Seccion Bioquimica

Montevideo, Uruguay

Seccion Bioquimica

Montevideo, Uruguay

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Esteves A.,Seccion Bioquimica
Journal of Biomolecular Structure and Dynamics | Year: 2013

Fatty acid (FA) binding proteins are small intracellular proteins whose members exhibit great diversity and low similarity at the primary structure level, but a highly conserved three-dimensional structure. Characterised by a high-affinity non-covalent binding of hydrophobic ligands, these proteins have a molecular mass of 14-15 kDa with a characteristic β-barrel structure. Members of this family have been identified along the zoological scale, with Platyhelminthes being the more primitive organisms where they have been reported. Two FA binding proteins (FABPs), EgFABP1 and EgFABP2, with 88% similarity have been identified in Echinococcus granulosus. In an effort to understand why two such similar proteins are expressed by this organism, we performed an in silico analysis of the binding capabilities of both proteins. The crystallographic structure of EgFABP1 was utilised as a template to model EgFABP2, and both were docked against palmitate, oleate, linoleate and arachidonate. The docked structures were submitted to 4 ns molecular dynamics simulations, and their protein-ligand interaction energies were measured. The collected data demonstrated that linoleate and arachidonate had the higher interaction energies when bound to EgFABP1 and that palmitate and linoleate had the higher interaction energies when bound to EgFABP2. External and internal binding surfaces were analysed, showing differences at both levels. Internal surface compositions suggested that both proteins could have preferences for certain FAs. Comparisons of the holo and apo forms of each protein indicated that the ligand imposed subtle, but specific modifications that could trigger surface signals. The differences found between the proteins under study suggest that they could have functional uniqueness in the parasite's metabolism. © 2013 Taylor & Francis.


Cousido-Siah A.,University of Strasbourg | Ayoub D.,University of Strasbourg | Ayoub D.,French National Center for Scientific Research | Berberian G.,Institute Investigacion Medica Mercedes y Martin Ferreyra | And 15 more authors.
Acta Crystallographica Section D: Biological Crystallography | Year: 2012

The protein ReP1-NCXSQ was isolated from the cytosol of squid nerves and has been shown to be required for MgATP stimulation of the squid nerve Na +/Ca2+ exchanger NCXSQ1. In order to determine its mode of action and the corresponding biologically active ligand, sequence analysis, crystal structures and mass-spectrometric studies of this protein and its Tyr128Phe mutant are reported. Sequence analysis suggests that it belongs to the CRABP family in the FABP superfamily. The X-ray structure at 1.28 Å resolution shows the FABP Β - barrel fold, with a fatty acid inside the barrel that makes a relatively short hydrogen bond to Tyr128 and shows a double bond between C9 and C10 but that is disordered beyond C12. Mass-spectrometric studies identified this fatty acid as palmitoleic acid, confirming the double bond between C9 and C10 and establishing a length of 16 C atoms in the aliphatic chain. This acid was caught inside during the culture in Escherichia coli and therefore is not necessarily linked to the biological activity. The Tyr128Phe mutant was unable to activate the Na+/Ca2+ exchanger and the corresponding crystal structure showed that without the hydrogen bond to Tyr128 the palmitoleic acid inside the barrel becomes disordered. Native mass-spectrometric analysis confirmed a lower occupancy of the fatty acid in the Tyr128Phe mutant. The correlation between (i) the lack of activity of the Tyr128Phe mutant, (ii) the lower occupancy/disorder of the bound palmitoleic acid and (iii) the mass-spectrometric studies of ReP1-NCXSQ suggests that the transport of a fatty acid is involved in regulation of the NCXSQ1 exchanger, providing a novel insight into the mechanism of its regulation. In order to identify the biologically active ligand, additional high-resolution mass-spectrometric studies of the ligands bound to ReP1-NCXSQ were performed after incubation with squid nerve vesicles both with and without MgATP. These studies clearly identified palmitic acid as the fatty acid involved in regulation of the Na+/Ca2+ exchanger from squid nerve. © 2012 International Union of Crystallography Printed in Singapore - all rights reserved.


