RESUMO
Tumor necrosis factor (TNF) is a major cytokine in inflammatory processes and its deregulation plays a pivotal role in several diseases. Here, we report that a zinc metalloprotease extracted from Bothrops moojeni venom (BmooMP-alpha-I) inhibits TNF directly by promoting its degradation. This inhibition was demonstrated by both in vitro and in vivo assays, using known TLR ligands. These findings are supported by molecular docking results, which reveal interaction between BmooMP-alpha-I and TNF. The major cluster of interaction between BmooMP-alpha-I and TNF was confirmed by the structural alignment presenting Ligand Root Mean Square Deviation LRMS = 1.05 Å and Interactive Root Mean Square Deviation IRMS = 1.01 Å, this result being compatible with an accurate complex. Additionally, we demonstrated that the effect of this metalloprotease on TNF is independent of cell cytotoxicity and it does not affect other TLR-triggered cytokines, such as IL-12. Together, these results indicate that this zinc metalloprotease is a potential tool to be further investigated for the treatment of inflammatory disorders involving TNF deregulation.
Assuntos
Bothrops , Venenos de Crotalídeos/metabolismo , Metaloendopeptidases/metabolismo , Simulação de Acoplamento Molecular , Proteínas de Répteis/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Zinco/metabolismo , Animais , Células Cultivadas , Venenos de Crotalídeos/química , Venenos de Crotalídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Metaloendopeptidases/química , Metaloendopeptidases/farmacologia , Camundongos Endogâmicos C57BL , Ligação Proteica , Conformação Proteica , Proteólise , Proteínas de Répteis/química , Proteínas de Répteis/farmacologia , Relação Estrutura-Atividade , Especificidade por Substrato , Receptores Toll-Like/agonistas , Receptores Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/química , Zinco/químicaRESUMO
In this paper, we describe the purification/characterization of BmooAi, a new toxin from Bothrops moojeni that inhibits platelet aggregation. The purification of BmooAi was carried out through three chromatographic steps (ion-exchange on a DEAE-Sephacel column, molecular exclusion on a Sephadex G-75 column, and reverse-phase HPLC chromatography on a C2/C18 column). BmooAi was homogeneous by SDS-PAGE and shown to be a single-chain protein of 15,000 Da. BmooAi was analysed by MALDI-TOF Spectrometry and revealed two major components with molecular masses 7824.4 and 7409.2 as well as a trace of protein with a molecular mass of 15,237.4 Da. Sequencing of BmooAi by Edman degradation showed two amino acid sequences: IRDFDPLTNAPENTA and ETEEGAEEGTQ, which revealed no homology to any known toxin from snake venom. BmooAi showed a rather specific inhibitory effect on platelet aggregation induced by collagen, adenosine diphosphate, or epinephrine in human platelet-rich plasma in a dose-dependent manner, whereas it had little or no effect on platelet aggregation induced by ristocetin. The effect on platelet aggregation induced by BmooAi remained active even when heated to 100°C. BmooAi could be of medical interest as a new tool for the development of novel therapeutic agents for the prevention and treatment of thrombotic disorders.
Assuntos
Bothrops/metabolismo , Venenos de Crotalídeos/isolamento & purificação , Venenos de Crotalídeos/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Bovinos , Cromatografia por Troca Iônica , Colágeno/farmacologia , Venenos de Crotalídeos/química , Epinefrina/farmacologia , Humanos , Dados de Sequência Molecular , Peso Molecular , Inibidores da Agregação Plaquetária/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Muscular necrosis is a serious consequence of Bothrops snake bites that may lead to permanent loss of tissue or function. Myonecrosis may be due to injury to blood vessels, destabilization and/or rupture of plasma membrane, and inflammatory mechanisms triggered by different proteins from the snake venom. In this work we describe the isolation and partial functional characterization of a myotoxin from B. alternatus snake venom. The myotoxin was isolated by a combination of ion exchange and gel filtration chromatography and displayed a molecular weight of approximately 15,000, as estimated by SDS-PAGE under reducing conditions. In non-reducing conditions a protein band of approximately 25,000 was also observed, suggesting that its native form is a homodimer. The myotoxin induced myonecrosis, but had no proteolytic and phospholipase A2 activities. The myotoxic activity was assessed on the basis of the histological and ultrastructural alterations induced by the toxin in the gastrocnemius skeletal muscle of Swiss mice. The toxin led to a series of drastic degenerative events characterized by extensive cellular destruction, loss of the arrangements of skeletal fibers, intense infiltration of inflammatory cells, fatty degeneration and hemorrhage. Electron microscopy analyses revealed that the myotoxin caused cell swelling, mitochondrial alterations and dilation of the sarcoplasmic reticulum, but did not affect the integrity of the muscle cell membranes. The myonecrosis caused by this toxin was related to the perturbation in the membrane permeability, intracellular alterations and inflammatory reaction.
