The SUMOylation system in Tryapanosoma brucei: proteomic studies and molecular tools for target proteins

VANINA E. ALVAREZ; PAULA A. IRIBARREN; LUCIA DI MARZIO; MARÍA A. BERAZATEGUI; JUAN J. CAZZULO

Caxambu - Minas Gerais

Encuentro; XXXII Annual Meeting of the Brazilian Society of Protozoology / XLIII Annual Meeting on Basic Research in Chagas Disease; 2016

Brazilian Society of Protozoology

Post-translationalmodification with the Small Ubiquitin-like Modifier (SUMO) is conserved ineukaryotic organisms and plays important regulatory roles in proteins affectingdiverse cellular processes. In Trypanosoma brucei, member of one of the earliest branchesin eukaryotic evolution, SUMO is essential for normal cell cycle progressionand is likely involved in the epigenetic control of genes crucial for parasitesurvival, such as those encoding the variant surface glycoproteins. Molecularpathways modulated by SUMO have started to be discovered by proteomic studies;however, characterization of functional consequences is limited to a reducednumber of targets. Here we present a proteomic strategy that allowed theidentification of SUMOylated proteins in T. brucei together with their acceptor sitesin an unambigous manner. To further validate this targets we developed abacterial strain engineered to produce SUMOylated proteins, by transferringSUMO from T. brucei togetherwith the enzymes essential for its activation and conjugation. Due to the lackof background in E. coli,this system is useful to express and identify SUMOylated proteins directly incell lysates by immunoblotting, and SUMOylated targets can be eventuallypurified for biochemical or structural studies. We applied this strategy todescribe the ability of TbSUMO to form chains in vitro and to detect the lysine residuesinvolved 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 generationand functional studies of SUMOylated proteins in trypanosomatids.