RESUMEN
Che-1 is a RNA polymerase II-binding protein involved in the transcription of E2F target genes and induction of cell proliferation. Here we show that Che-1 contributes to DNA damage response and that its depletion sensitizes cells to anticancer agents. The checkpoint kinases ATM/ATR and Chk2 interact with Che-1 and promote its phosphorylation and accumulation in response to DNA damage. These Che-1 modifications induce a specific recruitment of Che-1 on the TP53 and p21 promoters. Interestingly, it has a profound effect on the basal expression of p53, which is preserved following DNA damage. Notably, Che-1 contributes to the maintenance of the G2/M checkpoint induced by DNA damage. These findings identify a mechanism by which checkpoint kinases regulate responses to DNA damage.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de Ciclo Celular/fisiología , Proteínas de Unión al ADN/fisiología , Genes p53 , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/fisiología , Animales , Antineoplásicos/farmacología , Proteínas de la Ataxia Telangiectasia Mutada , División Celular , Quinasa de Punto de Control 2 , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Daño del ADN , Fase G2 , Humanos , Ratones , Células 3T3 NIH , Fosforilación , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
Major changes in medical, intensive care and organ transplantation practices are drastically increasing the risk of fungal opportunistic infections. We designed and set-up a MALDI-TOF MS-based assay to identify the most isolated and emerging therapy-refractory/uncommon fungi from cystic fibrosis (CF) and immunocompromised patients. Two-hundred and thirty isolates from 10 different genera (Aspergillus, Emericella, Fusarium, Geosmithia, Neosartorya, Penicillium, Pseudallescheria, Scedosporium, Talaromyces, Fomitopsis), investigated during routine diagnostic efforts, were correlated to 22 laboratory-adapted reference MALDI-TOF MS "proteomic phenotypes". A growth time-course at 30°C on Sabouraud agar medium was performed for the 22 "phenotypes" at 48, 72, 96 and 120h points. The best peptide extraction conditions for full recovery of conidia- or asci-producing multihyphal morph structures and the highest intra- and inter-class profiling correlation were identified for the 120h point spectra dataset, from which an engineered library derived (pre-analytical phase). Fingerprinting classifiers, selected by Wilcoxon/Kruskal-Wallis algorithm, were computed by Genetic Algorithm, Support Vector Machine, Supervised Neuronal Network and Quick Classifier model construction. MS identification (ID) of clinical isolates was referred to genotyping (GT) and, retrospectively, compared to routine morphotyping (MT) IDs (analytical phase). Proteomic phenotyping is revolutionizing diagnostic mycology as fully reflecting species/morph varieties but often overcoming taxonomic hindrance.