RESUMEN
The intracellular parasite Leishmania braziliensis is the causal agent of cutaneous and mucocutaneous leishmaniasis, a group of endemic diseases in tropical regions, including Latin America. New therapeutic targets are required to inhibit the pathogen without affecting the host. The enzyme nicotinamide/nicotinate mononucleotide adenylyltransferase (NMNAT; EC: 2.7.7.1/18) is a potential target, since it catalyzes the final step in the biosynthesis of nicotinamide adenine dinucleotide (NAD+), which is an essential metabolite in multiple cellular processes. In this work, we produced and evaluated the catalytic activity of the recombinant protein 6HisΔ241-249LbNMNAT to study the functional relevance of the exclusive insertion present in the enzyme of L. braziliensis (LbNMNAT), but absent in the primary structure of human NMNATs. Our results indicate that the 241-249 insertion constitutes a structural element that connects the protein structure Rossmann topology with the carboxyl-terminal domain of the enzyme. The removal of this region drastically decreases the solubility, and enzymatic activity of the recombinant, causing its inactivation. Molecular dynamics simulations were carried out with the wild-type and truncated enzymes to verify additional changes in their stability, which indicated a better stability in the wild-type protein. These findings constitute an initial step to identify a new inhibition mechanism for the development of focused pharmacological strategies on exclusive insertions from the LbNMNAT protein.
RESUMEN
Resumen Los sistemas de defensa anti-oxidante utilizados por el parásito intracelular Leishmania braziliensis durante el proceso de infección permiten eliminar especies reactivas de oxígeno y nitrógeno a expensas de equivalentes reductores derivados de la tripanotiona, evitando daños celulares del patógeno. Con el objetivo de identificar potenciales blancos moleculares para el desarrollo de fármacos contra este parásito, se realizó la detección de la enzima triparedoxina peroxidasa citoplasmática de L. braziliensis (LbTXNPxII), la cual es esencial para disminuir concentraciones tóxicas de peróxido de hidrógeno en el contexto de infección. Para esto se generaron anticuerpos policlonales en modelo aviar, partiendo de la clonación, expresión y purificación de la proteína recombinante 6xHis-SUMO-LbTXNPxII (37kDa) en el sistema heterólogo Escherichia coli. La proteína purificada se utilizó como antígeno para la producción de anticuerpos IgY, cuya implementación en estudios in situ permitió detectar y localizar la enzima LbTXNPxII endógena (22kDa) en el citoplasma de promastigotes fijados y verificar su interacción molecular con la nicotinamida/ nicotinato mononucleótido adenilil transferasa, enzima involucrada en la síntesis del NAD. De este modo, se reporta el desarrollo de una herramienta bioquímica para la identificación y estudio de la enzima LbTXNPxII y su participación en vías del metabolismo energético y de defensa anti-oxidante.
Abstract The antioxidant defense systems used by the intracellular parasite Leishmania braziliensis during the infection process make it possible to eliminate reactive oxygen and nitrogen species at the expense of reducing equivalents derived from trypanothione, avoiding cellular damage of the pathogen. In order to identify potential molecular targets for the development of drugs against this parasite, the cytoplasmic tryparedoxin peroxidase of L. braziliensis (LbTXNPxII), which is essential to reduce toxic concentrations of hydrogen peroxide in the context of infection, was carried out. In this regard, polyclonal antibodies were generated in an avian model, starting from the cloning, expression, and purification of the recombinant protein 6xHis-SUMO-LbTXNPxII (37kDa) in the heterologous system of Escherichia coli. The purified protein was used as an antigen for the production of IgY antibodies, whose implementation in in situ experiments allowed the detection and localization of the endogenous LbTXNPxII enzyme (22kDa) in the cytoplasm of fixed promastigotes, as well as the verification of its molecular interaction with nicotinamide/nicotinate mononucleotide adenylyltransferase, an enzyme involved in the synthesis of NAD. Thus, the development of a biochemical tool for the identification and study of the LbTXNPxII enzyme and its participation in energy metabolism and antioxidant defense pathways is reported.
Resumo Os sistemas de defesa antioxidante utilizados pelo parasita intracelular Leishmania braziliensis durante o processo de infecção, permitem a eliminação de espécies reativas de oxigênio e nitrogênio em detrimento de equivalentes redutores derivados de tripanotiona, evitando o dano celular do patógeno. Com o objetivo de identificar potenciais alvos moleculares para o desenvolvimento de drogas contra esse parasita, foi detectada a enzima citoplasmática triparedoxina peroxidase de L. braziliensis (LbTXNPxII), essencial para reduzir as concentrações tóxicas de peróxido de hidrogênio no contexto de infecção. Para isso, anticorpos policlonais foram gerados em modelo aviário, a partir da clonagem, expressão e purificação da proteína recombinante 6xHis-SUMO-LbTXNPxII (37kDa) no sistema heterólogo de Escherichia coli. A proteína purificada foi utilizada como antígeno para a produção de anticorpos IgY, cuja implementação em experimentos in situ permitiu a detecção e localização da enzima LbTXNPxII endógena (22kDa) no citoplasma de promastigotas fixos e verificar sua interação molecular com nicotinamida/nicotinato mononucleotídeo adenililtransferase, enzima envolvida na síntese de NAD. Assim, é relatado o desenvolvimento de uma ferramenta bioquímica para a identificação e estudo da enzima LbTXNPxII e sua participação no metabolismo energético e nas vias de defesa antioxidante.
