Análise peptidômica dos venenos de vespas sociais neotropicais

Os peptídeos possuem um papel essencial para as funções fisiológicas de animais, plantas e de alguns microrganismos, e frequêntemente apresentam-se como candidatos a novas drogas na sua forma natural, servindo como modelos para o “desenho” de peptídeos modificados, para uma obtenção de perfis farmacológicos melhores. Neste contexto, os venenos das vespas sociais são interessantes sob o ponto de vista da pesquisa, uma vez que são ricos em peptídeos policatiônicos envolvidos com processos inflamatórios (lise de membranas, Desgranulação de mastócitos, quimiotaxia, entre outros processos), além de efeitos antibióticos contra bactérias Gram-positivas e Gram-negativas. Com base nisso, o objetivo deste trabalho foi realizar uma bioprospecção dos diferentes componentes peptídicos presentes nos venenos das vespas sociais Agelaia pallipes pallipes e Agelaia vicina, através da identificação e da caracterização estrutural e funcional dos componentes peptídicos mais abundantes destes venenos. Para isso, os venenos das vespas supracitadas foram extraidos em acetonitrila, fracionados por cromatografia de fase reversa e analisado por espectrometria de massas (ESI-MS e ESI-MSn). Todos os peptídeos caracterizados foram sintetizados manualmente por estratégia Fmoc, para a realização de ensaios farmacológicos e fisiológicos. Além de abrir novas vertentes de estudos estruturais e funcionais, o presente projeto alcançou os objetivos inicialmente almejados, identificando e caracterizando estruturalmente doze peptídeos presentes nos venenos das duas espécies de vespas. As denominações e atividades biológicas dessas moléculas foram: Protonectina apresentou-se lítico para hemáceas, degranulador de leucócitos quimiotático, induziu o fenômeno de hiperalgesia e foi edematogênico; o peptídeo Protonectina (1-4)-OH apresentou-se hemolítico para eritrócitos e desgranulador...Peptides have an important role on physiologic functions of animals, plants and microorganisms. These natural peptides frequently are candidates for new drugs natural, and are used as models for the construction of modificated peptides in order to improve their pharmacological activities. From academic point of view, social wasp venoms are interesting once they are rich in policationic and polifunctional peptides that are involved with inflammatory processes (membrane lyses, mast cell degranulation, chemotaxis, and other processes), antimicrobial effects against Gram-positive and Gram-negative bacterias. Therefore, the main objective of this study was to bioprospect a diversity of peptidic compounds from the venoms of the neotropical social wasps Agelaia pallipes pallipes and Agelaia vicina through peptidomic analysis, identifying and characterizing these molecules structurally and functionally. For that, the venom from both wasps was extracted in MeCN 50% (v/v), analyzed and sequenced by ESI-MS, ESI-MSn. All characterized peptides were manually synthesized by Fmoc strategy and used for pharmacological and phisiological activities. This work has reached the initially poposed objectives by the indentification and functional characterization of twelve peptides from the social wasps venoms studied. Of these identified molecules, nine were isolated from the social wasp A. p. pallipes venom (Protonectin (1-4)-OH, Protonectin (1-5)-OH, Protonectin (1-6)-OH, Protonectin (7-12); Agelaia MP-I, Agelaia MP-II, Pallipin-I, Pallipin-II, and Pallipin-III), and from the venom of the social wasp A. vicina, four peptides were characterized (Protonectin (7-9)-OH, Protonectin, Protonectin (1-6), and Agelaia MP-I). These results show that the venom of both wasps are very similar to each other, probably due to the kinship of such species. Functionally, the peptide protonectin is lytic to erythrocytes... (Complete abstract click electronic access below)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Efeito das interações peptídeo-peptídeo e peptídeo-membrana nas atividades funcionais de toxinas peptídicas do veneno da vespa social Agelaia pallipes pallipes (Hymenoptera, Vespidae)

