Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 5 de 5
Filtrar
Más filtros

Banco de datos
Tipo de estudio
Tipo del documento
Asunto de la revista
País de afiliación
Intervalo de año de publicación
1.
EMBO Rep ; 22(12): e53007, 2021 12 06.
Artículo en Inglés | MEDLINE | ID: mdl-34605140

RESUMEN

While Epstein-Barr virus (EBV) establishes a life-long latent infection in apparently healthy human immunocompetent hosts, immunodeficient individuals are at particular risk to develop lymphoproliferative B-cell malignancies caused by EBV. A key EBV protein is the transcription factor EBV nuclear antigen 2 (EBNA2), which initiates B-cell proliferation. Here, we combine biochemical, cellular, and in vivo experiments demonstrating that the mitotic polo-like kinase 1 (PLK1) binds to EBNA2, phosphorylates its transactivation domain, and thereby inhibits its biological activity. EBNA2 mutants that impair PLK1 binding or prevent EBNA2 phosphorylation are gain-of-function mutants. They exhibit enhanced transactivation capacities, accelerate the proliferation of infected B cells, and promote the development of monoclonal B-cell lymphomas in infected mice. Thus, PLK1 coordinates the activity of EBNA2 to attenuate the risk of tumor incidences in favor of the establishment of latency in the infected but healthy host.


Asunto(s)
Infecciones por Virus de Epstein-Barr , Herpesvirus Humano 4 , Animales , Proteínas de Ciclo Celular , Infecciones por Virus de Epstein-Barr/complicaciones , Antígenos Nucleares del Virus de Epstein-Barr/genética , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Ratones , Fosforilación , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas , Latencia del Virus , Quinasa Tipo Polo 1
2.
PLoS Pathog ; 13(10): e1006664, 2017 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-28968461

RESUMEN

Epstein-Barr virus (EBV) infection converts resting human B cells into permanently proliferating lymphoblastoid cell lines (LCLs). The Epstein-Barr virus nuclear antigen 2 (EBNA2) plays a key role in this process. It preferentially binds to B cell enhancers and establishes a specific viral and cellular gene expression program in LCLs. The cellular DNA binding factor CBF1/CSL serves as a sequence specific chromatin anchor for EBNA2. The ubiquitous expression of this highly conserved protein raises the question whether additional cellular factors might determine EBNA2 chromatin binding selectively in B cells. Here we used CBF1 deficient B cells to identify cellular genes up or downregulated by EBNA2 as well as CBF1 independent EBNA2 chromatin binding sites. Apparently, CBF1 independent EBNA2 target genes and chromatin binding sites can be identified but are less frequent than CBF1 dependent EBNA2 functions. CBF1 independent EBNA2 binding sites are highly enriched for EBF1 binding motifs. We show that EBNA2 binds to EBF1 via its N-terminal domain. CBF1 proficient and deficient B cells require EBF1 to bind to CBF1 independent binding sites. Our results identify EBF1 as a co-factor of EBNA2 which conveys B cell specificity to EBNA2.


Asunto(s)
Linfocitos B/metabolismo , Cromatina/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Transactivadores/metabolismo , Proteínas Virales/metabolismo , Linfocitos B/virología , Línea Celular , Humanos , Regiones Promotoras Genéticas/inmunología , Unión Proteica , Secuencias Reguladoras de Ácidos Nucleicos/inmunología
3.
PLoS Pathog ; 11(5): e1004910, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-26024477

RESUMEN

Epstein-Barr virus (EBV) is a γ-herpesvirus that may cause infectious mononucleosis in young adults. In addition, epidemiological and molecular evidence links EBV to the pathogenesis of lymphoid and epithelial malignancies. EBV has the unique ability to transform resting B cells into permanently proliferating, latently infected lymphoblastoid cell lines. Epstein-Barr virus nuclear antigen 2 (EBNA-2) is a key regulator of viral and cellular gene expression for this transformation process. The N-terminal region of EBNA-2 comprising residues 1-58 appears to mediate multiple molecular functions including self-association and transactivation. However, it remains to be determined if the N-terminus of EBNA-2 directly provides these functions or if these activities merely depend on the dimerization involving the N-terminal domain. To address this issue, we determined the three-dimensional structure of the EBNA-2 N-terminal dimerization (END) domain by heteronuclear NMR-spectroscopy. The END domain monomer comprises a small fold of four ß-strands and an α-helix which form a parallel dimer by interaction of two ß-strands from each protomer. A structure-guided mutational analysis showed that hydrophobic residues in the dimer interface are required for self-association in vitro. Importantly, these interface mutants also displayed severely impaired self-association and transactivation in vivo. Moreover, mutations of solvent-exposed residues or deletion of the α-helix do not impair dimerization but strongly affect the functional activity, suggesting that the EBNA-2 dimer presents a surface that mediates functionally important intra- and/or intermolecular interactions. Our study shows that the END domain is a novel dimerization fold that is essential for functional activity. Since this specific fold is a unique feature of EBNA-2 it might provide a novel target for anti-viral therapeutics.


