RESUMO
This study investigates the valorization potential of yellowfin tuna (Thunnus albacares) tails to produce high-value commercial products. Firstly, the tuna tails were placed in a perforated stainless-steel cylinder, and hydraulic pressure was applied to separate the skin from the muscle in the tails. The extracted muscle was then utilized as a nitrogen source for the growth of the proteolytic enzyme producer Bacillus subtilis, while the skins were employed for gelatin extraction. The proteases from B. subtilis were partially purified and used to produce antioxidant peptides from the obtained gelatin. The gelatin formed a gel upon cooling, with gelling and melting temperatures of 16 °C and 22 °C, respectively, and a Bloom strength of approximately 160. Response Surface Methodology (RSM) was employed to determine the optimal hydrolysis conditions to achieve the highest antioxidant activity (35.96% measured as DPPH radical scavenging activity), which were 50 °C and 6.5 IU of enzyme. The findings emphasize the importance of an integrated approach to maximize the value of tuna by-products, promoting sustainability within the framework of a circular bioeconomy. Overall, these results contribute to the efficient utilization of tuna by-products, waste reduction, and enhanced economic viability of the tuna industry.
RESUMO
This study presents an approach that utilizes low-value agro-industrial by-products as culture media for producing high-value proteolytic enzymes. The objective was to assess the impact of six agro-industrial by-products as culture media on the production of proteolytic enzymes. Bacillus subtilis strains, confirmed through comprehensive biochemical, morphological, and molecular analyses, were isolated and identified. Enzymatic activity was evaluated using azocasein and casein substrates, and the molecular sizes of the purified extract components were determined. The results demonstrated that the isolated bacteria exhibited higher metabolic and enzymatic activity when cultured in media containing 1 % soybean oil cake or feather meal. Furthermore, higher concentrations of the culture media were found to hinder the production of protease. Optimal protease synthesis on soybean oil cake and feather meal media was achieved after 4 days, using both the azocasein and casein methods. Semi-purification of the enzymatic extract obtained from Bacillus subtilis in feather meal and soybean oil cake resulted in a significant increase in azocaseinolytic and caseinolytic activities. Gel electrophoresis analysis revealed multiple bands in the fractions with the highest enzymatic activity in soybean oil cake, indicating the presence of various enzymes with varying molecular sizes. These findings highlight the potential of utilizing low-value agro-industrial by-products as efficient culture media for the sustainable and economically viable production of proteolytic enzymes with promising applications in various industries.
RESUMO
BACKGROUND: In recent years, the rising global demand for cheese, the high cost and limited supply of calf rennet, and consumer choices have increased research into new alternatives to animal or recombinant chymosins for cheese making. Plant proteases with caseinolytic activity (CA) and milk-clotting activity (MCA) have been proposed as alternatives for milk clotting to obtain artisanal cheeses with new organoleptic properties. They have been named vegetable rennets (vrennets). The aim of this study was to evaluate the performance of two Solanum tuberosum aspartic proteases (StAP1 and StAP3) as vrennets for cheese making and to obtain a statistical model that could predict and optimize their enzymatic activity. RESULTS: To optimize the CA and MCA activities, a response surface methodology was used. Maximum values of CA and MCA for both enzymes were found at pH 5.0 and 30-35 °C. Analysis of the degradation of casein subunits showed that it is possible to tune the specificity of both enzymes by changing the pH. At pH 6.5, the αS - and ß- subunit degradation is reduced while conserving a significant MCA. CONCLUSION: The statistical models obtained in this work showed that StAP1 and StAP3 exert CA and MCA under pH and temperature conditions compatible with those used for cheese making. The casein subunit degradation percentages obtained also allowed us to select the best conditions for the degradation of the κ-casein subunit by StAPs. These results suggest that StAP1 and StAP3 are good candidates as vrennets for artisan cheese making. © 2023 Society of Chemical Industry.
Assuntos
Queijo , Solanum tuberosum , Animais , Solanum tuberosum/metabolismo , Queijo/análise , Caseínas/química , Quimosina/análise , Ácido Aspártico Endopeptidases , Peptídeo Hidrolases/metabolismo , Leite/químicaRESUMO
The interest for food-derived bioactive peptides, either from common or unconventional sources, has increased due to their potential therapeutic effect against a wide range of diseases. The study of such bioactive peptides using conventional methods is a long journey, expensive and time-consuming. Hence, bioinformatic approaches, which can not only help to predict the formation of bioactive peptides from any known protein source, but also to analyze the protein structure/function relationship, have gained a new meaning in this scientific field. Therefore, this review aims to provides an overview of conventional characterization methods and the most recent advances in the field of in silico approaches for predicting and screening promising food-derived bioactive peptides.
