Estudio de los patrones de ubiquitinación y la actividad de proteólisis específica del proteosoma en Giardia Intestinalis / Study of the ubiquitination patterns and the specific activity of the proteasome in Giardia Intestinalis

El parasito protozoario Giardia intestinalis es un agente causante de diarrea en humanos alrededor del mundo. Ademas de su importancia como patogeno es un eucariota que divergio tempranamente y constituye un importante modelo de estudio de procesos celulares basicos. Durante su ciclo de vida, G. intestinalis, presenta dos procesos de diferenciacion, la enquistacion y la exquistacion, los cuales involucran modificaciones intracelulares, transcripcion de nuevos genes, sintesis y probablemente degradacion especifica de proteinas. En nuestro laboratorio se ha demostrado la existencia y expresion de genes esenciales dentro del proceso de ubiquitinacion de proteinas en G. intestinalis. En el presente trabajo se realizo la purificacion de ubiquitina a partir de trofozoitos de G. intestinalis, se produjo un anticuerpo antiubiquitina especifico, se estandarizaron las condiciones para la deteccion y evaluacion de los patrones de ubiquitinacion y se identifico una proteina ubiquitinada. Por otro lado, se realizo la purificacion y caracterizacion parcial del 20S proteosoma de Giardia y se evidencio una acumulacion de conjugados proteicos de alto peso molecular en respuesta a la inhibicion del proteosoma. Durante el proceso de enquistacion, la actividad del proteosoma se incremento. Ademas, se encontro una actividad peptidasa similar al proteosoma en extractos crudos de proteina de Giardia intestinalis. Estos resultados demuestran la presencia de un sistema ubiquitina-proteosoma activo durante el estadio de trofozoito y durante el proceso de enquistacion en G. intestinalis. Ademas, nuestras observaciones sugieren la presencia otra actividad similar al proteosoma que podria estar involucrada en la via de proteolisis en este eucariota basal. / Abstract. The parasitic protozoan Giardia intestinalis is one of the major causes of diarrhoea in humans worldwide. Furthermore, G. intestinalis belongs to the earliest diverging eukaryotic lineage known and provides an important model for studying basic insights into key pathways. G. intestinalis undergo two differentiation events, encystation and excystation, which involves intracellular rearrangement, new gene transcription, protein synthesis and likely specific protein degradation. In our laboratory previous studies demonstrated the presence and expression of components of the ubiquitination system in G. intestinalis. In this work was isolated ubiquitin from trophozoites and was produced a specific antibody against this protein, using this antibody was standardized a method to evaluate the ubiquitination patterns and was possible to identify a protein ubiquitinated. On the other hand, was purified and characterized partially the 20S proteasome from Giardia and was evidenced a constitutive turnover of cellular proteins via the ubiquitin pathway after treatment with proteasome inhibitors. During the process of encystation, proteasome activity increased. In addition, was found a roteasome-like peptidase activity in G. intestinalis crude extracts which was about 10S and its activity was importantly increased during the encystation process. These results demonstrate the presence of an active ubiquitin-proteasome system during the trophozoite stage and during the encystation process of G. intestinalis with an interesting behaviour during this differentiation process and with the presence of interesting effects after proteasome specific inhibition in vivo. Furthermore, our observations suggest the presence of another proteasome-like activity that could be involved in the proteolytic pathway in this basal eukaryote.