Estudos estruturais e funcionais da interação entre derivados do ácido cinâmico e fosfolipase A2 homóloga do veneno de Bothrops jararacussu
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Data
2016-03-01
Autores
Cardoso, Fábio Florença [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Os acidentes ofídicos constituem um problema de saúde pública, afetando regiões de clima tropical e subtropical e áreas rurais e pobres de países da América Latina, África, Ásia e Oceania. No Brasil, o gênero Bothrops é responsável por cerca de 90% dos acidentes ofídicos notificados, cujo envenenamento é caracterizado por intensa mionecrose local ineficientemente neutralizada pela soroterapia. O veneno botrópico possui uma classe de proteínas miotóxicas estruturalmente semelhantes às fosfolipases A2 (PLA2), responsáveis por induzir lesões musculares por um mecanismo não-catalítico parcialmente explicado por diferentes hipóteses. Contudo, há evidências que os efeitos miotóxico e paralisante in vitro são decorrentes de sua atividade desestabilizadora de membranas e que atuam em sinergia com as PLA2 catalíticas no envenenamento. Neste estudo, foi desenvolvido um novo protocolo de purificação da miotoxina não-catalítica (PLA2 homólogas ou proteínas PLA2-like) botrópica BthTX-I, a qual foi avaliada em testes cristalográficos, calorimétricos, miográficos e morfológicos. Potenciais inibidores vegetais da classe dos cinamatos foram co-cristalizados com a BthTX-I e testados em inibir as lesões e paralisia musculares in vitro promovida pela toxina a fim de evoluir no conhecimento da relação estrutura/atividade das PLA2 homólogas miotóxicas. Dentre todos os compostos testados, os ácidos chicórico e caftárico apresentaram-se como excelentes inibidores da BthTX-I. Contudo, foi possível apenas realizar a análise estrutural do complexo BthTX-I/ácido chicórico (AC), o qual apresentou características diferentes de outros complexos estudados com outros inibidores e toxinas botrópicas. De acordo com as propostas mais recentes dos componentes estruturais envolvidos na atividade das PLA2 homólogas miotóxicas, bem como pela interface apresentada entre BthTX-I e AC, é possível que a interação proteína/inibidor gere uma mudança conformacional da toxina para um tetrâmero em solução, de modo que o alinhamento dos sítios de ancoragem à membrana (MDoS) e de ruptura da mesma (MDiS) estejam impedidos.
Snakebites are a public health problem, concerning tropical and subtropical regions, rural and poor areas of Latin America, Africa, Asia and Oceania countries. In Brazil, Bothrops genus accounts for about 90% of reported snakebites, whose envenomation is characterized by intense local myonecrosis inefficiently neutralized by antivenom. A class of myotoxic proteins found in Bothrops venoms which is structurally similar to phospholipases A2 (PLA2), is responsible for inducing muscle injuries by a non-catalytic mechanism, partially explained by different hypotheses. However, there are evidences that myotoxic and in vitro paralyzing effects are due to their destabilizing-membrane activity and they act in synergy with the catalytic PLA2 myotoxins in envenomation. In this study, it was developed a new protocol for purification of a non-catalytic botropic myotoxin (PLA2 homologues or PLA2-like proteins) BthTX-I, which was evaluated by crystallographic, calorimetric, myographic and morphologic assays. Potential plant inhibitors of cinnamates class were co-crystallized with BthTX-I and tested to inhibit in vitro paralysis and muscle injuries promoted by the toxin. Among all the compounds tested, the chicoric and caftaric acids presented excellent BthTX-I inhibition characteristiscs. However, only chicoric acid (CA) we were able to perform crystallographic experiments, which presented different structural characteristics compared to other ligands and bothropic toxins. According to the latest proposals of the structural components involved in the activity of PLA2 homologue myotoxins, as well as by the interface presented between BthTX-I and AC, it was possible to propose that the interaction between protein and the inhibitor leads to tetrameric oligomerization, preventing the alignment between both in membrane-docking site (MDoS) and membrane-disrupting site (MDiS).
Snakebites are a public health problem, concerning tropical and subtropical regions, rural and poor areas of Latin America, Africa, Asia and Oceania countries. In Brazil, Bothrops genus accounts for about 90% of reported snakebites, whose envenomation is characterized by intense local myonecrosis inefficiently neutralized by antivenom. A class of myotoxic proteins found in Bothrops venoms which is structurally similar to phospholipases A2 (PLA2), is responsible for inducing muscle injuries by a non-catalytic mechanism, partially explained by different hypotheses. However, there are evidences that myotoxic and in vitro paralyzing effects are due to their destabilizing-membrane activity and they act in synergy with the catalytic PLA2 myotoxins in envenomation. In this study, it was developed a new protocol for purification of a non-catalytic botropic myotoxin (PLA2 homologues or PLA2-like proteins) BthTX-I, which was evaluated by crystallographic, calorimetric, myographic and morphologic assays. Potential plant inhibitors of cinnamates class were co-crystallized with BthTX-I and tested to inhibit in vitro paralysis and muscle injuries promoted by the toxin. Among all the compounds tested, the chicoric and caftaric acids presented excellent BthTX-I inhibition characteristiscs. However, only chicoric acid (CA) we were able to perform crystallographic experiments, which presented different structural characteristics compared to other ligands and bothropic toxins. According to the latest proposals of the structural components involved in the activity of PLA2 homologue myotoxins, as well as by the interface presented between BthTX-I and AC, it was possible to propose that the interaction between protein and the inhibitor leads to tetrameric oligomerization, preventing the alignment between both in membrane-docking site (MDoS) and membrane-disrupting site (MDiS).
Descrição
Palavras-chave
Veneno de serpente, Bothrops, Fosfolipase A2 homóloga, Cristalografia, Miografia, Snake venom, Bothrops, Phospholipase A2 homologue, X-ray crystallography, Myography