Importin pathway could play an active role in protein translocation in protozoan pathogen Trypanosoma cruzi

LARI LEHTIÖ; MARIA LAURA KEVORKIAN; SILVIA FERNANDEZ VILLAMIL; EZEOGO OBAJI; SALOMÉ C. VILCHEZ LARREA

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

SAIC-SAIB-SAA-SABiol-SAP-SAI-SAFE-SAFIS-SABiofisica-SAH

In higher eukaryotes, active transport of proteins to the nucleus occurs through the nuclear pore complexes (NPCs), directed by nuclear localization signals (NLS). These are recognized by importin α in the cytoplasm which binds to importin β, responsible of facilitating translocation of this trimeric complex across the NPC. Importin α consists of two functional domains, a short basic N-terminal domain (the IBB domain) sufficient for importin β binding, which also acts as an autoinhibitor, and a NLS-binding domain comprised of armadillo (arm) repeats.In lower eukaryotes such as trypanosomatids, there is scarce information about nuclear protein targeting. Therefore, the aim of our work is the study of the importin pathway in Trypanosoma cruzi (T. cruzi), causative agent of Chagas Disease, using the N-terminal domain of the poly(ADP-ribose)polymerase from T. cruzi (TcPARP-NT) as a tool, which is known to be sufficient for nuclear translocation. We found a sequence in this parasite that encodes for a putative importin α (TcIMPα). This gene was cloned and expressed as a full length protein (TcIMPα FL) or lacking the IBB domain (TcIMPα ΔIBB). In order to verify interaction, a pull down assay using His-tag capture was made and binding of TcPARP-NT with TcIMPα ΔIBB was observed. Though in a Size Exclusion Chromatography this protein complex did not elute together, by the use of centrifugal filters, with a cut off higher than the TcPARP-NT molecular weight, binding was noticed Interaction was confirmed by Bio-Layer Interferometry that enables real-time, label-free analysis for determination of affinity which resulted in μM order. With a pull-down assay, it was observed that TcIMPα FL and TcPARP-NT do not bind, which reinforces the idea of importin α autoinhibition. Taken together, these results give us a first insight into the presence of an operative importin system in T. cruzi.