Trypanosoma cruzi bromodomain factor 3binds cytoskeletal and flagellar acetylated alpha-tubulin

ALONSO, VICTORIA LUCÍA; VILLANOVA, GABRIELA VANINA; RITAGLIATI, CARLA; CRIBB, PAMELA; SERRA, ESTEBAN

Mar del Plata

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

Sociedad Argentina de Protozoología

Bromodomains are highly conserved acetyl-lysine binding domains. They were originally found in proteins associated with chromatin and in nuclear acetyltransferases. The Trypanosoma cruzi genome encodes five Bromodomain Factors (TcBDF1-5). We describe here Bromodomain Factor 3 from Trypanosoma cruzi (TcBDF3), the first bromodomain containing protein reported outside the nucleus. TcBDF3 expression was observed using purified antibodies by western blot (WB) and immunofluorescence microscopy in all life cycle stages of T. cruzi. In epimastigotes and amastigotes it is located in the cytoplasm and in trypomastigotes and metacyclic trypomastigotes at the flagella. Subcellular localization of TcBDF3 was also determined by digitonin extraction and expression of TcBDF3 fused to fluorescent proteins in epimastigotes. Trypanosoma cruzi flagellum and subpellicular microtubules contain acetylated ��-tubulin. Microtubules can acquire different posttranslational modifications (PTMs), including acetylation, and it has been proposed that these PTMs can generate a code that can be read by microtubule-associated proteins similar to how the histone code dictates chromatin functions. We believe that TcBDF3 through its interaction with acetylated ��-tubulin could be involved in the remodeling of the cytoskeleton during the metacyclogenesis process. In order to study the possible interaction between TcBDF3 and ��-tubulin, we performed co-localization assays in isolated cytoskeletons and flagella from T. cruzi epimastigotes. Interaction between the two proteins was confirmed in vivo by co-immunoprecipitation assays and in vitro by Far western blot assays with synthetic acetylated tubulin peptides and recombinant TcBDF3. The immunoprecipitation experiments also revealed that TcBDF3 forms part of a multisubunit complex. In the last few years several authors, using high resolution mass spectrometry, identified thousands of acetylated proteins involved in  transcription, DNA repair, chromatin remodeling, cell cycle, splicing, metabolism, cytoskeletal dynamics, apoptosis, nuclear import, protein folding and cellular signaling in different organisms. It has even been proposed that acetylation could have the same regulatory relevance as phosphorylation, both inside and outside the nuclear compartment. The presence of a Bromodomain containing protein outside the nucleus supports this hypothesis.