RESUMO
Proteolytic enzymes are widely distributed in nature, playing essential roles in important biological functions. Recently, the use of plant proteases at the industrial level has mainly increased in the food industry (e.g., cheesemaking, meat tenderizing, and protein hydrolysate production). Current technological and scientific advances in the detection and characterization of proteolytic enzymes have encouraged the search for new natural sources. Thus, this work aimed to explore the milk-clotting and proteolytic properties of different tissues of Vallesia glabra. Aqueous extracts from the leaves, fruits, and seeds of V. glabra presented different protein profiles, proteolytic activity, and milk-clotting activity. The milk-clotting activity increased with temperature (30-65 °C), but this activity was higher in leaf (0.20 MCU/mL) compared with that in fruit and seed extracts (0.12 and 0.11 MCU/mL, respectively) at 50 °C. Proteolytic activity in the extracts assayed at different pH (2.5-12.0) suggested the presence of different types of active proteases, with maximum activity at acidic conditions (4.0-4.5). Inhibitory studies indicated that major activity in V. glabra extracts is related to cysteine proteases; however, the presence of serine, aspartic, and metalloproteases was also evident. The hydrolytic profile of caseins indicated that V. glabra leaves could be used as a rennet substitute in cheesemaking, representing a new and promising source of proteolytic enzymes.
Assuntos
Apocynaceae/enzimologia , Leite/química , Peptídeo Hidrolases/química , Folhas de Planta/enzimologia , Proteínas de Plantas/química , Proteólise , Sementes/enzimologia , Animais , Concentração de Íons de HidrogênioRESUMO
Pleurotus albidus, a naturally growing species in the Amazon region, has been considered a promising source of milk-clotting proteases. The production of such enzymes using lignocellulosic residues is a sustainable alternative to replace mammalian rennet. The application of P. albidus milk-clotting proteases in cheese making has not yet been reported in the scientific literature. The aim of this study was to characterize the milk-clotting proteases of P. albidus and use these enzymes in the production of Minas frescal cheese. For the production of coagulating proteases, the mushroom was grown in açaí seeds supplemented with rice bran (10%, w/w). The parameters affecting the production of coagulant, such as inoculum size, fermentation time, initial pH of cultivation medium and age of the inoculum were evaluated. The coagulant extract obtained under optimal production conditions was evaluated for optimal pH and temperature, pH and temperature stability, effect of ions and inhibitors. Significant production of coagulating proteases was obtained under the following conditions: inoculum size (2.5%), fermentation time (10 days), initial pH of the cultivation medium (6), and inoculum age (10 days). The coagulant exhibited significant catalytic activity in pH 5.0 at 55°C, with stability at 45°C and was completely inhibited by iodoacetic acid. The milk-clotting proteases of P. albidus were efficient for making Minas frescal cheese that presented 55.0% of moisture, 20.0% of lipids and 17.20% of protein. Pleurotus albidus is a potential source of milk-clotting proteases that can be applied in dairy industry for production of fresh Minas frescal cheese.(AU)
Assuntos
Pleurotus/química , Agentes de Coagulação , Peptídeo Hidrolases/análise , Queijo/análiseRESUMO
Pleurotus albidus, a naturally growing species in the Amazon region, has been considered a promising source of milk-clotting proteases. The production of such enzymes using lignocellulosic residues is a sustainable alternative to replace mammalian rennet. The application of P. albidus milk-clotting proteases in cheese making has not yet been reported in the scientific literature. The aim of this study was to characterize the milk-clotting proteases of P. albidus and use these enzymes in the production of Minas frescal cheese. For the production of coagulating proteases, the mushroom was grown in açaí seeds supplemented with rice bran (10%, w/w). The parameters affecting the production of coagulant, such as inoculum size, fermentation time, initial pH of cultivation medium and age of the inoculum were evaluated. The coagulant extract obtained under optimal production conditions was evaluated for optimal pH and temperature, pH and temperature stability, effect of ions and inhibitors. Significant production of coagulating proteases was obtained under the following conditions: inoculum size (2.5%), fermentation time (10 days), initial pH of the cultivation medium (6), and inoculum age (10 days). The coagulant exhibited significant catalytic activity in pH 5.0 at 55°C, with stability at 45°C and was completely inhibited by iodoacetic acid. The milk-clotting proteases of P. albidus were efficient for making Minas frescal cheese that presented 55.0% of moisture, 20.0% of lipids and 17.20% of protein. Pleurotus albidus is a potential source of milk-clotting proteases that can be applied in dairy industry for production of fresh Minas frescal cheese.
