Estudo da formação de domínios em bicamadas lipídicas induzidos por peptídeos antimicrobianos

Estudos de visualização de GUVs por microscopias de fluorescência e contraste de fase, realizados com três mastoparanos (MP-1, N-MP-1 e MPX), evidenciam que alguns destes peptídeos induzem a formação de regiões densas na superfície das GUVS que foram atribuídas a agregacão ou segregação lipídica. Evidências experimentais anteriores mostraram que estes peptídeos apresentam atividade interfacial e suas cargas líquidas +2, +3 e +4 conferem atividade lítica preferencial em vesículas aniônicas, bem como atividade antimicrobiana contra bactérias Gram negativas e Gram positivas. A formação de domínios poderia ser um mecanismo lítico alternativo para a atividade interfacial. Segregação lipídica induzida por estes peptídeos foi então avaliada por calorimetria diferencial de varredura (DSC), observando-se o impacto destes peptídeos nas propriedades termotrópicas de misturas de lipídios que são miméticas de bactérias Gram negativas. As misturas binárias utilizadas foram POPE:DOPG (3:1) e DPPE:DPPG (4:1 e 1:4). A transição de fase gel-líquida cristalina da mistura POPE:DOPG em 20mM PIPES (140mM NaCl + 1mM EDTA, pH=7,4) foi centrada em 14 oC com um ”ombro”em, apro-ximadamente, 16 oC indicando miscibilidade incompleta destes lipídios. Na razão molar de lipídio por peptídeo (L/P)=400, os três peptídeos estudados induziram aumento na temperatura de fusão (Tm) de suspensão de vesículas multilamelares. Destes peptídeos MP-1, com carga +2, mostrou a maior variação de Tm. As mudanças em Tm foram observadas ser dependente da relação L/P, sendo maior deslocamento de Tm em L/P=15. O aumento da temperatura de transição de fase indica interação preferencial destes...GUVs visualization studies by fluorescence and phase contrast microscopies, carried out with three mastoparan peptides (MP-1, N-MP-1 and MPX) provided evidences that some of these peptides induce dense regions in the GUV’s surfaces that were attributed to aggregation or lipid segregation. Previous experimental evidences showed that these peptides display inter-facial activity and their net charges +2, +3 and +4 confer preferential lytic activity in anionic vesicles as well as antimicrobial activity against Gram negative and Gram positive bacteria. The domain formation could be an alternative lytic mechanism for the interfacial activity. Lipid segregation induced by these peptides was then evaluated by differential scanning calorime-try (DSC), by observing the impact of these peptides on the thermotropic properties of lipid mixtures which are mimetics of the Gram negative bacteria. The binary mixtures used were POPE:DOPG (3:1) and DPPE:DPPG (4:1 and 1:4). The gel to liquid crystalline phase transi-tion of the mixture POPE:DOPG in 20 mM PIPES (140 mM NaCl, 1 mM EDTA, pH7.4) was centered in 14 oC with a shoulder in approximately 16 oC indicating incomplete miscibility. At lipid to peptide molar ratio (L/P)=400 the three peptides studied induced increase of the melting temperature Tm of multilamelar vesicle suspensions. From these peptides MP-1, with charge +2, showed the largest variation of Tm. The changes in Tm were observed to be dependent on the L/P ratio, displacing to larger temperature for L/P =15 ratio. The increase in the of the phase transition temperature indicate preferential interaction of these peptides with the anionic com-ponent of the mixture leading to the formation of domains enriched with the other component (POPE) which, in pure state, has the largest Tm. Other three peptides... (Complete abstract click electronic access below)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Estudo da formação de domínios em membranas modelos induzidos por peptídeos antimicrobianos e sua ação interfacial

