Estudos da interação da proteína adaptadora Grb2 (Growth Factor Receptor-Bound Protein 2) com os flavonoides morina e rutina
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Data
2017-04-13
Autores
Silva, Paulo Henrique da [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A proteína adaptadora Grb2 é uma importante reguladora da proteína quinase FGFR2 antes de estímulos extracelulares e é conhecida por formar complexos protéicos responsáveis por ativar a via de sinalização da MAPK, relacionada com a proliferação celular. Quando Grb2 é fosforilada ela se dissocia de FGFR2. Está por sua vez, adquire a capacidade de recrutar proteínas parceiras do citosol para iniciar vias de sinalização importantes para realização do metabolismo celular. A desfosforilação de Grb2 pela fosfatase Shp2 refaz o complexo FGFR2-Grb2 retomando controle sobre FGFR2. Desta maneira, devido a esta versatilidade em executar funções variadas dentro da célula, Grb2 torna-se um alvo importante para testar sua interação com moléculas conhecidas por apresentar propriedades farmacológicas. Sendo assim, as moléculas Morina (2’,3,4’,5,7-pentahidroxiflavona) e Rutina (Quercetina-3-O-α-L-Rhamnopiranosil-(1→6)-β-D-Glucopiranosídeo), foram escolhidas devido a suas propriedades anti-tumorais conhecidas na literatura e pela ausência de estudos em nível molecular que relacionem as propriedades destas moléculas com as proteínas que atuam nesta via de sinalização. Por conseguinte, utilizou-se espectroscopia de fluorescência em estado estacionário e ressonância magnética nuclear, com o objetivo de caracterizar a interação entre Morina e Rutina com Grb2. Através da determinação do perfil termodinâmico de interação destas moléculas com Grb2, obtidos por fluorescência, pudemos inferir um mecanismo entropicamente dirigido, compatível com interações hidrofóbicas para ambas moléculas, com constantes de associação compreendidos entre 104 e 105 M-1. Corroborando com os resultados, a ressonância magnética nuclear forneceu os epítopos da interação entre estas moléculas com Grb2 e mostrou por experimentos de competição que esta proteína liga-se mais fortemente à Morina que na Rutina. Posteriormente, os epítopos obtidos previamente, foram utilizados como guia para realizar simulações computacionais, como docking molecular, que indicaram que ambas moléculas ligam-se preferencialmente ao domínio SH2 de Grb2. Estes resultados contribuem para criação de uma base de conhecimento sobre o mecanismo de interação de moléculas variadas com proteínas, com o intuito em desenvolver fármacos mais eficientes de combate ao câncer.
Grb2 adaptor protein is an important regulator of FGFR2 before extracellular stimuli and is known to form complexes that activate MAPKinase pathway. When phosphorylated Grb2 dissociates from FGFR2 that gets able to recruit protein partners from the cytosol in order to start important signaling pathways inside cells. Dephosphorylating of Grb2 by Shp2 recue the FGFR2-Grb2 complex resulting in a control upon FGFR2. Because Grb2 shows to be versatile to performing functions inside the cell other than adaptor protein, it becomes an important target to test the interaction with molecules known to have anti-tumor properties. Therefore, the molecules Morin (2’,3,4’,5,7-pentahydroxyflavone) and Rutin (quercetin-3-O-α-L-Ramnopiranosil-(1→6)-β-D-glucopyranoside), were chosen because to its anti-inflammatory and anti-tumor properties known in the literature and because the absence of studies at the molecular level that brings up information about the properties of these molecules to specific protein targets. Thus, we have used static fluorescence spectroscopy and nuclear magnetic resonance in order to characterize the interaction between those molecules and Grb2. The thermodynamic profile got from fluorescence assays allow us to infer that the interaction between the molecules with Grb2 is entropically driven, compatible with hydrophobic interactions for both molecules where the equilibrium constants can be found between 104 and 105 M-1. Furthermore, nuclear magnetic resonance has provided us important information about the epitopes of the protein-ligand interaction, which have been used to do computation simulations to show that both molecules bind in the Grb2-SH2 domain. Competition assays have shown that Morin binds stronger then Rutin to Grb2.These results contribute to create knowledge about the interaction mechanism of these molecules with target proteins. Such characterization would help us to develop more effective drugs to treat cancer.
Grb2 adaptor protein is an important regulator of FGFR2 before extracellular stimuli and is known to form complexes that activate MAPKinase pathway. When phosphorylated Grb2 dissociates from FGFR2 that gets able to recruit protein partners from the cytosol in order to start important signaling pathways inside cells. Dephosphorylating of Grb2 by Shp2 recue the FGFR2-Grb2 complex resulting in a control upon FGFR2. Because Grb2 shows to be versatile to performing functions inside the cell other than adaptor protein, it becomes an important target to test the interaction with molecules known to have anti-tumor properties. Therefore, the molecules Morin (2’,3,4’,5,7-pentahydroxyflavone) and Rutin (quercetin-3-O-α-L-Ramnopiranosil-(1→6)-β-D-glucopyranoside), were chosen because to its anti-inflammatory and anti-tumor properties known in the literature and because the absence of studies at the molecular level that brings up information about the properties of these molecules to specific protein targets. Thus, we have used static fluorescence spectroscopy and nuclear magnetic resonance in order to characterize the interaction between those molecules and Grb2. The thermodynamic profile got from fluorescence assays allow us to infer that the interaction between the molecules with Grb2 is entropically driven, compatible with hydrophobic interactions for both molecules where the equilibrium constants can be found between 104 and 105 M-1. Furthermore, nuclear magnetic resonance has provided us important information about the epitopes of the protein-ligand interaction, which have been used to do computation simulations to show that both molecules bind in the Grb2-SH2 domain. Competition assays have shown that Morin binds stronger then Rutin to Grb2.These results contribute to create knowledge about the interaction mechanism of these molecules with target proteins. Such characterization would help us to develop more effective drugs to treat cancer.
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Palavras-chave
Grb2, FGFR2, Morina, Rutina, Morin, Rutin