INGEBI   02650
INSTITUTO DE INVESTIGACIONES EN INGENIERIA GENETICA Y BIOLOGIA MOLECULAR "DR. HECTOR N TORRES"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Searching for new cAMP effectors in Trypanosoma cruzi
Autor/es:
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:
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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.