Os venenos de Vespidae são importantes ferramentas para a defesa dos ninhos. Acidentes com ferroadas de vespas sociais são muito comuns e podem causar diversos sintomas nas vitimas. Esses venenos são ricos em peptídeos policatiônicos, geralmente envolvidos em processos inflamatórios. Os peptídeos mais abundantes encontrados nos venenos das vespas são os peptídeos quimiotáticos e mastoparanos, porém dentre os diversos componentes encontrados no veneno da vespa social A. p. pallipes ainda existem algumas toxinas pouco caracterizadas farmacologicamente. Entre elas encontrou-se os peptídeos Protonectina e Protonectina (1-6) com massas moleculares de 1209 Da (I-L-G-T-I-L-G-L-L-K-G-L-NH2) e 628 Da (I-L-G-T-I-L-NH2), respectivamente. Ao nível molecular, esses peptídeos interagem entre si, resultando na potenciação das atividades funcionais da Protonectina. Considerando-se a importância deste tipo de interação molecular para a indução de processos inflamatórios, os objetivos deste estudo foram: caracterizar a estrutura secundária desses peptídeos individualmente e também a estrutura supramolecular resultante de suas interações, observando os efeitos da mesma sobre as ações de degranulação de mastócitos, quimiotaxia e hemólise. Ambos os peptídeos foram sintetizados manualmente em fase sólida por química Fmoc. A Protonectina é um peptídeo anfifílico, enquanto que a Protonectina (1-6) é muito pequena para assumir estrutura secundária anfifílica. Análises de Dicroísmo Circular, revelaram que na presença de TFE, a Protonectina e Protonectina (1-6) tendem a apresentar estrutura secundária constituída de 36.7% e 17.6% na forma de hélicea, respectivamente. Entretanto, a mistura de ambos peptídeos na proporção de 1:1 resultou em uma estrutura supramolecular que apresentou 48.3% de hélice-a sugerindo, assim, uma possível interação entre esses dois peptídeos...Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Chemometric analysis of Hymenoptera toxins and defensins: A model for predicting the biological activity of novel peptides from venoms and hemolymph

When searching for prospective novel peptides, it is difficult to determine the biological activity of a peptide based only on its sequence. The trial and error approach is generally laborious, expensive and time consuming due to the large number of different experimental setups required to cover a reasonable number of biological assays. To simulate a virtual model for Hymenoptera insects, 166 peptides were selected from the venoms and hemolymphs of wasps, bees and ants and applied to a mathematical model of multivariate analysis, with nine different chemometric components: GRAVY, aliphaticity index, number of disulfide bonds, total residues, net charge, pI value, Boman index, percentage of alpha helix, and flexibility prediction. Principal component analysis (PCA) with non-linear iterative projections by alternating least-squares (NIPALS) algorithm was performed, without including any information about the biological activity of the peptides. This analysis permitted the grouping of peptides in a way that strongly correlated to the biological function of the peptides. Six different groupings were observed, which seemed to correspond to the following groups: chemotactic peptides, mastoparans, tachykinins, kinins, antibiotic peptides, and a group of long peptides with one or two disulfide bonds and with biological activities that are not yet clearly defined. The partial overlap between the mastoparans group and the chemotactic peptides, tachykinins, kinins and antibiotic peptides in the PCA score plot may be used to explain the frequent reports in the literature about the multifunctionality of some of these peptides. The mathematical model used in the present investigation can be used to predict the biological activities of novel peptides in this system, and it may also be easily applied to other biological systems. © 2011 Elsevier Inc

Profiling the peptidome of the venom from the social wasp Agelaia pallipes pallipes

The wasp Agelaia pallipes pallipes is one of the most aggressive species from the neotropical region, causing many stinging accidents every year, characterized by severe envenoming reactions. The identification of peptides is important for understanding the envenoming process; however, the tiny amount of venom produced by these insects makes this task a challenge, using classical analytical approaches. Thus, the venom was previously fractionated, and the sequences were obtained through the use of electrospray ionization with a tridimensional ion-trap and time-of-flight mass analysis under CID conditions. This approach permitted the sequence assignment of nine peptides. The presence of type -d and -w ions generated from the fragmentation of the side chains was used to resolve I/L ambiguity. The distinction between K and Q residues was achieved through esterification of the alpha- and epsilon-amino groups in the peptides, followed by mass spectrometry analysis. Six of these peptides were short, linear and polycationic, while the three other peptides presented a single disulfide bridge. The use of reduction and alkylation protocols, followed by ESI-IT-TOF/MS analysis under CID conditions, permitted easy sequencing of the three peptides presenting this post-translational modification. These peptides presented activity related to mast cell degranulation, hemolysis, or even the chemotaxis of leukocytes. (C) 2011 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Agelaia MP-I: A peptide isolated from the venom of the social wasp, Agelaia pallipes pallipes, enhances insulin secretion in mice pancreatic islets

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Peptides isolated from animal venoms have shown the ability to regulate pancreatic beta cell function. Characterization of wasp venoms is important, since some components of these venoms present large molecular variability, and potential interactions with different signal transduction pathways. For example, the well studied mastoparan peptides interact with a diversity of cell types and cellular components and stimulate insulin secretion via the inhibition of ATP dependent K+ (K-ATP) channels, increasing intracellular Ca2+ concentration. In this study, the insulin secretion of isolated pancreatic islets from adult Swiss mice was evaluated in the presence of synthetic Agelaia MP-I (AMP-I) peptide, and some mechanisms of action of this peptide on endocrine pancreatic function were characterized. AMP-I was manually synthesized using the Fmoc strategy, purified by RP-HPLC and analyzed using ESI-IT-TOF mass spectrometry. Isolated islets were incubated at increasing glucose concentrations (2.8, 11.1 and 22.2 mM) without (Control group: CTL) or with 10 mu M AMP-I (AMP-I group). AMP-I increased insulin release at all tested glucose concentrations, when compared with CTL (P < 0.05). Since molecular analysis showed a potential role of the peptide interaction with ionic channels, insulin secretion was also analyzed in the presence of 250 mu M diazoxide, a K-ATP channel opener and 10 mu M nifedipine, a Ca2+ channel blocker. These drugs abolished insulin secretion in the CTL group in the presence of 2.8 and 11.1 mM glucose, whereas. AMP-I also enhanced insulin secretory capacity, under these glucose conditions, when incubated with diazoxide and nifedipine. In conclusion, AMP-I increased beta cell secretion without interfering in K-ATP and L-type Ca2+ channel function, suggesting a different mechanism for this peptide, possibly by G protein interaction, due to the structural similarity of this peptide with Mastoparan-X, as obtained by modeling. (c) 2012 Elsevier Ltd. All rights reserved.604SI596602Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)FAPESP [2011/51684-1

