RESUMEN
The CHD1 gene, encoding the chromo-domain helicase DNA-binding protein-1, is one of the most frequently deleted genes in prostate cancer. Here, we examined the role of CHD1 in DNA double-strand break (DSB) repair in prostate cancer cells. We show that CHD1 is required for the recruitment of CtIP to chromatin and subsequent end resection during DNA DSB repair. Our data support a role for CHD1 in opening the chromatin around the DSB to facilitate the recruitment of homologous recombination (HR) proteins. Consequently, depletion of CHD1 specifically affects HR-mediated DNA repair but not non-homologous end joining. Together, we provide evidence for a previously unknown role of CHD1 in DNA DSB repair via HR and show that CHD1 depletion sensitizes cells to PARP inhibitors, which has potential therapeutic relevance. Our findings suggest that CHD1 deletion, like BRCA1/2 mutation in ovarian cancer, may serve as a marker for prostate cancer patient stratification and the utilization of targeted therapies such as PARP inhibitors, which specifically target tumors with HR defects.
Asunto(s)
ADN Helicasas/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Biomarcadores , Proteínas Portadoras/genética , Línea Celular Tumoral , Cromatina , Roturas del ADN de Doble Cadena , ADN Helicasas/deficiencia , ADN Helicasas/genética , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Endodesoxirribonucleasas , Humanos , Masculino , Proteínas Nucleares/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Neoplasias de la Próstata/genética , Reparación del ADN por RecombinaciónRESUMEN
PHF13 is a chromatin affiliated protein with a functional role in differentiation, cell division, DNA damage response and higher chromatin order. To gain insight into PHF13's ability to modulate these processes, we elucidate the mechanisms targeting PHF13 to chromatin, its genome wide localization and its molecular chromatin context. Size exclusion chromatography, mass spectrometry, X-ray crystallography and ChIP sequencing demonstrate that PHF13 binds chromatin in a multivalent fashion via direct interactions with H3K4me2/3 and DNA, and indirectly via interactions with PRC2 and RNA PolII. Furthermore, PHF13 depletion disrupted the interactions between PRC2, RNA PolII S5P, H3K4me3 and H3K27me3 and resulted in the up and down regulation of genes functionally enriched in transcriptional regulation, DNA binding, cell cycle, differentiation and chromatin organization. Together our findings argue that PHF13 is an H3K4me2/3 molecular reader and transcriptional co-regulator, affording it the ability to impact different chromatin processes.
Asunto(s)
Cromatina/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Histonas/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Animales , Línea Celular , Inmunoprecipitación de Cromatina , Cromatografía en Gel , Cristalografía por Rayos X , Regulación de la Expresión Génica , Humanos , Espectrometría de Masas , Ratones , Unión ProteicaRESUMEN
Recent studies involving several viral systems have highlighted the importance of cellular intrinsic defense mechanisms through nuclear antiviral proteins that restrict viral propagation. These factors include among others components of PML nuclear bodies, the nuclear DNA sensor IFI16, and a potential restriction factor PHF13/SPOC1. For several nuclear replicating DNA viruses, it was shown that these factors sense and target viral genomes immediately upon nuclear import. In contrast to the anticipated view, we recently found that incoming adenoviral genomes are not targeted by PML nuclear bodies. Here we further explored cellular responses against adenoviral infection by focusing on specific conditions as well as additional nuclear antiviral factors. In line with our previous findings, we show that neither interferon treatment nor the use of specific isoforms of PML nuclear body components results in co-localization between incoming adenoviral genomes and the subnuclear domains. Furthermore, our imaging analyses indicated that neither IFI16 nor PHF13/SPOC1 are likely to target incoming adenoviral genomes. Thus our findings suggest that incoming adenoviral genomes may be able to escape from a large repertoire of nuclear antiviral mechanisms, providing a rationale for the efficient initiation of lytic replication cycle.
