Functional characterization of TcCyc2, a cyclin from Trypanosoma cruzi

MARIANA POTENZA, ; SERGIO SCHENKMAN,; MARC LAVERRIERE,; MARÍA TERESA TELLEZ

Mar del Plata

Congreso; IX Congreso Argentino de Protozoología y Enfermedades Parasitarias; 2011

Soc. Argentina de Protozoología y Enfermedades Parasitarias

SUMMARY: Cell cycle in eukaryotes is a finely regulated process, in which cyclins and cyclin dependent kinases (CDKs) play main roles. Cyclins bind and activate CDKs, leading to these kinases trigger phosphorylation reactions that activate or inactivate several target proteins. At physiological levels, different cyclins bind different CDKs, in order to orchestrate the sequential and unidirectional events required for proper progression through the cell cycle. It was demonstrated in other eukaryotes that modulation of CDK activity by cyclins is carried out regulating in turn cyclin expression, degradation and/or localization, in a cell cycle phase dependent manner. As a result, cyclins activate specific CDKs at required times along the cycle, avoiding uncontrolled cell division. The cell cycle control in Trypanosomatids is under study, but the mechanisms that govern this process in Trypanosoma cruzi, the etiological agent of Chagas Disease, are still poorly understood. Ten sequences were annotated as putative cyclins in the genome of T. cruzi (TcCyc2 to TcCyc11), three of which were showed to interact with the cyclin related kinase TcCRK1 by our group (TcCyc2, TcCyc5 and TcCyc5). TcCyc2 could rescue a cyclin G1 deficient strain of Saccharomyces cerevisiae. This gene is not the archetypal G1 or G2/M cyclin, but its sequence shows similarity to PHO-like cyclins, involved in phosphate metabolism in yeast. In the other hand, it was demonstrated that down regulation of its homologue TbCyc2 arrest the cell cycle at G1 phase in Trypanosoma brucei. To further characterize TcCyc2 gene, we have over expressed this protein fused to HA tag in epimastigotes of T. cruzi. We detected the protein in the cytosol and showed no variation in its localization along the different phases of the cell cycle. The phenotype of parasites was analyzed by chemical synchronization with hydroxyurea (HU). We found that TcCyc2-HA over-expressing epimastigotes pass through G2/M boundary more efficiently than control parasites in synchronized cultures. It was also observed that TcCyc2-HA over-expressing epimastigotes divide faster; showing shorter doubling times in asynchronous, non-HU treated cultures compared to control parasites. Moreover, the size of TcCyc2-HA over-expressing epimastigotes was smaller than wild type parasites, which indicates a deregulation in the mechanisms of cell size control. Taken together, these results imply TcCyc2 in the cell division control in the parasite.