RESUMEN
Mind bomb (Mib) proteins are large, multi-domain E3 ligases that promote ubiquitination of the cytoplasmic tails of Notch ligands. This ubiquitination step marks the ligand proteins for epsin-dependent endocytosis, which is critical for in vivo Notch receptor activation. We present here crystal structures of the substrate recognition domains of Mib1, both in isolation and in complex with peptides derived from Notch ligands. The structures, in combination with biochemical, cellular, and in vivo assays, show that Mib1 contains two independent substrate recognition domains that engage two distinct epitopes from the cytoplasmic tail of the ligand Jagged1, one in the intracellular membrane proximal region and the other near the C terminus. Together, these studies provide insights into the mechanism of ubiquitin transfer by Mind bomb E3 ligases, illuminate a key event in ligand-induced activation of Notch receptors, and identify a potential target for therapeutic modulation of Notch signal transduction in disease.
Asunto(s)
Modelos Moleculares , Estructura Terciaria de Proteína , Receptores Notch/química , Ubiquitina-Proteína Ligasas/química , Secuencia de Aminoácidos , Animales , Western Blotting , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Línea Celular Tumoral , Cristalografía por Rayos X , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Epítopos/química , Epítopos/genética , Epítopos/metabolismo , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteína Jagged-1 , Ligandos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Mutación , Unión Proteica , Estructura Secundaria de Proteína , Receptores Notch/genética , Receptores Notch/metabolismo , Homología de Secuencia de Aminoácido , Proteínas Serrate-Jagged , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteína Wnt1RESUMEN
Recent work indicates that defects in late phases of the endosomal pathway caused by loss of function of the tumour suppressor gene lethal (2) giant discs (lgd) or the function of the ESCRT complexes I-III result in the ligand-independent activation of the Notch pathway in all imaginal disc cells in Drosophila melanogaster. lgd encodes a member of an uncharacterised protein family, whose members contain one C2 domain and four repeats of the DM14 domain. The function of the DM14 domain is unknown. We here report a detailed structure-function analysis of Lgd protein, which reveals that the DM14 domains are essential for the function of Lgd and act in a redundant manner. Moreover, our analysis indicates that the DM14 domain provides the specific function, whereas the C2 domain is required for the subcellular location of Lgd. We found that Lgd interacts directly with the ESCRT-III subunit Shrub through the DM14 domains. The interaction is required for the function of Shrub, indicating that Lgd contributes to the function of the ESCRT-III complex. Furthermore, our genetic studies indicate that the activation of Notch in ESCRT and lgd mutant cells occurs in a different manner and that the activity of Shrub and other ESCRT components are required for the activation of Notch in lgd mutant cells.