Molecular and Biochemical Characterization of Adenosine Deaminases acting on tRNA of Trypanosoma cruzi

LOBO M MAITE; BALOUZ VIRGINIA; DUCREY IVANA; BERTOTTI SANTIAGO; FLEMING MC IAN; ALFONZO D JUAN; BUSCAGLIA CARLOS A; CÁMARA M DE LOS MILAGROS

Resistencia

Congreso; Molecular and Biochemical Characterization of Adenosine Deaminases acting on tRNA of Trypanosoma cruzi; 2018

Sociedad Argentina de Protozoología

Trypanosoma cruzi is the protozoan causative of Chagas Disease, a major health and economic issue in Latin America. This parasite has a complex population, which is composed of multiple strains displaying considerable genetic drift and phenotypic diversity. In order to analyze the impact of codon usage variability in T. cruzi, we began the molecular characterization of the Adenosine Deaminases acting on tRNA, which is a heterodimer composed by TcADAT2 and TcADAT3. As a first approach, we analyzed TcADAT2 and TcADAT3 expression in the main developmental stages of T. cruzi at an RNA and protein level by Real-Time PCR and Western Blot, respectively, observing that both proteins are expressed along the parasites life cycle. Secondly and as to start the biochemical characterization of the heterodimer we performed in vitro DNA deamination assays using an Ames test in bacteria, in which we evaluated the capacity of the ADATs of deaminating DNA, observing that the heterodimer has the capacity to deaminate DNA. Furthermore, to evaluate the heterodimers capacity to edit tRNAs, we performed in vitro tRNA editing assays followed by TLC analysis evidencing that the heterodimer has tRNA editing activity. Finally, using a transgenic line approach in the CL Brener strain, we expressed ectopically FLAG-tagged TcADAT2 and TcADAT3 observing that TcADAT2 subcellular localization is partially nuclear and cytoplasmic while TcADAT3 is localized in the cytoplasm. Localization was confirmed by digitonin extraction assays followed by Western Blot analysis against the FLAG epitope. Moreover, overexpression of either protein in T. cruzi leads to an increase in the intracellular tRNA adenosine-to-inosine pool. These findings open a novel perspective on gene expression regulation, tRNA abundance and codon usage in Trypanosoma cruzi (and trypanosomatids in general).