CONICET | Buscador de Institutos y Recursos Humanos

Data from genomeannotation and mass spectrometry projects of pathogens has allowed theidentification of novel proteins, for which their putative functions cannot be predictedby sequence similarity. Trypanosoma cruzi,the etiological agent of Chagas disease, has a complex life cycle in which celldifferentiation and invasion are tightly coordinated. In this parasite, calciumand calcium binding proteins play important roles in the processes of mammaliancell infection and survival. Using a rational search engine, we selected an uncharacterized gene, named TcCALIwhich encodes a 103 amino acid protein with two putative calcium binding sites.Previously, we found that this protein is able to form disulphide bonds and hasa putative palmitoylation domain.We also found TcCALI localizes alongthe cytoplasm in all differential stages of the parasite and several proteinbands at higher molecular weight than expected are detected by western blot. Inorder to study TcCALI´s role on theparasite physiology, we searched for its associated components, to characterizethe metabolic environment of this protein with unknown function. To this aim,two experimental approaches were addressed. First, protein extracts from T. cruzi were immunoprecipitated usinganti-TcCALI antibodies and commercialkits consisting of recombinant Protein G covalently coupled to magnetic beads. Theresults showed that TcCALI associatesto one or more components, which probably modify the TcCALI conformation. On the other hand, yeast two hybrid assays areundergoing with the objective to identify TcCALIbinding partners from a cDNA library. For this, TcCALI and Lamin (negative control) were cloned separately in theshuttle vector pBMT116 and transfected to S.cereviseae (L40 strain). Selection of recombinant yeast was achievedplating transformants in minimal media. Future steps involve transfection of TcCALI expressing yeasts with a T. cruzi cDNA library cloned in thepVP16 vector.