Structure–function relationships of the peptide Paulistine: A novel toxin from the venom of the social wasp Polybia paulista

AbstractBackgroundThe peptide Paulistine was isolated from the venom of wasp Polybia paulista. This peptide exists under a natural equilibrium between the forms: oxidised — with an intra-molecular disulphide bridge; and reduced — in which the thiol groups of the cysteine residues do not form the disulphide bridge. The biological activities of both forms of the peptide are unknown up to now.MethodsBoth forms of Paulistine were synthesised and the thiol groups of the reduced form were protected with the acetamidemethyl group [Acm-Paulistine] to prevent re-oxidation. The structure/activity relationships of the two forms were investigated, taking into account the importance of the disulphide bridge.ResultsPaulistine has a more compact structure, while Acm-Paulistine has a more expanded conformation. Bioassays reported that Paulistine caused hyperalgesia by interacting with the receptors of lipid mediators involved in the cyclooxygenase type II pathway, while Acm-Paullistine also caused hyperalgesia, but mediated by receptors involved in the participation of prostanoids in the cyclooxygenase type II pathway.ConclusionThe acetamidemethylation of the thiol groups of cysteine residues caused small structural changes, which in turn may have affected some physicochemical properties of the Paulistine. Thus, the dissociation of the hyperalgesy from the edematogenic effect when the actions of Paulistine and Acm-Paulistine are compared to each other may be resulting from the influence of the introduction of Acm-group in the structure of Paulistine.General significanceThe peptides Paulistine and Acm-Paulistine may be used as interesting tools to investigate the mechanisms of pain and inflammation in future studies

Structural characterization of novel chemotactic and mastoparan peptides from the venom of the social wasp Agelaia pallipes pallipes by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

High-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) and high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) techniques were applied for the detection, purification, monitoring, and sequencing of two novel and biologically active peptides occurring at very low levels in the venom of the wasp Agelaia pallipes pallipes. These peptides were sequenced under LC/ESI-MS/MS conditions and designated as Agelaia-CP (I/L-L-G-T-I-L-G-L-L-K-G-I/L-NH2, MW 1207.8Da) and Agelaia-MP (I/L-N-W-L-K-L-G-K-A-I-I-D-A-I/L-NH2, MW 1565.0Da). The peptide Agelaia-CP showed no hemolytic activity, but it behaved as a mast cell degranulator and induced a potent chemotaxis in polymorphonucleated leukocyte (PMNL) cells, typical of a wasp chemotactic peptide. The peptide Agelaia-MP showed both powerful mast cell degranulation and hemolysis of washed rat red blood cells, and is thus assigned as a new member of the mastoparan family of peptides. Both peptides seem to be directly involved in the strong inflammatory reactions associated with wasp stings. © Copyright 2004 John Wiley & Sons, Ltd.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Investigating the effect of different positioning of lysine residues along the peptide chain of mastoparans for their secondary structures and biological activities

In order to investigate the effect of the different positions of the positive charges generated by the ionization of the side-chain of lysine residues, on the structure-activity relationship of the mastoparans, the peptides Protonectarina-MP (INWKALLDAAKKVL-NH2), Parapolybia-MP (INWKKMAATALKMI-NH2) and Asn-2-Polybia-MP I (INWKKLLDAAKQIL-NH2) and MK-578 (INWLKAKKVAGMIL-NH2) were investigated as models. Thus, the four peptides had their secondary structure studied and were submitted to assays of mast cell degranulation, hemolysis, and antibiosis. The results of the bioassays made clear that those peptides bearing the positive charges positioned at the positions 4/5 and/or from 11 to 13 are the most active ones; meanwhile, the localization of the positive charges in the middle of peptide chain resulted in a poorly active peptide. Thus, Protonectarina-MP, Parapolybia-MP, and Asn-2-Polybia-MP I presented physiologically important hemolysis and antibiosis, while MK-578 presented only a reduced antibiotic activity. Circular dichroism analysis were carried-out in different environments revealing that the anionic environment of a mixture of phosphatidylcholine and phosphatidylglycerol (70:30) liposomes favored the higher helical content of the four peptides in this study in relation to the zwiterionic environment of 100% phosphatidylcholine liposomes. The positioning of the lysine residues at the strategic positions (4/5 and 11-13), flanking and maintaining stable alpha-helix which extends from the 4th to the 13th residue along the peptide chain, seems to contribute to maximal lytic efficiency of the mastoparans, which in turn results in a more homogeneous hydrophobic surface in the amphipathic structure.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Mastoparan Effects In Skeletal Muscle Damage: An Ultrastructural View Until Now Concealed.

