Estudio biofísico y estructural de Na-FAR-1, miembro de una nueva familia de proteínas de nematodos que unen ácidos grasos y retinol

Los parásitos nematodos producen diversas proteínas solubles que unen lípidos (LBPs) estructuralmente distintas a las del huésped. Las funciones que cumplen se desconocen pero se hipotetiza que estarían involucradas en las funciones típicas internas de organismos multicelulares, como la utilización y transporte de compuestos no solubles, y en externas especializadas. Algunas de estas proteínas participarían en la modificación del entorno local en el tejido del huésped, posibilitando la modulación y la evasión de la respuesta inmune. Entre las LBPs producidas por nematodos se encuentran las FAR (Fatty Acid and Retinol binding proteins), una clase novedosa de proteínas que unen ácidos grasos y retinol. Tienen un tamaño aproximado de 19 kDa y sus estructuras que parecen ser ricas en alfa-hélices aún no han sido completamente dilucidadas. La comprensión del rol que cumple esta familia de proteínas tiene gran interés fisiopatológico ya que podrían desempeñar funciones relevantes en la biología de los parásitos que las producen y dadas las diferencias estructurales que presentarían con respecto a las LBPs de sus huéspedes, servirían como potenciales blancos para el diseño de nuevas terapias antiparasitarias. Con la finalidad de contribuir a la caracterización de las proteínas FAR y avanzar de este modo en la determinación de su función biológica, se llevaron a cabo estudios biofísicos y estructurales que permitieron resolver la estructura de Na-FAR-1 en solución por espectroscopía de resonancia magnética nuclear. Determinándose que consta de once hélices que conforman una cavidad interna de gran tamaño, donde podrían ubicarse ligandos hidrofóbicos. La estequiometría de unión de los complejos formados por Na-FAR-1 estaría dada por cuatro moléculas de ácido oleico por molécula de proteína, pero se limitaría a una única molécula de ligando en el caso del retinol y de los análogos fluorescentes de ácidos grasos empleados para su estudio. A su vez se evidenció que además de los ligandos esperados como ácidos grasos y retinol, esta proteína es capaz de unir fosfolípidos y diacilglicéridos. La amplia diversidad de unión a ligandos, sumada a su localización en el intestino del nematodo, indicarían que podría participar en el direccionamiento hacia los distintos tejidos de los lípidos ingeridos.Facultad de Ciencias Exacta

Identification of potassium phosphite responsive miRNAs and their targets in potato

Micro RNAs (miRNAs) are small single strand non-coding RNAs that regulate geneexpression at the post-transcriptional level, either by translational inhibition or mRNA degradation based on the extent of complementarity between the miRNA and its target mRNAs. Potato (Solanum tuberosum L.) is the most important horticultural crop in Argentina. Achieving an integrated control of diseases is crucial for this crop, where frequent agrochemical applications, particularly fungicides, are carried out. A promising strategy is based on promoting induced resistance through the application ofenvironmentally friendly compounds such as phosphites, inorganic salts of phosphorous acid. The use of phosphites in disease control management has provento be effective. Although the mechanisms underlying their effect remain unclear, we postulated that miRNAs could be involved. Therefore we performed next generation sequencing (NGS) in potato leaves treated and non treated with potassium phosphite (KPhi). We identified 25 miRNAs that were expressed differentially, 14 already annotated in miRBase and 11 mapped to the potato genome as potential new miRNAs. A prediction of miRNA targets showed genes related to pathogen resistance,transcription factors, and oxidative stress. We also analyzed in silico stress and phytohormone responsive cis-acting elements on differentially expressed pre miRNAs. Despite the fact that some of the differentially expressed miRNAs have been already identified, this is to our knowledge the first report identifying miRNAs responsive to abiocompatible stress resistance inducer such as potassium phosphite, in plants.Further characterization of these miRNAs and their target genes, might help toelucidate the molecular mechanisms underlying KPhi-induced resistance.Fil: Rey Burusco, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Daleo, Gustavo Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Feldman, Mariana Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentin

