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

Mar del Plata

Congreso; X Congreso Argentino de Protozoología y Enfermedades Parasitarias; 2014

Sociedad Argentina de Protozoología

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 (PC) and bloodstream (BS) 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. We obtained parasites with a single and a double replacement of SUMO alleles, achieving a significant improvement in the yield of the purification of conjugates compared to SUMO overexpression cell lines. Proteomic analysis of these samples, however, showed that the proteins in the samples 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). Specific digestion of the sample with Lys-C cleaves all proteins but SUMO. Additionally, the introduction of an Arg residue prior to the GG motif in the C-terminus of SUMO allows to map its acceptor Lys in substrates by locating lysines modified by SUMO diGly-remnant after a subsequent cleavage of SUMOylated peptides with trypsin. Meanwhile, since trypsin digestion creates a diGly signature that can also derive from other Ubls, containing an Arg residue N-terminal to the GlyGly motif, such as Ubiquitin or NEDD8, we started to work on a new scheme with the Thr residue prior to GlyGly motif being mutated to Lys. This enables to purify SUMO modified proteins and enrich for diGly-Lys containing peptides following their digestion with Lys-C (Tammsalu et al., 2014). This approach allows the identification of SUMOylated proteins together with their acceptor sites in an unambiguous manner.