Caracterização biofísico-química das proteínas FGFR2 e Grb2 e sua dinâmica de interação
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Data
2019-04-23
Autores
Tedesco, Jéssica Andrade
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Universidade Estadual Paulista (Unesp)
Resumo
FGFR2 (Fibroblast Growth Factor Receptor 2) é uma proteína receptora que apresenta um papel crucial na regulação do metabolismo celular, expressão gênica, crescimento, divisão e diferenciação celular. Vias de sinalização se iniciam com a ligação de fatores de crescimento aos seus receptores localizados na porção extracelular proteína FGFR2, induzindo sua fosforilação seguida de recrutamento de proteínas parceiras do citosol. Uma destas parceiras é a proteína adaptadora Grb2 (Growth Factor Receptor Bound Protein 2). O mecanismo de interação entre estas duas proteínas é fundamental para o funcionamento da via de sinalização MAPK (Mitogen Activated Protein Kinase). Elucidar tal mecanismo é crucial para o estudo da sinalização celular e seus comportamentos aberrantes. Experimentos TROSY-based de taxas de relaxação transversa foram realizados em colaboração com o Centro Nacional de Ressonância Magnética Nuclear (CNRMN). Os espectros 1D 1H do domínio quinase demonstram uma boa estruturação e dinâmica de acordo com a proteína. Tempos de relaxação aparente entre 16,2 e 23,5 ns foram encontrados, sendo compatíveis com proteínas que apresentam massa molecular entre 38,508 ± 7,709kDa, coerentes com a massa molecular de 40kDa do domínio quinase. Os espectros 2D 15N da interação entre FGFR2 e Grb2 mostram uma perda de sinal relacionado os domínios SH3-Grb2 quando comparados aos espectros da proteína Grb2 pura. Esta perda de sinal demonstra que ambos os domínios C- e N-terminal SH3-Grb2 estão participando da interação com FGFR2, enquanto o domínio SH2-Grb2 permanece dinâmico. Experimentos de DSC e DLS foram realizados com Grb2 em diferentes pHs para a determinação de possíveis alterações na sua estrutura devido à mudança entre pHs ácidos e bases. Constatou-se boa conformação, congruente com resultados observados na literatura, para pHs básicos. No entanto, em pHs ácidos foi observado um aumento na turbidez da solução impossibilitando a realização das análises. Tal comportamento gera duas hipóteses: a agregação da proteína devido à mudança significativa na carga total da proteína e a formação de transições de fase líquido-líquido.
FGFR2 (Fibroblast Growth Factor Receptor 2) is a receptor protein playing a crucial role in regulating cellular metabolism, genetic expression, growth, division and cellular differentiation. Signaling upstream starts when growth factors bind to receptors localized on the extracellular portion of FGFR2 which induce its phosphorylation followed by recruitment of protein partners from cytosol. One of these partners is the adaptor protein Grb2 (Growth Factor Receptor Bound Protein 2) which interaction mechanism between those proteins is fundamental for the health operation of MAPK (Mitogen Activated Protein Kinase). Elucidating such mechanism is crucial for the study of that signaling pathway and its aberrant behavior. TROSY-based studies of transverse relaxation rates were performed in collaboration with Centro Nacional de Ressonância Magnética Nuclear (CNRMN). 1D 1H spectrums of kinase domain demonstrate a good structure and dynamic of the protein. Apparent relaxation times between 16,2 and 23,5 ns were found, being compatible with proteins which present molecular weight of 38,508 ± 7,709kDa, coherent with the 40kDa molecular weight of kinase domain. 2D spectrums of FGFR2-Grb2 interaction showed a loss of signal related to SH3 domains in the compared spectrums of 15N isotopic labeled Grb2 and the mixture of labeled Grb2 and cytosolic FGFR2. This signal loss demonstrates that both N- and C-terminal Grb2-SH3 domains take place on the interaction with FGFR2 while Grb2-SH2 domain remains dynamic. This result adds a new figure to the FGFR2-Grb2 interaction tale which early was thought to be a role exclusively done by C-terminus Grb2-SH3 domain. DSC and DLS experiments were carried on with Grb2 in different pH buffers to determine possible alterations on its structure due to acid and basic pH changes. On basic pH, Grb2 presents good conformation being consistent with results observed on the literature. However, on acid pH, an increase in the turbidity of the solution was observed, making impossible the analysis. Such behavior leads us to two hypotheses: aggregation of the protein due to significant change on the net charge and the formation of liquid-liquid phase transition.
FGFR2 (Fibroblast Growth Factor Receptor 2) is a receptor protein playing a crucial role in regulating cellular metabolism, genetic expression, growth, division and cellular differentiation. Signaling upstream starts when growth factors bind to receptors localized on the extracellular portion of FGFR2 which induce its phosphorylation followed by recruitment of protein partners from cytosol. One of these partners is the adaptor protein Grb2 (Growth Factor Receptor Bound Protein 2) which interaction mechanism between those proteins is fundamental for the health operation of MAPK (Mitogen Activated Protein Kinase). Elucidating such mechanism is crucial for the study of that signaling pathway and its aberrant behavior. TROSY-based studies of transverse relaxation rates were performed in collaboration with Centro Nacional de Ressonância Magnética Nuclear (CNRMN). 1D 1H spectrums of kinase domain demonstrate a good structure and dynamic of the protein. Apparent relaxation times between 16,2 and 23,5 ns were found, being compatible with proteins which present molecular weight of 38,508 ± 7,709kDa, coherent with the 40kDa molecular weight of kinase domain. 2D spectrums of FGFR2-Grb2 interaction showed a loss of signal related to SH3 domains in the compared spectrums of 15N isotopic labeled Grb2 and the mixture of labeled Grb2 and cytosolic FGFR2. This signal loss demonstrates that both N- and C-terminal Grb2-SH3 domains take place on the interaction with FGFR2 while Grb2-SH2 domain remains dynamic. This result adds a new figure to the FGFR2-Grb2 interaction tale which early was thought to be a role exclusively done by C-terminus Grb2-SH3 domain. DSC and DLS experiments were carried on with Grb2 in different pH buffers to determine possible alterations on its structure due to acid and basic pH changes. On basic pH, Grb2 presents good conformation being consistent with results observed on the literature. However, on acid pH, an increase in the turbidity of the solution was observed, making impossible the analysis. Such behavior leads us to two hypotheses: aggregation of the protein due to significant change on the net charge and the formation of liquid-liquid phase transition.
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Palavras-chave
FGFR2, Grb2, Interação, Dinâmica, RMN, DSC, DLS, Interaction, Dynamic, NMR