Iriarte A.,Laboratorio Of Organizacion Y Evolucion Del Genoma | Iriarte A.,Laboratorio Of Evolucion | Sanguinetti M.,Seccion Bioquimica | Fernandez-Calero T.,Institute Pasteur Of Montevideo | And 3 more authors.
Gene | Year: 2012

Aspergillus is a genus of mold fungi that includes more than 200 described species. Many members of the group are relevant pathogens and other species are economically important. Only one species has been analyzed for codon usage, and this was performed with a small number of genes. In this paper, we report the codon usage patterns of eight completely sequenced genomes which belong to this genus. The results suggest that selection for translational efficiency and accuracy are the major factors shaping codon usage in all of the species studied so far, and therefore they were active in the last common ancestor of the group. Composition and molecular distances analyses show that highly expressed genes evolve slower at synonymous sites. We identified a conserved core of translationally optimal codons and study the tRNA gene pool in each genome. We found that the great majority of preferred triplets match the respective cognate tRNA with more copies in the respective genome. We discuss the possible scenarios that can explain the observed differences among the species analyzed. Finally we highlight the biotechnological application of this research regarding heterologous protein expression. © 2012 Elsevier B.V.


Iriarte A.,Laboratorio Of Organizacion Y Evolucion Del Genoma | Iriarte A.,Laboratorio Of Evolucion | Baraibar J.D.,Laboratorio Of Organizacion Y Evolucion Del Genoma | Diana L.,Seccion Bioquimica | And 4 more authors.
Journal of Biomolecular Structure and Dynamics | Year: 2014

Many studies have explored the mechanisms involved in relative amino acid usage (RAAU) in a variety of prokaryotes and eukaryotes. A strong bias was observed in highly expressed genes (HEGs) of endosymbiotic bacteria. By means of correspondence analysis, we studied the major trends affecting internal variability of RAAU in Mollicutes. The principal trend is related to the usage of smaller, less aromatic, and GC-rich coded amino acids in HEGs. Given the nature of the genetic code, these properties are linked among them. Expectedly, we found a slow evolutionary rate of HEGs, which is likely driven by purifying selection. On the other hand, the rest of the genes accumulate rapid changes as a result of the extreme mutational bias toward A + T of the genomes and genetic drift, increasing internal variability. Amino acid changes across the phylogeny of the group were traced in order to estimate the mean molecular weight and aromaticity trends in each branch. Finally, we compared amino acid usage bias within and between Mollicutes and the free-living Firmicutes. © 2013 Taylor & Francis.


Geisinger A.,Seccion Bioquimica | Geisinger A.,Institute Investigaciones Biologicas Clemente Estable | Rodriguez-Casuriaga R.,Institute Investigaciones Biologicas Clemente Estable
Cytogenetic and Genome Research | Year: 2010

Mammalian spermatogenesis is nowadays still poorly understood at the molecular level, mainly due to the heterogeneous nature of testes, which contain a high number of different cell types, and to the lack of spermatogenic cell culture systems for in vitro studies. As a consequence, the development and/or application of methodological approaches aiming at the enrichment or purification of specific testicular cell types are of great interest and have been addressed by scientists for at least 4 decades. Among the many applications that flow cytometry (FC) has gained since its invention, analysis and sorting of spermatogenic cell populations represent a promising strategy to efficiently overcome testis heterogeneity drawback. Surprisingly, FC has been only rarely used as a preparative method for downstream gene expression studies in specific spermatogenic stages. This work aims to provide an overview of FC for spermatogenic studies including preparation of testicular single cell suspensions, dyes for DNA staining, and our own experience with rodent testis material. Copyright © 2010 S. Karger AG, Basel.