Resonancia magnética nuclear

La Resonancia Magnética Nuclear (RMN) es una técnica espectroscópica muy poderosa que nos permite estudiar a nivel atómico moléculas de interés biológico. Complementaria a la técnica de rayos X, constituye una herramienta fundamental para la determinación de estructuras de proteínas y otras biomoléculas en solución. Sin embargo, a diferencia de otras técnicas del campo de la biología estructural, brinda información sobre procesos dinámicos, lo cual representa una de sus características más sobresalientes y en consecuencia, su versatilidad determina que sea ampliamente utilizada. Respecto a los alcances y aplicaciones, podemos mencionar desde la elucidación de compuestos pequeños en la química orgánica, la determinación de estructuras de moléculas biológicas y sus interacciones, y el estudio cinético de reacciones; hasta el diseño racional de drogas y el diagnóstico por imágenes en medicina. En este capítulo nos enfocaremos en el uso de RMN para el análisis de estructuras de proteínas en solución. Dada la gran complejidad subyacente a esta técnica, nos ajustaremos a brindar nociones básicas que permitan al estudiante comprender cómo se arriba a un conjunto de estructuras tridimensionales, a partir de información experimental. Desarrollaremos las explicaciones de una manera descriptiva, lo más despojada de formalismos posible, con la intención de acercar al lector inexperto al creciente número de publicaciones científicas que emplean RMN, facilitando su abordaje. Para un estudio riguroso, recomendamos la lectura de los trabajos citados en la sección de Bibliografía. Adicionalmente, discutiremos en forma breve algunas aplicaciones como el análisis de interacción entre biomoléculas y el estudio de proteínas en células (in cell NMR).Facultad de Ciencias Exacta

Análise estrutural e funcional de proteínas solúveis que ligam lipídios de parasitas helmintos

Los parásitos helmintos producen y secretan una gran variedad de proteínas que unen lípidos (LBPs, del inglés lipid binding proteins) que podrían participar en la obtención de nutrientes tales como ácidos grasos y colesterol desde el hospedador. Asimismo, se postula que las LBPs podrían intervenir en la regulación de la respuesta inmune del hospedador. Conocer más acerca de las estructuras de estas proteínas, así como de sus interacciones con ligandos y membranas, es claramente pertinente para comprender las interacciones parásito-hospedador que ellas pudieran mediar. Por otra parte, dichos estudios permitirán profundizar en el conocimiento de los mecanismos de infección helmíntica y en el papel que estas proteínas juegan en la biología de los helmintos en general. Asimismo, esta información podría contribuir al establecimiento de medidas terapéuticas y de prevención de las enfermedades causadas por estos parásitos.Helminth parasites produce and secrete a great variety of lipid binding proteins (LBPs) that may participate in the acquisition of nutrients such as fatty acids and cholesterol from their host. It is also postulated that LBPs might interfere in the regulation of the host's immune response. Knowing more about the structure of these proteins as well as their interactions with ligands and membranes is important in order to understand the host-parasite interaction that they could mediate. On the other hand, these studies will contribute to obtain further knowledge about the mechanisms of helminth infection and the role that these proteins play in helminth biology. Moreover, this information would be useful to set new therapeutic and prevention measures for the diseases caused by these parasites.Os parasitas helmintos produzem e secretam uma grande variedade de proteínas que ligam lipídios, LBPs (Lipid Binding Proteins, por sua sigla em inglês), que poderiam estar envolvidas na obtenção de nutrientes tais como ácidos graxos e colesterol a partir do hospedeiro. Do mesmo modo, é postulado que as LBPs poderiam intervir na regulação da resposta imune do hospedeiro. Saber mais sobre as estruturas dessas proteínas, bem como sobre as suas interações com ligantes e membranas é claramente pertinente para compreender as interações parasita-hospedeiro que elas pudessem mediar. Além disso, estes estudos irão permitir um melhor entendimento dos mecanismos de infecção helmíntica e o papel que estas proteínas desempenham na biologia de helmintos em geral. Também, essa informação poderia ajudar a estabelecer medidas terapêuticas e de prevenção das doenças provocadas por esses parasitas.Fil: Franchini, Gisela Raquel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Corsico, Betina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Pórfido, Jorge Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Silva, Valeria.Fil: Ibañez Shimabukuro, Marina.Fil: Rey Burusco, Florencia

1H, 13C and 15N chemical shift assignments of Na-FAR-1, a helix-rich fatty acid and retinol binding protein of the parasitic nematode Necator americanus