Assuntos
Agentes de Coagulação , Peptídeo Hidrolases/análise , Pleurotus/química , Queijo/análiseRESUMO
The fungal genus Pyrenochaetopsis has received particular attention because of its different lifestyles, such as numerous plant pathogenic, saprophytic, and endophytic species. Its ability to infect plant cells relies heavily upon secreted peptidases. Here, we investigated the biochemical properties and catalytic specificity of a new serine peptidase secreted by the filamentous fungus Pyrenochaetopsis sp. We found that while this neutral serine peptidase displayed optimal activity at a pH of 7.0 and temperature of 45 °C, it tolerated a wide range of pH conditions and temperatures lower than 45 °C. Its peptidase activity was depressed by some metallic ions (such as aluminum, cobalt, and copper (II) chloride) and enhanced by others (such as sodium, lithium, magnesium, potassium, calcium, and manganese). Lastly, the enzyme showed the greatest specificity for non-polar amino acids, particularly leucine and isoleucine, and moderate specificity for basic and neutral polar amino acids. It displayed the least specificity for acidic residues.
Assuntos
Ascomicetos/enzimologia , Biocatálise , Serina Proteases/química , Serina Proteases/metabolismo , Inibidores Enzimáticos/farmacologia , Guanidina/farmacologia , Metais/farmacologia , Inibidores de Serina Proteinase/farmacologia , Especificidade por Substrato , Tensoativos/farmacologia , Temperatura , Ureia/farmacologiaRESUMO
The aim of this study was to determine the antifungal activity of the proteolytic fraction P1G10 from Vasconcellea cundinamarcencis (ex-Carica candamarcensis) against Botrytis cinerea, the causative agent of pre- and postharvest damaging disease in fruit and vegetables. The survival of B. cinerea at different concentrations of P1G10 showed that 1â¯mg/mL inhibited 50% of mycelium growth after 72â¯h incubation. The kinetic of growth inhibition fits the Weibull distribution function, and the data was confirmed by the IC50 survival assay. The study shows that P1G10 inhibits conidia germination and germ tube elongation of B. cinerea relative to untreated conidia. Hypersensitivity to cell wall-perturbing agents (Calcofluor white and Congo red) was observed in mycelium cells treated with P1G10. In addition, P1G10 exhibited inhibitory effect on the adhesion of conidia, provoked alterations in membrane integrity and induced production of reactive oxygen species accompanied by cellular damage. Our results highlight the effect of P1G10 on mycelium growth, cell wall alterations, membrane integrity and adhesion. P1G10 emerges as promising antifungal to control disease causing agents in the food agroindustry.
Assuntos
Botrytis/efeitos dos fármacos , Carica/química , Microbiologia de Alimentos , Látex/química , Extratos Vegetais/farmacologia , Antifúngicos/farmacologia , Parede Celular/efeitos dos fármacos , Frutas/microbiologia , Micélio/crescimento & desenvolvimento , Proteólise , Esporos Fúngicos/efeitos dos fármacosRESUMO
La adhesión entre las resinas hidrófilas y la dentina se encuentra sometida a una degradación permanente cuya intensidad aumenta en función del tiempo transcurrido. Esto es producto de la actividad de las metaloproteinasas, de las catepsinas y otras enzimas colagenolíticas, responsables de la destrucción paulatina de las fibras colágenas de la capa híbrida. La mayoría de las estrategias para inhibir estas enzimas han sido ensayos de laboratorio, mientras que las investigaciones clínicas son escasas. En este trabajo se analiza la literatura relacionada con las estrategias sugeridas para prevenir la degradación del colágeno de la capa híbrida en la interfaz resina-dentina (AU)
The bonds between hidrophylic resins and dentin are subjected to a time-dependent collagenolytic degradation. Endogenous enzymes such as matrix metalloproteinases, catepsins and other enzymes are responsible for the hybrid layer destruction. The majority of strategies developed to inhibit these enzymes are laboratory evaluations while clinical studies are scarce. This review examines the literature related to the strategies suggested to prevent collagen degradation of the hybrid layer in resin-dentin interfaces (AU)
Assuntos
Resinas Compostas , Adesivos Dentinários , Peptídeo Hidrolases , Clorexidina , Colágeno , Ácido Edético , MetacrilatosRESUMO
Multi-domain inhibitors capable to block the activity of different classes of proteases are not very common in nature. However, these kinds of molecules are attractive systems for biomedical or biotechnological applications, where two or more different targets need to be neutralized. SmCI, the Sabellastarte magnifica Carboxypeptidase Inhibitor, is a tri-domain BPTI-Kunitz inhibitor capable to inhibit serine proteases and A-like metallocarboxypeptidases. The BPTI-Kunitz family of proteins includes voltage gated channel blockers and inhibitors of serine proteases. SmCI is therefore, the only BPTI-Kunitz protein capable of inhibiting metallocarboxypeptidases. The X-ray structure of the SmCI-carboxypeptidase A complex previously obtained by us, revealed that this enzyme interacts with SmCI N-tail. In the complex, the reactive loops for serine protease inhibition remain fully exposed to the solvent in each domain, suggesting SmCI can simultaneously interact with multiple serine proteases. The twofold goals of this study were: i) to establish serine proteases-SmCI binding stoichiometry, given that the inhibitor is comprised of three potential binding domains; and ii) to determine whether or not SmCI can simultaneously bind both classes of enzymes, to which it binds individually. Our experimental approach included a variety of techniques for the study of protein-protein interactions, using as model enzymes pancreatic trypsin, elastase and carboxypeptidase A. In particular, we combined information obtained from gel filtration chromatography, denaturing electrophoresis, nuclear magnetic resonance spectroscopy and enzyme inhibition assays. Our results show that SmCI is able to bind three trypsin molecules under saturating conditions, but only one elastase interacts with the inhibitor. Additionally, we demonstrated that SmCI can bind serine proteases and carboxypeptidases at the same time (at least in the ratio 1:1:1), becoming the first protease inhibitor that simultaneously blocks these two mechanistic classes of enzymes.
