Estudos de síntese, conformação e atividade biológica da gomesina e análogos através de diferentes metodologias
Data
2013
Autores
Domingues, Tatiana Moreira 
Orientadores
Miranda, Antonio de
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
O peptideo antimicrobiano gomesina (Gm) foi inicialmente isolado da hemolinfa da Acanthoscurria gomesiana, uma aranha caranguejeira encontrada no Brasil. Fortemente cationico, possui 18 residuos de aminoacidos (pGlu1-Cys-Arg-Arg-Leu5-Cys-Tyr-Lys-Gln-Arg10-Cys-Val-Thr-Tyr-Cys15-Arg-Gly-Arg-NH2) e duas ligacoes de dissulfeto nas posicoes Cys2,15 e Cys6,11. Trata-se de um PAM de largo espectro de acao, cuja atividade litica foi avaliada como elevada e rapida. Gm e analogos foram sintetizados pelo metodo em fase solida e utilizando-se a estrategia t-Boc. Em seguida, os peptideos foram clivados, purificados e caracterizados, alcancando-se um indice de pureza satisfatorio. Por meio de ensaios biologicos (de atividade antimicrobiana e hemolitica), estudos espectroscopicos e de cinetica de vazamento de carboxifluoresceina (CF), a Gm e todos os seus analogos foram avaliados, tendo sido possivel tracar uma relacao estrutura/atividade para esses peptideos, que comprova a importancia da manutencao das ligacoes de dissulfeto pelos analogos da Gm para o desempenho de uma boa atividade litica. Com base nesses primeiros resultados, a Gm e seu analogo menos ativo ([Ser2,6,11,15]-Gm) foram selecionados para analises mais detalhadas, utilizando-se, para tanto, modelos de membrana. Assim sendo, bicamadas lipidicas (LUVs), micelas e monomeros de SDS (dodecil sulfato de sodio) foram os sistemas mimeticos escolhidos para investigar o mecanismo de acao desses peptideos. A analise dos dados de calorimetria de titulacao isotermica (ITC) e de cinetica de vazamento de CF indicou forte interacao da Gm com as LUVs e rapida acao de desestruturacao dessas bicamadas, em um processo entalpicamente dirigido. Atraves da utilizacao das tecnicas de espalhamento de luz estatico e dinamico, e microscopia otica de contraste de fase, foi possivel detectar a formacao de grandes agregados lipidicos, mesmo em baixas razoes peptideo/lipidio. Com o intuito de minimizar a agregacao lipidica, um lipidio modificado contendo o polimero polietilenoglicol (PE-PEG2000) foi adicionado as vesiculas. A Gm tambem apresentou intensa ligacao exotermica (ITC) e uma alta taxa de permeabilizacao dessas bicamadas modificadas, alem de reduzida formacao de agregados. O analogo [Ser2,6,11,15]-Gm exibiu semelhante atividade a da Gm em interacao com LUVs contendo 50 mol% de POPG e uma diminuicao acentuada dessa atividade para LUVs com 25 mol% de POPG, reafirmando a importancia da atracao eletrostatica para esses peptideos. Alem disso, espectros de CD confirmaram que a Gm e seu analogo linear adotam conformacao em dobra beta quando em contato com lipossomas e SDS acima e abaixo da CMC (concentracao micelar critica). Em outros estudos, analises de fluorescencia e absorbancia de analogos ciclicos da Gm, marcados com um residuo de Trp, em presenca de diferentes concentracoes de SDS, apontaram para uma maior interacao desses peptideos com o SDS monomerico, resultando em um maior blueshift do espectro de emissao do Trp, bem como em um aumento na absorbancia das amostras, maior ou igual ao causado pelo SDS na forma micelar. O uso de dois sistemas mimeticos de membrana diferentes, bem como as diversas metodologias empregadas, foi fundamental para uma ampla investigacao das etapas requeridas pela Gm para causar a ruptura das biomembranas. Alem disso, esses estudos foram importantes na deteccao e caracterizacao da formacao de agregados durante a interacao da Gm com moleculas anfifilicas anionicas, agregacao essa que possivelmente se estende a outros peptideos cationicos, e que muitas vezes e negligenciada.
