Interaction between FAK/αB-crystalline is important for viability of the glioblastoma cells

Glioblastoma multiforme is a tumor of the central nervous system. Focal Adhesion Kinase (FAK) and αB-crystalline are two proteins involved in glioblastoma development. In this study, we investigated whether the FAK/αB-crystalline interaction is important for glioblastoma cells, we aimed to investigate the interaction of these two proteins in the glioblastoma multiforme cell line U87-MG. Two peptides named FP01 peptide (derived from αB-crystalline) and FP02 peptide (derived from FAK) were synthesized for this study. Treatment of U87-MG with the peptides FP01 and FP02 in the concentration at 50 µM reduced the viability cellular to around 41% and 51%, respectively. Morphological alterations in the cells treated with the peptides when compared to the control were observed. This study suggests that the interaction between FAK and αB-crystalline is important for the viability of glioblastoma cells.

Keywords
Glioblastoma; FAK; αB-crystalline; Peptides; Protein interaction

Authorship

Mariany Lopes da Costa Folly

performed experiments

analyzed data

Departamento de Ciências Básicas da Vida, Universidade Federal de Juiz de Fora Campus Governador Valadares (UFJF-GV), MG, Brazil, E-mail: mariany.lopes@ufjf.brUniversidade Federal de Juiz de ForaBrazilMG, BrazilDepartamento de Ciências Básicas da Vida, Universidade Federal de Juiz de Fora Campus Governador Valadares (UFJF-GV), MG, Brazil, E-mail: mariany.lopes@ufjf.br

http://orcid.org/0000-0001-9523-6187

Luana Lulio

performed experiments

Universidade Federal de Juiz de Fora, Campus Governador Valadares (UFJF-GV), MG, Brazil, E-mail: luanolulio@hotmail.comUniversidade Federal de Juiz de ForaBrazilMG, BrazilUniversidade Federal de Juiz de Fora, Campus Governador Valadares (UFJF-GV), MG, Brazil, E-mail: luanolulio@hotmail.com

http://orcid.org/0009-0004-7336-390X

Raquel Tognon-Ribeiro

provided new tools and reagents

Departamento de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas, Universidade Federal de Alfenas (UNIFAL-MG), MG, Brazil, E-mail: raquel.ribeiro@unifal-mg.edu.brUniversidade Federal de AlfenasBrazilMG, BrazilDepartamento de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas, Universidade Federal de Alfenas (UNIFAL-MG), MG, Brazil, E-mail: raquel.ribeiro@unifal-mg.edu.br

http://orcid.org/0000-0002-4184-706X

João Eustáquio Antunes

conceived and supervised the study

designed experiments

provided new tools and reagents

analyzed data

wrote the manuscript

Departamento de Farmácia, Universidade Federal de Juiz de Fora, Campus Governador Valadares (UFJF-GV), MG, Brazil, E-mail: joao.antunes@ufjf.brUniversidade Federal de Juiz de ForaBrazilMG, BrazilDepartamento de Farmácia, Universidade Federal de Juiz de Fora, Campus Governador Valadares (UFJF-GV), MG, Brazil, E-mail: joao.antunes@ufjf.br

http://orcid.org/0000-0003-1693-5823

Michelle Bueno de Moura Pereira ^**Correspondence: M. B. M. Pereira. Departamento de Ciências Básicas da Vida. Universidade Federal de Juiz de Fora (Campus Governador Valadares). Rua Manoel Byrro, 241 - Vila Bretas. CEP: 35032-620, Governador Valadares, MG, Brasil. Phone: 0055-33-98407-2355. Email: michelle.antunes@ufjf.br.

conceived and supervised the study

designed experiments

provided new tools and reagents

analyzed data

wrote the manuscript

http://orcid.org/0000-0003-2065-1700

*Correspondence: M. B. M. Pereira. Departamento de Ciências Básicas da Vida. Universidade Federal de Juiz de Fora (Campus Governador Valadares). Rua Manoel Byrro, 241 - Vila Bretas. CEP: 35032-620, Governador Valadares, MG, Brasil. Phone: 0055-33-98407-2355. Email: michelle.antunes@ufjf.br.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

SCIMAGO INSTITUTIONS RANKINGS

Figures

Figures (5)

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FIGURE 1
FAK interacts with αB-crystalline in glioblastoma cells. (a) Representative immunoblots of three independent experiments showing extracts from gliblastoma cells probed with anti-FAK and anti-αB-crystalline (CryAB) antibodies. Actin served as loading control. (b) Representative (n=3) anti-FAK immunoblot of αB-crystalline immunoprecipitates (IP). Anti-αB-crystalline immunoblot served as loading control. SN (supernatant).

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FIGURE 2
Interaction region of FP01 and FP02 peptides and proteins FAK and αB-crystalline by molecular docking. (a) HPLC chromatographic profile and ESI MS/MS spectrum of the peptide named FP01 of the αB-crystalline protein and of the peptide named FP02 of the FAK protein (b) Molecular docking between the FAT domain of FAK and the ACD domain of αB-crystalline. Ribbon representation of the ACD domain of αB-crystalline with the peptide FP01 (SPEELKVKVL) in yellow and ribbon representation of the FAK FAT domain with the peptide FP02 (NDKVYENVTG) in purple. (c) Frontal image of the model highlighting the contact surface between the FP01 peptide and the α 3 and α 4 helices of the FAK FAT domain. (d) In yellow, the region of the FP02 peptide present in the α1 helix of the FAT domain of FAK. (e) Oblique image of the model with indications of the interaction interface involved, highlighting the contact surface between the FP02 peptide and the β6 + 7 and β3 sheets of the αB-crystalline ACD domain. (f) In yellow, the region of the FP01 peptide present in the β4 sheet of the ACD domain of αB-crystalline.

