RESUMEN
A new visualizing method was developed for monitoring protein-protein (P-P) interactions in live mammalian cells. P-P interactions are visualized by directing localization of a bait protein to endosomes. This method is sufficiently robust to analyze signal-dependent P-P interactions such as calcium-dependent protein interactions. We visualized interactions between activated calmodulin and calmodulin-binding proteins, and observed oscillatory interactions via time-lapse imaging. In addition, this new method can simultaneously monitor multiple P-P interactions in a single live cell, which allows comparison of interactions between several prey proteins and a single bait protein. We observed that CaMKK1 and CaMKIIalpha bind calmodulin with distinct binding affinities in live cell, which indicates that calcium signaling is fine-tuned by distinct activation patterns of CaM kinases. This method will enable investigation of cellular processes based on dynamic P-P interactions.
Asunto(s)
Señalización del Calcio , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Calmodulina/metabolismo , Procesamiento de Imagen Asistido por Computador/métodos , Técnicas del Sistema de Dos Híbridos , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/química , Calmodulina/química , Células HeLa , Humanos , Microscopía ConfocalRESUMEN
Reactivation of the p53 pathway by a potential therapeutic antagonist, which inhibits HDM2 and HDMX, is an attractive strategy for drug development in oncology. Developing blockers towards conserved hydrophobic pockets of both HDMs has mainly focused on small synthetic compounds; however, this approach has proved challenging. Here we describe an approach to generate a potent HDM dual inhibitor, p53LZ2, by rational protein grafting of the p53 transactivation domain onto a homodimeric leucine zipper. p53LZ2 shows tight binding affinity to both HDMs compared with wild-type p53 in vitro. X-ray crystallographic, comparative modelling and small-angle X-ray scattering studies of p53LZ2-HDM complexes show butterfly-shaped structures. A cell-permeable TAT-p53LZ2 effectively inhibits the cancer cell growth in wild-type but not mutant p53 by arresting cell cycle and inducing apoptosis in vitro. Thus, p53LZ2, designed by rational grafting, shows a potential therapeutic approach against cancer.
Asunto(s)
Leucina Zippers/genética , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Recombinantes/farmacología , Proteína p53 Supresora de Tumor/genética , Secuencia de Aminoácidos , Animales , Apoptosis/genética , Puntos de Control del Ciclo Celular/genética , Proteínas de Ciclo Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Femenino , Células HCT116 , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Simulación de Dinámica Molecular , Complejos Multiproteicos/ultraestructura , Trasplante de Neoplasias , Neoplasias/tratamiento farmacológico , Ingeniería de Proteínas , Estructura Terciaria de Proteína , Proteínas Recombinantes/ultraestructura , Alineación de Secuencia , Trasplante Heterólogo , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/ultraestructuraRESUMEN
Interferon regulatory factor 3 (IRF3) is activated in response to various environmental stresses including viral infection and DNA-damaging agents. However, the biological function of IRF3 in cell growth is not well understood. We demonstrated that IRF3 markedly inhibited growth and colony formation of cells. IRF3 blocked DNA synthesis and induced apoptosis. Based on this negative control of cell growth by IRF3, we examined whether functional loss of IRF3 may contribute to oncogenic transformation. IRF3 activity was specifically inhibited by expression of its dominant negative mutant. This mutant lacks a portion of the DNA binding domain like IRF3a, an alternative splice form of IRF3 in the cells. This dominant negative inhibition blocked expression of specific IRF3 target genes. Mutant IRF3 efficiently transformed NIH3T3 cells, as demonstrated by anchorage-independent growth in soft agar and tumorigenicity in nude mice. These results imply that IRF3 may function as a tumor suppressor and suggest a possible role for the relative levels of IRF3 and its dominant negative mutant in tumorigenesis.