Asunto(s)
Adenoviridae/genética , Proteínas de Unión al ADN/inmunología , Genoma Viral , Proteínas Nucleares/inmunología , Fosfoproteínas/inmunología , Factores de Transcripción/inmunología , Adenoviridae/fisiología , Infecciones por Adenoviridae/inmunología , Línea Celular Tumoral , Técnica del Anticuerpo Fluorescente Indirecta , Interacciones Huésped-Patógeno , Humanos , Interferones/farmacología , Microscopía Fluorescente , Neutrófilos/citología , Neutrófilos/virología , Proteínas Nucleares/genética , Replicación ViralRESUMEN
Recessive mutations in the MPV17 gene cause mitochondrial DNA depletion syndrome, a fatal infantile genetic liver disease in humans. Loss of function in mice leads to glomerulosclerosis and sensineural deafness accompanied with mitochondrial DNA depletion. Mutations in the yeast homolog Sym1, and in the zebra fish homolog tra cause interesting, but not obviously related phenotypes, although the human gene can complement the yeast Sym1 mutation. The MPV17 protein is a hydrophobic membrane protein of 176 amino acids and unknown function. Initially localised in murine peroxisomes, it was later reported to be a mitochondrial inner membrane protein in humans and in yeast. To resolve this contradiction we tested two new mouse monoclonal antibodies directed against the human MPV17 protein in Western blots and immunohistochemistry on human U2OS cells. One of these monoclonal antibodies showed specific reactivity to a protein of 20 kD absent in MPV17 negative mouse cells. Immunofluorescence studies revealed colocalisation with peroxisomal, endosomal and lysosomal markers, but not with mitochondria. This data reveal a novel connection between a possible peroxisomal/endosomal/lysosomal function and mitochondrial DNA depletion.
Asunto(s)
Anticuerpos Monoclonales/química , Endosomas/metabolismo , Lisosomas/metabolismo , Proteínas de la Membrana/genética , Proteínas Mitocondriales/genética , Peroxisomas/metabolismo , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/aislamiento & purificación , Línea Celular Tumoral , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Endosomas/ultraestructura , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Técnica del Anticuerpo Fluorescente , Expresión Génica , Humanos , Lisosomas/ultraestructura , Proteínas de la Membrana/metabolismo , Ratones , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Proteínas Mitocondriales/metabolismo , Mutación , Osteoblastos/metabolismo , Osteoblastos/ultraestructura , Peroxisomas/ultraestructura , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Hepatitis B virus (HBV) is hyperendemic to southern Africa, with genotype A of HBV being the predominant genotype, and subgenotype A1 prevailing. Infection with this subgenotype is associated with rapid disease progression, and high frequency of hepatocellular carcinoma development. The objectives of our study was to construct recombinant 1.28 mer replication competent HBV DNA plasmids of subgenotypes A1, A2 and D3 containing authentic endogenous HBV promoters and to follow their replication in vitro after transfection of Huh7 cells. We found that subgenotype D3 replicated at a lower level, as measured by HBsAg and HBV DNA levels, when compared to cells transfected with genotype A. There was no difference in the intracellular and extracellular HBsAg between cells transfected with subgenotypes A1 or A2. Cells transfected with subgenotype A1 had higher levels of intracellular replicative intermediates and HBcAg, and lower extracellular expression of HBeAg from days 1 to 3, when compared to cells transfected with subgenotype A2. In conclusion, the generation of these replication competent clones is an important step in the functional characterization of subgenotypes of HBV circulating in Africa and their comparison to strains circulating in other geographical regions of the world.
Asunto(s)
Virus de la Hepatitis B/genética , Virus de la Hepatitis B/fisiología , Regiones Promotoras Genéticas , Replicación Viral , Línea Celular , Genotipo , Antígenos de la Hepatitis B/análisis , Hepatocitos/virología , Humanos , Plásmidos , TransfecciónRESUMEN
Little is known about immediate phases after viral infection and how an incoming viral genome complex counteracts host cell defenses, before the start of viral gene expression. Adenovirus (Ad) serves as an ideal model, since entry and onset of gene expression are rapid and highly efficient, and mechanisms used 24-48 hours post infection to counteract host antiviral and DNA repair factors (e.g. p53, Mre11, Daxx) are well studied. Here, we identify an even earlier host cell target for Ad, the chromatin-associated factor and epigenetic reader, SPOC1, recently found recruited to double strand breaks, and playing a role in DNA damage response. SPOC1 co-localized with viral replication centers in the host cell nucleus, interacted with Ad DNA, and repressed viral gene expression at the transcriptional level. We discovered that this SPOC1-mediated restriction imposed upon Ad growth is relieved by its functional association with the Ad major core protein pVII that enters with the viral genome, followed by E1B-55K/E4orf6-dependent proteasomal degradation of SPOC1. Mimicking removal of SPOC1 in the cell, knock down of this cellular restriction factor using RNAi techniques resulted in significantly increased Ad replication, including enhanced viral gene expression. However, depletion of SPOC1 also reduced the efficiency of E1B-55K transcriptional repression of cellular promoters, with possible implications for viral transformation. Intriguingly, not exclusive to Ad infection, other human pathogenic viruses (HSV-1, HSV-2, HIV-1, and HCV) also depleted SPOC1 in infected cells. Our findings provide a general model for how pathogenic human viruses antagonize intrinsic SPOC1-mediated antiviral responses in their host cells. A better understanding of viral entry and early restrictive functions in host cells should provide new perspectives for developing antiviral agents and therapies. Conversely, for Ad vectors used in gene therapy, counteracting mechanisms eradicating incoming viral DNA would increase Ad vector efficacy and safety for the patient.