Asunto(s)
Antígenos Nucleares del Virus de Epstein-Barr/química , Proteínas Mutantes/química , Transactivadores/genética , Activación Transcripcional , Proteínas Virales/química , Adulto , Secuencia de Aminoácidos , Western Blotting , Cristalografía por Rayos X , Antígenos Nucleares del Virus de Epstein-Barr/genética , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Técnicas para Inmunoenzimas , Inmunoprecipitación , Datos de Secuencia Molecular , Proteínas Mutantes/genética , Mutación/genética , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Proteínas Virales/genética
4.
J Biol Chem ; 287(22): 18863-71, 2012 May 25.
Artículo en Inglés | MEDLINE | ID: mdl-22496454

RESUMEN

Archaeal promoters consist of a TATA box and a purine-rich adjacent upstream sequence (transcription factor B (TFB)-responsive element (BRE)), which are bound by the transcription factors TATA box-binding protein (TBP) and TFB. Currently, only a few activators of archaeal transcription have been experimentally characterized. The best studied activator, Ptr2, mediates activation by recruitment of TBP. Here, we present a detailed biochemical analysis of an archaeal transcriptional activator, PF1088, which was identified in Pyrococcus furiosus by a bioinformatic approach. Operon predictions suggested that an upstream gene, pf1089, is polycistronically transcribed with pf1088. We demonstrate that PF1088 stimulates in vitro transcription by up to 7-fold when the pf1089 promoter is used as a template. By DNase I and hydroxyl radical footprinting experiments, we show that the binding site of PF1088 is located directly upstream of the BRE of pf1089. Mutational analysis indicated that activation requires the presence of the binding site for PF1088. Furthermore, we show that activation of transcription by PF1088 is dependent upon the presence of an imperfect BRE and is abolished when the pf1089 BRE is replaced with a BRE from a strong archaeal promoter. Gel shift experiments showed that TFB recruitment to the pf1089 operon is stimulated by PF1088, and TFB seems to stabilize PF1088 operator binding even in the absence of TBP. Taken together, these results represent the first biochemical evidence for a transcriptional activator working as a TFB recruitment factor in Archaea, for which the designation TFB-RF1 is suggested.


Asunto(s)
Archaea/genética , Factores de Transcripción/genética , Transcripción Genética , Secuencia de Bases , Cartilla de ADN , Ensayo de Cambio de Movilidad Electroforética
5.
Appl Environ Microbiol ; 76(10): 3308-13, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-20363792

RESUMEN

Pyrococcus furiosus is a model organism for analyses of molecular biology and biochemistry of archaea, but so far no useful genetic tools for this species have been described. We report here a genetic transformation system for P. furiosus based on the shuttle vector system pYS2 from Pyrococcus abyssi. In the redesigned vector, the pyrE gene from Sulfolobus was replaced as a selectable marker by the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene (HMG-CoA) conferring resistance of transformants to the antibiotic simvastatin. Use of this modified plasmid resulted in the overexpression of the HMG-CoA reductase in P. furiosus, allowing the selection of strains by growth in the presence of simvastatin. The modified shuttle vector replicated in P. furiosus, but the copy number was only one to two per chromosome. This system was used for overexpression of His(6)-tagged subunit D of the RNA polymerase (RNAP) in Pyrococcus cells. Functional RNAP was purified from transformed cells in two steps by Ni-NTA and gel filtration chromatography. Our data provide evidence that expression of transformed genes can be controlled from a regulated gluconeogenetic promoter.


Asunto(s)
Vectores Genéticos/genética , Pyrococcus furiosus/genética , Transformación Genética , Acilcoenzima A , Cromatografía de Afinidad , ARN Polimerasas Dirigidas por ADN/aislamiento & purificación , ARN Polimerasas Dirigidas por ADN/metabolismo , Dosificación de Gen , Regulación Bacteriana de la Expresión Génica , Plásmidos/genética
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA