RESUMO
Hemorrhage induced by snake venom metalloproteases (SVMPs) results from proteolysis, capillary disruption, and blood extravasation. HF3, a potent SVMP of Bothrops jararaca, induces hemorrhage at pmol doses in the mouse skin. To gain insight into the hemorrhagic process, the main goal of this study was to analyze changes in the skin peptidome generated by injection of HF3, using approaches of mass spectrometry-based untargeted peptidomics. The results revealed that the sets of peptides found in the control and HF3-treated skin samples were distinct and derived from the cleavage of different proteins. Peptide bond cleavage site identification in the HF3-treated skin showed compatibility with trypsin-like serine proteases and cathepsins, suggesting the activation of host proteinases. Acetylated peptides, which originated from the cleavage at positions in the N-terminal region of proteins in both samples, were identified for the first time in the mouse skin peptidome. The number of peptides acetylated at the residue after the first Met residue, mostly Ser and Ala, was higher than that of peptides acetylated at the initial Met. Proteins cleaved in the hemorrhagic skin participate in cholesterol metabolism, PPAR signaling, and in the complement and coagulation cascades, indicating the impairment of these biological processes. The peptidomic analysis also indicated the emergence of peptides with potential biological activities, including pheromone, cell penetrating, quorum sensing, defense, and cell-cell communication in the mouse skin. Interestingly, peptides generated in the hemorrhagic skin promoted the inhibition of collagen-induced platelet aggregation and could act synergistically in the local tissue damage induced by HF3.
Assuntos
Bothrops , Venenos de Crotalídeos , Camundongos , Animais , Venenos de Crotalídeos/toxicidade , Venenos de Crotalídeos/química , Metaloproteases/química , Metaloproteases/metabolismo , Metaloproteases/farmacologia , Hemorragia/induzido quimicamente , Venenos de Serpentes/toxicidade , Venenos de Serpentes/química , Peptídeos , Bothrops/metabolismoRESUMO
Cancer is a serious health problem due to the complexity of establishing an effective treatment. The purpose of this work was to evaluate the activity of a triazaspirane as a migration and invasion inhibitor in PC3 prostatic tumor cells through a possible negative regulation of the FAK/Src signal transduction pathway and decreased secretion of metalloproteinases 2 and 9. Molecular docking analysis was performed using Moe 2008.10 software. Migration (wound-healing assay) and invasion (Boyden chamber assay) assays were performed. In addition, the Western blot technique was used to quantify protein expression, and the zymography technique was used to observe the secretion of metalloproteinases. Molecular docking showed interactions in regions of interest of the FAK and Src proteins. Moreover, the biological activity assays demonstrated an inhibitory effect on cell migration and invasion, an important suppression of metalloproteinase secretion, and a decrease in the expression of p-FAK and p-Src proteins in treated PC3 cells. Triazaspirane-type molecules have important inhibitory effects on the mechanisms associated with metastasis in PC3 tumor cells.
Assuntos
Neoplasias da Próstata , Masculino , Humanos , Células PC-3 , Simulação de Acoplamento Molecular , Linhagem Celular Tumoral , Neoplasias da Próstata/patologia , Processos Neoplásicos , Movimento Celular , Metaloproteases/farmacologia , Invasividade NeoplásicaRESUMO
Staphylococcus lugdunensis produces lugdulysin, a metalloprotease that may contribute to its virulence. This study aimed to evaluate the biochemical aspects of lugdulysin and investigate its effect on Staphylococcus aureus biofilms. The protease was isolated and characterized for its optimal pH and temperature, hydrolysis kinetics, and influence of metal cofactor supplementation. The protein structure was determined via homology modeling. The effect on S. aureus biofilms was assessed by the micromethod technique. The protease optimal pH and temperature were 7.0 and 37 °C, respectively. EDTA inhibited protease activity, confirming it as a metalloprotease. Lugdulysin activity was not recovered by divalent ion supplementation post-inhibition, and supplementation with divalent ions did not change enzymatic activity. The isolated enzyme was stable for up to 3 h. Lugdulysin significantly inhibited the formation and disrupted preestablished protein-matrix MRSA biofilm. This preliminary study indicates that lugdulysin has a potential role as a competition mechanism and/or modulation of staphylococcal biofilm.
