RESUMEN
ASPP (apoptosis-stimulating proteins of p53) proteins bind PP-1c (protein phosphatase 1) and regulate p53 impacting cancer cell growth and apoptosis. Here we determine the crystal structure of the oncogenic ASPP protein, iASPP, bound to PP-1c. The structure reveals a 1:1 complex that relies on interactions of the iASPP SILK and RVxF motifs with PP-1c, plus interactions of the PP-1c PxxPxR motif with the iASPP SH3 domain. Small-angle X-ray scattering analyses suggest that the crystal structure undergoes slow interconversion with more extended conformations in solution. We show that iASPP, and the tumor suppressor ASPP2, enhance the catalytic activity of PP-1c against the small-molecule substrate, pNPP as well as p53. The combined results suggest that PxxPxR binding to iASPP SH3 domain is critical for complex formation, and that the modular ASPP-PP-1c interface provides dynamic flexibility that enables functional binding and dephosphorylation of p53 and other diverse protein substrates.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/metabolismo , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína Fosfatasa 1/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Secuencias de Aminoácidos , Compuestos de Anilina/metabolismo , Sitios de Unión , Biocatálisis , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Compuestos Organofosforados/metabolismo , Unión Proteica , Conformación Proteica , Proteína Fosfatasa 1/química , Dispersión del Ángulo Pequeño , Proteína p53 Supresora de Tumor/metabolismo , Difracción de Rayos XRESUMEN
Calnexin is a type 1 integral endoplasmic reticulum (ER) membrane molecular chaperone with a highly conserved C-terminal domain oriented to the cytoplasm. Protein N-myristoylation plays an important role in a wide variety of cellular signal transduction pathways and it is catalyzed by N-myristoyltransferase (NMT), a cytoplasmic and ER associated enzyme. Here using yeast two-hybrid screen, Western blot analysis, immunoprecipitation, immunolocalization and cellular fractionation we discovered that N-myristoyltransferase 1 interacts with calnexin at the ER. These observations point at a previously unrecognized contribution of calnexin to the retention of NMT1 at the ER membrane.
Asunto(s)
Aciltransferasas/metabolismo , Calnexina/metabolismo , Retículo Endoplásmico/metabolismo , Fibroblastos/metabolismo , Fracciones Subcelulares/metabolismo , Animales , Animales Recién Nacidos , Sitios de Unión , Células Cultivadas , Activación Enzimática , Ratones , Unión Proteica , Especificidad por Sustrato , Distribución TisularRESUMEN
The endoplasmic reticulum has an intricate network of pathways built to deal with the secretory and integral membrane protein synthesis demands of the cell, as well as adaptive responses set up for the endoplasmic reticulum to rely on when stressed. These pathways are both essential and complex, and because of these 2 factors, several situations can lead to a dysfunctional endoplasmic reticulum and result in a dysfunctional cell with the potential to contribute to the progression of disease. The endoplasmic reticulum has been implicated in several metabolic, neurodegenerative, inflammatory, autoimmune, and renal diseases and disorders, and in particular, cardiovascular diseases. The role of the endoplasmic reticulum in cardiovascular disease shows how the change in function of a particular microscopic organelle can lead to macroscopic changes in the form of disease.