Animal venoms have been valuable sources for development of new drugs and important tools to understand cellular functioning in health and disease. The venom of Polybia paulista, a neotropical social wasp belonging to the subfamily Polistinae, has been sampled by headspace solid phase microextraction and analyzed by gas chromatography-mass spectrometry. Recent study has shown that mastoparan, a major basic peptide isolated from the venom, reproduces the myotoxic effect of the whole venom. In this study, Polybia-MPII mastoparan was synthesized and studies using transmission electron microscopy were carried out in mice tibial anterior muscle to identify the subcellular targets of its myotoxic action. The effects were followed at 3 and 24 h, 3, 7, and 21 days after mastoparan (0.25 mug/muL) intramuscular injection. The peptide caused disruption of the sarcolemma and collapse of myofibril arrangement in myofibers. As a consequence, fibers presented heteromorphic amorphous masses of agglutinated myofilaments very often intermingled with denuded sarcoplasmic areas sometimes only surrounded by a persistent basal lamina. To a lesser extent, a number of fibers apparently did not present sarcolemma rupture but instead appeared with multiple small vacuoles. The results showed that sarcolemma, sarcoplasmic reticulum (SR), and mitochondria were the main targets for mastoparan. In addition, a number of fibers showed apoptotic-like nuclei suggesting that the peptide causes death both by necrosis and apoptosis. This study presents a hitherto unexplored view of the effects of mastoparan in skeletal muscle and contributes to discuss how the known pharmacology of the peptide is reflected in the sarcolemma, SR, mitochondria, and nucleus of muscle fibers, apparently its subcellular targets.71220-

Effect of the aspartic acid D2 on the affinity of Polybia-MP1 to anionic lipid vesicles

Polybia-MP1 (IDWKKLLDAAKQIL-NH2), a helical peptide extracted from the venom of a Brazilian wasp, has broad-spectrum antimicrobial activities without being hemolytic or cytotoxic. This peptide has also displayed anticancer activity against cancer cell cultures. Despite its high selectivity, MP1 has an unusual low net charge (Q = +2). The aspartic residue (D2) in the N-terminal region plays an important role in its affinity and selectivity; its substitution by asparagine (D2N mutant) led to a less selective peptide. Aiming to explore the importance of this residue for the peptides' affinity, we compared the zwitterionic and anionic vesicle adsorption activity of Polybia-MP1 versus its D2N mutant and also mastoparan X (MPX). The adsorption, electrostatic, and conformational free energies were assessed by circular dichroism (CD) and fluorescence titrations using large unilamellar vesicles (LUVs) at the same conditions in association with measurement of the zeta potential of LUVs in the presence of the peptides. The adsorption free energies of the peptides, determined from the partition coefficients, indicated higher affinity of MP1 to anionic vesicles compared with the D2N mutant and MPX. The electrostatic and conformational free energies of MP1 in anionic vesicles are less favorable than those found for the D2N mutant and MPX. Therefore, the highest affinity of MP1 to anionic vesicles is likely due to other energetic contributions. The presence of D2 in MP1 makes these energetic components 1.2 and 1.5 kcal/mol more favorable compared with the D2N mutant and MPX, respectively.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Paulistine—The Functional Duality of a Wasp Venom Peptide Toxin