Assuntos
Carboxipeptidases/antagonistas & inibidores , Carboxipeptidases/metabolismo , Poliquetos/enzimologia , Inibidores de Proteases/química , Serina Proteases/metabolismo , Animais , Cinética , Espectroscopia de Ressonância Magnética , Tripsina/química , Tripsina/metabolismoRESUMO
This study evaluated the use of three solid brewery wastes: brewer's spent grain, hot trub and residual brewer's yeast, as alternative media for the cultivation of lactic acid bacteria to evaluate their potential for proteolytic enzyme production. Initially, a mixture experimental design was used to evaluate the effect of each residue, as well as different mixtures (with the protein content set at 4%) in the enzyme production. At predetermined intervals, the solid and liquid fractions were separated and the extracellular proteolytic activity was determined. After selecting the best experimental conditions, a second experiment, factorial experimental design, was developed in order to evaluate the protein content in the media (1 to 7%) and the addition of fermentable sugar (glucose, 1 to 7%). Among the wastes, residual yeast showed the highest potential for the production of extracellular enzymes, generating a proteolytic extract with 2.6 U/mL in 3 h. However, due to the low content of the fermentable sugars in the medium, the addition of glucose also had a positive effect, increasing the proteolytic activity to 4.9 U/mL. The best experimental conditions of each experimental design were reproduced for comparison, and the enzyme content was separated by ethanol precipitation. The best medium produced a precipitated protein with proteolytic activity of 145.5 U/g.
RESUMO
Protease-activated receptors (PARs) are G protein-coupled receptors, which are activated by proteolytical cleavage of the amino-terminus and act as sensors for extracellular proteases. We hypothesized that PAR-1 and PAR-2 can be modulated by inflammatory stimulus in human dental pulp cells. PAR-1 and PAR-2 gene expression in human pulp tissue and MDPC-23 cells were analyzed by quantitative polymerase chain reaction. Monoclonal PAR-1 and PAR-2 antibodies were used to investigate the cellular expression of these receptors using Western blot, flow cytometry, and confocal microscopy in MDPC-23 cells. Immunofluorescence assays of human intact and carious teeth were performed to assess the presence of PAR-1 and PAR-2 in the dentin-pulp complex. The results show for the first time that human odontoblasts and MDPC-23 cells constitutively express PAR-1 and PAR-2. PAR-2 activation increased significantly the messenger RNA expression of matrix metalloproteinase (MMP)-2, MMP-9, MMP-13, and MMP-14 in MDPC-23 cells ( P < 0.05), while the expression of these enzymes decreased significantly in the PAR-1 agonist group ( P < 0.05). The high-performance liquid chromatography and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry analysis showed the presence of MMP-13 activity cleaving PAR-1 at specific, noncanonical site TLDPRS42↓F43LL in human dental pulp tissues. Also, we detected a presence of a trypsin-like activity cleaving PAR-2 at canonical site SKGR20↓S21LIGRL in pulp tissues. Confocal microscopy analysis of human dentin-pulp complex showed intense positive staining of PAR-1 and PAR-2 in the odontoblast processes in dentinal tubules of carious teeth compared to intact ones. The present results support the hypothesis of activation of the upregulated PAR-1 and PAR-2 by endogenous proteases abundant during the inflammatory response in dentin-pulp complex.
