Different strategies for proteomic identification of SUMOylated proteins in Trypanosoma brucei

PAULA A. IRIBARREN; MARÍA A. BERAZATEGUI; JULIO C. BAYONA; TRIIN TAMMSALU; RON T. HAY; JUAN J. CAZZULO; VANINA E. ALVAREZ

Rosario

Congreso; L Reunión Anual.Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2014

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular-SAIB

SUMOylation is a post-translational modification involving the covalent attachment of a small ubiquitin-like protein called SUMO to a variety of proteins. In T. brucei, SUMO resulted essential for procyclic and bloodstream forms. Moreover, there is an association between SUMO and the expression of the variable surface glycoproteins. The aim of this work is to compare different strategies for proteomic identification of SUMO conjugates. We generated different cell lines with SUMO chromosomal tagging that enable its expression at physiological levels and provide tags for tandem affinity purification of the conjugates. In this way, we achieved a significant improvement in the yield of the purification but the proteins identified by MS did not differ significantly from the control. To reduce the purification of contaminants we applied a complementary approach utilizing Lysine-deficient SUMO (Matic et al.,2010) with the introduction of an R residue prior to the GG motif in the C-terminus of SUMO to map its acceptor K in substrates. Meanwhile, since this strategy creates a GG signature that can also derive from other Ubls, we started to work on a new scheme with a K residue prior to the GG motif. This enables to enrich for GG-K containing peptides following their digestion with Lys-C allowing the identification of the acceptor sites in an unambiguous manner (Tammsalu et al.,2014). COMUNICACIÓN ORAL