RESUMEN
Objetivo Determinar estrutural básica e modelagem molecular por via comparativa da giroxina, uma serinoprotease trombina símile isolada do veneno da Crotalus durissus terrificus. Métodos As análises de similaridade de estrutura primária e secundária foram geradas através de sistemas logarítmicos e probabilísticos de modo a encontrar um molde proteico da mesma família da giroxina com capacidade de servir de molde para gerar estruturas tridimensionais comparativas da proteína selecionada. Resultados A giroxina é uma proteína com 241 aminoácidos, de caráter ácido, instável e de melhor ação em meio aquoso. Possui um peso molecular de 26.1kDa e 6 prováveis pontes de dissulfeto. Conclusões A giroxina é uma proteína trombina símile com ação na dissolução de coágulos e seu estudo estrutura-atividade pode trazer novos fármacos para embolias pulmonares, infartos cardíacos e trombose de membros baixos. Novos estudos observam a interação da giroxina com receptores PAR-1 de astrócitos o que pode indicar uma nova classe de medicamentos para tratamento de síndromes neurodegenerativas, tais como a síndrome de Parkinson.
Objective To determination basic structure and molecular modeling by comparative means of gyroxin, a serinoproteases trombinelike isolated from the Crotalus durissus terrifius venom. Methods Analysis of similarity of primary and secondary structures generated through logaritmic and probabilistic systems to find a protein model of the same family with capacity to be used as template to create tridimensional comparative structures of the selected protein. Results The gyroxin is a 241 aminoacids protein, of acid qualities, instable and have better activity in water solutions. It has 26.1kDa of molecular weight and 6 probable dissulfide bonds. Conclusions Gyroxin is a trombine-like protein acting to dissolve blood clots and her structure-activity study may bring new drugs to treat pulmonary embolism, cardiac arrest and lower members tromboses. New studies appoint the interaction between gyroxin and PAR-1 astrocytes receptors who may indicate a new class of neurodegeneratives syndromes drugs, such as Parkinson's syndrome.
RESUMEN
Gyroxin is a serine protease displaying a thrombin-like activity found in the venom of the South American rattlesnake Crotalus durissus terrificus. Typically, intravenous injection of purified gyroxin induces a barrel rotation syndrome in mice. The serine protease thrombin activates platelets aggregation by cleaving and releasing a tethered N-terminus peptide from the G-protein-coupled receptors, known as protease-activated receptors (PARs). Gyroxin also presents pro-coagulant activity suggested to be dependent of PARs activation. In the present work, the effects of these serine proteases, namely gyroxin and thrombin, on PARs were comparatively studied by characterizing the hydrolytic specificity and kinetics using PARs-mimetic FRET peptides. We show for the first time that the short (sh) and long (lg) peptides mimetizing the PAR-1, -2, -3, and -4 activation sites are all hydrolyzed by gyroxin exclusively after the Arg residues. Thrombin also hydrolyzes PAR-1 and -4 after the Arg residue, but hydrolyzes sh and lg PAR-3 after the Lys residue. The kcat/KM values determined for gyroxin using sh and lg PAR-4 mimetic peptides were at least 2150 and 400 times smaller than those determined for thrombin, respectively. For the sh and lg PAR-2 mimetic peptides the kcat/KM values determined for gyroxin were at least 6500 and 2919 times smaller than those determined for trypsin, respectively. The kcat/KM values for gyroxin using the PAR-1 and -3 mimetic peptides could not be determined due to the extreme low hydrolysis velocity. Moreover, the functional studies of the effects of gyroxin on PARs were conducted in living cells using cultured astrocytes, which express all PARs. Despite the ability to cleavage the PAR-1, -2, -3, and -4 peptides, gyroxin was unable to activate the PARs expressed in astrocytes as determined by evaluating the cytosolic calcium mobilization. On the other hand, we also showed that gyroxin is able to interfere with the activation of PAR-1 by thrombin or by synthetic PAR-1 agonist in cultured astrocytes. Taken together, the data presented here allow us showing that gyroxin cleaves PARs-mimetic peptides slowly and it does not induce activation of PARs in astrocytes. Although gyroxin does not mobilize calcium it was shown to interfere with PARs activation by thrombin and PAR-1 agonist. The determination of gyroxin enzymatic specificity and kinetics on PAR-1, -2, -3, and -4 will potentially help to fill the gap in the knowledge in this field, as the PARs are still believed to have a key role for the gyroxin biological effects.
Asunto(s)
Venenos de Crotálidos/química , Crotalus , Receptores Proteinasa-Activados/metabolismo , Serina Proteasas/metabolismo , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Calcio/metabolismo , Coagulantes/química , Citosol/metabolismo , Hidrólisis , Masculino , Ratones , Receptores Proteinasa-Activados/antagonistas & inhibidores , Transducción de Señal , América del Sur , Trombina/química , Tripsina/metabolismoRESUMEN
Gyroxin, a thrombin-like enzyme isolated from Crotalus durissus terrificus venom and capable of converting fibrinogen into fibrin, presents coagulant and neurotoxic activities. The aim of the present study was to evaluate such coagulant and toxic properties. Gyroxin was isolated using only two chromatographic steps - namely gel filtration (Sephadex G-75) and affinity (Benzamidine Sepharose 6B) - resulting in a sample of high purity, as evaluated by RP-HPLC C2/C18 and electrophoretic analysis that showed a molecular mass of 30 kDa. Gyroxin hydrolyzed specific chromogenic substrates, which caused it to be classified as a serine proteinase and thrombin-like enzyme. It was stable from pH 5.5 to 8.5 and inhibited by Mn²+, Cu²+, PMSF and benzamidine. Human plasma coagulation was more efficient at pH 6.0. An in vivo toxicity test showed that only behavioral alterations occurred, with no barrel rotation. Gyroxin was not able to block neuromuscular contraction in vitro, which suggests that its action, at the studied concentrations, has no effect on the peripheral nervous system.