Proteomic Study of SUMOylated proteins in Trypanosoma brucei

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

Buenos Aires

Conferencia; Molecular mechanisms in cell signaling and gene expression; 2013

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular-SAIB

SUMOylation is a regulatory post translational modification involving the covalent attachment of a small ubiquitin-like protein called SUMO to a variety of proteins, participating in diverse cellular processes. The functional consequences of SUMO attachment are based on the alteration of the target interaction surface. SUMO resulted essential for procyclic (PC) and bloodstream (BS) forms of Trypanosoma brucei. There is strong evidence supporting the association of SUMO with the expression of the variable surface glycoprotein, responsible for the evasion of the immune response. We intend to accomplish the proteomic identification of SUMO targets in T. brucei by performing SUMO chromosomal tagging. This enables SUMO expression at physiological levels avoiding competition with the endogenous form and providing tags for tandem affinity purification of SUMO conjugates. In addition, we have applied an approach to reduce unspecific purification of contaminant proteins by a LysC-based strategy allowing the identification of the modified lysine at the same time. We succeeded in obtaining PC and BS clones with a double allele knock-in of this SUMO variant. Immunofluorescence and Western blot analysis showed that the tagged SUMO forms have a nuclear localization and display a characteristic SUMOylation pattern. We are now purifying SUMOylated proteins to obtain their proteomic profile.