Study of SUMO peptidases in Trypanosoma brucei

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

Buenos Aires

Congreso; EMBO MEETING: Ubiquitin and UBLs. At the crossroads from chromatin to protein; 2014

EMBO-CONICET

Trypanosomatids protozoa cause serious neglected diseases to man, affecting a high number of people worldwide. Among the diseases caused by trypanosomatids are Chagas disease in South America (caused by Trypanosoma cruzi), African sleeping sickness (caused by Trypanosoma brucei) and leishmaniasis (caused by different species from the genus Leishmania). Protein post-translational modification by SUMO is present in T. brucei, resulting essential in both procyclic (PCF)1 and bloodstream (BSF)2 stages of its life cycle. Furthermore it has been suggested that SUMOylation might be involved in the regulation of parasite antigenic variation of the surface glycoprotein coat, protecting the parasite from the host immune response (López Farfán D et al., manuscript under revision). Although a preliminary list of SUMO modified proteins in T.brucei has been obtained by proteomic analysis, the cellular processes regulated by SUMO and the conjugation and deconjugation pathways in the parasite have not yet been fully characterized. Therefore, the essential role SUMO deconjugating enzymes play in the regulation and dynamics of SUMOylation, and the identification of inhibitors for these enzymes, turn SUMO deconjugating enzymes into interesting drug targets in parasites3. The aim of this work is to identify in silico the complete set of T. brucei SUMO peptidases as well as to start a cellular and biochemical characterization of these enzymes. We have first performed BLAST searches against T. brucei genome using known SUMO proteases as queries, resulting in 7 genes encoding putative peptidases with their active site residues conserved. We determined the subcellular localization of these candidate proteases by endogenous protein tagging with a Flag epitope at the C-terminus and subsequent immunofluorescence analysis using commercial anti-Flag antibodies. We are currently optimizing the recombinant expression of the proteins in bacteria to obtain reasonable amounts of the purified enzymes allowing us to perform biochemical studies. We plan to assess their activity as peptidases, processing SUMO precursor, as well as their isopeptidase activity by removing SUMO from conjugated targets. 1. Liao S., Wang T., Fan K. and TU X. (2010) The small ubiquitin-like modifier (SUMO) is essential in cell cycle regulation in Trypanosoma brucei. Nature. 414:759-763. 2. Obado S. O., Bot C., Echeverry M. C., Bayona J. C., Alvarez V. E., Taylor M. C. and Kelly J. M. (2011) Centromere-associated topoisomerase activity in bloodstream form Trypanosoma brucei. Nucleic Acids Res. Feb; 39 (3): 1023-1033. 3. Ponder EL, Albrow VE, Leader BA, Békés M, Mikolajczyk J, Fonović UP, Shen A, Drag M, Xiao J, Deu E, Campbell AJ, Powers JC, Salvesen GS, Bogyo M. (2011) Functional characterization of a SUMO deconjugating protease of Plasmodium falciparum using newly identified small molecule inhibitors. Chem Biol. Jun 24;18(6):711-21.