RESUMEN
Chromatin assembly factor 1 (CAF1) consisting of p150, p60 and p48 is known to assemble histones onto newly synthesized DNA and thus maintain the chromatin structure. Here, we show that CAF1 expression was induced in human cytomegalovirus (HCMV)-infected cells, concomitantly with global chromatin decondensation. This apparent conflict was thought to result, in part, from CAF1 mislocalization to compartments of HCMV DNA synthesis through binding of its largest subunit p150 to viral immediate-early protein 2 (IE2). p150 interaction with p60 and IE2 facilitated HCMV DNA synthesis. The IE2Q548R mutation, previously reported to result in impaired HCMV growth with unknown mechanism, disrupted IE2/p150 and IE2/histones association in our study. Moreover, IE2 interaction with histones partly depends on p150, and the HCMV-induced chromatin decondensation was reduced in cells ectopically expressing the p150 mutant defective in IE2 binding. These results not only indicate that CAF1 was hijacked by IE2 to facilitate the replication of the HCMV genome, suggesting chromatin assembly plays an important role in herpesviral DNA synthesis, but also provide a model of the virus-induced chromatin instability through CAF1.
Asunto(s)
Factor 1 de Ensamblaje de la Cromatina/metabolismo , Citomegalovirus/metabolismo , Interacciones Huésped-Patógeno , Proteínas Inmediatas-Precoces/metabolismo , Transactivadores/metabolismo , Sustitución de Aminoácidos , Línea Celular , Cromatina/metabolismo , Factor 1 de Ensamblaje de la Cromatina/análisis , Ensamble y Desensamble de Cromatina , ADN Viral/metabolismo , Histonas/metabolismo , Humanos , Proteínas Inmediatas-Precoces/análisis , Proteínas Inmediatas-Precoces/genética , Unión Proteica , Subunidades de Proteína/análisis , Subunidades de Proteína/metabolismo , Transactivadores/análisis , Transactivadores/genética , Replicación ViralRESUMEN
It remains unclear what determines the subcellular localization of hepatitis B virus (HBV) core protein (HBc) and particles. To address this fundamental issue, we have identified four distinct HBc localization signals in the arginine rich domain (ARD) of HBc, using immunofluorescence confocal microscopy and fractionation/Western blot analysis. ARD consists of four tight clustering arginine-rich subdomains. ARD-I and ARD-III are associated with two co-dependent nuclear localization signals (NLS), while ARD-II and ARD-IV behave like two independent nuclear export signals (NES). This conclusion is based on five independent lines of experimental evidence: i) Using an HBV replication system in hepatoma cells, we demonstrated in a double-blind manner that only the HBc of mutant ARD-II+IV, among a total of 15 ARD mutants, can predominantly localize to the nucleus. ii) These results were confirmed using a chimera reporter system by placing mutant or wild type HBc trafficking signals in the heterologous context of SV40 large T antigen (LT). iii) By a heterokaryon or homokaryon analysis, the fusion protein of SV40 LT-HBc ARD appeared to transport from nuclei of transfected donor cells to nuclei of recipient cells, suggesting the existence of an NES in HBc ARD. This putative NES is leptomycin B resistant. iv) We demonstrated by co-immunoprecipitation that HBc ARD can physically interact with a cellular factor TAP/NXF1 (Tip-associated protein/nuclear export factor-1), which is known to be important for nuclear export of mRNA and proteins. Treatment with a TAP-specific siRNA strikingly shifted cytoplasmic HBc to nucleus, and led to a near 7-fold reduction of viral replication, and a near 10-fold reduction in HBsAg secretion. v) HBc of mutant ARD-II+IV was accumulated predominantly in the nucleus in a mouse model by hydrodynamic delivery. In addition to the revised map of NLS, our results suggest that HBc could shuttle rapidly between nucleus and cytoplasm via a novel TAP-dependent NES.
Asunto(s)
Proteínas de la Cápside/metabolismo , Núcleo Celular/metabolismo , Virus de la Hepatitis B/metabolismo , Virión/metabolismo , Transporte Activo de Núcleo Celular/genética , Transporte Activo de Núcleo Celular/fisiología , Alanina/genética , Sustitución de Aminoácidos/fisiología , Animales , Arginina/genética , Humanos , Ratones , Proteínas del Núcleo Viral/química , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/metabolismo , Internalización del Virus , Liberación del Virus/fisiologíaRESUMEN
A series of 3-O-acylated (-)-epigallocatechins were synthesized and their inhibition of steroid 5α-reductase was studied. They were prepared from the reaction of EGCG with tert-butyldimethylsilyl chloride followed by reductive cleavage of the ester bond. The resultant (-)-epigallocatechins penta-O-tert-butyldimethylsilyl ether was esterified with different fatty acids then desilylated to provide the corresponding products. The activity of 3-O-acylated (-)-epigallocatechins increased with the increasing carbon numbers of the fatty acid moiety, reaching maximum for 16 carbon atoms (compound 4h) with an IC50 of 0.53 µM, which was â¼12-fold more potent than EGCG (IC50=6.29 µM). Introduction of monounsaturated fatty acid provided the most potent compound 6 (IC50=0.48 µM), which showed moderate anti-tumor activity in vivo.