Assuntos
Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Staphylococcus lugdunensis , Humanos , Staphylococcus aureus , Antibacterianos/farmacologia , Biofilmes , Metaloproteases/farmacologia , Peptídeo Hidrolases , Testes de Sensibilidade MicrobianaRESUMO
BmooMPα-I has kininogenase activity, cleaving kininogen releasing bradykinin and can hydrolyze angiotensin I at post-proline and aspartic acid positions, generating an inactive peptide. We evaluated the antihypertensive activity of BmooMPα-I in a model of two-kidney, one-clip (2K1C). Wistar rats were divided into groups: Sham, who underwent sham surgery, and 2K1C, who suffered stenosis of the right renal artery. In the second week of hypertension, we started treatment (Vehicle, BmooMPα-I and Losartan) for two weeks. We performed an electrocardiogram and blood and heart collection in the fourth week of hypertension. The 2K1C BmooMPα-I showed a reduction in blood pressure (systolic pressure: 131 ± 2 mmHg; diastolic pressure: 84 ± 2 mmHg versus 174 ± 3 mmHg; 97 ± 4 mmHg, 2K1C Vehicle, p < 0.05), improvement in electrocardiographic parameters (Heart Rate: 297 ± 4 bpm; QRS: 42 ± 0.1 ms; QT: 92 ± 1 ms versus 332 ± 6 bpm; 48 ± 0.2 ms; 122 ± 1 ms, 2K1C Vehicle, p < 0.05), without changing the hematological profile (platelets: 758 ± 67; leukocytes: 3980 ± 326 versus 758 ± 75; 4400 ± 800, 2K1C Vehicle, p > 0.05), with reversal of hypertrophy (left ventricular area: 12.1 ± 0.3; left ventricle wall thickness: 2.5 ± 0.2; septum wall thickness: 2.3 ± 0.06 versus 10.5 ± 0.3; 2.7 ± 0.2; 2.5 ± 0.04, 2K1C Vehicle, p < 0.05) and fibrosis (3.9 ± 0.2 versus 7.4 ± 0.7, 2K1C Vehicle, p < 0.05). We concluded that BmooMPα-I improved blood pressure levels and cardiac remodeling, having a cardioprotective effect.
Assuntos
Bothrops , Venenos de Crotalídeos , Hipertensão Renovascular , Animais , Ratos , Pressão Sanguínea , Venenos de Crotalídeos/farmacologia , Frequência Cardíaca , Hipertensão Renovascular/tratamento farmacológico , Metaloproteases/farmacologia , Ratos Wistar , Remodelação VentricularRESUMO
Extracellular proteolytic enzymes are produced by a variety of pathogenic microorganisms, and contribute to host colonization by modulating virulence. Here, we present a first characterization of leptolysin, a Leptospira metalloprotease of the pappalysin family identified in a previous exoproteomic study. Comparative molecular analysis of leptolysin with two other pappalysins from prokaryotes, ulilysin and mirolysin, reveals similarities regarding calcium, zinc, and arginine -binding sites conservation within the catalytic domain, but also discloses peculiarities. Variations observed in the primary and tertiary structures may reflect differences in primary specificities. Purified recombinant leptolysin of L. interrogans was obtained as a ~50 kDa protein. The protease exhibited maximal activity at pH 8.0 and 37°C, and hydrolytic activity was observed in the presence of different salts with maximum efficiency in NaCl. Substrate specificity was assessed using a small number of FRET peptides, and showed a marked preference for arginine residues at the P1 position. L. interrogans leptolysin proteolytic activity on proteinaceous substrates such as proteoglycans and plasma fibronectin was also evaluated. All proteins tested were efficiently degraded over time, confirming the protease´s broad-spectrum activity in vitro. In addition, leptolysin induced morphological alterations on HK-2 cells, which may be partially attributed to extracellular matrix (ECM) degradation. Hemorrhagic foci were observed in the dorsal skin of mice intradermally injected with leptolysin, as a plausible consequence of ECM disarray and vascular endothelium glycocalyx damage. Assuming that leptospiral proteases play an important role in all stages of the infectious process, characterizing their functional properties, substrates and mechanisms of action is of great importance for therapeutic purposes.
Assuntos
Leptospira , Metaloproteases , Animais , Arginina/metabolismo , Leptospira/química , Leptospira/metabolismo , Leptospirose , Metaloproteases/metabolismo , Metaloproteases/farmacologia , Camundongos , Peptídeo Hidrolases/metabolismoRESUMO
Snakebite envenoming is still a worrying health problem in countries under development, being recognized as a neglected disease by the World Health Organization. In Latin America, snakes from the genus Bothrops are widely spread and in Brazil, the Bothrops moojeni is a medically important species. The pharmacological effects of bothropic snake venoms include pain, blisters, bleeding, necrosis and even amputation of the affected limb. Snake venom metalloproteinases are enzymes abundantly present in venom from Bothrops snakes. These enzymes can cause hemorrhagic effects and lead to myonecrosis due to ischemia. Here, we present BmooMP-I, a new P-I class of metalloproteinase (this class only has the catalytic domain in the mature form) isolated from B. moojeni venom. This protein is able to express fibrinogenolytic and gelatinase activities, which play important roles in the prey's immobilization and digestion, and also induces weak hemorrhagic effect. The primary sequence assignment was done by a novel method, SEQUENCE SLIDER, which combines crystallographic, bioinformatics and mass spectrometry data. The high-resolution crystal structure reveals the monomeric assembly and the conserved metal binding site H141ExxH145xxG148xxH151 with the natural substitution Gly148Asp that does not interfere in the zinc coordination. The presence of a structural calcium ion on the surface of the protein, which can play an important role in the stabilization of hemorrhagic toxins, was observed in the BmooMP-I structure. Due to the relevant local and systemic effects of snake venom metalloproteinases, studies involving these proteins help to better understand the pathological effects of snakebite envenoming.
