The metallocarboxypeptidase M32 family in trypanosomatids.

NIEMIROWICZ, GABRIELA TERESA; OPPENHEIMER, FLORENCIA MAIA; BOUVIER, LEÓN ALBERTO; FRASCH, ALEJANDRA PAULA; CAZZULO, JUAN JOSÉ

Penang

Conferencia; The 9th General Meeting of the International Proteolysis Society (IPS 2015 Proteolysis Conference); 2015

International Proteolysis Society

Metallocarboxypeptidases (MCP) of the M32 family, while broadly distributed among prokaryotic organisms,can only be found in a few eukaryotes including trypanosomatids. Among these organisms are human and animal pathogens of medical relevance such as Trypanosoma brucei and T. cruzi, the respective causative agents of Sleeping Sickness and Chagas disease. The genomes of these parasites encode M32 MCPs which share 72% identity. Both orthologues have a cytosolic localization and are expressed in all parasite cultured forms. The enzymes display a preference for Arg/Lys residues at P1´ but exhibit marked differences at P1. To further explore MCPs substrate specificity, we employed four positional scanning synthetic combinatorial libraries of fluorescence resonance energy transfer (FRET) peptides. Our results indicated that TbMCP-1 has a restricted selectivity for Phe in P1 compared to TcMCP-1, which presented a wider range of substrate utilization. The S2, S3 and S4 subsites of both MCPs could accommodate a broad range of residues. In order to study the physiological role of M32 MCPs in trypanosomatid biology, we performed RNAi knockdown experiments in procyclic form T. brucei trypomastigotes. Preliminary results showed an increase in the duplication times of the obtained cell lines. Phenotype analysis through microscopic methodologies are currently being undertaken to address the observed slower growth rates.