It has been reported that Paulistine in the venom of the wasp Polybia paulista co-exists as two different forms: an oxidized form presenting a compact structure due to the presence of a disulfide bridge, which causes inflammation through an apparent interaction with receptors in the 5-lipoxygenase pathway, and a naturally reduced form (without the disulfide bridge) that exists in a linear conformation and which also causes hyperalgesia and acts in the cyclooxygenase type II pathway. The reduced peptide was acetamidomethylated (Acm-Paulistine) to stabilize this form, and it still maintained its typical inflammatory activity. Oxidized Paulistine docks onto PGHS2 (COX-2) molecules, blocking the access of oxygen to the heme group and inhibiting the inflammatory activity of Acm-Paulistine in the cyclooxygenase type II pathway. Docking simulations revealed that the site of the docking of Paulistine within the PGHS2 molecule is unusual among commercial inhibitors of the enzyme, with an affinity potentially much higher than those observed for traditional anti-inflammatory drugs. Therefore, Paulistine causes inflammatory activity at the level of the 5-lipooxygenase pathway and, in parallel, it competes with its reduced form in relation to the activation of the cyclooxygenase pathway. Thus, while the reduced Paulistine causes inflammation, its oxidized form is a potent inhibitor of this activity

Proposta de melhorias e de indicadores na gestão sustentável de resíduos provenientes de navios no Porto de Lisboa

Trabalho final de mestrado para obtenção do grau de Mestre em Engenharia da Qualidade e AmbienteO transporte marítimo é essencial para a economia mundial, uma vez que mais do 90% do comércio mundial é efetuado por via marítima (IMO, 2019). O transporte marítimo de matérias-primas e mercadorias é considerado o mais económico, o mais eficiente e com menos impactes ambientais, no entanto não é isento de impactes ambientais negativos. Os mesmos resultam dos resíduos gerados pelos navios (RGN) e resíduos da carga (RC), da emissão de quantidades significativas de gases com efeito estufa (GEE) e de outros gases poluentes. Para evitar a poluição ambiental marinha causada pela descarga ilegal de substâncias e resíduos nocivos para o mar, em 1973, foi adotada uma Convenção Internacional: o protocolo MARPOL 73/78. Da mesma forma, a União Europeia (UE) adotou a diretiva 2000/59/CE, sobre meios portuários de receção de resíduos (MPRR), cujos principais objetivos são impedir a descarga de RGN e RC e da carga no mar, e a proteção do meio marinho. O Porto de Lisboa (PL) é o responsável pela gestão de RGN e RC, garantindo a sua gestão até ao destino final adequado. De acordo com os requisitos exigidos pela Diretiva 2000/59/CE, o PL implementou um sistema de gestão de RGN, cujos objetivos são os seguintes: reduzir as descargas de RGN e da carga no mar; fornecer MPRR adequados a todos e novos tipos de resíduos, em condições de segurança; melhorar o sistema de receção de resíduos; incentivar a descarga de resíduos nos MPRR e a proteção do meio marinho. O objetivo deste estudo é quantificar os RGN e RC descarregados no PL e analisar o sistema de gestão de resíduos (SGR) do porto, a fim de estabelecer indicadores e propostas de melhoria. O tratamento dos dados dos resíduos em conjunto com os das inspeções realizadas aos navios e aos MPRR forneceu informação relevante para a criação de indicadores sobre produção de resíduos a bordo dos navios e do sistema de gestão dos mesmos. Aplicou-se uma análise SWOT, para identificar possíveis barreiras na gestão e contribuir para um processo de melhoria contínua. As principais oportunidades de melhoria identificadas são: implementação das normas ISO 9001:2015 e 14001:2015, integração navio-porto; incentivar as descargas nos MPRR; o aumento das inspeções aos navios e a fiscalização nos terminais; sensibilizar e formar a todas as partes integrantes; incrementar o número de MPRR por tipologia e o controlo dos abastecimentos aos navios. Concluiu-se que, apesar dos avanços na redução da poluição causada pelo transporte marítimo, ainda há um grande desafio pela frente no PL.The protection of the environment has become a major issue since global pollution has reached such alarming levels. This pollution comes mainly from the industrialization of the world trade and also from means of transport. Maritime transport is essential for the global economy as more than 90% of the world trade is by ship. Even though maritime transport is considered the most economical, efficient and environmentally friendly means of transport, it has introduced negative environmental impacts due to the ship generated waste (SGW), the significant amount of greenhouse gas emissions, the fossil fuel consumption and the transport of animal products from third countries. In order to prevent ocean pollution caused by the illegal disposal of hazardous substances and waste, an International Convention was adopted in 1973, the Protocol of MARPOL 73/78. Additionally, the European Union (EU) adopted the Directive 2000/59/CE on Port Reception Facilities (PRF) whose main objectives are to avoid the disposal of SGW into the ocean and to protect the marine environment. The Port of Lisbon (PL) is responsible for the ship generated waste management (SGWM) of the ships which call at PL securing its delivery to the adequate final destination. The PL complies with the regulations required by the EU Directive 2000/59/CE on PRF and it has implemented a SGWM system whose objectives are as follows: to reduce SGW and cargo waste disposal into the sea; to provide adequate PRF to both all the waste and new residues in safe conditions, to improve the waste collection service; to encourage waste disposal to PRFs and to protect the marine environment. The purpose of this study is to quantify the waste coming from ships and cargo discharged at PL and to analyse the waste management system of the Port so as to improve it. To do this a SWOT analysis was carried out in order to identify possible management barriers and also to contribute to a continuous improvement process. The waste data processing together with ship inspections provided us with relevant information for the development of indicators of both waste generation on board and the management system of such waste. Some of the main opportunities for improvement identified are: implementation of the ISO 9001:2015 and 14001:2015 standards, port-ship integration, the improvement of management in the parties involved, to encourage waste disposal in PRF, the increase of ship inspections and audits in terminals, to raise awareness and train all the parties involved, to increase the number of waste reception facilities according to the different waste types and the supervision of ship supplies. It was concluded that in spite of the progress towards the reduction in pollution caused by maritime transport, there is still a great challenge before us.N/