Assuntos
Bothrops/metabolismo , Venenos de Crotalídeos/enzimologia , Metaloproteases/química , Metaloproteases/farmacologia , Sequência de Aminoácidos , Animais , Cálcio/química , Cátions/química , Biologia Computacional , Venenos de Crotalídeos/química , Venenos de Crotalídeos/isolamento & purificação , Venenos de Crotalídeos/farmacologia , Cristalização , Cristalografia por Raios X , Bases de Dados de Proteínas , Fibrinogênio/metabolismo , Gelatina/metabolismo , Hemorragia/enzimologia , Espectrometria de Massas , Metaloproteases/isolamento & purificação , Camundongos , Modelos Moleculares , Alinhamento de Sequência , Análise de Sequência de Proteína , Pele/enzimologia , Pele/metabolismoRESUMO
Chronic wounds are a major health problem that cause millions of dollars in expenses every year. Among all the treatments used, active wound treatments such as enzymatic treatments represent a cheaper and specific option with a fast growth category in the market. In particular, bacterial and plant proteases have been employed due to their homology to human proteases, which drive the normal wound healing process. However, the use of these proteases has demonstrated results with low reproducibility. Therefore, alternative sources of proteases such as snake venom have been proposed. Here, we performed a functional mining of proteases from rattlesnakes (Crotalus ornatus, C. molossus nigrescens, C. scutulatus, and C. atrox) due to their high protease predominance and similarity to native proteases. To characterize Crotalus spp. Proteases, we performed different protease assays to measure and confirm the presence of metalloproteases and serine proteases, such as the universal protease assay and zymography, using several substrates such as gelatin, casein, hemoglobin, L-TAME, fibrinogen, and fibrin. We found that all our venom extracts degraded casein, gelatin, L-TAME, fibrinogen, and fibrin, but not hemoglobin. Crotalus ornatus and C. m. nigrescens extracts were the most proteolytic venoms among the samples. Particularly, C. ornatus predominantly possessed low molecular weight proteases (P-I metalloproteases). Our results demonstrated the presence of metalloproteases capable of degrading gelatin (a collagen derivative) and fibrin clots, whereas serine proteases were capable of degrading fibrinogen-generating fibrin clots, mimicking thrombin activity. Moreover, we demonstrated that Crotalus spp. are a valuable source of proteases that can aid chronic wound-healing treatments.
Assuntos
Venenos de Crotalídeos/enzimologia , Crotalus/metabolismo , Metaloproteases , Proteínas de Répteis , Serina Proteases , Ferimentos e Lesões/tratamento farmacológico , Animais , Fibrinólise/efeitos dos fármacos , Humanos , Metaloproteases/química , Metaloproteases/farmacologia , Reprodutibilidade dos Testes , Proteínas de Répteis/química , Proteínas de Répteis/farmacologia , Serina Proteases/química , Serina Proteases/farmacologia , Ferimentos e Lesões/metabolismo , Ferimentos e Lesões/patologiaRESUMO
Snake venom metalloproteinases (SVMPs) are key toxins involved in local inflammatory reactions after snakebites. This study aimed to investigate the effect of SVMP domains on the alterations in leukocyte-endothelium interactions in the microcirculation of mouse cremaster muscle. We studied three toxins: BnP1, a PI-toxin isolated from Bothrops neuwiedi venom, which only bears a catalytic domain; Jararhagin (Jar), a PIII-toxin isolated from Bothrops jararaca venom with a catalytic domain, as well as ECD-disintegrin and cysteine-rich domains; and Jar-C, which is produced from the autolysis of Jar and devoid of a catalytic domain. All these toxins induced an increase in the adhesion and migration of leukocytes. By inhibiting the catalytic activity of Jar and BnP1 with 1.10-phenanthroline (oPhe), leukocytes were no longer recruited. Circular dichroism analysis showed structural changes in oPhe-treated Jar, but these changes were not enough to prevent the binding of Jar to collagen, which occurred through the ECD-disintegrin domain. The results showed that the catalytic domain of SVMPs is the principal domain responsible for the induction of leukocyte recruitment and suggest that the other domains could also present inflammatory potential only when devoid of the catalytic domain, as with Jar-C.
