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El etiquetado bioortogonal en tándem descubre proteínas nacientes modificadas endógenamente por co-translación
Yanping Zhu1,2, Lianne I Willems1, Daniela Salas1,3
1Department of Chemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.
Journal of the American Chemical Society
|September 2, 2020
Resumen
Este estudio identifica proteínas endógenas modificadas por O-GlcNAc durante la traducción, revelando un nuevo mecanismo para la homeostasis de proteínas. Estos hallazgos avanzan en nuestra comprensión de O-GlcNAc
Área de la Ciencia:
- La bioquímica
- Biología molecular
- Biología celular
Sus antecedentes:
- La modificación O-GlcNAc de los residuos de serina/treonina es crucial para la fisiología y la salud celular.
- O-GlcNAc está emergiendo como un regulador clave de la homeostasis de las proteínas celulares.
- El trabajo previo indicó que la modificación de O-GlcNAc puede ocurrir cotranslacionalmente, evitando la degradación del polipéptido naciente.
Objetivo del estudio:
- Desarrollar estrategias para el etiquetado e identificación de las cadenas de polipéptidos nacientes modificadas endógenamente por O-GlcNAc.
- Investigar el papel de la O-GlcNAc cotranslacional en el control de la calidad de las proteínas y la proteostasis.
- Identificar proteínas endógenas específicas que se someten a una modificación cotranslacional de O-GlcNAc.
Principales métodos:
- Ingeniería metabólica en tándem utilizando O-propargilo-puromicina (OPP) para etiquetar las cadenas nacientes.
- Ingeniería metabólica de los sacarídeos con tetra-O-acetil-2-N-azidoacetil-2-desoxi-galactopiranosa (Ac4GalNAz) para el objetivo O-GlcNAc.
- Estrategias de ligadura quimio-selectiva secuencial para el etiquetado robusto.
- Enriquecimiento en dos pasos de las cadenas nacentes glucosiladas seguido de la proteómica de escopeta.
Principales resultados:
- Éxito en el etiquetado e identificación de proteínas endógenas modificadas cotranslacionalmente por O-GlcNAc.
- Validación de la O-GlcNAcilación cotranslacional para tres proteínas identificadas.
- Demostración de estrategias para aislar analíticos endógenos de baja abundancia de mezclas complejas.
Conclusiones:
- Desarrollo de nuevos métodos para aislar e identificar las proteínas modificadas cotranslacionalmente por O-GlcNAc.
- Proporcionó información sobre el papel de O-GlcNAc en el control de la calidad de las proteínas citoplasmáticas.
- Se abrieron caminos para estudiar las funciones más amplias de las modificaciones de proteínas cotranslacionales en la proteostasis.
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