The fatty acid and retinol-binding (FAR) proteins are a family of unusual helix-rich lipid binding proteins found exclusively in nematodes, and are secreted by a range of parasites of humans, animals and plants. Na-FAR-1 is from the parasitic nematode Necator americanus, an intestinal blood-feeding parasite of humans. Sequence-specific 1H, 13C and 15N resonance assignments have been obtained for the recombinant 170 amino acid protein, using three-dimensional triple-resonance heteronuclear magnetic resonance experiments. Backbone assignments have been obtained for 99.3 % of the non-proline HN/N pairs (146 out of 147). The amide resonance of T45 was not observed, probably due to rapid exchange with solvent water. A total of 96.9 % of backbone resonances were identified, while 97.7 % assignment of amino acid sidechain protons is complete. All Hα(166), Hβ(250) and Hγ(160) and 98.4 % of the Hδ (126 out of 128) atoms were assigned. In addition, 99.4 % Cα (154 out of 155) and 99.3 % Cβ (143 out of 144) resonances have been assigned. No resonances were observed for the NHn groups of R93 NεHε, arginine, Nη1H2, Nη2H2, histidine Nδ1Hδ1, Nε1Hε1 and lysine Nζ3H3. Na-FAR-1 has a similar overall arrangement of α-helices to Ce-FAR-7 of the free-living Caeorhabditis elegans, but with an extra C-terminal helix

1H, 13C and 15N chemical shift assignments of Na-FAR-1, a helix-rich fatty acid and retinol binding protein of the parasitic nematode Necator americanus

The fatty acid and retinol-binding (FAR) proteins are a family of unusual helix-rich lipid binding proteins found exclusively in nematodes, and are secreted by a range of parasites of humans, animals and plants. Na-FAR-1 is from the parasitic nematode Necator americanus, an intestinal blood-feeding parasite of humans. Sequence-specific 1H, 13C and 15N resonance assignments have been obtained for the recombinant 170 amino acid protein, using three-dimensional triple-resonance heteronuclear magnetic resonance experiments. Backbone assignments have been obtained for 99.3 % of the non-proline HN/N pairs (146 out of 147). The amide resonance of T45 was not observed, probably due to rapid exchange with solvent water. A total of 96.9 % of backbone resonances were identified, while 97.7 % assignment of amino acid sidechain protons is complete. All Hα(166), Hβ(250) and Hγ(160) and 98.4 % of the Hδ (126 out of 128) atoms were assigned. In addition, 99.4 % Cα (154 out of 155) and 99.3 % Cβ (143 out of 144) resonances have been assigned. No resonances were observed for the NHn groups of R93 NεHε, arginine, Nη1H2, Nη2H2, histidine Nδ1Hδ1, Nε1Hε1 and lysine Nζ3H3. Na-FAR-1 has a similar overall arrangement of α-helices to Ce-FAR-7 of the free-living Caeorhabditis elegans, but with an extra C-terminal helix.Facultad de Ciencias MédicasInstituto de Investigaciones Bioquímicas de La Plat

Diversity in the structures and ligand binding sites of nematode fatty acid and retinol binding proteins revealed by Na-FAR-1 from Necator americanus

Fatty acid and retinol binding proteins (FARs) comprise a family of unusual α-helix rich lipid binding proteins found exclusively in nematodes. They are secreted into host tissues by parasites of plants, animals and humans. The structure of a FAR protein from the free-living nematode Caenorhabditis elegans is available, but this protein (Ce-FAR-7) is from a subfamily of FARs that does not appear to be important at the host-parasite interface. We have therefore examined Na-FAR-1 from the blood-feeding intestinal parasite of humans, Necator americanus . The three dimensional structure of Na-FAR-1 in its ligand-free and ligand-bound forms, determined by nuclear magnetic resonance spectroscopy (NMR) and X-ray crystallography, respectively, reveals an a-helical fold similar to Ce-FAR-7, but Na-FAR-1 possesses a larger and more complex internal ligand binding cavity and an additional C-terminal a-helix. Titration of apo -Na-FAR-1 with oleic acid, analysed by NMR chemical shift perturbation, reveals that at least four distinct protein:ligand complexes can be formed. Na-FAR-1, and possibly other FARs, may have a wider repertoire for hydrophobic ligand binding, as confirmed here by our finding that a range of neutral and polar lipids co-purify with the bacterial recombinant protein. Finally, we show by immunohistochemistry that Na-FAR-1 is present in adult worms with a tissue distribution indicative of possible roles in nutrient acquisition by the parasite and in reproduction in the male