Assuntos
Domínio Catalítico/fisiologia , Leucócitos/patologia , Metaloproteases/farmacologia , Venenos de Serpentes/enzimologia , Músculos Abdominais/irrigação sanguínea , Animais , Bothrops , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Endotélio/metabolismo , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Metaloproteases/química , Camundongos , MicrocirculaçãoRESUMO
Atroxlysin-III (Atr-III) was purified from the venom of Bothrops atrox. This 56-kDa protein bears N-linked glycoconjugates and is a P-III hemorrhagic metalloproteinase. Its cDNA-deduced amino acid sequence reveals a multidomain structure including a proprotein, a metalloproteinase, a disintegrin-like and a cysteine-rich domain. Its identity with bothropasin and jararhagin from Bothrops jararaca is 97% and 95%, respectively. Its enzymatic activity is metal ion-dependent. The divalent cations, Mg2+ and Ca2+, enhance its activity, whereas excess Zn2+ inhibits it. Chemical modification of the Zn2+-complexing histidine residues within the active site by using diethylpyrocarbonate (DEPC) inactivates it. Atr-III degrades plasma fibronectin, type I-collagen, and mainly the α-chains of fibrinogen and fibrin. The von Willebrand factor (vWF) A1-domain, which harbors the binding site for GPIb, is not hydrolyzed. Platelets interact with collagen via receptors for collagen, glycoprotein VI (GPVI), and α2ß1 integrin. Neither the α2ß1 integrin nor its collagen-binding A-domain is fragmented by Atr-III. In contrast, Atr-III cleaves glycoprotein VI (GPVI) into a soluble ~55-kDa fragment (sGPVI). Thereby, it inhibits aggregation of platelets which had been stimulated by convulxin, a GPVI agonist. Selectively, Atr-III targets GPVI antagonistically and thus contributes to the antithrombotic effect of envenomation by Bothrops atrox.
Assuntos
Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Venenos de Crotalídeos/enzimologia , Crotalinae , Metaloproteases/farmacologia , Glicoproteínas da Membrana de Plaquetas/biossíntese , Sequência de Aminoácidos , Animais , Crotalinae/metabolismo , Matriz Extracelular , Metaloproteases/química , Metaloproteases/genética , Metaloproteases/isolamento & purificação , Modelos Moleculares , Filogenia , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Glicoproteínas da Membrana de Plaquetas/química , Conformação Proteica , Proteólise , Relação Estrutura-AtividadeRESUMO
Manifestations of local tissue damage, such as hemorrhage and myonecrosis, are among the most dramatic effects of envenomation by viperid snakes. Snake venom metalloproteinases (SVMPs) of the P-III class are main players of the hemorrhagic effect due to their activities in promoting blood vessel disruption. Hemorrhagic Factor 3 (HF3), a P-III class SVMP from Bothrops jararaca, shows a minimum hemorrhagic dose of 240 fmol on rabbit skin. The aim of this study was to assess the effects of a sub-cytotoxic dose of HF3 (50â¯nM) on the proteomic profile of C2C12 differentiated cells (myotubes) in culture, and on the peptidomic profile of the culture supernatant. Quantitative proteomic analysis using stable-isotope dimethyl labeling showed differential abundance of various proteins including enzymes involved in oxidative stress and inflammation responses. Identification of peptides in the supernatant of HF3-treated myotubes revealed proteolysis and pointed out potential new substrates of HF3, including glyceraldehyde-3-phosphate dehydrogenase, and some damage-associated molecular patterns (DAMPs). These experiments demonstrate the subtle effects of HF3 on muscle cells and illustrate for the first time the early proteolytic events triggered by HF3 on myotubes. Moreover, they may contribute to future studies aimed at explaining the inflammation process, hemorrhage and myonecrosis caused by SVMPs. SIGNIFICANCE: One of the main features of viperid snake envenomation is myotoxicity at the bite site, which, in turn is often associated with edema, blistering and hemorrhage, composing a complex pattern of local tissue damage. In this scenario, besides muscle cells, other types of cells, components of the extracellular matrix and blood vessels may also be affected, resulting in an outcome of deficient muscle regeneration. The main venom components participating in this pathology are metalloproteinases and phospholipases A2. Muscle necrosis induced by metalloproteinases is considered as an indirect effect related to ischemia, due to hemorrhage resulted from damage to the microvasculature. The pathogenesis of local effects induced by Bothrops venoms or isolated toxins has been studied by traditional methodologies. More recently, proteomic and peptidomic approaches have been used to study venom-induced pathogenesis. Here, in order to investigate the role of metalloproteinase activity in local tissue damage, we asked whether the hemorrhagic metalloproteinase HF3, at sub-cytotoxic levels, could alter the proteome of C2C12 myotubes in culture, thereby providing an insight into the mechanisms for the development of myonecrosis. Our results from mass spectrometric analyses showed subtle, early changes in the cells, including differential abundance of some proteins and proteolysis in the culture supernatant. The data illustrate the potential ability of metalloproteinases to trigger early systemic responses progressing from local cells and up to tissues.
