1Tc makes this element to be the first described non-LTR retroelement that has an internal promoter-ribozyme dual function. The L1Tc nucleotides located downstream of the ribozyme catalytic motif appear to inhibit its activity. This inhibition may be influenced by the existence of a specific L1Tc RNA conformation that is recognized by RNase P. © 2011 The Author(s).


PubMed | CSIC - Biological Research Center and Institute Parasitologia y Biomedicina Lopez Neyra CSIC
Type: Journal Article | Journal: Nucleic acids research | Year: 2016

Leishmania infantum is a protozoan parasite that is phagocytized by human macrophages. The host macrophages kill the parasite by generating oxidative compounds that induce DNA damage. We have identified, purified and biochemically characterized a DNA polymerase from L. infantum (LiPol), demonstrating that it is a DNA-dependent DNA polymerase involved in translesion synthesis of 8oxoG, abasic sites and thymine glycol lesions. Stably transfected L. infantum parasites expressing LiPol were significantly more resistant to oxidative and interstrand cross-linking agents, e.g. hydrogen peroxide, cisplatin and mitomycin C. Moreover, LiPol-overexpressing parasites showed an increased infectivity toward its natural macrophage host. Therefore, we propose that LiPol is a translesion synthesis polymerase involved in parasite DNA damage tolerance, to confer resistance against macrophage aggression.


Maranon C.,Institute Parasitologia y Biomedicina Lopez Neyra CSIC | Egui A.,Institute Parasitologia y Biomedicina Lopez Neyra CSIC | Carrilero B.,Hospital Virgen Of La Arrixaca | Thomas M.C.,Institute Parasitologia y Biomedicina Lopez Neyra CSIC | And 4 more authors.
Microbes and Infection | Year: 2011

CD8+ cytotoxic T lymphocyte (CTL) response is critical for controlling the infection of the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease. Since only a few CD8 antigens have been described in Chagas disease patients, the identification of new class I-restricted epitopes is urgently needed for the development of immunotherapies against T. cruzi infection. In this study, bioinformatic methods were used to predict HLA-A*02:01-binders, and 30 peptides were selected, synthesized and tested for HLA-A*02:01 binding. Among them, sixteen peptides with medium-to-high affinity were assayed for their recognition by CTL from HSP70-immunized or T. cruzi-infected transgenic B6-A2/Kb mice. Our results show that four immunodominant epitopes (HSP70210-8, HSP70255-63, HSP70316-24 and HSP70345-53) are contained in the T. cruzi HSP70 antigen. Indeed two of them (HSP70210-8 and HSP70316-24) were also recognized by CTL of HLA-A*02:01+ Chagas disease patients, indicating that these peptides are processed and displayed as MHC class I epitopes during the natural history of T. cruzi infection. The HLA-A*02:01 restriction was evidenced using peptide-pulsed K562-A2 cells as antigen-presenting cells. Both cytotoxic and cytokine-secreting activities were detected in response to the former two peptides and, moreover, 10/12 patients (83%) recognized at least one of these two HSP70-derived CD8+ epitopes. © 2011 Institut Pasteur.


PubMed | University of La Laguna and Institute Parasitologia y Biomedicina Lopez Neyra CSIC
Type: Journal Article | Journal: Protist | Year: 2015

RNA helicases are ubiquitous enzymes that participate in almost all aspects of RNA processing, including RNA and RNA-protein complex remodelling. In trypanosomatids, which post-transcriptionally regulate gene expression, the formation of different kinds of ribonucleoprotein granules under stress conditions modulates the parasites RNA metabolism. This paper describes the isolation of a putative DEVH-box RNA helicase produced by promastigotes of Leishmania braziliensis. Using a Cy3-labelled dT30 oligo, FISH showed the localization of this protein to mRNA granules under starvation stress conditions. The central region of the protein was shown to be responsible for this behaviour.


PubMed | Baruch S Blumberg Institute, Cornell University, Institute Parasitologia y Biomedicina Lopez Neyra CSIC and CIBER ISCIII
Type: Journal Article | Journal: Antimicrobial agents and chemotherapy | Year: 2016