1H, 13C and 15N chemical shift assignments of Na-FAR-1, a helix-rich fatty acid and retinol binding protein of the parasitic nematode Necator americanus

The fatty acid and retinol-binding (FAR) proteins are a family of unusual helix-rich lipid binding proteins found exclusively in nematodes, and are secreted by a range of parasites of humans, animals and plants. Na-FAR-1 is from the parasitic nematode Necator americanus, an intestinal blood-feeding parasite of humans. Sequence-specific 1H, 13C and 15N resonance assignments have been obtained for the recombinant 170 amino acid protein, using three-dimensional triple-resonance heteronuclear magnetic resonance experiments. Backbone assignments have been obtained for 99.3 % of the non-proline HN/N pairs (146 out of 147). The amide resonance of T45 was not observed, probably due to rapid exchange with solvent water. A total of 96.9 % of backbone resonances were identified, while 97.7 % assignment of amino acid sidechain protons is complete. All Hα(166), Hβ(250) and Hγ(160) and 98.4 % of the Hδ (126 out of 128) atoms were assigned. In addition, 99.4 % Cα (154 out of 155) and 99.3 % Cβ (143 out of 144) resonances have been assigned. No resonances were observed for the NHn groups of R93 NεHε, arginine, Nη1H2, Nη2H2, histidine Nδ1Hδ1, Nε1Hε1 and lysine Nζ3H3. Na-FAR-1 has a similar overall arrangement of α-helices to Ce-FAR-7 of the free-living Caeorhabditis elegans, but with an extra C-terminal helix.Facultad de Ciencias MédicasInstituto de Investigaciones Bioquímicas de La Plat

Two crystal forms of a helix-rich fatty acid- and retinol-binding protein, Na-FAR-1, from the parasitic nematode Necator americanus

Na-FAR-1 is an unusual -helix-rich fatty acid- and retinol-binding protein from Necator americanus, a blood-feeding intestinal parasitic nematode of humans. It belongs to the FAR protein family, which is unique to nematodes; no structural information is available to date for FAR proteins from parasites. Crystals were obtained with two different morphologies that corresponded to different space groups. Crystal form 1 exhibited space group P432 (unit-cell parameters a = b = c = 120.80 Å, α = β = γ = 90°) and diffracted to 2.5 Å resolution, whereas crystal form 2 exhibited space group F23 (unit-cell parameters a = b = c = 240.38 Å, α = β = γ = 90°) and diffracted to 3.2 Å resolution. Crystal form 2 showed signs of significant twinning.Instituto de Investigaciones Bioquímicas de La Plat

1H, 13C and 15N chemical shift assignments of Na-FAR-1, a helix-rich fatty acid and retinol binding protein of the parasitic nematode Necator americanus

The fatty acid and retinol-binding (FAR) proteins are a family of unusual helix-rich lipid binding proteins found exclusively in nematodes, and are secreted by a range of parasites of humans, animals and plants. Na-FAR-1 is from the parasitic nematode Necator americanus, an intestinal blood-feeding parasite of humans. Sequence-specific 1H, 13C and 15N resonance assignments have been obtained for the recombinant 170 amino acid protein, using three-dimensional triple-resonance heteronuclear magnetic resonance experiments. Backbone assignments have been obtained for 99.3 % of the non-proline HN/N pairs (146 out of 147). The amide resonance of T45 was not observed, probably due to rapid exchange with solvent water. A total of 96.9 % of backbone resonances were identified, while 97.7 % assignment of amino acid sidechain protons is complete. All Hα(166), Hβ(250) and Hγ(160) and 98.4 % of the Hδ (126 out of 128) atoms were assigned. In addition, 99.4 % Cα (154 out of 155) and 99.3 % Cβ (143 out of 144) resonances have been assigned. No resonances were observed for the NHn groups of R93 NεHε, arginine, Nη1H2, Nη2H2, histidine Nδ1Hδ1, Nε1Hε1 and lysine Nζ3H3. Na-FAR-1 has a similar overall arrangement of α-helices to Ce-FAR-7 of the free-living Caeorhabditis elegans, but with an extra C-terminal helix.Facultad de Ciencias MédicasInstituto de Investigaciones Bioquímicas de La Plat