Assuntos
Venenos de Crotalídeos/farmacologia , Metaloproteases/farmacologia , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Proteômica , Animais , Bothrops , Linhagem Celular , Venenos de Crotalídeos/química , Metaloproteases/química , Camundongos , Fibras Musculares Esqueléticas/patologiaRESUMO
In this work, we examined the proteolytic and phospholipase A2 (PLA2) activities of venom from the opisthoglyphous colubrid Pseudoboa neuwiedii. Proteolytic activity (3 and 10⯵g of venom) was comparable to that of Bothrops neuwiedii venom but less than Bothrops atrox. This activity was inhibited by EDTA and 1,10-phenanthroline but only slightly affected (≤30% inhibition) by PMSF and AEBSF, indicating it was mediated by snake venom metalloproteinases (SVMPs). The pH and temperature optima for proteolytic activity were 8.0 and 37⯰C, respectively. The venom had no esterase activity, whereas PLA2 activity was similar to B. atrox, greater than B. neuwiedii but less than B. jararacussu. SDS-PAGE revealed venom proteins >100â¯kDa, 45-70â¯kDa, 21-24â¯kDa and ~15â¯kDa, and mass spectrometry of protein bands revealed SVMPs, cysteine-rich secretory proteins (CRISPs) and PLA2, but no serine proteinases. In gelatin zymography, the most active bands occurred at 65-68â¯kDa (seen with 0.05-0.25⯵g of venom). Caseinolytic activity occurred at 50-66â¯kDa and was generally weaker than gelatinolytic activity. RP-HPLC of venom yielded 15 peaks, five of which showed gelatinolytic activity; peak 7 was the most active and apparently contained a P-III class SVMP. The venom showed α-fibrinogenase activity, without affecting the ß and γ chains; this activity was inhibited by EDTA and 1,10-phenanthroline. The venom did not clot rat citrated plasma but reduced the rate and extent of coagulation after plasma recalcification. In conclusion, P. neuwiedii venom is highly proteolytic and could potentially affect coagulation in vivo by degrading fibrinogen via SVMPs.
Assuntos
Colubridae/fisiologia , Peptídeo Hidrolases/metabolismo , Fosfolipases A2/metabolismo , Proteínas de Répteis/metabolismo , Venenos de Serpentes/enzimologia , Animais , Anticoagulantes/química , Anticoagulantes/metabolismo , Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Brasil , Colubridae/crescimento & desenvolvimento , Esterases/química , Esterases/metabolismo , Esterases/farmacologia , Concentração de Íons de Hidrogênio , Masculino , Metaloproteases/química , Metaloproteases/metabolismo , Metaloproteases/farmacologia , Peso Molecular , Peptídeo Hidrolases/química , Peptídeo Hidrolases/farmacologia , Inibidores de Fosfolipase A2/farmacologia , Fosfolipases A2/química , Fosfolipases A2/farmacologia , Inibidores de Proteases/farmacologia , Proteólise/efeitos dos fármacos , Ratos Wistar , Proteínas de Répteis/antagonistas & inibidores , Proteínas de Répteis/química , Proteínas de Répteis/farmacologia , Venenos de Serpentes/metabolismo , Venenos de Serpentes/farmacologia , Especificidade por Substrato , TemperaturaRESUMO
Snake venom metalloproteinases (SVMPs) are predominant in viperid venoms, which provoke hemorrhage and affect hemostasis and thrombosis. P-I class enzymes consist only of a single metalloproteinase domain. Despite sharing high sequence homology, only some of them induce hemorrhage. They have direct fibrin(ogen)olytic activity. Their main biological substrate is fibrin(ogen), whose Aα-chain is degraded rapidly and independently of activation of plasminogen. It is important to understand their biochemical and physiological mechanisms, as well as their applications, to study the etiology of some human diseases and to identify sites of potential intervention. As compared to all current antiplatelet therapies to treat cardiovascular events, the SVMPs have outstanding biochemical attributes: (a) they are insensitive to plasma serine proteinase inhibitors; (b) they have the potential to avoid bleeding risk; (c) mechanistically, they are inactivated/cleared by α2-macroglobulin that limits their range of action in circulation; and (d) few of them also impair platelet aggregation that represent an important target for therapeutic intervention. This review will briefly highlight the structure-function relationships of these few direct-acting fibrinolytic agents, including, barnettlysin-I, isolated from Bothrops barnetti venom, that could be considered as potential agent to treat major thrombotic disorders. Some of their pharmacological advantages are compared with plasmin.
Assuntos
Fibrinolíticos/farmacologia , Hemostasia/efeitos dos fármacos , Metaloproteases/farmacologia , Venenos de Serpentes/enzimologia , Serpentes , Sequência de Aminoácidos , Animais , Domínio Catalítico , Fibrinolíticos/química , Fibrinolíticos/uso terapêutico , Humanos , Metaloproteases/química , Metaloproteases/uso terapêutico , Especificidade da Espécie , Relação Estrutura-AtividadeRESUMO
In this study, the apoptosis inducing effects of baltergin as well as its influence on cell adhesion and migration on muscles cells in vitro were studied. Morphological analysis made by scanning electron and phase contrast microscopy demonstrated typical futures of programmed cell death, apoptosis. This mechanism was confirmed by fluorescence staining, molecular analysis of endonuclease activity and increased mRNA expression level of two representative genes (p53 and bax). On the other hand, baltergin exert an inhibition effect on myoblast cell adhesion and migration in vitro probably through a mechanism that involves the interaction of this enzyme with cell integrins. In conclusion, our results suggest that the absence of appropriate extracellular matrix contacts triggers anoikis. Therefore, this is the first report that demonstrated the mechanism of programmed cell death triggered by baltergin, a PIII metalloprotease isolated from Bothrops alternatus venom, in a myoblast cell line.
