Caracterização biofísica da delta-1-pirrolina-5- carboxilato desidrogenase de Trypanosoma cruzi

Abstract

Chagas Disease is a sickness that affects the population present of Latin America and it is classified by the World Health Organization as a Neglected Tropical Diseases. Chagas disease is caused by the flagellated parasite Trypanosoma cruzi, which belong to the same family as Trypanosoma brucei and Leishmania sp., and has a complex life cycle, going from an invertebrate host to a vertebrate one. In order to survive and proliferate in these host changes, T. cruzi must adapt itself to osmotic and oxidative stresses, changes in the environmental ion composition and shifts in energy sources. To perform this adaptation, the amino acid Lproline has presented an important and essential participation that affects the protozoan life cycle, such as support of the mitochondrial metabolism, the host-cell invasion and metacyclogenesis. T. cruzi 1-Delta-Pyrroline-5-Carboxylate Dehydrogenase (TcP5CDH) is involved in the catabolism of proline holding a major role in its conversion by transforming pyrroline-5-carboxylate into L-glutamate (the second step of the catabolic path) and, thus, seeming to be a promising molecular target for new drug development. The amino acids sequence of PP5CDH was used for conservation analysis, secondary structure prediction, identification of functional domains, and building of tertiary structure computer models with the techniques of Molecular Modeling and Molecular Docking. The TcP5CDH (MW: 60 kDa) was expressed in a heterologous fashion in Escherichia coli, and purified with affinity and size exclusion chromatography, resulting in approximately 2 mg/L of expression. The Dynamic Light Scattering assays where carried out with the recombinant P5CDH in the concentrations of 0.5, 1.0, 1.5 e 2.0 mg/mL, and presented an apparent molecular weight of 223,4 kDa (Rh: 12,01 nm), 246,4 kDa (Rh: 12,53 nm), 310,5 kDa (Rh: 13,83 nm) e 312,0 kDa (Rh: 12,13,86 nm), respespectively. The Circular Dichroism spectroscopy was performed with 0.2 mg/mL of TcP5CDH in the presence and absence of 100 μM of NAD+, L-Glu, and its inhibitor Disulfiram, presenting a Tm of Tm 60,01 ºC, 59,76 ºC, 57,76 ºC e 58,18 ºC, showing that TcP5CDH has a more thermic stability without ligands. Also, a deconvolution was made showing that TcP5CDH has 23% of alfa-helix, 12,3% of antiparallel beta-sheetst and 12,4% parallel beta-sheets, 18,3% of turns 41,7% of disorganized structures. These results will contribute to the understanding of the pathway of L- proline in T. cruzi and the possible future development of new drugs.