RESUMEN
The growing incidence rate of breast cancer, coupled with cellular chemotherapeutic resistance, has made this disease one of the most prevalent cancers among women worldwide. Despite the recent efforts to understand the underlying cause of the resistance due to mutation, there are no feasible tactics to overcome this bottleneck. This issue could be addressed by the concept of polypharmacology-disguising drugs present in the pharmacopeia for novel purposes (drug repurposing). Of note, we have proposed a multi-modal computational drug-repositioning stratagem to predict drugs possessing anti-proliferative effect. Our results suggest that Ombitasvir, a Hepatitis C NS5B polymerase inhibitor, could be "repurposed" for the control and prevention of beta-tubulin-driven breast cancers. J. Cell. Biochem. 118: 1412-1422, 2017. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Anilidas/farmacología , Antineoplásicos/farmacología , Antivirales/farmacología , Neoplasias de la Mama/genética , Carbamatos/farmacología , Reposicionamiento de Medicamentos/métodos , Tubulina (Proteína)/genética , Anilidas/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Carbamatos/uso terapéutico , Biología Computacional/métodos , Resistencia a Antineoplásicos/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Simulación del Acoplamiento Molecular , Mutación , Paclitaxel , Polifarmacología , Prolina , ValinaRESUMEN
Mowat-Wilson syndrome (MWS) is a severe neurodevelopmental disorder caused by heterozygous variants in the gene encoding transcription factor ZEB2. Affected individuals present with structural brain abnormalities, speech delay and epilepsy. In mice, conditional loss of Zeb2 causes hippocampal degeneration, altered migration and differentiation of GABAergic interneurons, a heterogeneous population of mainly inhibitory neurons of importance for maintaining normal excitability. To get insights into GABAergic development and function in MWS we investigated ZEB2 haploinsufficient induced pluripotent stem cells (iPSC) of MWS subjects together with iPSC of healthy donors. Analysis of RNA-sequencing data at two time points of GABAergic development revealed an attenuated interneuronal identity in MWS subject derived iPSC with enrichment of differentially expressed genes required for transcriptional regulation, cell fate transition and forebrain patterning. The ZEB2 haploinsufficient neural stem cells (NSCs) showed downregulation of genes required for ventral telencephalon specification, such as FOXG1, accompanied by an impaired migratory capacity. Further differentiation into GABAergic interneuronal cells uncovered upregulation of transcription factors promoting pallial and excitatory neurons whereas cortical markers were downregulated. The differentially expressed genes formed a neural protein-protein network with extensive connections to well-established epilepsy genes. Analysis of electrophysiological properties in ZEB2 haploinsufficient GABAergic cells revealed overt perturbations manifested as impaired firing of repeated action potentials. Our iPSC model of ZEB2 haploinsufficient GABAergic development thus uncovers a dysregulated gene network leading to immature interneurons with mixed identity and altered electrophysiological properties, suggesting mechanisms contributing to the neuropathogenesis and seizures in MWS.
RESUMEN
In recent years, workflows coupling DNA affinity purifications from crude nuclear extracts with quantitative mass spectrometry-based proteomics have enabled comprehensive mapping of protein-DNA interactions in an unbiased manner. Here, we describe a detailed protocol for one such method in which affinity purifications with extracts from cells or tissues of interest are combined with a chemical stable isotope labeling method, dimethyl labeling, to identify specific interaction partners for (hydroxy)methylated and non-methylated DNA sequences of interest.
Asunto(s)
5-Metilcitosina/análogos & derivados , 5-Metilcitosina/química , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Marcaje Isotópico/métodos , Proteoma/metabolismo , Biología Computacional/métodos , ADN/análisis , Epigénesis Genética , Humanos , Oxidación-Reducción , Proteoma/análisisRESUMEN
DNA methylation is an epigenetic modification, which regulates gene expression during cellular differentiation. This important function is thought to be carried out by transcriptional regulators, which are "readers" and effectors of this mark. In recent years, quantitative mass spectrometry-based interaction proteomics technology has emerged as a powerful tool to identify readers for methylated and unmethylated DNA in different cellular contexts. Furthermore, recent technology enables proteome-wide quantification of absolute affinities between proteins and methylated and unmethylated DNA in the context of crude nuclear extracts. Finally, recently developed locus-specific interaction proteomics approaches and modifications thereof facilitate an unbiased proteome characterization of methylated and unmethylated genomic loci in vivo. We summarize these recent findings in this review, and we argue that the integration of all these technologies, with also genomic sequencing-based approaches, will eventually result in a more detailed understanding of the link between DNA methylation and the regulation of transcription in health and disease.