New Insight into the Mechanism of Action of Wasp Mastoparan Peptides: Lytic Activity and Clustering Observed with Giant Vesicles

Antimicrobial peptides of the mastoparans family exert their bactericidal activity by binding to lipid membranes, inducing pores or defects and leaking the internal contents of vesicles and cells. However, this does not seem to be the only mechanism at play, and they might be important in the search for improved peptides with lower undesirable side effects. This work deals with three mastoparans peptides, Polybia-MP-1(MP-1), N2-Polybia-MP-1 (N-MP-1), and Mastoparan X (MPX), which exhibit high sequence homology. They all have three lysine residues and amidated C termini, but because of the presence of two, one, and no aspartic acid residues, respectively, they have +2, +3, and +4 net charges at physiological pH. Here we focus on the effects of these mastoparans peptides on anionic model membranes made of palmitoleyoilphosphatidylcholine (POPC) and palmitoleyoilphosphatidylglycerol (POPG) at 1:1 and 3:1 molar ratios in the presence and in the absence of saline buffer. Zeta potential experiments were carried out to measure the extent of the peptides' binding and accumulation at the vesicle surface, and CD spectra were acquired to quantify the helical structuring of the peptides upon binding. Giant unilamellar vesicles were observed under phase contrast and fluorescence microscopy. We found that the three peptides induced the leakage of GUVs at a gradual rate with many characteristics of the graded mode. This process was faster in the absence of saline buffer. Additionally, we observed that the peptides induced the formation of dense regions of phospholipids and peptides on the GUV surface. This phenomenon was easily observable for the more charged peptides.(MPX > N-MP-1 > MP-1) and in the absence of added salt. Our data suggest that these mastoparans accumulate on the bilayer surface and induce a transient interruption to its barrier properties, leaking the vesicle contents. Next, the bilayer recovers its continuity, but this happens in an inhomogeneous way, forming a kind of ply with peptides sandwiched between two juxtaposed membranes. Eventually, a peptide-lipid aggregate forming a lump is formed at high peptide-to-lipid ratios.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Influence of the Bilayer Composition on the Binding and Membrane Disrupting Effect of Polybia-MP1, an Antimicrobial Mastoparan Peptide with Leukemic T-Lymphocyte Cell Selectivity