Asunto(s)
3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/metabolismo , Inhibidores de 5-alfa-Reductasa/farmacología , Antineoplásicos/farmacología , Catequina/análogos & derivados , Inhibidores de 5-alfa-Reductasa/síntesis química , Inhibidores de 5-alfa-Reductasa/química , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Catequina/síntesis química , Catequina/química , Catequina/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Masculino , Ratones , Ratones SCID , Modelos Moleculares , Estructura Molecular , Estereoisomerismo , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
T cell factor 4 (TCF4) interacts with beta-catenin in the WNT signaling pathway and transactivates downstream target genes involved in cancer progression. To identify proteins that regulate TCF4-mediated biological responses, we performed a yeast two-hybrid screen to search for a TCF4-binding protein(s) and found that MAD2B interacts with TCF4. We confirmed that MAD2B is a TCF4-binding protein by co-immunoprecipitation. Using the TOPFLASH reporter assay, we found that MAD2B blocks TCF4-mediated transactivation. The MAD2B binding regions of TCF4 were identified by TCF4 deletion mapping and electrophoretic mobility shift assay analysis. TCF4 and MAD2B interactions abolished the DNA binding ability of TCF4. Knockdown of MAD2B in SW480 colorectal cancer cells led to the conversion of epithelial cells to a mesenchymal fibroblastoid phenotype (epithelial-mesenchymal transdifferentiation). An E-cadherin promoter reporter analysis showed that MAD2B modulates TCF4-mediated E-cadherin expression. MAD2B knockdown blocked E-cadherin expression and significantly induced mesenchymal markers, such as N-cadherin and vimentin. Mesenchymal induction was accompanied by F-actin redistribution and the appearance of a fibroblastoid phenotype. MAD2B knockdown also increased both mRNA and protein levels of Slug, a known TCF4-induced E-cadherin transcriptional repressor. A chromatin immunoprecipitation assay showed that MAD2B silencing enhances the ability of TCF4 to bind the Slug promoter. Thus, MAD2B is a novel TCF4-interacting protein. This study provides the first evidence for the involvement of MAD2B in TCF4-mediated epithelial-mesenchymal transdifferentiation.
Asunto(s)
Transdiferenciación Celular , Proteínas de Unión al ADN/metabolismo , Epitelio/patología , Mesodermo/patología , Proteínas/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Western Blotting , Cadherinas/genética , Cadherinas/metabolismo , Línea Celular , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/genética , Ensayo de Cambio de Movilidad Electroforética , Epitelio/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HCT116 , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Proteínas Mad2 , Mesodermo/metabolismo , Microscopía Confocal , Unión Proteica , Proteínas/genética , ARN Interferente Pequeño/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción de la Familia Snail , Factor de Transcripción 4 , Factores de Transcripción/genética , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
BACKGROUND: Nrf1 [p45 nuclear factor-erythroid 2 (p45 NF-E2)-related factor 1], a member of the CNC-bZIP (CNC basic region leucine zipper) family, is known to be a transcriptional activator by dimerization with distinct partners, such as Maf, FosB, c-Jun, JunD, etc. The transcriptional roles of CNC-bZIP family are demonstrated to be involved in globin gene expression as well as the antioxidant response. For example, CNC-bZIP factors can regulate the expression of detoxification proteins through AREs, such as expression of human gamma-glutamylcysteine synthetases (GCS), glutathione S-transferases (GST), UDP-glucuronosyl transferase (UDP-GT), NADP (H) quinone oxidoreductase (NQOs), etc. To further explore other factor(s) in cells related to the function of Nrf1, we performed a yeast two-hybrid screening assay to identify any Nrf1-interacting proteins. In this study, we isolated a cDNA encoding residues 126-475 of MCRS2 from the HeLa cell cDNA library. Some functions of MCRS1 and its splice variant-MSP58 and MCRS2 have been previously identified, such as transforming, nucleolar sequestration, ribosomal gene regulation, telomerase inhibition activities, etc. Here, we demonstrated MCRS2 can function as a repressor on the Nrf1-mediated transactivation using both in vitro and in vivo systems. RESULTS: To find other proteins interacting with the CNC bZIP domain of Nrf1, the CNC-bZIP region of Nrf1 was used as a bait in a yeast two-hybrid screening assay. MCRS2, a splicing variant of p78/MCRS1, was isolated as the Nrf1-interacting partner from the screenings. The interaction