Assuntos
Anoikis/efeitos dos fármacos , Bothrops/metabolismo , Venenos de Crotalídeos/enzimologia , Metaloproteases/farmacologia , Mioblastos/efeitos dos fármacos , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Venenos de Crotalídeos/isolamento & purificação , Venenos de Crotalídeos/farmacologia , Metaloproteases/isolamento & purificação , Camundongos , Camundongos Endogâmicos C3H , Microscopia Eletrônica de Varredura , Microscopia de Contraste de Fase , Mioblastos/citologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genética , Regulação para Cima , Proteína X Associada a bcl-2/biossíntese , Proteína X Associada a bcl-2/genéticaRESUMO
Breast cancer is a highly malignant carcinoma and remains the second leading cause of mortality among women. The antitumor effects of metalloproteinases and disintegrins from snake venom on various types of cancer cells have been investigated. In this study, we evaluated the antitumor and antiangiogenic effects on MDA-MB-231 human breast cancer cells and endothelial cells induced by Bothropoidin, a disintegrin-like metalloproteinase isolated from Bothrops pauloensis snake venom. At 24h after treatment at 100µg/mL, Bothropoidin exerted a moderate cytotoxic effect of 30% on MDA-MB-231 versus 10% cytotoxicity against MCF10A (a non-tumorigenic breast cell line), a significant difference that suggests a possible preference by this protein for targets in cancer cells. Early and late apoptosis of MDA-MB-231 was observed after Bothropoidin treatment (10µg/mL and 40µg/mL). Furthermore, this toxin inhibited not only the adhesion of MDA-MB-231 cells in a dose-dependent manner but also cell migration by approximately 45%. In addition, Bothropoidin decreased endothelial cells viability and adhesion in Matrigel and inhibited in vitro angiogenesis in Matrigel stimulated by bFGF, showing significantly fewer formed vessels. The results demonstrated that Bothropoidin has potent in vitro antitumor and antiangiogenic effect and represents a biotechnological tool for elucidating the antitumor effect of disintegrins-like metalloproteinases in cancer cells.
Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Bothrops , Venenos de Crotalídeos/enzimologia , Metaloproteases/farmacologia , Inibidores da Angiogênese/isolamento & purificação , Animais , Antineoplásicos/isolamento & purificação , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Metaloproteases/isolamento & purificaçãoRESUMO
Snake venom metalloproteinases (SVMPs) play key biological roles in prey immobilization and digestion. The majority of these activities depend on the hydrolysis of relevant protein substrates in the tissues. Hereby, we describe several isoforms and a cDNA clone sequence, corresponding to PII SVMP homologues from the venom of the Central American pit viper Bothriechis lateralis, which have modifications in the residues of the canonical sequence of the zinc-binding motif HEXXHXXGXXH. As a consequence, the proteolytic activity of the isolated proteins was undetectable when tested on azocasein and gelatin. These PII isoforms comprise metalloproteinase and disintegrin domains in the mature protein, thus belonging to the subclass PIIb of SVMPs. PII SVMP homologues were devoid of hemorrhagic and in vitro coagulant activities, effects attributed to the enzymatic activity of SVMPs, but induced a mild edema. One of the isoforms presents the characteristic RGD sequence in the disintegrin domain and inhibits ADP- and collagen-induced platelet aggregation. Catalytically-inactive SVMP homologues may have been hitherto missed in the characterization of snake venoms. The presence of such enzymatically-inactive homologues in snake venoms and their possible toxic and adaptive roles deserve further investigation.
