Estudos estruturais com fosfolipases A2 homólogas de veneno botrópicos em presença de íons com importância funcional
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2012-03-16
Autores
Borges, Rafael Junqueira [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
As fosfolipases A2 (PLA2s) de veneno de serpente são uma das principais toxinas responsavéis pela miotoxicidade e necrose muscular observadas nos acidentes ofídicos. Essas toxinas podem ser separadas em Asp49-PLA2s, cujo mecanismo é catalítico e dependente de cálcio, e as PLA2s homólogas, cuja estrutura é semelhante, porém atividade em mecanismo não catalítico e independente de cálcio. Apesar de os detalhes deste mecanismo não serem bem conhecidos, estudos funcionais ressaltam importância da região do C-terminal e estudos cristalográficos propõem, mais especificamente, um sítio miotóxico composto por resíduos básicos e um hydrophobic knuckle composto por hidrofóbicos, ambos situados na região C-terminal da toxina. O presente trabalho tem objetivo de oferecer mais detalhes acerca do mecanismo de ação miotóxico independente de cálcio. Para tanto, realizamos estudos cristalográficos com miotoxinas botrópicas BthTX-II (uma Asp- PLA2s), BthTX-I e PrTX-I (duas PLA2s homólogas), em presença de íons relevantes para suas atividades. Recentemente, foi demonstrado que íons, tal como manganês, cálcio, zinco entre outros, interferem na atividade biológica de PLA2s e PLA2s homólogas. Assim, estudo cristalográfico da PrTX-I e BthTX-I com íons manganês e zinco foram elucidados no estado nativo e complexado. Experimentos in vivo indicam que tais íons promoveriam efeito inibitório da miotoxicidade. Enquanto os íons manganês não foram localizados no modelo cristalográfico, os íons zinco foram identificados interagindo apenas no modelo nativo com uma His48 e com as His120 de ambos monômeros. Estudos comparativos destas estruturas com a PrTX-II (PLA2s homóloga) com ácidos graxos foi desenvolvido e guiaram para elaboração de nova hipótese do mecanismo de ação independente de...
Phospholipases A2 (PLA2s) from snake venom are among the main toxins responsibles for miotoxicity and muscular necrosis observed in offidic accidents. These toxins can be separated in Asp49-PLA2s, whose catalytic mechanism of action is dependent of calcium, and in homologue PLA2s or PLA2s-like, whose terciary structure is similar, although its activity is not catalytic in a calcium independent mechanism of action. Despite the fact that this calcium independent mechanism is not well known, functional studies highlight the C-terminal region and structural studies points to a myotoxic site, composed by basic residues, and a hydrophobic knuckle, composed by aromatic and hydrophobic, in the C-terminal region. The present study aims to gather more knowledge about this myotoxic calcium independent mechanism of action. For this purpose, the botropic myotoxins BthTX-II (an Asp49- PLA2), BthTX-I and PrTX-I (homologue PLA2s) were studied in the presence of divalent ions by Xray crystallography. Recently, it was demonstrated divalent ions, such as manganese, zinc and calcium, interfere with the biological activity of these toxins. The crystallographic structure of PrTX-I and BthTX-I with manganese and zinc ions in a native and complexed form were elucidated. In vivo studies demonstrated that these ions inhibit the myotoxic activity of these proteins. Although manganese ions were not found in the structures, zinc ions were found interacting with His48 and the C-terminal residue His120. Comparative studies of this structure and PrTX-II (a homologue PLA2) complexed with stearic acid were performed and support a new hypothesis for the myotoxic mechanism of action for homologue PLA2s which includes both myotoxic site and hydrophobic knuckle and, for the first time, His120. Differently from most PLA2s of snake venom... (Complete abstract click electronic access below)
Phospholipases A2 (PLA2s) from snake venom are among the main toxins responsibles for miotoxicity and muscular necrosis observed in offidic accidents. These toxins can be separated in Asp49-PLA2s, whose catalytic mechanism of action is dependent of calcium, and in homologue PLA2s or PLA2s-like, whose terciary structure is similar, although its activity is not catalytic in a calcium independent mechanism of action. Despite the fact that this calcium independent mechanism is not well known, functional studies highlight the C-terminal region and structural studies points to a myotoxic site, composed by basic residues, and a hydrophobic knuckle, composed by aromatic and hydrophobic, in the C-terminal region. The present study aims to gather more knowledge about this myotoxic calcium independent mechanism of action. For this purpose, the botropic myotoxins BthTX-II (an Asp49- PLA2), BthTX-I and PrTX-I (homologue PLA2s) were studied in the presence of divalent ions by Xray crystallography. Recently, it was demonstrated divalent ions, such as manganese, zinc and calcium, interfere with the biological activity of these toxins. The crystallographic structure of PrTX-I and BthTX-I with manganese and zinc ions in a native and complexed form were elucidated. In vivo studies demonstrated that these ions inhibit the myotoxic activity of these proteins. Although manganese ions were not found in the structures, zinc ions were found interacting with His48 and the C-terminal residue His120. Comparative studies of this structure and PrTX-II (a homologue PLA2) complexed with stearic acid were performed and support a new hypothesis for the myotoxic mechanism of action for homologue PLA2s which includes both myotoxic site and hydrophobic knuckle and, for the first time, His120. Differently from most PLA2s of snake venom... (Complete abstract click electronic access below)
Descrição
Palavras-chave
Biologia molecular, Fosfolipases, Serpente peçonhenta - Veneno, Venenos - Analise, Molecular biology, Phospholipases, Poisonous snakes
Como citar
BORGES, Rafael Junqueira. Estudos estruturais com fosfolipases A2 homólogas de veneno botrópicos em presença de íons com importância funcional. 2012. 112 f. Dissertação (mestrado) - Universidade Estadual Paulista Júlio de Mesquita Filho, Instituto de Biociências de Botucatu, 2012.