Evaluación de un candidato a transportador de NAD+ en el parásito protozoario Trypanosoma cruzi

dc.contributor.advisorRamírez Hernández, Maria Helena
dc.contributor.authorChacón Gómez, Miguel Esteban
dc.contributor.researchgroupLibbiq Unspa
dc.date.accessioned2022-07-25T12:33:44Z
dc.date.available2022-07-25T12:33:44Z
dc.date.issued2021
dc.descriptionilustraciones, fotografías, graficasspa
dc.description.abstractTrypanosoma cruzi causa la enfermedad de Chagas, patología distribuida globalmente y carente de tratamientos efectivos, lo que hace pertinente la búsqueda de estrategias alternas de control. La dinámica del dinucleótido de nicotinamida y adenina (NAD+) es determinante en la homeostasis celular, y, las proteínas que participan en ella son promisorios blancos farmacológicos. La síntesis del NAD+ es citosólica en T. cruzi, por lo que un sistema intracelular de distribución debe existir; proteínas de la Familia de Transportadores Mitocondriales (MCF) cumplen esta función en eucariotas, y se espera que el parásito cuente con homólogos capaces de movilizar al dinucleótido. Tres secuencias candidato a transportador de NAD+ de T. cruzi fueron caracterizadas; el estudio in silico de las proteínas TcNdt1 y TcNdt2 mostró que estructuralmente conservan características de la MCF, y por docking molecular se determinó que están dotadas de elementos capaces de interactuar específicamente con el dinucleótido. Por su parte, el candidato TcNdt3 es una proteína atípica de la MCF con una duplicación de sus elementos estructurales. En los 3 candidatos se predice una localización mitocondrial o glicosomal, al igual que la presencia sitios blanco de modificaciones postraduccionales. Ensayos de complementación realizados con las cepas Δndt1 y Δndt2 de Saccharomyces cerevisiae mostraron que TcNdt1 y TcNdt2 reestablecen el crecimiento rezagado de los mutantes en medio no fermentable, comprobando su actividad transportadora de NAD+. De forma complementaria, se desarrolló el sistema de expresión MISTIC que media la inserción de proteínas en membranas de E. coli, y se adelantaron ensayos piloto de transporte del dinucleótido. Empleando un antígeno recombinante generado en E. coli se produjeron IgY que permitieron la detección por inmunofluorescencia de la TcNdt2 endógena sobre epimastigotes de T. cruzi, indicando que presenta localización posiblemente glicosomal asociada a tráfico vesicular. Adicionalmente, se evaluó la expresión de un fragmento de la RVG del virus de la rabia en S. cerevisiae, donde fue posible obtener un patrón de reconocimiento diferencial en la inmunodetección, el cual es atribuible a la expresión de la recombinante en el modelo eucariota. Los resultados obtenidos en este estudio constituyen un aporte importante para entender la simplificación en el metabolismo del NAD+ en parásitos intracelulares, y sus relaciones con el hospedero. (Texto tomado de la fuente)spa
dc.description.abstractTrypanosoma cruzi is the etiological agent of Chagas disease, a disease that has a worldwide distribution and lacks effective treatments, making it pertinent to look for alternate control strategies. The nicotinamide-adenine nucleotide (NAD+) dynamics determines cell homeostasis, and the proteins involved in it are interesting pharmacological targets. NAD+ synthesis is cytosolic in T. cruzi, and therefore, an intracellular distribution system should exist; proteins belonging to the Mitochondrial Carrier Family (MCF) fulfill this role in eukaryotes, ant it is expected that the parasite has MCF homologues able to transport the dinucleotide. Three NAD+ carrier candidate sequences from T. cruzi were studied; in silico analysis of the TcNdt1 and TcNdt2 protein showed that typical structural features from the MCF are conserved in these proteins, and, through molecular docking, it was found that they are both endowed with structural elements able to interact specifically with the dinucleotide. On the other hand, the TcNdt3 candidate is an atypical MCF protein that shows a complete duplication of its structural elements. The 3 candidate sequences are predicted to have a mitochondrial or