Identification of calcineurin Echinococcus sp: Targets of Eg-FKBP-FK506 complex

NICOLAO MA. CELESTE; CUMINO ANDREA C.

Mar del Plata

Congreso; LVI Reunión Científica Anual de la Sociedad Argentina de Investigaciones Clínicas; 2011

Sociedad Argentina de Investigaciones Clínicas

Calcineurin (CaN) is a eukaryotic Ca- and calmodulin-dependent serine/threonine protein phosphatase (PP2B), potently inhibited by immunosuppressant drugs, cyclosporin A (CsA) and FK506, in the presence of their respective cytoplasmic immunophilin proteins, cyclophilin and FKBP, both described in Echinococcus sp. (Colebrook et al 2004; Cumino et. al., 2010). Previous experiments in our laboratory have showed that FK506 and CsA possess dose-dependent protoscolicidal activity in E. granulosus. CaN is a heterodimeric protein consisting of a catalytic subunit A, which contains an active site dinuclear metal center and a tightly associated, regulatory Ca-binding subunit B. We identified and analyzed in silico ortologous in Echinococcus genome: Eg-CaN-A and Eg-CaN-B (as well as their corresponding orthologous to E. multilocularis), both archetypal proteins with all residues implicated in the binding of pharmacological ligands, and in interactions with possible targets. The primary sequence of both subunits and heterodimeric quaternary structure is highly conserved in Echinococcus sp. Eg-CaN-A contains a CaM (calmodulin)-binding domain (PD343921), a CaN-B-binding domain (PD862669) and an autoinhibitory domain (PD322644). The CaN-B subunit itself is a CaM-like Ca+2-binding protein that contains four high-affinity Ca+2-binding EF hands. Binding of Ca+2 to CaN-B stimulates phosphatase activity, although to a lesser extent than when CaM is also present. Finally, CnB is also required for the inhibition of calcineurin by cyclophilin-cyclosporin A (Cyp-CsA) complex. Although the mechanisms of the estrogen receptor-independent effects of tamoxifen (TAM) remain a subject of debate, but it is clear from that TAM has a wide range of effects cell physiology implicated in regulates the transcriptional response to calcium. In E. granulosus, protoscolicidal treatment with TAM, showed induction of intracellular calcium release, the alteration of cellular membrane properties and the inhibition of P-glycoprotein (Nicolao et al., 2010). Transcriptional profiling studies showed that TAM reduces the Eg-canB expression, consistently with these observations, in other cellular systems, TAM could involve in the estrogen-dependent regulation of CaN. These findings highlight the potential of identification of downstream target proteins, a promising target for chemotherapy of cystic echinococcosis to define novel drug targets and elucidate conserved elements of signal transduction cascades.