The aminoglycoside Geneticin (G418) is known to inhibit cell culture proliferation, via virus-specific mechanisms, of two different virus genera from the family Flaviviridae. Here, we tried to determine whether Geneticin can selectively alter the switching of the nucleotide 1 to 570 RNA region of hepatitis C virus (HCV) and, if so, whether this inhibits viral growth. Two structure-dependent RNases known to specifically cleave HCV RNA were tested in the presence or absence of the drug. One was the Synechocystis sp. RNase P ribozyme, which cleaves the tRNA-like domain around the AUG start codon under high-salt buffer conditions; the second was Escherichia coli RNase III, which recognizes a double-helical RNA switch element that changes the internal ribosome entry site (IRES) from a closed (C) conformation to an open (O) one. While the drug did not affect RNase P activity, it did inhibit RNase III in the micromolar range. Kinetic studies indicated that the drug favors the switch from the C to the O conformation of the IRES by stabilizing the distal double-stranded element and inhibiting further processing of the O form. We demonstrate that, because the RNA in this region is highly conserved and essential for virus survival, Geneticin inhibits HCV Jc1 NS3 expression, the release of the viral genomic RNA, and the propagation of HCV in Huh 7.5 cells. Our study highlights the crucial role of riboswitches in HCV replication and suggests the therapeutic potential of viral-RNA-targeted antivirals.


Risso V.A.,University of Granada | Manssour-Triedo F.,University of Granada | Delgado-Delgado A.,University of Granada | Arco R.,University of Granada | And 7 more authors.
Molecular Biology and Evolution | Year: 2015

Local protein interactions ("molecular context" effects) dictate amino acid replacements and can be described in terms of site-specific, energetic preferences for any different amino acid. It has been recently debated whether these preferences remain approximately constant during evolution or whether, due to coevolution of sites, they change strongly. Such research highlights an unresolved and fundamental issue with far-reaching implications for phylogenetic analysis and molecular evolution modeling. Here, we take advantage of the recent availability of phenotypically supported laboratory resurrections of Precambrian thioredoxins and β-lactamases to experimentally address the change of site-specific amino acid preferences over long geological timescales. Extensive mutational analyses support the notion that evolutionary adjustment to a new amino acid may occur, but to a large extent this is insufficient to erase the primitive preference for amino acid replacements. Generally, site-specific amino acid preferences appear to remain conserved throughout evolutionary history despite local sequence divergence. We show such preference conservation to be readily understandable in molecular terms and we provide crystallographic evidence for an intriguing structural-switch mechanism: Energetic preference for an ancestral amino acid in a modern protein can be linked to reorganization upon mutation to the ancestral local structure around the mutated site. Finally, we point out that site-specific preference conservation naturally leads to one plausible evolutionary explanation for the existence of intragenic global suppressor mutations. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.


Calahorro A.J.,University of Granada | Penas-Sanjuan A.,University of Jaén | Melguizo M.,University of Jaén | Fairen-Jimenez D.,Northwestern University | And 4 more authors.
Inorganic Chemistry | Year: 2013

We report the synthesis of a novel ligand, 3,3′-(1,2,4,5-tetrazine-3, 6-diyl)dibenzoic acid (1). In this fragment, we have introduced two carboxylate groups with the aim of using this ligand as a linker to construct three-dimensional metal-organic frameworks (MOFs). We have been successful in the formation of zinc (2) and lanthanum (3) MOFs. The zinc compound is a two-dimensional structure, while the lanthanum material is a three-dimensional MOF with interesting channels. We include the luminescence and adsorption studies of these materials. Moreover, we have evaluated the in vitro toxicity of this novel ligand, concluding that it can be considered negligible. © 2013 American Chemical Society.


PubMed | University of Granada, University of Maryland Baltimore County and Institute Parasitologia y Biomedicina Lopez Neyra CSIC
Type: Journal Article | Journal: Molecular biology and evolution | Year: 2015

Local protein interactions (molecular context effects) dictate amino acid replacements and can be described in terms of site-specific, energetic preferences for any different amino acid. It has been recently debated whether these preferences remain approximately constant during evolution or whether, due to coevolution of sites, they change strongly. Such research highlights an unresolved and fundamental issue with far-reaching implications for phylogenetic analysis and molecular evolution modeling. Here, we take advantage of the recent availability of phenotypically supported laboratory resurrections of Precambrian thioredoxins and -lactamases to experimentally address the change of site-specific amino acid preferences over long geological timescales. Extensive mutational analyses support the notion that evolutionary adjustment to a new amino acid may occur, but to a large extent this is insufficient to erase the primitive preference for amino acid replacements. Generally, site-specific amino acid preferences appear to remain conserved throughout evolutionary history despite local sequence divergence. We show such preference conservation to be readily understandable in molecular terms and we provide crystallographic evidence for an intriguing structural-switch mechanism: Energetic preference for an ancestral amino acid in a modern protein can be linked to reorganization upon mutation to the ancestral local structure around the mutated site. Finally, we point out that site-specific preference conservation naturally leads to one plausible evolutionary explanation for the existence of intragenic global suppressor mutations.

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