between Nrf1 and MCRS2 was confirmed in vitro by GST pull-down assays and in vivo by co-immunoprecipitation. Further, the Nrf1-MCRS2 interaction domains were mapped to the residues 354-447 of Nrf1 as well as the residues 314-475 of MCRS2 respectively, by yeast two-hybrid and GST pull-down assays. By immunofluorescence, MCRS2-FLAG was shown to colocalize with HA-Nrf1 in the nucleus and didn't result in the redistribution of Nrf1. This suggested the existence of Nrf1-MCRS2 complex in vivo. To further confirm the biological function, a reporter driven by CNC-bZIP protein binding sites was also shown to be repressed by MCRS2 in a transient transfection assay. An artificial reporter gene activated by LexA-Nrf1 was also specifically repressed by MCRS2. CONCLUSION: From the results, we showed MCRS2, a new Nrf1-interacting protein, has a repression effect on Nrf1-mediated transcriptional activation. This was the first ever identified repressor protein related to Nrf1 transactivation.
Asunto(s)
Proteínas Nucleares/metabolismo , Factor Nuclear 1 de Respiración/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/metabolismo , Transactivadores/metabolismo , Células Cultivadas , ADN Complementario/genética , ADN Complementario/metabolismo , Técnica del Anticuerpo Fluorescente , Genes Reporteros , Células HeLa , Humanos , Proteínas Nucleares/genética , Factor Nuclear 1 de Respiración/genética , Proteínas de Unión al ARN/genética , Proteínas Represoras/genética , Transactivadores/genética , Activación Transcripcional , TransfecciónRESUMEN
Daxx, a human cell death-associated protein, was isolated as a Tcf4-interacting protein, using a yeast two-hybrid screen. Co-immunoprecipitation in HEK-293T cells and yeast two-hybrid screen in Y190 cells were performed to identify the interaction between Tcf4 with Daxx and to map the binding regions of Tcf4. In the nucleus, Daxx reduced DNA binding activity of Tcf4 and repressed Tcf4 transcriptional activity. Overexpression of Daxx altered the expression of genes downstream of Tcf4, including cyclin D1 and Hath-1, and induced G1 phase arrest in colon cancer cells. A reduction in Daxx protein expression was also observed in colon adenocarcinoma tissue when compared with normal colon tissue. This evidence suggests a possible physiological function of Daxx, via interaction with Tcf4, to regulate proliferation and differentiation of colon cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Neoplasias del Colon/patología , Neoplasias del Colon/fisiopatología , Proteínas Nucleares/fisiología , Factores de Transcripción TCF/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Sitios de Unión , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Proteínas Co-Represoras , Neoplasias del Colon/genética , ADN Complementario/genética , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Fase G1 , Expresión Génica , Genes bcl-1 , Humanos , Técnicas In Vitro , Chaperonas Moleculares , Complejos Multiproteicos , Proteínas Nucleares/genética , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de Transcripción TCF/genética , Proteína 2 Similar al Factor de Transcripción 7 , Técnicas del Sistema de Dos Híbridos , beta Catenina/metabolismoRESUMEN
BACKGROUND AND PURPOSE: The adenomatous polyposis coli (APC) and mismatch repair (MMR) pathways are both involved in the tumorigenesis of hereditary colorectal cancers. Chemoprevention focuses on the APC pathway in the absence of information concerning MMR targets. This study compared the anticancer effects of sulindac, celecoxib, curcumin, and nifedipine in MMR-deficient cell lines, in order to determine the most appropriate chemopreventive agent for long-term use in patients with hereditary colorectal cancer. METHODS: Five human colorectal cell lines (SW480, HCT116, LoVo, SW48, and HCT15) and an endometrial cancer cell line (HEC-1-A) were used for susceptibility testing. Tests included assays for growth inhibition, cell-cycle arrest, and apoptosis. RESULTS: Sulindac, celecoxib, curcumin, and nifedipine all displayed dose- and time-dependent anti-proliferation activities. Celecoxib was the most effective anti-proliferative agent, and increased the G0/G1 phase proportion in the cell cycle after treatment more significantly than the other agents in all cell lines. Curcumin displayed a more potent apoptosis-inducing activity than the other agents in treated cells. CONCLUSIONS: The tested drugs were effective against colorectal and endometrial cancer cell lines. Celecoxib is more potent with fewer side effects than sulindac. Nifedipine's observed chemopreventive efficacy may complement its known therapeutic application in patients with hypertension.