Assuntos
Polpa Dentária/citologia , Odontoblastos/enzimologia , Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Adulto , Western Blotting , Cromatografia Líquida de Alta Pressão , Citometria de Fluxo , Humanos , Inflamação/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Microscopia Confocal , RNA Mensageiro/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Regulação para CimaRESUMO
Human kallikrein 6 (KLK6) is highly expressed in the central nervous system and with elevated level in demyelinating disease. KLK6 has a very restricted specificity for arginine (R) and hydrolyses myelin basic protein, protein activator receptors and human ionotropic glutamate receptor subunits. Here we report a previously unreported activity of KLK6 on peptides containing clusters of basic amino acids, as in synthetic fluorogenic peptidyl-Arg-7-amino-4-carbamoylmethylcoumarin (peptidyl-ACC) peptides and FRET peptides in the format of Abz-peptidyl-Q-EDDnp (where Abz=ortho-aminobenzoic acid and Q-EDDnp=glutaminyl-N-(2,4-dinitrophenyl) ethylenediamine), in which pairs or sequences of basic amino acids (R or K) were introduced. Surprisingly, KLK6 hydrolyzed the fluorogenic peptides Bz-A-R↓R-ACC and Z-R↓R-MCA between the two R groups, resulting in non-fluorescent products. FRET peptides containing furin processing sequences of human MMP-14, nerve growth factor (NGF), Neurotrophin-3 (NT-3) and Neurotrophin-4 (NT-4) were cleaved by KLK6 at the same position expected by furin. Finally, KLK6 cleaved FRET peptides derived from human proenkephalin after the KR, the more frequent basic residues flanking enkephalins in human proenkephalin sequence. This result suggests the ability of KLK6 to release enkephalin from proenkephalin precursors and resembles furin a canonical processing proteolytic enzyme. Molecular models of peptides were built into the KLK6 structure and the marked preference of the cut between the two R of the examined peptides was related to the extended conformation of the substrates.
Assuntos
Calicreínas/metabolismo , Cinética , Peptídeo Hidrolases/metabolismo , Peptídeos/química , Aminoácidos Básicos/química , Aminoácidos Básicos/genética , Encefalinas/química , Encefalinas/metabolismo , Transferência Ressonante de Energia de Fluorescência , Furina/química , Furina/metabolismo , Humanos , Hidrólise , Calicreínas/química , Calicreínas/genética , Metaloproteinase 14 da Matriz/química , Metaloproteinase 14 da Matriz/metabolismo , Modelos Moleculares , Fator de Crescimento Neural/química , Fator de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/metabolismo , Neurotrofina 3 , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Peptídeos/metabolismo , Conformação Proteica , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Proteólise , Especificidade por SubstratoRESUMO
Submerged and solid-state bioprocesses have been extensively explored worldwide and employed in a number of important studies dealing with microbial cultivation for the production of enzymes. The development of these production technologies has facilitated the generation of new enzyme-based products with applications in pharmaceuticals, food, bioactive peptides, and basic research studies, among others. The applicability of microorganisms in biotechnology is potentiated because of their various advantages, including large-scale production, short time of cultivation, and ease of handling. Currently, several studies are being conducted to search for new microbial peptidases with peculiar biochemical properties for industrial applications. Bioprospecting, being an important prerequisite for research and biotechnological development, is based on exploring the microbial diversity for enzyme production. Limited information is available on the production of specific proteolytic enzymes from bacterial and fungal species, especially on the subgroups threonine and glutamic peptidases, and the seventh catalytic type, nonhydrolytic asparagine peptide lyase. This gap in information motivated the present study about these unique biocatalysts. In this study, the biochemical and biotechnological aspects of the seven catalytic types of proteolytic enzymes, namely aspartyl, cysteine, serine, metallo, glutamic, and threonine peptidase, and asparagine peptide lyase, are summarized, with an emphasis on new studies, production, catalysis, and application of these enzymes.