Peptídeos antimicrobianos agem diretamente na matriz lipídica da membrana celular perturbando o empacotamento lipídico, criando tensões elásticas e desequilíbrio de massa, que são relaxados pela formação de poros ou defeitos que dão origem ao processo lítico. Polybia-MP1, ou simplesmente MP1, com sequência de aminoácidos IDWKKLLDAAKQIL-NH2, extraído da vespa nativa Polybia paulista, é um exemplo destes peptídeos. Além de potente atividade antimicrobiana ele inibe a proliferação celular em culturas de células de câncer de próstata e bexiga. Este efeito inibitório acredita-se ser devido à presença simultânea de aminofosfolipídios fosfatidilserina (PS) e fosfatidiletanolamina (PE) nas monocamadas externas das membranas destas células. Investigação da atividade lítica em vesículas unilamelares gigantes (GUVs) revelaram que o peptídeo induziu a formação de regiões fluorescentes densas que foram atribuídas à agregação peptídeo/lipídio ou então à segregação lipídica. A atividade permeabilizadora do MP1 foi também fortemente modulada quando as GUVs continham frações de aminofosfolipídios PS e PE. Foi observado que a permeabilidade induzida pelo MP1 aumentou dramaticamente para vesículas de PC/PE/PS (7:1:2) permitindo influxo de moléculas de 10 kDa. Esta tese investigou a habilidade do MP1 em perturbar o empacotamento lipídico usando monocamadas lipídicas como modelo de membrana. Foram utilizados três fosfolipídios: PC, PE e PS contendo duas cadeias acíclicas. Isotermas de compressão foram obtidas usando cuba de Langmuir conjugadas com microscópios de fluorescência e de ângulo de Brewster. Por meio de isotermas de compressão de filme lipídico foram investigadas a preferência do peptídeo pelas fases lipídicas e seu efeito na coexistência de fases e nas alterações induzidas na forma, tamanho e número de domínios sólidos. Utilizando cadeias acíclicas derivadas de ácidos mirístico e palmítico ligados às cabeças polares PC, PE e PS, foi possível explorar o papel das cabeças polares e o efeito do comprimento das caudas no impacto do peptídeo nos filmes lipídicos. Estes experimentos mostraram que MP1 induziu mudanças na forma e no tamanho dos domínios sólidos. Estas alterações foram dependentes das cabeça polar e das condições da subfase: água pura e solução aquosa de NaCl. É importante destacar que em condições de subfase nas quais pares iônicos intermoleculares entre os aspárticos e lisinas podem ocorrer, em água e em baixa concentração de sal, o peptídeo co-cristalizou com lipídios de DPPC induzindo significativa mudança na forma de “triskelion” para formas alongadas e no tamanho dos domínios sólidos. Mudanças na forma e no tamanho foram também observados para DMPE e DMPS, mas com mecanismos diferentes. Devido a grande preferência de MP1 por filmes de PS foi observado que parte do filme peptídeo/lipídio foi expelido para a subfase em valores de pressão lateral compatíveis com a de bicamadas, sugerindo micelização do filme lipídico. Estas evidências de mudanças no empacotamento lipídico induzidas pelo peptídeo foram reforçadas investigando o efeito do MP1 no influxo de carboxifluoresceína em GUVs de PC/PS e PC/SM/Chol contendo ou não PS. Nesta última composição, domínios líquido-ordenados (Lo) podem ser visualizados e na presença de PS foi observado a formação de regiões densas na região líquido-desordenado (Ld) na membrana. Na presença de PS o MP1 induziu poros ou defeitos que permaneceram abertos na escala de tempo de minutos. Evidências para segregação lipídicas foram obtidas por experimentos de calorimetria diferencial de varredura (DSC) usando vesículas de DPPS puro e de misturas POPC/DPPS. Os resultados obtidos mostraram a indução de uma fase rica de peptídeo e lipídios aniônico e outra rica de lipídio zwitteriônico e perturbação no empacotamento lipídico de vesículas de lipídio aniônico puro.Antimicrobial peptides act directly on the lipid matrix of the cell membrane by perturbing the lipid packing, creating elastic stresses and mass imbalance, that are relieved by the formation of pores or defects resulting in the lytic activity. Polybia MP1, or shortly MP1, with aminoacid sequence IDWKKLLDAAKQIL-NH2, extracted from the native wasp Polybia paulista is an example of these peptides. Beyond a potent antibacterial activity, it inhibits cell proliferation in prostate and bladder cancer cell cultures. This inhibitory effect is believed to be due to the simultaneous presence of aminophospholipids: phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the outer leaflet of these cell membranes. Investigations of MP1 lytic activity in giant unilamellar vesicles (GUVs) revealed that the peptide induced some dense fluorescent regions on the vesicles that were interpreted as being due to peptide/lipid aggregation or to lipid segregation. The MP1 permeabilizing activity was also strongly modulated when fractions of aminophospholipids PS and PE were present in the lipid composition of these GUVs. It was observed that the permeability induced by MP1 increased dramatically for vesicles of PC/PE/PS (7:1:2) allowing influx of up to 10 kDa molecules. This thesis investigated the ability of MP1 in disturbing lipid packing using lipid monolayers as model membranes. Three diacylated phospholipids: PC, PE and PS were used. Compression isotherms obtained with Langmuir trough in conjunction with fluorescence and Brewster angle microscopies were used to investigate preferences of the peptide for lipid phases and its effects on the phase coexistence and on the changes of the shape, size and number of solid domains. Using diacyl chains derived from myristic and palmitic acids attached to PC, PE and PS polar heads it was possible to explore the role played by head groups and effect of the acyl chain lengths on the impact of the peptide in lipid films. These experiments showed that MP1 induced changes in both the shape and the size of solid domains. These changes were dependent on the head group and on the subphase conditions: pure water and aqueous solution of NaCl. It is remarkable that in subphase conditions in which intermolecular ionic pairs among aspartics and lysines are likely to occur, water and low salt concentration, the peptide was able to co-crystalize with DPPC lipids and induce significant changes in the shapes from “triskelion” to elongated and in the sizes of the solid domains. Change in the shape and size were also observed to DMPS and DMPE, but with different mechanisms. Due to the high preference of MP1 for PS films it was observed that part of the lipid/peptide films were irreversibly lost to the subphase, at lateral pressures compatible with that of membranes, suggesting micellization of the lipid film. These evidences for changes in the lipid packing induced by the peptide were reinforced by investigating the effect of MP1 on the influx of carboxyfluorescein in GUVs of PC/PS and of PC/SM/Chol containing or not PS. In this last composition liquid ordered (Lo) domains can be observed. In the presence of domains Lo and PS MP1 induced changes in the permeability and the formation of aggregates. The pores or defects opened in the PS containing vesicles remains opened in a minute time scale. In addition, evidences for lipid segregation was obtained from differential scan calorimetry (DSC) experiments using pure DPPS and mixed POPC/DPPS lipid vesicles showing the induction of a domain rich in peptide/anionic lipid complex and another rich in zwitterionic ones and perturbation on the lipid packing of pure anionic vesicles.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

