Estudo estrutural da interação da crotoxina e suas subunidades com membranas modelo
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Data
2020-02-19
Autores
Natal Junior, Carlos Roberto
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Universidade Estadual Paulista (Unesp)
Resumo
A crotoxina (CTX) é a mais abundante toxina presente no veneno da Crotalus durissus terrificus, representando cerca de 60% do peso seco do veneno bruto desta serpente. A CTX possui atividades neurotóxica, miotóxica e coagulante, sendo a principal responsável pelo elevado número de óbito em acidentes ofídicos causados por serpentes do gênero Crotalus. Além dessas características, foram descobertas ações da crotoxina com possíveis aplicações terapêuticas, por conta de seus efeitos anti-inflamatórios, analgésicos, imunomodulatórios e antitumorais. A CTX é um heterodímero composto por duas subunidades, uma ácida (CA) e outra básica (CB), sendo esta última uma fosfolipase A2 com atividade pré-sináptica de bloqueio da transmissão neuromuscular que provoca paralisias motora e respiratória nos animais. Embora as estruturas cristalográficas e por espalhamento de raios X a baixo ângulo (SAXS) de CTX e CB tenham sido resolvidas, pouco se sabe sobre a ação destas nas membranas celulares bem como da localização dos sítios de interação destas proteínas com as membranas alvo. Lipídios DMPC e DMPG foram utilizadas para se estudar a interação entre estas proteínas e membranas modelo. Técnicas de biofísica molecular, tais como espectroscopia de fluorescência estática e resolvida no tempo e espalhamento de raios X a baixo ângulo foram usadas para estudar o comportamento das proteínas na presença dos lipossomos. Os dados indicam ausência de interação entre CA e CTX sobre os lipídios isoteriônicos (DMPC), enquanto houve interação da CB com ambos sistemas lipídicos (DMPC e DMPG). Nas amostras de membranas modelo com carga negativa (DMPG), foi possível identificar interação envolvendo as proteínas CA e CTX com essas membranas, as quais não haviam interagido com as vesículas de DMPC, sendo que a proteína CA não provocou a lise da bicamada, enquanto a CTX desencadeou a lise dos lipídios. Além disso, os resultados sugerem que CA se dissocia de CB quando o heterodímero CTX se aproxima dos lipossomos.
Crotoxin (CTX) is the most abundant toxin present in Crotalus durissus terrificus venom, representing about 60% of its dry weight. CTX has neurotoxic, myotoxic and coagulant activities, being the main responsible for the high death rate in snakebite accidents caused by Crotalus snakes. Additionally, crotoxin actions with possible therapeutic applications were recently discovered, due to its anti-inflammatory, analgesic, immunomodulatory and antitumor effects. CTX is a heterodimer composed of two subunits, one acidic (CA) and the other basic (CB). CB is a phospholipase A2 with presynaptic activity blocking neuromuscular transmission that causes motor and respiratory paralysis in animals. Although the crystallographic and small angle X-ray scattering (SAXS) structures of CTX and CB have been resolved, their mechanism of action on cell membranes and the exact protein interaction sites with membranes are poorly understood. Lipids DMPC and DMPG were used to study the interaction on between these proteins and model membranes. Molecular biophysics techniques, such as static and time-resolved fluorescence spectroscopy and small angle X-ray scattering, were used to study the behavior of these proteins in presence of liposomes. The data obtained indricothere absence of interaction between CA and CTX with zwitterionic lipids (DMPC), while pointed the interaction between CB with both lipid systems (DMPC and DMPG). In the negatively charged model membrane samples (DMPG), it was possible to identify the interaction of CA and CTX with these membranes, which did not interact with the DMPC vesicles. CA did not cause bilayer lysis, while CTX triggered lipid lysis. Finally, the results suggested that CA dissociates from CB when CTX heterodimer approaches the liposomes.
Crotoxin (CTX) is the most abundant toxin present in Crotalus durissus terrificus venom, representing about 60% of its dry weight. CTX has neurotoxic, myotoxic and coagulant activities, being the main responsible for the high death rate in snakebite accidents caused by Crotalus snakes. Additionally, crotoxin actions with possible therapeutic applications were recently discovered, due to its anti-inflammatory, analgesic, immunomodulatory and antitumor effects. CTX is a heterodimer composed of two subunits, one acidic (CA) and the other basic (CB). CB is a phospholipase A2 with presynaptic activity blocking neuromuscular transmission that causes motor and respiratory paralysis in animals. Although the crystallographic and small angle X-ray scattering (SAXS) structures of CTX and CB have been resolved, their mechanism of action on cell membranes and the exact protein interaction sites with membranes are poorly understood. Lipids DMPC and DMPG were used to study the interaction on between these proteins and model membranes. Molecular biophysics techniques, such as static and time-resolved fluorescence spectroscopy and small angle X-ray scattering, were used to study the behavior of these proteins in presence of liposomes. The data obtained indricothere absence of interaction between CA and CTX with zwitterionic lipids (DMPC), while pointed the interaction between CB with both lipid systems (DMPC and DMPG). In the negatively charged model membrane samples (DMPG), it was possible to identify the interaction of CA and CTX with these membranes, which did not interact with the DMPC vesicles. CA did not cause bilayer lysis, while CTX triggered lipid lysis. Finally, the results suggested that CA dissociates from CB when CTX heterodimer approaches the liposomes.
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Palavras-chave
Veneno de serpentes, Fosfolipase A2, Crotalus, Membranas modelo, Snake venom, Phospholipase A2, Model membranes