CONICET | Buscador de Institutos y Recursos Humanos

ADRIANA V. JÄGER, JAVIER G. DE GAUDENZI, BÁRBARA MC CORMACK, JESICA G. MILD, DANIEL MUSIKANT, SERGIO PANTANO, DANIEL L. ALTSCHULER Y MARTIN M. EDREIRA

Lugar:

Woods Hole, MA.

Reunión:

Congreso; Molecular Parasitology Meeting XXIV; 2013

Resumen:

cAMP has been implicated as secondary messenger in a wide range of cellular processes. In eukaryotic cells, cAMP effectors include PKA and Epac. Although several studies suggested a role of cAMP in the development of T. cruzi life cycle, little is known about cAMP mechanism of action in the parasite. Early studies demonstrated a negative role for cAMP in cell proliferation. To this point, a PKA version of T. cruzi (TcPKA) has been cloned and characterized. However, inhibition of TcPKA instead of the expected rescue, led to epimastigote death. This result strongly suggests the existence of a PKA-independent pathway, even though no genomic sequence for Epac has been found. In order to identify new cAMP effectors, we carried out in silico studies using the predicted T. cruzi proteome. A combination of search methods let us identify 27 proteins with putative cAMP binding domains (CBDs). Phylogenetic analysismade from coding sequence alignments showed that these proteins are segregated into two main branches: one containing protein kinases and the other gathering hypothetical proteinswith no assigned function. On the other hand, phylogenetic analysis of theCBDs presented a homogeneousdistribution, consistent with the conservation of a nucleotide-binding domain. To gain structural insights on the topology of T. cruzi?s CBDs we generated comparative models of the two strongest hits using PKA-RIα, the closest mammalian homologue with known structure bound to cAMP, as template. Pull-down and displacement assays using a cAMP-Agarose resin and free cAMP, allowed us to experimentally validate these candidates as bona fide cAMP-binding proteins. Therefore, we were able to identify new putative effectors of the PKA-independent pathway in T. cruzi biology.