glycosomal localization, and throughout the TcNdt1, TcNdt2 and TcNdt3 sequences post-translational modification sites are predicted. Complementation assays carried out with the Saccharomyces cerevisiae mutant strains Δndt1 and Δndt2, showed that the TcNdt1 and TcNdt2 sequences reestablish yeast cell growth on a non-fermentable media, supporting that the T. cruzi proteins are functional NAD+ carriers. As a complementary approach, the MISTIC expression system, that mediates recombinant protein insertion in E. coli membranes, was developed, and pilot dinucleotide transport assays were performed. Using a recombinant antigen produced in E. coli, specific IgY against TcNdt2 were raised, which allowed for the endogenous carrier recognition through immunofluorescence on T. cruzi epimastigotes, showing that the NAD+ carrier has a glycosomal localization, linked to the vesicular transport. Further, the expression of a recombinant fragment derived from the rabies virus RVG protein was evaluated in S. cerevisiae, and at the immunodetection a differential recognition pattern was obtained, which can be attributed to the recombinant protein production in the eukaryotic system. The results obtained in this study make for an important contribution for the understanding of the NAD+ metabolism simplification that occurred in the intracellular parasites, and for the understanding of the host-parasite interactions.eng
dc.description.degreelevelMaestríaspa
dc.description.degreenameMagíster en Ciencias - Bioquímicaspa
dc.description.researchareaBioquímica y Biología Molecular de Parásitosspa
dc.format.extentxix, 112 páginasspa
dc.format.mimetypeapplication/pdfspa
dc.identifier.instnameUniversidad Nacional de Colombiaspa
dc.identifier.reponameRepositorio Institucional Universidad Nacional de Colombiaspa
dc.identifier.repourlhttps://repositorio.unal.edu.co/spa
dc.identifier.urihttps://repositorio.unal.edu.co/handle/unal/81733
dc.language.isospaspa
dc.publisherUniversidad Nacional de Colombiaspa
dc.publisher.branchUniversidad Nacional de Colombia - Sede Bogotáspa
dc.publisher.departmentDepartamento de Químicaspa
dc.publisher.facultyFacultad de Cienciasspa
dc.publisher.placeBogotá, Colombiaspa
dc.publisher.programBogotá - Ciencias - Maestría en Ciencias - Bioquímicaspa
dc.relation.indexedRedColspa
dc.relation.indexedLaReferenciaspa
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dc.rights.accessrightsinfo:eu-repo/semantics/openAccessspa
dc.rights.licenseAtribución-NoComercial-CompartirIgual 4.0 Internacionalspa
dc.rights.urihttp://creativecommons.org/licenses/by-nc-sa/4.0/spa
dc.subject.ddc570 - Biología::572 - Bioquímicaspa
dc.subject.otherParásitosspa
dc.subject.otherParasiteseng
dc.subject.proposalProteínas recombinantesspa
dc.subject.proposalAnticuerposspa
dc.subject.proposalBioinformáticaspa
dc.subject.proposalRecombinant proteineng
dc.subject.proposalBioinformaticseng
dc.subject.proposalAntibodieseng
dc.subject.proposalMembranaspa
dc.subject.proposalMembraneeng
dc.subject.proposalIntracelularspa
dc.subject.proposalIntracellulareng
dc.subject.proposalEnsayos de complementaciónspa
dc.subject.proposalComplementation assayseng
dc.subject.unescoTripanosomiasiseng
dc.subject.unescoBiotecnologíaspa
dc.subject.unescoBiotechnologyeng
dc.titleEvaluación de un candidato a transportador de NAD+ en el parásito protozoario Trypanosoma cruzi
dc.title.translatedEvaluation of a NAD+ carrier candidate in the protozoan parasite Trypanosoma cruzispa
dc.typeTrabajo de grado - Maestríaspa
dc.type.coarhttp://purl.org/coar/resource_type/c_bdccspa
dc.type.coarversionhttp://purl.org/coar/version/c_ab4af688f83e57aaspa
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dc.type.driverinfo:eu-repo/semantics/masterThesisspa
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dc.type.versioninfo:eu-repo/semantics/acceptedVersionspa
dcterms.audience.professionaldevelopmentEstudiantesspa
dcterms.audience.professionaldevelopmentInvestigadoresspa
dcterms.audience.professionaldevelopmentMaestrosspa
oaire.accessrightshttp://purl.org/coar/access_right/c_abf2spa

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