Assuntos
Bactérias/enzimologia , Proteínas de Bactérias , Proteínas Fúngicas , Fungos/enzimologia , Peptídeo Hidrolases , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Catálise , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Peptídeo Hidrolases/biossíntese , Peptídeo Hidrolases/química , Peptídeo Hidrolases/isolamento & purificaçãoRESUMO
Secretome analysis can be described as a subset of proteomics studies consisting in the analysis of the molecules secreted by cells or tissues. Dengue virus (DENV) infection can lead to a broad spectrum of clinical manifestations, with the severe forms of the disease characterized by hemostasis abnormalities and liver injury. The hepatocytes are a relevant site of viral replication and a major source of plasma proteins. Until now, we had limited information on the small molecules secreted by hepatic cells after infection by DENV. In the present study, we analysed a fraction of the secretome of mock- and DENV-infected hepatic cells (HepG2 cells) containing molecules with <10kDa, using different proteomic approaches. We identified 175 proteins, with 57 detected only in the samples from mock-infected cells, 59 only in samples from DENV-infected cells, and 59 in both conditions. Most of the peptides identified were derived from proteins larger than 10kDa, suggesting a proteolytic processing of the secreted molecules. Using in silico analysis, we predicted consistent differences between the proteolytic processing occurring in mock and DENV-infected samples, raising, for the first time, the hypothesis that differential proteolysis of secreted molecules would be involved in the pathogenesis of dengue. BIOLOGICAL SIGNIFICANCE: Since the liver, one of the targets of DENV infection, is responsible for producing molecules involved in distinct biological processes, the identification of proteins and peptides secreted by hepatocytes after infection would help to a better understanding of the physiopathology of dengue. Proteomic analyses of molecules with <10kDa secreted by HepG2 cells after infection with DENV revealed differential proteolytic processing as an effect of DENV infection.
Assuntos
Vírus da Dengue , Fígado/metabolismo , Proteólise , Proteômica/métodos , Dengue/metabolismo , Células Hep G2 , Hepatócitos/química , Hepatócitos/virologia , Humanos , Fígado/virologiaRESUMO
The snake venom metalloproteinases (SVMPs) are abundant enzymes in the venoms of viper snakes and responsible for most of the symptoms of envenoming. Their actions are related to the hydrolysis of extracellular matrix components, plasma proteins and hydrolysis or binding to cell surface proteins resulting in the activation or inhibition of their activity. In this work, the main objective was to analyze the implications of the structural and functional diversity of B. neuwiedi venom SVMPs, evaluating their effects on mammal’s hemostatic disorders and against prey or predator of snakes. Initially, we elucidated the partial amino acid sequence of class P-I SVMPs (which presented only 78% similarity to each other), in addition to obtaining the crystal of one. Subsequently we identified different degrees of hemorrhage induced by class P-III SVMPs (with hemorrhagic minimum doses of: 3.08 μg for Ic, 5.67 μg for IIb and 7.55 μg for IIc). In addition, different actions of the SVMPs tested (both P-I and P-III) were observed against extracellular matrix components (adhesion and cleavage) and against endothelial cells (adhesion and viability). It was also observed in the coagulation experiment, the presence of SVMPs that specifically acted on mammalian blood, as well as others that acted only on bird blood, demonstrating a selective action of SVMPs. With this we conclude that there must be the contribution of other factors, besides the structural domains, to the functional diversity of SVMPs. Therefore, the functional diversity of SVMPs contributed to the adaptive role of snake venoms. Key words: Bothrops, metalloproteinases, hemostasis, proteolytic e
As metaloproteinases de venenos de serpentes (SVMPs) são enzimas abundantes em venenos de espécies da família Viperidae e responsáveis por grande parte dos sintomas do envenenamento. Sua ação está relacionada com a proteólise dos componentes da matriz extracelular, proteínas plasmáticas e também com hidrólise ou ligação a proteínas de superfície celular induzindo a ativação ou inibição da atividade das mesmas. Neste trabalho, o principal objetivo foi analisar as implicações da diversidade estrutural e funcional das SVMPs do veneno de B. neuwiedi, avaliando seus efeitos em distúrbios hemostáticos de mamíferos e frente a presa ou predador de serpentes. Inicialmente, elucidamos a sequência parcial de aminoácidos das SVMPs de classe P-I (que apresentaram apenas 78% de similaridade entre si), além de obter o cristal de uma. Posteriormente identificamos diferentes graus de hemorragia induzidos pelas SVMPs de classe P-III (com doses mínimas hemorrágicas de: 3,08 μg para Ic; 5,67 μg para IIb e 7,55 μg para IIc). Além disso, foram observadas diferentes ações das SVMPs testadas (tanto P-I quanto PIII) frente aos componentes de matriz extracelular (adesão e clivagem) e frente às células endoteliais (adesão e viabilidade). Observou-se também, no experimento de coagulação, a presença de SVMPs que atuaram especificamente no sangue de mamífero, assim como outras que atuaram apenas em sangue de ave, demonstrando uma ação seletiva das SVMPs. Deste modo, concluímos que deve haver a contribuição de outros fatores, além dos domínios estruturais, para a diversidade funcional de SVMPs. Não obstante, a diversidade funcional das SVMPs contribuiu para o papel adaptativo dos venenos ofídicos.
