RESUMEN
The DEK oncoprotein regulates cellular chromatin function via a number of protein-protein interactions. However, the biological relevance of its unique pseudo-SAP/SAP-box domain, which transmits DNA modulating activities in vitro, remains largely speculative. As hypothesis-driven mutations failed to yield DNA-binding null (DBN) mutants, we combined random mutagenesis with the Bacterial Growth Inhibition Screen (BGIS) to overcome this bottleneck. Re-expression of a DEK-DBN mutant in newly established human DEK knockout cells failed to reduce the increase in nuclear size as compared to wild type, indicating roles for DEK-DNA interactions in cellular chromatin organization. Our results extend the functional roles of DEK in metazoan chromatin and highlight the predictive ability of recombinant protein toxicity in E. coli for unbiased studies of eukaryotic DNA modulating protein domains.
Asunto(s)
Cromatina/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , ADN/metabolismo , Escherichia coli/efectos de los fármacos , Mutación con Pérdida de Función , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas Recombinantes/toxicidad , Sesgo , Núcleo Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Cromatina/química , Cromatina/genética , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/toxicidad , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Genoma Bacteriano/efectos de los fármacos , Genoma Bacteriano/genética , Humanos , Mutagénesis , Nucleosomas/química , Nucleosomas/genética , Nucleosomas/metabolismo , Proteínas Oncogénicas/química , Proteínas Oncogénicas/toxicidad , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/toxicidad , Proteínas de Unión a Poli-ADP-Ribosa/química , Proteínas de Unión a Poli-ADP-Ribosa/toxicidad , Dominios Proteicos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Pruebas de Toxicidad/métodosRESUMEN
In two proof-of-concept studies, we established and validated the Bacterial Growth Inhibition Screen (BGIS), which explores recombinant protein toxicity in Escherichia coli as a largely overlooked and alternative means for basic characterization of functional eukaryotic protein domains. By applying BGIS, we identified an unrecognized RNA-interacting domain in the DEK oncoprotein (this study) and successfully combined BGIS with random mutagenesis as a screening tool for loss-of-function mutants of the DNA modulating domain of DEK [1]. Collectively, our findings shed new light on the phenomenon of recombinant protein toxicity in E. coli. Given the easy and rapid implementation and wide applicability, BGIS will extend the repertoire of basic methods for the identification, analysis and unbiased manipulation of proteins.
Asunto(s)
Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/toxicidad , Pruebas de Toxicidad/métodos , Animales , Sesgo , Biocatálisis , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/toxicidad , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/toxicidad , Escherichia coli/genética , Humanos , Mutación con Pérdida de Función , Proteínas Oncogénicas/química , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Proteínas Oncogénicas/toxicidad , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/toxicidad , Proteínas de Unión a Poli-ADP-Ribosa/química , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/toxicidad , Dominios Proteicos/genética , ARN/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/toxicidad , Receptores de la Familia Eph/química , Receptores de la Familia Eph/genética , Receptores de la Familia Eph/metabolismo , Receptores de la Familia Eph/toxicidad , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Reproducibilidad de los Resultados , Factores de Tiempo , Pruebas de Toxicidad/normasRESUMEN
SET is a multi-functional protein in proliferating cells. Some of the proposed functions of SET suggest an important nuclear role. However, the nuclear import pathway of SET is also unknown and the function of SET in neurons is unclear. Presently, using cortical neurons, we report that the nuclear import of SET is mediated by an impalpha/impbeta-dependent pathway. Nuclear localization signal, (168)KRSSQTQNKASRKR(181), in SET interacts with impalpha3, which recruits impbeta to form a ternary complex, resulting in efficient transportation of SET into nucleus. By in vitro nuclear import assay based on digitonin-permeabilized neurons, we further demonstrated that the nuclear import of SET relies on Ran GTPase. We provide evidence that this nuclear localization of SET is important in neuronal survival. Under basal conditions, SET is predominately nuclear. However, upon death induced by genotoxic stress, endogenous SET decreases in the nucleus and increases in the cytoplasm. Consistent with a toxic role of SET in the cytoplasm, targeted expression of SET to the cytoplasm exacerbates death compared to wild type SET expression which is protective following DNA damage. Taken together, our results indicate that SET is imported into the nucleus through its association with impalpha3/impbeta, and that localization of SET is important in regulation of neuronal death.