Nuclear Translocation of proteins via importin pathway in protozoan pathogen Trypanosoma cruzi

MARÍA LAURA KEVORKIAN; EZEOGO OBAJI; SALOMÉ VILCHEZ LARREA; LARI LEHTIÖ; FERNÁNDEZ VILLAMIL, SILVIA H.

Buenos Aires

Congreso; Reunión Conjunta de Socidades de Biociencias; 2017

SAIC, SAIB, SAI, SAB, SAA, SAFE, SAFIS, SAH, SAP

In higher eukaryotes, active transport of proteins to the nucleus occurs through the nuclear pore complexes (NPCs), directed by nuclearlocalization signal (NLS) sequences. The NLS is recognizedby importin α in the cytoplasm that then binds to importin β whichtransports this trimeric complex across the NPC. The importin αconsists of two functional domains, a short basic N-terminal domain(the IBB domain) sufficient for importin β binding, which also actsas an autoinhibitory domain, and a NLS-binding domain comprisedof armadillo (arm) repeats. There is limited amount of informationabout nuclear protein targeting in lower eukaryotes such as trypanosomatids.Therefore, we aimed to study the importin pathway inTrypanosoma cruzi (T. cruzi), causative agent of Chagas Disease,using the N-terminal domain of the poly(ADP-ribose)polymerasefrom T. cruzi (TcPARP-NT) as a tool, which is known to be sufficientfor nuclear translocation. Through a bioinformatic approach wefound a sequence encoding for a putative importin α (TcIMPα) inthis parasite. This gene was cloned and expressed as a full lengthprotein (TcIMPα FL) or lacking the IBB domain (TcIMPα ΔIBB). A pulldown assay using His-tag capture confirmed binding of TcPARP-NTwith TcIMPα ΔIBB. In a Size Exclusion Chromatography this proteincomplex did not elute together, but by using centrifugal filters, witha cut off higher than the TcPARP-NT molecular weight, binding wasobserved. In addition, interaction of TcIMPα with TcPARP was confirmedby Bio-Layer Interferometry that enables real-time, label-freeanalysis for determination of affinity, which resulted in μM dissociationconstant. It was established that TcIMPα FL and TcPARP-NT donot bind reinforcing the idea of importin α autoinhibition, also presentin the parasite. Taken together, these results give us a first insightinto the presence of an operative importin system in T. cruzi whichcould play an active role in protein translocation.