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Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission
Published on: April 22, 2021
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Análisis de la interacción proteína-proteína en una sola célula viva mediante el uso de un sistema FRET basado en la
Seong-Hyun Park1, Wooseok Ko2, Hyun Soo Lee2
1Center for Biofunctional Molecules, Department of Chemistry , Yonsei University , Seoul 03722 , Republic of Korea.
Journal of the American Chemical Society
|February 2, 2019
Resumen
La proteína de choque térmico 70 (Hsp70) se une a Bax para suprimir la apoptosis. Los nuevos métodos FRET revelan cómo las sustancias que inducen la apoptosis interrumpen esta interacción Hsp70-Bax, ofreciendo información sobre la regulación de la muerte celular.
Área de la Ciencia:
- Biología molecular
- Biología celular
- La bioquímica
Sus antecedentes:
- La proteína de choque térmico 70 (Hsp70) es un regulador clave de la apoptosis, conocido por unirse a Bax e inhibir la muerte celular programada.
- Los mecanismos precisos por los cuales el complejo Hsp70-Bax se disocia durante la inducción de la apoptosis no se comprenden completamente.
Objetivo del estudio:
- Para aclarar los mecanismos moleculares subyacentes a la disociación de Bax de Hsp70 durante la apoptosis.
- Desarrollar y utilizar un nuevo sistema de transferencia de energía por resonancia Förster (FRET) para estudiar las interacciones proteína-proteína en tiempo real dentro de las células vivas.
Principales métodos:
- Desarrollo de un sistema FRET utilizando Hsp70 fusionado con proteína fluorescente amarilla (YFP) y Bax diseñado con un aminoácido fluorescente (ANAP) a través de la expansión del código genético.
- Imágenes de células vivas y análisis dependiente del tiempo de las señales FRET para monitorear las interacciones Hsp70-Bax.
- Investigar los efectos de varias sustancias que inducen la apoptosis, incluidos los activadores de Bax, los activadores de p53, los ligandos de muerte y los inhibidores de Bcl-2, en el complejo Hsp70-Bax.
Principales resultados:
- Los activadores de Bax dirigidos a los sitios desencadenantes de Bax inhibían la interacción Hsp70-Bax, mientras que un activador específico dirigido al sitio S184 terminal C no lo hacía.
- La inhibición de la interacción Hsp70-Hsp40 también bloqueó la interacción Bax-Hsp70.
- Los activadores de p53, los ligandos de muerte y los inhibidores de Bcl-2 promueven la disociación de Bax de Hsp70 a través de vías distintas que implican la reactivación de p53 o la activación de las proteínas BH3 solamente.
Conclusiones:
- El estudio empleó con éxito un nuevo sistema FRET para diseccionar los mecanismos de disociación del complejo Hsp70-Bax durante la apoptosis.
- Los diferentes agentes que inducen la apoptosis utilizan distintas vías moleculares para interrumpir la interacción Hsp70-Bax, destacando la complejidad de la regulación de la apoptosis.
- El sistema FRET desarrollado es una herramienta valiosa para investigar otras interacciones dinámicas proteína-proteína en sistemas biológicos.
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