THE SUMOYLATION SYSTEM IN TRYAPANOSOMA BRUCEI: PROTEOMIC STUDIES AND MOLECULAR TOOLS FOR TARGET PROTEINS

ALVAREZ VE; IRIBARREN PA; DI MARZIO LA; BERAZATEGUI MA; CAZZULO JJ

Caxambu

Congreso; XXXII Reunião Anual da Sociedade Brasileira de Protozoologia; 2016

Sociedade Brasileira de Protozoología

Post-translational modification with the Small Ubiquitin-like Modifier (SUMO) is conserved in eukaryotic organisms and plays important regulatory roles in proteins affecting diverse cellular processes. In Trypanosoma brucei, member of one of the earliest branches in eukaryotic evolution, SUMO is essential for normal cell cycle progression and is likely involved in the epigenetic control of genes crucial for parasite survival, such as those encoding the variant surface glycoproteins. Molecular pathways modulated by SUMO have started to be discovered by proteomic studies; however, characterization of functional consequences is limited to a reduced number of targets. Here we present a proteomic strategy that allowed the identification of SUMOylated proteins in T. brucei together with their acceptor sites in an unambigous manner. To further validate this targets we developed a bacterial strain engineered to produce SUMOylated proteins, by transferring SUMO from T. brucei together with the enzymes essential for its activation and conjugation. Due to the lack of background in E. coli, this system is useful to express and identify SUMOylated proteins directly in cell lysates by immunoblotting, and SUMOylated targets can be eventually purified for biochemical or structural studies. We applied this strategy to describe the ability of TbSUMO to form chains in vitro and to detect the lysine residues involved in this process. Furthermore, to validate targets, we applied an in vitro deconjugation assay using the T. brucei SUMO-specific protease capable of reverting the pattern of modification. This system represents a valuable tool for target validation, mutant generation and functional studies of SUMOylated proteins in trypanosomatids.