Bromodomain factor 1 of Trypanosoma cruzi is targeted to the glycosomes by an N-terminal sequence

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

Mar del Plata

Congreso; Congreso Argentino de Protozoología y enfermedades Parasitarias; 2011

Sociedad Argentina de Protozoología

The bromodomain is a protein domain involved in the recognition of acetylated lysines. It represents an evolutionarily conserved module, present almost exclusively in nuclear proteins. Lysine modification is a reversible and highly regulated posttranslational modification. New proteomics technology has enabled the identification of thousands of acetylated proteins distributed among the different compartments and involved in several processes. One of the most surprising findings has been that metabolic enzymes are highly represented among the acetylome. Ninety percent of the proteins involved in central metabolism were reported to be acetylated and biochemical studies on a handful of enzymes showed that acetylation has multiple effects, increasing the activity of some enzymes while inhibiting the activity of others. These efforts have uncovered a stunning complexity of the acetylome that potentially rivals that of the phosphoproteome. Trypanosoma cruzi Bromodomain factor 1 is a 295 amino acids protein which contains a bromodomain in the N-terminal half of the protein. Western blot analysis of T. cruzi lysates with anti-TcBDF1, immunofluorescence microscopy of the different life cycle stages and immunoelectron microscopy of epimastigotes confirm TcBDF1s non-nuclear localization. Co-localization assays with several markers suggest a glycosomal location. The glycosome is a peroxisome-like organelle specific to trypanosomatids, which contains most of the glycolitic enzymes and other enzymatic systems. Glycosomal proteins are encoded in the nucleus necessitating organellar protein import. The aminoacid sequence of TcBDF1 was analyzed with the PeroxisomeDB server, which recognized in its N-terminus a peroxisome-targeting signal type 2 (PTS-2), one of the topogenic signals that direct glycosomal proteins into the matrix. To determine if the first 27 amino acids present in TcBDF1 are responsible of its import to the glycosome, we transiently transfected epimastigotes with constructs coding the whole protein (BDF1), a truncated version which lacks the first 27 amino acids (BDF1-deltaN) or only the N-terminus targeting signal (BDF1PTS2), fused to the Red Fluorescent Protein. The intracellular localization of the different fusion proteins was determined by fluorescence microscopy. Furthermore, the transfectants were subjected to immunofluorescence microscopy using antibodies against BDF1 and the glicosome-specific protein hexokinase (HK). These results confirm that BDF1 possesses a PTS-2 in its N-terminus, responsible of directing the protein to the glycosomes.