Estudo da formação de domínios em membranas modelos induzidos por peptídeos antimicrobianos e sua ação interfacial
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Data
2016-05-13
Autores
Alvares, Dayane dos Santos [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
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.
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.
Descrição
Palavras-chave
Polybia-MP1, Atividade antitumoral, Peptídeo antimicrobiano, Monocamadas lipídicas, DSC, GUVs, BAM, Microscopia de fluorescência, Aminofosfolipídios, Fosfatidiletanolamina, Fostatidilserina, Antitumor activity, Antimicrobial peptide, Monolayers, Fluorescence microscopy, Brewster angle microscopy, Aminophospholipids, Phosphatidylethanolamine, Phosphatidylserine