Molecular characterization of a specific L-proline permease from Trypanosoma cruzi

SILBER, ARIEL; SAYE, MELISA; MIRANDA, MARIANA; CAMARA, MARIA DE LOS MILAGROS; PEREIRA, CA

Caxambú

Congreso; XXVIII Reunión Anual de la Sociedad Brasilera de Protozoología; 2012

SBPz

In trypanosomatids, proline is a key amino acid since constitutes a main carbon and energysource. Recently, new roles in the parasite biology have been assigned to this metabolite. InTrypanosoma cruzi, proline also provides the energy to support the host-cell invasion process inmetacyclic trypomastigotes and the infection progression, particularly participating in thedifferentiation from intracellular epimastigotes to trypomastigotes. It is also noticeable that theaccumulation of free proline constitutes a defense mechanism against oxidative stress. Thisaccumulation is determined mainly by the rate three independent events: degradation,biosynthesis and transport. In this work we identified a proline permease called TcAAP1, whichbelongs to the Amino Acids/Auxin Permeases superfamily (TcAAAP). This protein of 476 aa,has 9-10 transmembrane spanners, with an N-terminal domain of 90 aa completely variable interms of primary structure. This feature constitutes a characteristic of all the TcAAAP family.Using deletion mutant yeast strains, lacking the general amino acid permease (GAP1) and theproline permease (PUT4), and the plasmid pDR196-TcAAP1, complementation assays wereperformed. Yeasts transformed with TcAAP1 were able to grow in minimal medium using prolineas the single nitrogen source. In addition, yeast transformed with TcAAP1 showed a prolinetransport rate up to 10-folds higher than controls. Finally, a TcAAP1 overexpression model wasconstructed in T. cruzi epimastigotes. In this model the subcellular localization of TcAAP1 will beestablished using the endogenous tri-FLAG tag, and also the parasites will be tested for in vitrodifferentiation assays and for oxidative stress resistance. Supported by::FAPESP-CNPq-INBEQMEDI-CONICET-ANPCyT