Assuntos
Metaloproteases/isolamento & purificação , Peptídeos/isolamento & purificação , Venenos de Serpentes/química , Viperidae , Adulto , Sequência de Aminoácidos , Animais , Coagulação Sanguínea/efeitos dos fármacos , Caseínas/metabolismo , Clonagem Molecular , DNA Complementar/genética , Edema , Gelatina/metabolismo , Hemorragia , Humanos , Metaloproteases/química , Metaloproteases/genética , Metaloproteases/farmacologia , Camundongos , Modelos Moleculares , Peptídeos/química , Peptídeos/genética , Peptídeos/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Domínios Proteicos , Proteólise , Zinco/metabolismoRESUMO
Coagulopathies following snakebite are triggered by pro-coagulant venom toxins, in which metalloproteases play a major role in envenomation-induced coagulation disorders by acting on coagulation cascade, platelet function and fibrinolysis. Considering this relevance, here we describe the isolation and biochemical characterization of moojenactivase (MooA), a metalloprotease from Bothrops moojeni snake venom, and investigate its involvement in hemostasis in vitro. MooA is a glycoprotein of 85,746.22 Da, member of the PIIId group of snake venom metalloproteases, composed of three linked disulfide-bonded chains: an N-glycosylated heavy chain, and two light chains. The venom protease induced human plasma clotting in vitro by activating on both blood coagulation factors II (prothrombin) and X, which in turn generated α-thrombin and factor Xa, respectively. Additionally, MooA induced expression of tissue factor (TF) on the membrane surface of peripheral blood mononuclear cells (PBMC), which led these cells to adopt pro-coagulant characteristics. MooA was also shown to be involved with production of the inflammatory mediators TNF-α, IL-8 and MCP-1, suggesting an association between MooA pro-inflammatory stimulation of PBMC and TF up-regulation. We also observed aggregation of washed platelets when in presence of MooA; however, the protease had no effect on fibrinolysis. Our findings show that MooA is a novel hemostatically active metalloprotease, which may lead to the development of coagulopathies during B. moojeni envenomation. Moreover, the metalloprotease may contribute to the development of new diagnostic tools and pharmacological approaches applied to hemostatic disorders.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Bothrops/metabolismo , Coagulantes/farmacologia , Venenos de Crotalídeos/enzimologia , Fator Xa/metabolismo , Leucócitos/efeitos dos fármacos , Metaloendopeptidases/farmacologia , Metaloproteases/farmacologia , Protrombina/metabolismo , Tromboplastina/metabolismo , Adulto , Sequência de Aminoácidos , Animais , Coagulantes/isolamento & purificação , Venenos de Crotalídeos/isolamento & purificação , Venenos de Crotalídeos/farmacologia , Estabilidade Enzimática , Feminino , Humanos , Concentração de Íons de Hidrogênio , Mediadores da Inflamação/metabolismo , Cinética , Leucócitos/metabolismo , Masculino , Metaloendopeptidases/isolamento & purificação , Metaloproteases/isolamento & purificação , Pessoa de Meia-Idade , Temperatura , Adulto JovemRESUMO
This study describes the biochemical and functional characterization of a new metalloproteinase named BbMP-1, isolated from Bothrops brazili venom. BbMP-1 was homogeneous on SDS-PAGE, presented molecular mass of 22,933Da and pI 6.4. The primary structure was partially elucidated with high identity with others metalloproteinases from Viperidae venoms. The enzymatic activity on azocasein was evaluated in different experimental conditions (pH, temperature). A significant reduction in enzyme activity after exposure to chelators of divalent cations (EDTA), reducing agents (DTT), pH less than 5.0 or temperatures higher than 45 °C was observed. BbMP-1 showed activity on fibrinogen degrading Aα chain quickly and to a lesser extent the Bß chain. Also demostrated to be weakly hemorrhagic, presenting however, significant myotoxic and edematogenic activity. The in vitro activity of BbMP-1 against Plasmodium falciparum showed an IC50 of 3.2 ± 2.0 µg/mL. This study may help to understand the pathophysiological effects induced by this group of toxin and their participation in the symptoms observed in cases of snake envenomation. Moreover, this result is representative for this group of proteins and shows the biotechnological potential of BbMP-1 by the demonstration of its antiplasmodial activity.
Assuntos
Antiparasitários/farmacologia , Bothrops/metabolismo , Venenos de Crotalídeos/enzimologia , Metaloproteases/química , Plasmodium falciparum/efeitos dos fármacos , Animais , Antiparasitários/química , Antiparasitários/isolamento & purificação , Caseínas/química , Caseínas/metabolismo , Eletroforese em Gel de Poliacrilamida , Fibrinogênio/química , Fibrinogênio/metabolismo , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Masculino , Metaloproteases/isolamento & purificação , Metaloproteases/farmacologia , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , TemperaturaRESUMO
BACKGROUND: Malignant melanoma is a less common but highly dangerous form of skin cancer; it starts in the melanocytes cells found in the outer layer of the skin. Jararhagin toxin, a metalloproteinase isolated from Bothrops jararaca snake venom acts upon several biological processes, as inflammation, pain, platelet aggregation, proliferation and apoptosis, though not yet approved for use, may one day be employed to treat tumors. METHODS: B16F10 murine melanoma cells were treated with jararhagin (jara), a disintegrin-like metalloproteinase isolated from Bothrops jararaca snake venom, and jari (catalytic domain inactivated with 1,10-phenanthroline). Viability and adhesion cells were evaluated by MTT assay. The expression of caspase-3 active, phases of the cell cycle and apoptosis were assessed by flow cytometry. We analyze in vivo the effects of jararhagin on melanoma growth, apoptosis and metastasis. RESULTS: The tumor cells acquired round shapes, lost cytoplasmic expansions, formed clusters in suspension and decreased viability. Jari was almost 20 times more potent toxin than jara based on IC50 values and on morphological changes of the cells, also observed by scanning electron microscopy. Flow cytometry analysis showed 48.3% decrease in the proliferation rate of cells and 47.2% increase in apoptosis (jara) and necrosis (jari), following 1.2 µM jara and 0.1 µM jari treatments. Caspase-3 activity was increased whereas G0/G1 cell cycle phase was on the decline. Proliferative rate was assessed by staining with 5,6-carboxyfluoresceindiacetate succinimidyl ester, showing a significant decrease in proliferation at all concentrations of both toxins. CONCLUSIONS: In vivo treatment of the toxins was observed reduction in the incidence of nodules, and metastasis and antiproliferative inhibition capacity. This data strengthens the potential use jararhagin as an anti-neoplastic drug.
