INVESTIGADORES
LOOS Julia Alexandra
congresos y reuniones científicas
Título:
Fuctional characterization and expression of Echinococcus granulosus Calcineurin.
Autor/es:
M. CELESTE NICOLAO; JULIA A. LOOS; ANDREA C. CUMINO
Lugar:
Guayaquil
Reunión:
Congreso; XXI Congreso Latinoamericano de Parasitología (FLAP); 2013
Resumen:
Introduction. Calcineurin is a Ca2+/calmodulin-activated serine/threonine-phosphatase (PP2B) with
critical role in the coupling of calcium signals to cellular responses. It is a heterodimeric protein
consisting of a catalytic (CaN-A) and a regulatory calcium-binding subunit (CaN-B), and is
inhibited by cyclophilin-cyclosporin A and FKBP-FK506 complexes. Its inactivation blocks
different cellular processes resulting in cell death. In this study we focused on the functional
characterization of the catalytic subunit of calcineurin in the Echinococcus granulosus larval stages.
Materials and methods. E. granulosus can-A, can-B and chp genes were identified in
protoscoleces and cysts by RT-PCR and sequencing (GenBank KC146906, HQ454284 and
KC146907, respectively). Bioinformatic analysis revealed that their predicted proteins possess all
residues implicated in the binding of pharmacological ligands. Also, we performed in toto
immunofluorescence assays in protoscoleces to determine CaN-A localization and its
association in exocytic processes using the FM4-64 fluorescent dye. We determined the CaN-A
PP2B activity in basal conditions, with activators (Cl2Ni) and inhibitors (CsA) from protoscolex and
metacestode protein extracts in presence of imidazol and phosphatases inhibitors.
Results. We demonstrated in protoscoleces that in low calcium conditions (BAPTA/ClLa) Eg-can-
B and Eg-chp expressions were down-regulated respect to controls. Eg-CaN-A represents an active
protein phosphatase, since PP2B was the 60% of the total serine/threonine phosphatase activity. Eg-
CaN-A was mainly immunolocalized in tegument, suckers and excretory bladder. Eg-CaNA
expression was higher in CsA-treated protoscoleces and lower in BAPTA/ClLa-treated
protoscoleces compared with controls. Finally, we examined the unloading kinetics of FM4-64 after
stimulation with KCl and HCl, demonstrating that the exocytosis was significantly reduced in CsAtreated
larvae, in agreement with other invertebrate species.
Conclusion. We have carried out a functional analysis of the E. granulosus calcineurin which
coordinate multiple calcium-dependent dephosphorylation processes in metazoa. These findings
contribute to elucidate conserved elements of signal transduction cascades and define novel drug
targets.