Hyperalgesic and edematogenic effects of peptides isolated from the venoms of honeybee (Apis mellifera) and neotropical social wasps (Polybia paulista and Protonectarina sylveirae)

Stings by bees and wasps, including Brazilian species, are a severe public health problem. The local reactions observed after the envenoming includes typical inflammatory response and pain. Several studies have been performed to identify the substances, including peptides that are responsible for such phenomena. The aim of the present study is to characterize the possible nociceptive (hyperalgesic) and edematogenic effects of some peptides isolated from the venoms of the honeybee (Apis mellifera) and the social wasps Polybia paulista and Protonectarina sylveirae, in addition to characterize some of the mechanisms involved in these phenomena. For this purpose, different doses of the peptides mellitin (Apis mellifera), Polybia-MP-I, N-2-Polybia-MP-I (Polybia paulista), Protonectarina-MP-NH2 and Protonectarina-MP-OH (Protonectarina sylveirae) were injected into the hind paw of mice. Hyperalgesia and edema were determined after peptide application, by using an electronic von Frey apparatus and a paquimeter. Carrageenin and saline were used as controls. Results showed that melittin, Polybia-MP-I, N-2-Polybia-MP-I, Protonectarina-MP-NH(2) and Protonectarina-MP-OH peptides produced a dose- and time-related hyperalgesic and edematogenic responses. Both phenomena are detected 2 h after melittin, Polybia-MP-I, N-2-Polybia-MP-I injection; their effects lasted until 8 h. In order to evaluate the role of prostanoids and the involvement of lipidic mediators in hyperalgesia induced by the peptides, indomethacin and zileuton were used. Results showed that zileuton blocked peptide-induced hyperalgesia and induced a decrease of the edematogenic response. on the other hand, indomethacin did not interfere with these phenomena. These results indicate that melittin, Polybia-MP-I, N-2-Polybia-MP-I, Protonectarina-MP-NH(2), and Protonectarina-MP-OH peptides could contribute to inflammation and pain induced by insect venoms.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Agelaia MP-I: A peptide isolated from the venom of the social wasp, Agelaia pallipes pallipes, enhances insulin secretion in mice pancreatic islets

Peptides isolated from animal venoms have shown the ability to regulate pancreatic beta cell function. Characterization of wasp venoms is important, since some components of these venoms present large molecular variability, and potential interactions with different signal transduction pathways. For example, the well studied mastoparan peptides interact with a diversity of cell types and cellular components and stimulate insulin secretion via the inhibition of ATP dependent K + (K ATP) channels, increasing intracellular Ca 2+ concentration. In this study, the insulin secretion of isolated pancreatic islets from adult Swiss mice was evaluated in the presence of synthetic Agelaia MP-I (AMP-I) peptide, and some mechanisms of action of this peptide on endocrine pancreatic function were characterized. AMP-I was manually synthesized using the Fmoc strategy, purified by RP-HPLC and analyzed using ESI-IT-TOF mass spectrometry. Isolated islets were incubated at increasing glucose concentrations (2.8, 11.1 and 22.2 mM) without (Control group: CTL) or with 10 μM AMP-I (AMP-I group). AMP-I increased insulin release at all tested glucose concentrations, when compared with CTL (P < 0.05). Since molecular analysis showed a potential role of the peptide interaction with ionic channels, insulin secretion was also analyzed in the presence of 250 μM diazoxide, a K ATP channel opener and 10 μM nifedipine, a Ca 2+ channel blocker. These drugs abolished insulin secretion in the CTL group in the presence of 2.8 and 11.1 mM glucose, whereas AMP-I also enhanced insulin secretory capacity, under these glucose conditions, when incubated with diazoxide and nifedipine. In conclusion, AMP-I increased beta cell secretion without interfering in K ATP and L-type Ca 2+ channel function, suggesting a different mechanism for this peptide, possibly by G protein interaction, due to the structural similarity of this peptide with Mastoparan-X, as obtained by modeling. © 2012 Elsevier Ltd