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Craspase es una proteasa activada por ARN guiada por CRISPR
Chunyi Hu1, Sam P B van Beljouw2,3, Ki Hyun Nam4
1Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.
Science (New York, N.Y.)
|August 25, 2022
Resumen
La caspasa guiada por CRISPR (Craspase) se activa mediante la unión al ARN objetivo, lo que permite la actividad de la proteasa contra Csx30. Este sistema autorregulador desactiva la función de la proteasa después de la escisión del ARN objetivo.
Área de la Ciencia:
- Biología molecular
- La bioquímica
- Biología estructural
Sus antecedentes:
- El sistema CRISPR-Cas tipo III-E utiliza el complejo efector gRAMP/Cas7-11.
- Este complejo se asocia con TPR-CHAT/Csx29 para formar la enzima Craspase.
- Craspase funciona como una caspase guiada por CRISPR con actividades de orientación de ARN y proteasa.
Objetivo del estudio:
- Para aclarar los mecanismos de la escisión del ARN objetivo y la activación de la proteasa en Craspase.
- Para identificar el sustrato proteico endógeno de Craspase.
- Para entender la capacidad de autorregulación del sistema Craspase.
Principales métodos:
- Microscopía cryoelectrónica (cryo-EM) para capturar instantáneas estructurales de Craspase.
- Ensayos bioquímicos para estudiar la unión al ARN, la escisión y la actividad de la proteasa.
- Identificación y caracterización del sustrato proteico de Craspase.
Principales resultados:
- El emparejamiento de ARN guía-objetivo en la región 5' desplaza un bucle de entrada, iniciando cambios conformacionales.
- Estos cambios activan alostéricamente la díada catalítica de la proteasa y crean una bolsa de unión al sustrato.
- Csx30 fue identificado como el sustrato endógeno, proteolizado específicamente por Craspase activado.
- La actividad de la proteasa es inhibida por la escisión del ARN objetivo por gRAMP y no activada por secuencias específicas de ARN.
Conclusiones:
- Craspase es una nueva proteasa activada por ARN con mecanismos intrínsecos de autorregulación.
- El sistema demuestra un control preciso sobre la actividad de la proteasa, vinculado al reconocimiento y la escisión del ARN objetivo.
- Esta investigación proporciona información estructural y mecanicista sobre la diversificación del sistema CRISPR-Cas.
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