Assuntos
Antineoplásicos/uso terapêutico , Bothrops , Venenos de Crotalídeos/uso terapêutico , Melanoma/tratamento farmacológico , Metaloendopeptidases/uso terapêutico , Neoplasias Cutâneas/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Adesão Celular/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Venenos de Crotalídeos/isolamento & purificação , Venenos de Crotalídeos/farmacologia , Melanoma/metabolismo , Metaloendopeptidases/isolamento & purificação , Metaloendopeptidases/farmacologia , Metaloproteases/farmacologia , Metaloproteases/uso terapêutico , Camundongos , Agregação Plaquetária , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/uso terapêutico , Neoplasias Cutâneas/metabolismo , Veneno de Bothrops jararaca , Melanoma Maligno CutâneoRESUMO
Snake venom metalloproteinases (SVMPs) are major components in most viperid venoms that induce disturbances in the hemostatic system and tissues of animals envenomated by snakes. These disturbances are involved in human pathology of snake bites and appear to be essential for the capture and digestion of snake's prey and avoidance of predators. SVMPs are a versatile family of venom toxins acting on different hemostatic targets which are present in venoms in distinct structural forms. However, the reason why a large number of different SVMPs are expressed in some venoms is still unclear. In this study, we evaluated the interference of five isolated SVMPs in blood coagulation of humans, birds and small rodents. P-III class SVMPs (fractions Ic, IIb and IIc) possess gelatinolytic and hemorrhagic activities, and, of these, two also show fibrinolytic activity. P-I class SVMPs (fractions IVa and IVb) are only fibrinolytic. P-III class SVMPs reduced clotting time of human plasma. Fraction IIc was characterized as prothrombin activator and fraction Ic as factor X activator. In the absence of Ca2+, a firm clot was observed in chicken blood samples with fractions Ic, IIb and partially with fraction IIc. In contrast, without Ca2+, only fraction IIc was able to induce a firm clot in rat blood. In conclusion, functionally distinct forms of SVMPs were found in B. neuwiedi venom that affect distinct mechanisms in the coagulation system of humans, birds and small rodents. Distinct SVMPs appear to be more specialized to rat or chicken blood, strengthening the current hypothesis that toxin diversity enhances the possibilities of the snakes for hunting different prey or evading different predators. This functional diversity also impacts the complexity of human envenoming since different hemostatic mechanisms will be targeted by SVMPs accounting for the complexity of the response of humans to venoms.
Assuntos
Venenos de Crotalídeos/enzimologia , Hemostáticos/química , Metaloproteases/química , Proteínas de Répteis/química , Adaptação Biológica , Sequência de Aminoácidos , Animais , Bothrops , Galinhas , Venenos de Crotalídeos/isolamento & purificação , Venenos de Crotalídeos/farmacologia , Fator X/química , Feminino , Hemostáticos/isolamento & purificação , Hemostáticos/farmacologia , Humanos , Masculino , Metaloproteases/isolamento & purificação , Metaloproteases/farmacologia , Camundongos , Proteólise , Protrombina/química , Ratos , Proteínas de Répteis/isolamento & purificação , Proteínas de Répteis/farmacologia , Mordeduras de SerpentesRESUMO
The present study aimed to evaluate the proteolytic and biological activities of a new metalloproteinase from B. moojeni venom. The purification of BmooMP α -II was carried out through two chromatographic steps (ion-exchange and affinity). BmooMP α -II is a monomeric protein with an apparent molecular mass of 22.5 kDa on SDS-PAGE 14% under nonreducing conditions. The N-terminal sequence (FSPRYIELVVVADHGMFTKYKSNLN) revealed homology with other snake venom metalloproteinases, mainly among P-I class. BmooMP α -II cleaves A α -chain of fibrinogen followed by B ß -chain, and does not show any effect on the γ -chain. Its optimum temperature and pH for the fibrinogenolytic activity were 30-50°C and pH 8, respectively. The inhibitory effects of EDTA and 1,10-phenantroline on the fibrinogenolytic activity suggest that BmooMP α -II is a metalloproteinase. This proteinase was devoid of haemorrhagic, coagulant, or anticoagulant activities. BmooMP α -II caused morphological alterations in liver, lung, kidney, and muscle of Swiss mice. The enzymatically active protein yet inhibited collagen, ADP, and ristocetin-induced platelet aggregation in a concentration-dependent manner. Our results suggest that BmooMP α -II contributes to the toxic effect of the envenomation and that more investigations to elucidate the mechanisms of inhibition of platelet aggregation may contribute to the studies of snake venom on thrombotic disorders.