T. cruzi Bromodomain Factor 3 binds acetylated α tubulin and migrates to the flagellum during metacyclogenesis

ALONSO, VICTORIA LUCÍA; VILLANOVA GABRIELA VANINA; RITAGLIATI, CARLA; MOTTA, MARIA CRISTINA M.; CRIBB, PAMELA; ESTEBAN, SERRA

Mendoza

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular; 2012

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular

T. cruzi Bromodomain Factor 3 binds acetylated α tubulin and migrates to the flagellum during metacyclogenesis Victoria L. Alonso1,2, Gabriela V. Villanova1,2, Carla Ritagliati2, Cristina M. Motta3, Pamela Cribb1,2 and Esteban Serra1,2 1FBioyF, UNR, Argentina. 2IBR, CONICET, Argentina. 3 Instituto de Biofísica Carlos Chagas Filho, UFRJ, Brazil. Bromodomains are highly conserved acetyl-lysine binding domains found in proteins associated with chromatin and nuclear acetyltransferases. The Trypanosoma cruzi genome encodes at least four Bromodomain Factors. We describe here Bromodomain Factor 3 (TcBDF3), which localized exclusively at the cytoplasm. TcBDF3 cytolocalization was determined using purified antibodies by western blot and immunofluorescence in all life cycle stages of T. cruzi. In epimastigotes and amastigotes it was detected at the cytoplasm, the flagellum and the flagelar pocket and in trypomastigotes only at the flagellum. Subcellular localization of TcBDF3 was also determined by digitonin extraction, ultrastructural immunocytochemistry and expression of TcBDF3 fused to Cyan Fluorescent Protein. The expression of a truncated version of the protein having only the bromodomain leads to growth retardation and lower yields of trypomastigotes in in vitro metacyclogenesis experiments. Tubulin can acquire different posttranslational modifications which modulate microtubule functions. Acetylated α tubulin has been found in the axoneme of flagella and cilia. To study the possible interaction between TcBDF3 and α tubulin we performed colocalization assays in isolated cytoskeletons and flagella from T. cruzi epimastigotes and trypomastigotes. Interaction between the two proteins was confirmed in vivo by co-immunoprecipitation and in vitro by far western blot assays with synthetic acetylated α tubulin peptides and recombinant TcBDF3. The results suggest that TcBDF3 may be involved in the remodeling of the cytoskeleton during metacyclogenesis.