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In Vitro Analysis of E3 Ubiquitin Ligase Function
Published on: May 14, 2021
Bases estructurales para la selección y orientación del sustrato fosfodependiente por la SCFCdc4 ubiquitina ligasa
Stephen Orlicky1, Xiaojing Tang, Andrew Willems
1Program in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, M5G 1X5, Toronto, Ontario, Canada.
Cell
|January 30, 2003
Resumen
El sistema de ubiquitina elimina con precisión los reguladores del ciclo celular como Sic1. Los investigadores encontraron que el Cdc4
Área de la Ciencia:
- * Biología molecular.
- * Biología celular.
- * La bioquímica es la bioquímica.
Sus antecedentes:
- * La progresión del ciclo celular depende de la degradación regulada de las proteínas a través del sistema de ubiquitina.
- * Los inhibidores de la cinasa dependiente de ciclina (CDK), como Sic1, son objetivos críticos para la degradación.
- * El complejo de ubiquitina ligasa SCF ((Cdc4) se dirige a Sic1 para ubiquitinación y degradación.
Objetivo del estudio:
- * Para dilucidar el mecanismo molecular del reconocimiento y ubiquitinación de Sic1 por SCF (Cdc4).
- * Comprender el papel de la fosforilación de Sic1 en su unión a Cdc4.
- * Investigar cómo las características estructurales de Sic1 y Cdc4 influyen en el reconocimiento del sustrato.
Principales métodos:
- * Cristalografía de rayos X para determinar la estructura del complejo Skp1-Cdc4 unido a un fosfopéptido.
- * Ensayos bioquímicos para evaluar afinidades de unión y tasas de ubiquitinación.
- * Ingeniería de proteínas para modificar la especificidad del sustrato de Cdc4.
Principales resultados:
- * Un núcleo de Cdc4-phosphodegron (CPD) (Leu-Leu-pThr-Pro) se une al dominio de la hélice WD40 de Cdc4.4.
- * Sic1 contiene múltiples sitios CPD de baja afinidad con discordancia estructural con el sitio de unión Cdc4.
- * La Cdc4 rediseñada exhibe una especificidad reducida, lo que permite la ubiquitinación de Sic1 menos fosforilado.
Conclusiones:
- * El reconocimiento de Sic1 por SCF (Cdc4) implica un modo de unión de equilibrio con múltiples sitios de baja afinidad.
- * Un umbral de fosforilación para el reconocimiento de Sic1 se explica por la dinámica de enlace.
- * Comprender este mecanismo proporciona información sobre el control del ciclo celular y las vías de degradación de las proteínas.
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