Modification of proteins by the covalent attachment of ubiquitin is a key regulatory mechanism of many cellular processes including protein degradation by the 26S proteasome. Deubiquitination, reversal of this modification, must also regulate the fate and function of ubiquitin-conjugated proteins. Deubiquitinating enzymes (DUBs) catalyze the removal of ubiquitin from ubiquitin-conjugated substrate proteins as well as from its precursor proteins. DUBs occupy the largest family of enzymes in the ubiquitin system, implying their diverse function in regulation of the ubiquitin-mediated pathways. Here, we use the recently completed genome sequences of  T. cruzi (TcruziDB http://TcruziDB.org) in an in silico analysis to explore the diversity of DUBs in this parasite. To identify novel members of the DUB family of cysteine proteinases, we used the BLAST algorithm to screen the T. cruzi genome databases looking for DNA sequences encoding putative proteins similar to previously described family members. According to our exhaustive analysis of the T. cruzi genome, we have at least 25 members that contain both the conserved cysteine (Cys) and histidine boxes essential for catalysis. Most of these proteins have also been annotated in the protease database MEROPS (http://www.merops.ac.uk) as members of the C19 family of cysteine proteases. As a preliminary step to study the physiological role of DUBs in T. cruzi, we examined by semi-quantitative RT-PCR the expression pattern of DUB-10, -12, -14 and -15 in epimastigote from T. cruzi I (Colombian strain) and T. cruzi II (CL, CL-Brenner, Berenice-62, Berenice-78 and Y). Our results demonstrated that whereas level of DUB-10 and -12 remained constant in both T. cruzi I and T. cruzi II strains, DUB-14 is only produced at significant levels by Berenice 78, Colombian and Y strains. Finally, transcripts for DUB-15 were not detected in Berenice 62, CL and CL-Brener strains. These results provide evidence of the extreme complexity and diversity of the DUBs in T. cruzi and open the possibility to explore the relevance of their multiple components in the regulation of ubiquitin-mediated pathways in this parasite.
Supported by FAPEMIG, CNPq, UFOP.