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

The effect of acidic residues and amphipathicity on the lytic activities of mastoparan peptides studied by fluorescence and CD spectroscopy

Some mastoparan peptides extracted from social wasps display antimicrobial activity and some are hemolytic and cytotoxic. Although the cell specificity of these peptides is complex and poorly understood, it is believed that their net charges and their hydrophobicity contribute to modulate their biological activities. We report a study, using fluorescence and circular dichroism spectroscopies, evaluating the influence of these two parameters on the lytic activities of five mastoparans in zwitterionic and anionic phospholipid vesicles. Four of these peptides, extracted from the venom of the social wasp Polybia paulista, present both acidic and basic residues with net charges ranging from +1 to +3 which were compared to Mastoparan-X with three basic residues and net charge +4. Previous studies revealed that these peptides have moderate-to-strong antibacterial activity against Gram-positive and Gram-negative microorganisms and some of them are hemolytic. Their affinity and lytic activity in zwitterionic vesicles decrease with the net electrical charges and the dose response curves are more cooperative for the less charged peptides. Higher charged peptides display higher affinity and lytic activity in anionic vesicles. The present study shows that the acidic residues play an important role in modulating the peptides' lytic and biological activities and influence differently when the peptide is hydrophobic or when the acidic residue is in a hydrophilic peptide.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

