Protein acetylation and non-nuclear bromodomains: possible atypical pathways from Trypanosoma cruzi

SERRA EC; CRIBB P; RITAGLIATI C; ALONSO VL; VILLANOVA VG

MAr del Plata

Mesa redonda; IX congreso Argentino de Protozoologia y Enfermedades Parasitarias; 2011

Lysine acetylation is a reversible and highly regulated posttranslational modification (PTM) which is recognized specifically by a protein domain called bromodomain. The bromodomain is an evolutionarily conserved module, present almost exclusively in nuclear proteins. New proteomics technology has enabled the identification of thousands of acetylated proteins distributed among the different cell-compartments and involved in a variety of processes including transcription, DNA repair, chromatin remodeling, cell cycle, splicing, metabolism, cytoskeletal dynamics, apoptosis, nuclear import, protein folding and cellular signaling (Choudhary et al, 2009). Surprisingly, around ninety percent of the proteins involved in central metabolism were reported to be acetylated and biochemical experiments showed that acetylation increased the activity of some metabolic enzymes while inhibited the activity of others (Wang et al, 2010). These findings led to the proposal that acetylation-related mechanisms are crucial for cell development (Norris et al, 2009). Trypanosoma cruzi genome encodes five Bromodomain Factors (TcBDF1-5). TcBDF2, as all bromodomain-containing proteins described until now, is located inside the nucleus, where it binds H4 histone acetylated in K10 and K14 and participates in chromatin remodeling (Villanova et al, 2009). In contrast, both TcBDF1 and TcBDF3 show a very atypical out-of-nucleus localization, a feature completely unexpected for this type of proteins. BDF1´s non-nuclear localization was analyzed by western blot analysis of total lysates, nuclear and non-nuclear extracts and cytoplasmatic sub-fractions of epimastigotes, immunofluorescence microscopy of the different life cycle stages and immunoelectron microscopy of epimastigotes. Co-localization assays with several markers suggest a glycosomal location. BDF1 N-terminus shows a peroxisome-targeting signal type 2 (PTS-2), one of the topogenic signals that direct proteins into the glycosomal matrix. We prepared a truncated version of TcBDF1 which lacks the first 27 amino acids bearing the putative PTS-2 (TcBDF1DN) fused to RFP. When transfected, TcBDF1DN expression was widespread in the cytoplasm, and no co-localization with the glycolitic enzyme hexokynase, previously shown with TcBDF1 was evidenced, thus confirming that PTS-2 at the N-terminus of TcBDF1 is responsible for directing the protein to the glycosome. TcBDF3 expression was studied using purified antibodies by western blot (WB) and immunofluorescence. 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 subpelicullar microtubules contain acetylated alpha-tubulin. Microtubules can acquire post translational modifications including acetylation and it has been proposed that these PTMs can generate a code that can be read by microtubule-associated proteins in a way resembling how the histone code dictates chromatin functions (Verhey and Gaertig, 2007). We propose that TcBDF3, through its interaction with acetylated alpha-tubulin, could be involved in the remodeling of the cytoskeleton during the metacyclogenesis process. In order to study the possible interaction between TcBDF3 and alfa-tubulin we performed co-localization assays in isolated cytoskeletons and flagella from 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. It has even been proposed that acetylation could have the same regulatory relevance as phosphorylation, both inside and outside the nuclear compartment. However, no module capable of recognizing this modification has been described, besides the nuclear bromodomains. The fact that T. cruzi has two non-nuclear bromodomain-containing proteins suggests the existence in this parasite of divergent ancient protein recognition pathways, not described in eukaryotic cells until now.