This study shows that MP-1, a peptide from the venom of the Polybia paulista wasp, is more toxic to human leukemic T-lymphocytes than to human primary lymphocytes. By using model membranes and electrophysiology measurements to investigate the molecular mechanisms underlying this selective action, the porelike activity of MP-1 was identified with several bilayer compositions. The highest average conductance was found in bilayers formed by phosphatidylcholine or a mixture of phosphatidylcholine and phosphatidylserine (70:30). The presence of cholesterol or cardiolipin substantially decreases the MP-1 pore activity, suggesting that the membrane fluidity influences the mechanism of selective toxicity. The determination of partition coefficients from the anisotropy of Tip indicated higher coefficients for the anionic bilayers. The partition coefficients were found to be 1 order of magnitude smaller when the bilayers contain cholesterol or a mixture of cholesterol and sphingomyelin. The blue shift fluorescence, anisotropy values, and Stern-Volmer constants are indications of a deeper penetration of MP-1 into anionic bilayers than into zwitterionic bilayers. Our results indicate that MP-1 prefers to target leukemic cell membranes, and its toxicity is probably related to the induction of necrosis and not to DNA fragmentation. This mode of action can be interpreted considering a number of bilayer properties like fluidity, lipid charge, and domain formation. Cholesterol-containing bilayers are less fluid and less charged and have a tendency to form domains. In comparison to healthy cells, leukemic T-lymphocyte membranes are deprived of this lipid, resulting in decreased peptide binding and lower conductance. We showed that the higher content of anionic lipids increases the level of binding of the peptide to bilayers. Additionally, the absence of cholesterol resulted in enhanced pore activity. These findings may drive the selective toxicity of MP-1 to Jurkat cells.Council for Scientific and Technological Development (CNPq) [154550/2006-0, 477780/2010-5, 301064/2004-0, 306821/2009-5]Council for Scientific and Technological Development (CNPq)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [06/57122-7, 07/03657-0, 2010/52077-9, 2010/11823-0]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP

Interactions of mast cell degranulating peptides with model membranes: A comparative biophysical study

In the last decade, there has been renewed interest in biologically active peptides in fields like allergy, autoimmume diseases and antibiotic therapy. Mast cell degranulating peptides mimic G-protein receptors, showing different activity levels even among homologous peptides. Another important feature is their ability to interact directly with membrane phospholipids, in a fast and concentration-dependent way. The mechanism of action of peptide HR1 on model membranes was investigated comparatively to other mast cell degranulating peptides (Mastoparan, Eumenitin and Anoplin) to evidence the features that modulate their selectivity. Using vesicle leakage, single-channel recordings and zeta-potential measurements, we demonstrated that HR1 preferentially binds to anionic bilayers, accumulates, folds, and at very low concentrations, is able to insert and create membrane spanning ion-selective pores. We discuss the ion selectivity character of the pores based on the neutralization or screening of the peptides charges by the bilayer head group charges or dipoles. (C) 2009 Elsevier Inc. All rights reserved.WinEDR package[154550/2006-0]WinEDR packageCouncil for Scientific and Technological Development (CNPq)[142566/2005-5]Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)CAPES[301064/2004-0]Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)CAPES[310559/2006-5]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP[06/57122-7]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP[07/09657-0