RESUMO
Jellyfishes are amongst the most familiar of venomous marine animals. Jellyfish encounters with humans are common, although subsequent envenomation has varying toxicity ranging from usually mild symptoms to sometimes lethal consequences. Jellyfish venoms are of biotechnological importance, with toxins displaying antimicrobial, analgesic and anti-tumor activities. Furthermore, proteolytic enzymes have also been described. Recently, the proteomic profile of putative toxins isolated from nematocysts of the hydromedusae Olindias sambaquiensis was reported, which included protease and phospholipase enzymes. Here we present the results of a study to experimentally confirm enzymatic activity in extract of O. sambaquiensis tentacles. Specimens of O. sambaquiensis were collected at the coast of São Sebastião, SP, Brazil. A procedure to generate tentacle extract was standardised, yielding high quantities of proteins of different molecular masses. The extract was assayed for the presence of metalloproteases, serine proteases and phospholipase A2 activities using substrates that cover a wide range of each class of these enzymes and also specific substrates for metalloproteases ADAMs and MMPs. The extract showed activity towards all substrates confirming previous proteomic evidence for the presence of these enzymes. In addition, proteolytic activity was also detected by zymography assays on casein and gelatin. However, when comparing these activities to snake venoms, which are rich in proteases and phospholipases, the metalloprotease activity detected was lower than the high levels observed in Bothrops jararaca venom, but levels of serine protease and phospholipase A2 enzyme activity was comparable to the one observed in venoms of Bothrops snakes, known for its strong anti-coagulant and myotoxic activities due to serine proteases and phospholipases A2 molecules, respectively. These data validate the functional properties of proteins characterised previously by proteomics and provide a better understanding of the toxin arsenal of this underexplored venomous animal, indicating the role of active serine proteases and phospholipases A2 in the action of the venom.
As águas-vivas ou medusas estão entre os animais peçonhentos marinhos mais populares. Acidentes envolvendo medusas são comuns e sua toxicidade para humanos pode variar de sintomas leves até envenenamentos fatais em alguns casos. Os venenos de medusas são de importância biotecnológica, apresentando toxinas com atividade antimicrobiana, analgésica e antitumoral. Além disso, enzimas proteolíticas também já foram descritas. Recentemente o perfil proteômico de toxinas putativas dos nematocistos isolados da hidromedusa Olindias sambaquiensis foi caracterizado, identificando a presença de proteases e fosfolipases. Nesse trabalho nós apresentamos os resultados de um estudo que confirma a presença de atividade enzimática no extrato de tentáculos de O. sambaquiensis. Os espécimes foram coletados na costa de São Sebastião, SP, Brasil. O procedimento para obtenção do extrato de tentáculos foi padronizado, rendendo grande quantidade de proteínas de diferentes massas moleculares. O extrato foi avaliado quanto a presença de atividade de metaloproteases, serino proteases e fosfolipases A2, utilizando substratos genéricos para essas enzimas e também substratos específicos para as metaloproteases do tipo ADAMs e MMPs. O extrato apresentou atividade sobre todos os substratos, confirmando evidências proteômicas anteriores. Além disso, a atividade proteolítica também foi detectada por ensaios de zimografia em caseína e gelatina. Quando comparamos essas atividades com venenos de serpentes, que são abundantes em proteases e fosfolipases, a atividade de metaloproteases obtida foi bem menor que os altos índices demonstrados pelo veneno de Bothrops jararaca, entretanto, as atividades observadas para serino proteases e fosfolipases A2 foram comparáveis à atividade observada em venenos de serpentes Bothrops, conhecidas pela sua forte atividade anticoagulante e miotóxica devido a presença de serino proteases e fosfolipases A2, respectivamente. Esses resultados validam as propriedades funcionais das proteínas caracterizadas previamente pelo proteoma e fornecem um melhor entendimento do arsenal tóxico desse animal peçonhento pouco explorado, indicando o papel das serino proteases e fosfolipases A2 na ação do veneno.
RESUMO
KLK7 substrate specificity was evaluated by families of fluorescence resonance energy transfer (FRET) peptides derived from Abz-KLFSSK-Q-EDDnp (Abz=ortho-aminobenzoic acid and Q-EDDnp=glutaminyl-N-[2,4-dinitrophenyl] ethylenediamine), by one bead-one peptide FRET peptide library in PEGA resin, and by the FRET peptide libraries Abz-GXX-Z-XX-Q-EDDnp (Z and X are fixed and random natural amino acids, respectively). KLK7 hydrolyzed preferentially F, Y or M, and its S1' and S2' subsites showed selectivity for hydrophilic amino acids, particularly R and K. This set of specificities was confirmed by the efficient kininogenase activity of KLK7 on Abz-MISLM(↓)KRPPGFSPF(↓)RSSRI-NH2 ((↓)indicates cleavage), hydrolysis of somatostatin and substance P and inhibition by kallistatin. The peptide Abz-NLY(↓)RVE-Q-EDDnp is the best synthetic substrate so far described for KLK7 [kcat/Km=455 (mMs)(-1)] that was designed from the KLK7 substrate specificity analysis. It is noteworthy that the NLYRVE sequence is present in human semaphorin 6B. KLK7 is activated by GAGs, inhibited by neutral salts, and activated by high concentration of kosmotropic salt. Pyroglutamic acid inhibited KLK7 (Ki=33mM) and is present in skin moisturizing factor (124mM). The KLK7 specificity described here and elsewhere reflects its participation in patho-physiological events in skin, the gastrointestinal tract and central nervous system, where KLK7 is significantly expressed.