The antimicrobial peptide gomesin (Gm) was initially isolated from the hemolymph of Acanthoscurria gomesiana, a spider crab found in Brazil. It is strongly cationic, containing 18 amino acid residues (pGlu1 -Cys-Arg-Arg-Leu5-Cys-Tyr-Lys-GlnArg10-Cys-Val-Thr-Tyr-Cys15-Arg-Gly-Arg-NH2) and two disulfide bridges at positions Cys2,15 and Cys6,11. This PAM has a broad spectrum of action, whose lytic activity was considered as high and fast. Gm and analogues were synthesized by solid phase method and t-Boc strategy. Then, peptides were cleaved, purified and characterized, yielding a satisfactory percentage of purity. Via biological assays (antimicrobial and hemolytic activities), spectroscopic and carboxyfluorescein (CF) leakage studies, Gm and its analogues were evaluated. In that way, it was possible to extract a structure/activity relationship of these peptides, which reaffirmed the importance of the disulfide bridges to maintain a good lytic activity of Gm analogues. Based on these initial results, Gm and its less active analogue ([Ser2, 6,11,15]-Gm) were selected for more detailed analysis, which were evaluated using membrane models. Hence, lipid bilayers (LUVs), and SDS micelles and monomers were chosen as mimetic systems to investigate the mechanism of action of these peptides. Data analysis of ITC and kinetics of CF leakage indicated a strong interaction of Gm with LUVs, and also a fast disruption of these bilayers, in an enthalpy driven process. Through the use of static and dynamic light scattering and phase contrast microscopy techniques, it was possible to detect the formation of large lipid aggregates, even at low peptide/lipid ratios. In order to minimize the lipid aggregation, a modified lipid containing a polyethylene glycol polymer (PE-PEG2000) was added to vesicles. Gm also showed a strong exothermic interaction (ITC) and a high permeation rate of these modified bilayers, besides reduction in the aggregates formed. The analogue [Ser2, 6,11,15]-Gm exhibited similar activity as Gm in interaction with 50 mol% of POPG LUVs and a decrease in this activity for 25 mol% of POPG LUVs, reaffirming the importance of electrostatic attraction for these peptides. CD spectra confirmed that Gm and its linear analogue adopt beta-turn motif when in contact with liposomes and SDS above and below the CMC. In other studies, fluorescence and absorbance analyses of cyclic analogues of Gm, labeled with a Trp residue, in the presence of different concentrations of SDS, indicated a better interaction of these peptides with monomeric SDS, which caused a blueshift in the Trp emission spectra, and an increase in the sample absorbance, greater than or equal to that caused by SDS in micellar form. The use of two different membrane mimetic systems, as well as the different methodologies employed, was essential for a full investigation of the steps required by Gm to cause the disruption of biomembranes. Moreover, these studies have been important to detect and characterize the aggregates formed during the interaction of Gm with anionic biomolecules, whose event is possibly extended to other cationic peptides and often unnoticed.
The antimicrobial peptide gomesin (Gm) was initially isolated from the hemolymph of Acanthoscurria gomesiana, a spider crab found in Brazil. It is strongly cationic, containing 18 amino acid residues (pGlu1 -Cys-Arg-Arg-Leu5-Cys-Tyr-Lys-GlnArg10-Cys-Val-Thr-Tyr-Cys15-Arg-Gly-Arg-NH2) and two disulfide bridges at positions Cys2,15 and Cys6,11. This PAM has a broad spectrum of action, whose lytic activity was considered as high and fast. Gm and analogues were synthesized by solid phase method and t-Boc strategy. Then, peptides were cleaved, purified and characterized, yielding a satisfactory percentage of purity. Via biological assays (antimicrobial and hemolytic activities), spectroscopic and carboxyfluorescein (CF) leakage studies, Gm and its analogues were evaluated. In that way, it was possible to extract a structure/activity relationship of these peptides, which reaffirmed the importance of the disulfide bridges to maintain a good lytic activity of Gm analogues. Based on these initial results, Gm and its less active analogue ([Ser2, 6,11,15]-Gm) were selected for more detailed analysis, which were evaluated using membrane models. Hence, lipid bilayers (LUVs), and SDS micelles and monomers were chosen as mimetic systems to investigate the mechanism of action of these peptides. Data analysis of ITC and kinetics of CF leakage indicated a strong interaction of Gm with LUVs, and also a fast disruption of these bilayers, in an enthalpy driven process. Through the use of static and dynamic light scattering and phase contrast microscopy techniques, it was possible to detect the formation of large lipid aggregates, even at low peptide/lipid ratios. In order to minimize the lipid aggregation, a modified lipid containing a polyethylene glycol polymer (PE-PEG2000) was added to vesicles. Gm also showed a strong exothermic interaction (ITC) and a high permeation rate of these modified bilayers, besides reduction in the aggregates formed. The analogue [Ser2, 6,11,15]-Gm exhibited similar activity as Gm in interaction with 50 mol% of POPG LUVs and a decrease in this activity for 25 mol% of POPG LUVs, reaffirming the importance of electrostatic attraction for these peptides. CD spectra confirmed that Gm and its linear analogue adopt beta-turn motif when in contact with liposomes and SDS above and below the CMC. In other studies, fluorescence and absorbance analyses of cyclic analogues of Gm, labeled with a Trp residue, in the presence of different concentrations of SDS, indicated a better interaction of these peptides with monomeric SDS, which caused a blueshift in the Trp emission spectra, and an increase in the sample absorbance, greater than or equal to that caused by SDS in micellar form. The use of two different membrane mimetic systems, as well as the different methodologies employed, was essential for a full investigation of the steps required by Gm to cause the disruption of biomembranes. Moreover, these studies have been important to detect and characterize the aggregates formed during the interaction of Gm with anionic biomolecules, whose event is possibly extended to other cationic peptides and often unnoticed.
Descrição
Citação
DOMINGUES, Tatiana Moreira. Estudos de síntese, conformação e atividade biológica da gomesina e análogos através de diferentes metodologias. 2013. 143 f. Tese (Doutorado em Biologia Molecular) – Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP). São Paulo, 2013.