Study of a novel putative Calcium binding protein from Trypanosoma cruzi

MARIANA POTENZA,; DI RENZO AGOSTINA; TELLEZ IÑÒN M.TERESA

Rosario

Congreso; XXVI Reuniòn Anual de la Sociedad Argentina de Protozoologia; 2013

Sociedad Argentina de Protozoologia

CO12- Study of a novel putative Calcium binding protein from Trypanosoma cruzi Understanding the role of calcium-related genes in Trypanosoma cruzi biology, the causative of Chagas disease, could broaden the scope in research of new drug targets against this parasite. Evidences have shown that Ca2+ may play an important role in T. cruzi differentiation and mammalian infection; processes that assure parasite survival and life cycle continuity. In order to define new Ca2+-mediated signal transduction pathways specific for trypanosomatids, we start to study some uncharacterized gene products that present putative calcium binding domains. We perform a search at the TritrypDB web site, a database containing the annotation and analysis of some pathogenic trypanosomatid genome projects. As a search engine, we use the terms ?hypothetical protein?, ?calcium ion binding?, ?proteome evidence? and ?phosphorylation proteome evidence?. As a result, four proteins were found and one of them was selected for further study. This 103 amino acid sequence, named TcCalI, presents several EF-hand motifs and one palmitoylation site as predicted by bioinformatic tools. We cloned and expressed TcCalI in bacteria and then raised antibodies against the purified recombinant protein. Homo-dimerization of TcCalI via inter-molecular disulphide bond formation is suggested in polyacrylamide gel electrophoresis followed by the addition of dithiothreitol. By western blot, we found that TcCalI is expressed in all the three stages of T. cruzi, at the predicted molecular weight and as well as two higher bands. We determined the sub-cellular localization of TcCalI all around the cytoplasm and along the flagellum in epimastigotes and trypomastigotes forms. To better define the nature of the TcCalI, Ca2+ binding assays, immuneprecipitation experiments and yeast two-hybrid approaches are under way, in order to identify those possible counterparts that shed light about the function of this interesting protein.