Functional characterization of a hybrid FMSR protein from Trypanosoma cruzi Dm28c

GONZALEZ, LN; BIROCCO, F; SASONI, N; GUERRERO, SA; IGLESIAS, AA; ARIAS, DG

Salta

Congreso; SAIB LV Reunión Anual; 2019

SAIB

Methionine is an amino acidsusceptible to being oxidized to methionine sulfoxide (MetSO). The reduction ofMetSO to methionine is catalyzed by methionine sulfoxide reductase (MSR), anenzyme present in almost all organisms. In trypanosomatids, the study ofantioxidant systems has been mainly focused on the involvement oftrypanothione, a specific redox component in these organisms. Trypanosoma cruzi, the etiological agentof Chagas disease, is auxotroph by methionine. Through an analysis of itsgenome project, we identified a coding sequence of free methionine sulfoxidereductase (fMSR), a protein only present in unicellular organisms. In T. cruzi, this protein is constituted bya GAF-like domain fused to TIP41 domain (homologous to yeast TIP41 proteininvolved in the TOR pathway negative regulation). The encoding sequence for theGAF domain was expressed in Escherichiacoli, and the corresponding recombinant protein was purified andfunctionally characterized. The recombinant protein exhibited MSR activity withL-Met(R)SO and T. cruzi tryparedoxins and thioredoxin as the reducing substrates.Our results supported that this enzyme has non-saturation ping-pong kinetics.In addition, based on the fact that the GAF domain is present in proteinscapable to bind nucleotides, we evaluated the effect of AMP, ADP or ATP on itsMSR activity. We observed an inhibitory effect at low substrate concentration(in the presence of MgCl2), mainly by ATP. On the other hand, weperformed a yeast complementation assay using a Δtip41 mutant. The results showed that TcfMSR (both isolated TIP41 domain as the full protein) couldcompensate the sensitive to rapamycin phenotype. This result indicates that theTcfMSR is active acting in the yeastTOR pathway. These results suggest that the TcfMSRprotein is a possible link between the redox metabolism and the TOR pathway in Trypanosoma cruzi. Granted by ANPCyT(PICT2016-1778 and PICT2017-2268)