Study of SUMOylation in Trypanosoma brucei

PAULA A. IRIBARREN; MARÍA A. BERAZATEGUI; JUAN JOSÉ CAZZULO; JULIO C. BAYONA; VANINA E. ALVAREZ

Riva del Garda

Congreso; Ubiquitin and ubiquitin-like proteins: From structure to function; 2013

European Molecular Biology Organization (EMBO)

Trypanosomatids are protozoa responsible for human diseases affecting a high number of people. In Trypanosoma brucei, the etiological agent of african trypanosomiasis, SUMO resulted essential for procyclic(PC) and bloodstream(BS) forms. There is also strong evidence that supports the association of SUMO with the expression of the variable surface glycoprotein, responsible for the evasion of the immune response. The aim of this work is to accomplish the proteomic identification of SUMO targets in T. brucei by performing SUMO chromosomal tagging. This strategy enables SUMO expression at physiological levels, avoids the competition with the endogenous form, while providing tags for tandem affinity purification of SUMO and SUMO conjugates. In addition, we have applied a complementary approach described by Matic et al. to reduce unspecific purification of contaminant proteins by a LysC based strategy that allows the identification of the modified lysine by proteomic analysis at the same time. We have successfully obtained PC and BS clones with a double allele knock-in of this SUMO variant. Immunofluorescence and Western blot analysis showed that in both cell lines tagged SUMO forms have a nuclear localization and display a characteristic SUMOylation pattern. Currently we are purifying SUMOylated proteins to obtain their proteomic profile.