Harispe L.,Seccion Bioquimica | Garcia G.,Catedra de Inmunologia | Arbildi P.,Catedra de Inmunologia | Pascovich L.,Catedra de Inmunologia | And 4 more authors.
Acta Tropica | Year: 2010

Glutathione transferases (GSTs) are believed to be a major detoxification system in helminths. We describe the expression and functional analysis of EgGST, a cytosolic GST from Echinococcus granulosus, related to the Mu-class of mammalian enzymes. EgGST was produced as an enzymatically active dimeric protein (rEgGST), with highest specific activity towards the standard substrate 1-chloro-2,4-dinitrobenzene (CDNB; 2.5 μmol min -1 mg -1), followed by ethacrynic acid. Interestingly, rEgGST displayed glutathione peroxidase activity (towards cumene hydroperoxide), and conjugated reactive carbonyls (trans-2-nonenal and trans,trans-2,4-decadienal), indicating that it may intercept damaging products of lipid peroxidation. In addition, classical GST inhibitors (cybacron blue, triphenylthin chloride and ellagic acid) and a number of anthelmintic drugs (mainly, hexachlorophene and rafoxanide) were found to interfere with glutathione-conjugation to CDNB; suggesting that they may bind to EgGST. Considered globally, the functional properties of rEgGST are similar to those of putative orthologs from Echinococcus multilcularis and Taenia solium, the other medically important cestodes. Interestingly, our results also indicate that differences exist between these closely related cestode GSTs, which probably reflect specific biological functions of the molecules in each parasitic organism. © 2009 Elsevier B.V. All rights reserved.


PubMed | Seccion Bioquimica
Type: Journal Article | Journal: Acta tropica | Year: 2010

Glutathione transferases (GSTs) are believed to be a major detoxification system in helminths. We describe the expression and functional analysis of EgGST, a cytosolic GST from Echinococcus granulosus, related to the Mu-class of mammalian enzymes. EgGST was produced as an enzymatically active dimeric protein (rEgGST), with highest specific activity towards the standard substrate 1-chloro-2,4-dinitrobenzene (CDNB; 2.5 micromol min(-1)mg(-1)), followed by ethacrynic acid. Interestingly, rEgGST displayed glutathione peroxidase activity (towards cumene hydroperoxide), and conjugated reactive carbonyls (trans-2-nonenal and trans,trans-2,4-decadienal), indicating that it may intercept damaging products of lipid peroxidation. In addition, classical GST inhibitors (cybacron blue, triphenylthin chloride and ellagic acid) and a number of anthelmintic drugs (mainly, hexachlorophene and rafoxanide) were found to interfere with glutathione-conjugation to CDNB; suggesting that they may bind to EgGST. Considered globally, the functional properties of rEgGST are similar to those of putative orthologs from Echinococcus multilcularis and Taenia solium, the other medically important cestodes. Interestingly, our results also indicate that differences exist between these closely related cestode GSTs, which probably reflect specific biological functions of the molecules in each parasitic organism.


PubMed | Seccion Bioquimica
Type: Journal Article | Journal: Journal of biomolecular structure & dynamics | Year: 2013

Fatty acid (FA) binding proteins are small intracellular proteins whose members exhibit great diversity and low similarity at the primary structure level, but a highly conserved three-dimensional structure. Characterised by a high-affinity non-covalent binding of hydrophobic ligands, these proteins have a molecular mass of 14-15 kDa with a characteristic -barrel structure. Members of this family have been identified along the zoological scale, with Platyhelminthes being the more primitive organisms where they have been reported. Two FA binding proteins (FABPs), EgFABP1 and EgFABP2, with 88% similarity have been identified in Echinococcus granulosus. In an effort to understand why two such similar proteins are expressed by this organism, we performed an in silico analysis of the binding capabilities of both proteins. The crystallographic structure of EgFABP1 was utilised as a template to model EgFABP2, and both were docked against palmitate, oleate, linoleate and arachidonate. The docked structures were submitted to 4 ns molecular dynamics simulations, and their protein-ligand interaction energies were measured. The collected data demonstrated that linoleate and arachidonate had the higher interaction energies when bound to EgFABP1 and that palmitate and linoleate had the higher interaction energies when bound to EgFABP2. External and internal binding surfaces were analysed, showing differences at both levels. Internal surface compositions suggested that both proteins could have preferences for certain FAs. Comparisons of the holo and apo forms of each protein indicated that the ligand imposed subtle, but specific modifications that could trigger surface signals. The differences found between the proteins under study suggest that they could have functional uniqueness in the parasites metabolism.

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