A gene knock-in strategy for the identification of SUMO targets in Trypanosoma brucei by proteomic analysis

IRIBARREN, P.; BAYONA, J.C.; CAZZULO, J.J.; ALVAREZ, V.E.

Mar del Plata

Congreso; IX Congreso Argentino de Protozoología y Enfermedades Parasitarias; 2011

Sociedad Argentina de Protozoología

SUMOylation is a post translational modification that involves the covalent attachment of a small ubiquitin-like protein called SUMO to a variety of proteins. In eukaryotic organisms this modification has shown to be important for the regulation of several biological processes, such as DNA repair and replication, cell cycle progression, RNA metabolism and translational initiation, among others. Previous studies performed in our laboratory also pointed out the importance of SUMO in the biology of Trypanosoma cruzi, as inferred from a global map of SUMOylated proteins obtained by a proteomic approach. Furthermore, we and others have demonstrated that this system is essential in Trypanosoma brucei procyclic (PC) and bloodstream forms (BS). The aim of this work is to identify SUMOylated proteins in the PC and BS forms of T. brucei. We have designed an improved strategy that enables SUMO production at physiological levels and avoids the competition with the endogenous form. This strategy consists in the replacement of both SUMO gene alleles with an N-terminal tagged version of SUMO, allowing a tandem affinity purification and mass spectrometry identification of SUMO targets. In addition, we will try to identify the modified lysine residue in the target proteins generating an additional cell line carrying a second modification at the C-terminal end of SUMO. The introduction of a trypsin cleavage site near the C-terminus of SUMO will produce a signature peptide that can be unambiguously identified by mass spectrometry. We have successfully obtained PC hemicigotes for both SUMO variants. Immunofluorescence and Western blot analysis showed that in both cell lines tagged SUMO forms have a nuclear localization and display a characteristic SUMOylation pattern, similar to the endogenous SUMO. Currently we are performing a second allele knock-in in order to obtain fully replaced SUMO cell lines for the large scale proteomic analysis.