Interaction of a synthetic antimicrobial peptide with model membrane by fluorescence spectroscopy

Static and time-resolved fluorescence of tryptophan and ortho-aminobenzoic acid was used to investigate the interaction of the synthetic antimicrobial peptide L1A (IDGLKAIWKKVADLLKNT-NH2) with POPC and POPC:POPG. N-acetylated (Ac-L1A) and N-terminus covalently bonded ortho-aminobenzoic acid (Abz-L1A-W8V) were also used. Static fluorescence and quenching by acrylamide showed that the peptides adsorption to the lipid bilayers was accompanied by spectral blue shift and by a decrease in fluorescence quenching, indicating that the peptides moved to a less polar environment probably buried in the lipidic phase of the vesicles. These results also suggest that the loss of the N-terminus charge allowed deeper fluorophore insertion in the bilayer. Despite the local character of spectroscopic information, conclusions can be drawn about the peptides as a whole. The dynamic behaviors of the peptides are such that the mean intensity lifetimes, the long correlation time and the residual anisotropy at long times increased when the peptides adsorb in lipid vesicles, being larger in anionic vesicles. From the steady-state increase in fluorescence intensity and anisotropy, we observed that the partition coefficient of peptides L1A and its Abz analog in both types of vesicles are higher than the acetylated analog; moreover, the affinity to the anionic vesicle is higher than to the zwitterionic.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

Protonectin peptides target lipids, act at the interface and selectively kill metastatic breast cancer cells while preserving morphological integrity

© 2021 Elsevier Inc. All rights reserved.Despite the need for innovative compounds as antimicrobial and anticancer agents, natural sources of peptides remain underexplored. Protonectin (PTN), a cationic dodecapeptide of pharmacological interest, presents large hydrophobicity that is associated with the tendency to aggregate and supposedly influences bioactivity. A disaggregating role was assigned to PTN' N-terminal fragment (PTN1-6), which enhances the bioactivity of PTN in a 1:1 mixture (PTN/PTN1-6). Spectroscopic techniques and model membranes (phospholipid bilayers and SDS micelles) revealed that environment-dependent aggregation is reduced for PTN/PTN1-6, but cytotoxicity of PTNs on MDA-MB-231 breast cancer showed the same CC50 values around 16 µM and on MCF-10A epithelial breast cells 6 to 5-fold higher values, revealing a selective interaction. Since PTN1-6 lacks activity on breast cells, its presence should differently affect PTN activity, suggesting that aggregation could modulate activity depending on the membrane characteristics. Indeed, increased partitioning and lytic activity of PTN/PTN1-6 were found in model membranes independently of charge density, but affected by the curvature tendency. PTN and PTN/PTN1-6 do not alter morphology and roughness of cancer cells, indicating a superficial interaction with membranes and consistent with results obtained in NMR experiments. Our results indicate that aggregation of PTNs depends on the membrane characteristics and modulates the activity of the peptides.This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP [Funding Projects Nos. 2012/24259-0, 2014/08372-7, 2016/50178-8] and Fundação para a Ciência e a Tecnologia, Portugal – FCT I. P. [Funding PTDC/BBB-BQB/1693/2014]. DSA has a postdoctoral fellowship (FAPESP grant# 2015/25620-7) and also thanks CAPES for former scholarships. DBM and FDO acknowledge, respectively, CAPES scholarships and FCT I.P. fellowship [PD/BD/135046/2017]. The Brazilian BioscienceNational Laboratory (LNBio) is acknowledged for the NMR timemachine [proposal RMN-23300].info:eu-repo/semantics/publishedVersio

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