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FIGURE 3
Treatment with peptides FP01 or FP02 reduces viability celullar in glioblastoma cells (a) Cytotoxic effect of FP01 peptide at different concentrations (10µM, 25µM, 50µM) at 24 hours, represented as a percentage in relation to the control group (PBS buffer). (b) Cytotoxic effect of FP01 peptide at different concentrations (10µM, 25µM, 50µM) at 48 hours, represented as a percentage in relation to the control group (PBS buffer). (c) Cytotoxic effect of FP02 peptide at different concentrations (10µM, 25µM, 50µM) at 24 hours, represented as a percentage in relation to the control group (PBS buffer). (d) Cytotoxic effect of FP02 peptide at different concentrations (10µM, 25µM, 50µM) at 48 hours, represented as a percentage in relation to the control group (PBS buffer). *p < 0.0001 versus PBS. Red dated line indicates the minimum percentage of significant cell viability reduction.

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FIGURE 4
Treatment with peptides FP01 or FP02 promoves apoptosis in glioblastoma cells. (a) Representative immunoblots of three independent experiments showing the expression of c-Casp3 after treatment with the peptides FP01 or FP02. Actin served as loading control.

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FIGURE 5
Treatment with peptides FP01 or FP02 alters morphology of glioblastoma cells. Representative image of U87-MG glioblastoma cells treated for 24 hours with PBS buffer (negative control) (panel A), cells treated with 50 µM TAT peptide (panel B), cells treated with 50 µM FP01 peptide (panel C) and cells treated with 50 µM FP02 peptide (panel D). 40x magnification.

FIGURE 1 FAK interacts with αB-crystalline in glioblastoma cells. (a) Representative immunoblots of three independent experiments showing extracts from gliblastoma cells probed with anti-FAK and anti-αB-crystalline (CryAB) antibodies. Actin served as loading control. (b) Representative (n=3) anti-FAK immunoblot of αB-crystalline immunoprecipitates (IP). Anti-αB-crystalline immunoblot served as loading control. SN (supernatant).

FIGURE 2 Interaction region of FP01 and FP02 peptides and proteins FAK and αB-crystalline by molecular docking. (a) HPLC chromatographic profile and ESI MS/MS spectrum of the peptide named FP01 of the αB-crystalline protein and of the peptide named FP02 of the FAK protein (b) Molecular docking between the FAT domain of FAK and the ACD domain of αB-crystalline. Ribbon representation of the ACD domain of αB-crystalline with the peptide FP01 (SPEELKVKVL) in yellow and ribbon representation of the FAK FAT domain with the peptide FP02 (NDKVYENVTG) in purple. (c) Frontal image of the model highlighting the contact surface between the FP01 peptide and the α 3 and α 4 helices of the FAK FAT domain. (d) In yellow, the region of the FP02 peptide present in the α1 helix of the FAT domain of FAK. (e) Oblique image of the model with indications of the interaction interface involved, highlighting the contact surface between the FP02 peptide and the β6 + 7 and β3 sheets of the αB-crystalline ACD domain. (f) In yellow, the region of the FP01 peptide present in the β4 sheet of the ACD domain of αB-crystalline.

FIGURE 3 Treatment with peptides FP01 or FP02 reduces viability celullar in glioblastoma cells (a) Cytotoxic effect of FP01 peptide at different concentrations (10µM, 25µM, 50µM) at 24 hours, represented as a percentage in relation to the control group (PBS buffer). (b) Cytotoxic effect of FP01 peptide at different concentrations (10µM, 25µM, 50µM) at 48 hours, represented as a percentage in relation to the control group (PBS buffer). (c) Cytotoxic effect of FP02 peptide at different concentrations (10µM, 25µM, 50µM) at 24 hours, represented as a percentage in relation to the control group (PBS buffer). (d) Cytotoxic effect of FP02 peptide at different concentrations (10µM, 25µM, 50µM) at 48 hours, represented as a percentage in relation to the control group (PBS buffer). *p < 0.0001 versus PBS. Red dated line indicates the minimum percentage of significant cell viability reduction.

FIGURE 4 Treatment with peptides FP01 or FP02 promoves apoptosis in glioblastoma cells. (a) Representative immunoblots of three independent experiments showing the expression of c-Casp3 after treatment with the peptides FP01 or FP02. Actin served as loading control.

FIGURE 5 Treatment with peptides FP01 or FP02 alters morphology of glioblastoma cells. Representative image of U87-MG glioblastoma cells treated for 24 hours with PBS buffer (negative control) (panel A), cells treated with 50 µM TAT peptide (panel B), cells treated with 50 µM FP01 peptide (panel C) and cells treated with 50 µM FP02 peptide (panel D). 40x magnification.

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E-mail: bjps@usp.br

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Interaction between FAK/αB-crystalline is important for viability of the glioblastoma cells

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[1] *Correspondence: M. B. M. Pereira. Departamento de Ciências Básicas da Vida. Universidade Federal de Juiz de Fora (Campus Governador Valadares). Rua Manoel Byrro, 241 - Vila Bretas. CEP: 35032-620, Governador Valadares, MG, Brasil. Phone: 0055-33-98407-2355. Email: michelle.antunes@ufjf.br.