Asunto(s)
Antineoplásicos/farmacología , Ciclo Celular/efectos de los fármacos , Neoplasias Colorrectales/patología , Neoplasias Endometriales/patología , Apoptosis/efectos de los fármacos , Celecoxib , Línea Celular Tumoral , Quimioprevención/métodos , Curcumina/farmacología , Femenino , Humanos , Nifedipino/farmacología , Pirazoles/farmacología , Sulfonamidas/farmacología , Sulindac/farmacologíaRESUMEN
With the aim of identifying potential cellular proteins that mediate the transcriptional regulation of YY1, a HeLa cDNA library was screened using the yeast two-hybrid system. A previously unknown protein interacting with YY1 was identified and named YY1AP. By using the 5'-rapid amplification of cDNA ends technique, the full-length cDNA of YY1AP was cloned and sequenced. The cDNA was 2253 bp in length and encoded an open reading frame of 750 amino acids. The chromosomal gene was made up of 10 exons separated by nine introns and is localized on chromosome 1 (1q21.3). Northern blot analysis revealed that YY1AP is ubiquitously expressed in various human tissues and cancer cell lines. Co-immunoprecipitation and immunostaining of cells further indicated that YY1AP co-localizes with YY1 in the nucleus. Furthermore, YY1AP was shown to be capable of enhancing the transcriptional activation of an YY1 responsive promoter. Subsequent analysis by glutathione S-transferase pull-down assay showed that YY1AP contained two YY1 binding regions. The transactivation region of YY1AP would seem to be localized within the section of amino acids 260-345. It is proposed that YY1AP is a novel co-activator of YY1.
Asunto(s)
ADN Complementario/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/fisiología , Transactivadores/química , Transactivadores/fisiología , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Northern Blotting , Western Blotting , Proteínas de Ciclo Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Clonación Molecular , Factores de Unión al ADN Específico de las Células Eritroides , Exones , Biblioteca de Genes , Glutatión Transferasa/metabolismo , Células HeLa , Humanos , Intrones , Luciferasas/metabolismo , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Plásmidos/metabolismo , Pruebas de Precipitina , Regiones Promotoras Genéticas , Unión Proteica , Estructura Terciaria de Proteína , Distribución Tisular , Activación Transcripcional , Transfección , Técnicas del Sistema de Dos Híbridos , Factor de Transcripción YY1RESUMEN
Yin Yang 1 (YY1) is a highly conserved and multifunctional transcription factor. The diverse activities of YY1 are regulated and sometimes modified by interaction with various other proteins. By using a yeast two-hybrid screening system, SAP30 was identified as a protein that associates with YY1 and it is able to enhance YY1-mediated repression in a dose-dependent manner. SAP30 is a 30kDa nuclear protein and is a component of the human histone deacetylase complex. In this study, the interaction of SAP30 and YY1 was confirmed both by in vitro and in vivo assays. The interaction domains between YY1 and SAP30 were mapped to the C-terminal segment of YY1 (295-414) and the C-terminal 91 amino acid region of SAP30. The observation that YY1, SAP30, and HDAC1 form a complex in vivo provides evidence that YY1 also recruits HDAC1 indirectly via its binding to SAP30. These results describe a novel mechanism for YY1-mediated repression.
Asunto(s)
Histona Desacetilasas/metabolismo , Factores de Transcripción/metabolismo , Sitios de Unión , Factores de Unión al ADN Específico de las Células Eritroides , Células HeLa , Histona Desacetilasa 1 , Histona Desacetilasas/química , Histona Desacetilasas/genética , Humanos , Técnicas In Vitro , Sustancias Macromoleculares , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Transcripción Genética , Técnicas del Sistema de Dos Híbridos , Factor de Transcripción YY1 , Dedos de ZincRESUMEN
Using a transactivation-defective p53 derivative as bait, STK15, a centrosome-associated oncogenic serine/threonine kinase, was isolated as a p53 partner. The p53-STK15 interaction was confirmed further by co-immunoprecipitation and GST pull-down studies. In co-transfection experiments, p53 suppressed STK15-induced centrosome amplification and cellular transformation in a transactivation-independent manner. The suppression of STK15 oncogenic activity by p53 might be explained in part by the finding that p53 inhibited STK15 kinase activity via direct interaction with the latter's Aurora box. Taken together, these findings revealed a novel mechanism for the tumor suppressor function of p53.