Assuntos
Glicosaminoglicanos/farmacologia , Calicreínas/metabolismo , Peptídeo Hidrolases/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Biocatálise/efeitos dos fármacos , Transferência Ressonante de Energia de Fluorescência , Humanos , Hidrólise/efeitos dos fármacos , Cinética , Cininogênios/metabolismo , Dados de Sequência Molecular , Concentração Osmolar , Ácido Pirrolidonocarboxílico/farmacologia , Semaforinas/metabolismo , Serpinas/metabolismo , Somatostatina/metabolismo , Substância P/metabolismo , Especificidade por Substrato , Fatores de TempoRESUMO
The snake venom metalloproteinases (SVMPs) are abundant enzymes in the venoms of viper snakes and responsible for most of the symptoms of envenoming. Their actions are related to the hydrolysis of extracellular matrix components, plasma proteins and hydrolysis or binding to cell surface proteins resulting in the activation or inhibition of their activity. In previous studies we recently found a diversity of SVMPs transcripts produced after stimulation of Bothrops neuwiedi venom gland. In this work, our aim was to characterize the different metalloproteinases in the Bothrops neuwiedi venom affecting hemostasis. Initially, the presence of distinct SVMPs in the venom was accessed by twodimensional electrophoresis and reverse phase chromatography. To obtain fractions enriched with SVMPs and characterize their functional activities, the venom was fractionated into molecular exclusion column, associated with anion exchange column and the resulting pools tested for enzymatic, hemorrhagic and fibrinolytic activities, blood clotting, reactivity with anti-SVMPs antibodies and mass spectrometry. This approach allowed the identification of six SVMPs, four class P-III and two class P-I. The SVMPs class P-III had gelatinolytic and hemorrhagic activities, of these, two had yet fibrinolytic activity. The two P-I were only fibrinolytic. Regarding coagulation activity, at least one P-III was characterized as prothrombin activator and one P-III as factor X activator. We conclude that there is a high variety of SVMPs in Bothrops neuwiedi snake venom, which disturb different hemostatic mechanisms and contribute to symptoms caused by this snake envenoming.
As metaloproteinases de venenos de serpentes (SVMPs) são enzimas abundantes em venenos de espécies da família Viperidae e responsáveis por grande parte dos sintomas do envenenamento. Sua ação está relacionada com a proteólise dos componentes da matriz extracelular, proteínas plasmáticas e também com hidrólise ou ligação a proteínas de superfície celular induzindo a ativação ou inibição da atividade das mesmas. Em estudos com serpentes do “Complexo Bothrops neuwiedi”, verificamos recentemente a diversidade dos transcritos de SVMPs produzidos após estímulo da glândula de veneno. Neste trabalho, o principal objetivo foi caracterizar as diferentes metaloproteinases presentes no veneno de Bothrops neuwiedi com ações na hemostasia. Inicialmente, confirmamos a presença de distintas SVMPs no veneno através de eletroforese bidimensional e cromatografia em coluna de fase reversa. Para a obtenção de frações enriquecidas com SVMPs e caracterização de suas atividades funcionais, o veneno foi fracionado em coluna de exclusão molecular, associado à coluna de troca aniônica e os pools resultantes testados quanto às atividades enzimática, hemorrágica, fibrinolítica, coagulante, reatividade com anticorpos anti-SVMPs e espectrometria de massas. Dessa forma, foi possível a identificação de seis pools contendo SVMPs, sendo quatro da classe P-III e dois da classe P-I. As SVMPs de classe P-III apresentaram atividades gelatinolítica e hemorrágica, destas, duas ainda apresentaram atividade fibrinolítica. As duas P-I foram apenas fibrinolíticas. Com relação à atividade coagulante, foram caracterizadas ao menos uma P-III ativadora de protrombina e uma P-III ativadora de fator X. Com isso concluímos que há grande variedade de SVMPs no veneno de Bothrops neuwiedi, as quais agem em diferentes mecanismos hemostáticos e contribuem para os sintomas causados nos envenenamentos por essa serpente.