Asunto(s)
Adenoviridae/metabolismo , Infecciones por Adenovirus Humanos/metabolismo , Proteínas de Unión al ADN/metabolismo , Inmunidad Innata , Proteolisis , Factores de Transcripción/metabolismo , Adenoviridae/genética , Proteínas E1B de Adenovirus/genética , Proteínas E1B de Adenovirus/metabolismo , Proteínas E4 de Adenovirus/genética , Proteínas E4 de Adenovirus/metabolismo , Infecciones por Adenovirus Humanos/genética , Proteínas de Unión al ADN/genética , Células HEK293 , Humanos , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Factores de Transcripción/genéticaRESUMEN
Survival time-associated plant homeodomain (PHD) finger protein in Ovarian Cancer 1 (SPOC1, also known as PHF13) is known to modulate chromatin structure and is essential for testicular stem-cell differentiation. Here we show that SPOC1 is recruited to DNA double-strand breaks (DSBs) in an ATM-dependent manner. Moreover, SPOC1 localizes at endogenous repair foci, including OPT domains and accumulates at large DSB repair foci characteristic for delayed repair at heterochromatic sites. SPOC1 depletion enhances the kinetics of ionizing radiation-induced foci (IRIF) formation after γ-irradiation (γ-IR), non-homologous end-joining (NHEJ) repair activity, and cellular radioresistance, but impairs homologous recombination (HR) repair. Conversely, SPOC1 overexpression delays IRIF formation and γH2AX expansion, reduces NHEJ repair activity and enhances cellular radiosensitivity. SPOC1 mediates dose-dependent changes in chromatin association of DNA compaction factors KAP-1, HP1-α and H3K9 methyltransferases (KMT) GLP, G9A and SETDB1. In addition, SPOC1 interacts with KAP-1 and H3K9 KMTs, inhibits KAP-1 phosphorylation and enhances H3K9 trimethylation. These findings provide the first evidence for a function of SPOC1 in DNA damage response (DDR) and repair. SPOC1 acts as a modulator of repair kinetics and choice of pathways. This involves its dose-dependent effects on DNA damage sensors, repair mediators and key regulators of chromatin structure.
Asunto(s)
Cromatina/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Línea Celular , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Rayos gamma , Heterocromatina , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Tolerancia a Radiación , Reparación del ADN por Recombinación , Proteínas Represoras/metabolismo , Proteína 28 que Contiene Motivos TripartitoRESUMEN
Human spermatogonia (Spg) and their fetal precursors express fibroblast growth factor receptor 3 (FGFR3). To further elucidate the role of FGFR3 in the control of Spg self-renewal, proliferation, and/or differentiation, and to narrow down the FGFR3-positive cell type(s) in the normal adult human testis, tissue sections and whole mount preparations of seminiferous tubules were analyzed combining immunofluorescence and confocal fluorescence microscopy. FGFR3 protein was chiefly observed in cellular membranes and cytoplasmic vesicles of a subpopulation of type A Spg, which comprised the chromatin rarefaction zone-containing type A(dark). Cytoplasmic expression of FGFR3 and nuclear expression of proliferation-associated antigen KI-67 were mutually exclusive. Similarly, FGFR3-positive Spg were negative for Doublesex and Mab-3 related transcription factor 1 (DMRT1). By contrast, undifferentiated embryonic cell transcription factor 1 (UTF1) and survival time-associated PHD finger in ovarian cancer 1 protein (SPOC1) were co-expressed in the nuclei of FGFR3-positive Spg. Whole mounted seminiferous tubules illustrated the clonogenic arrangement of the FGFR3/UTF1 double-positive Spg, which mainly occurred as pairs or quadruplets and, different from the KIT-positive Spg, showed no overlap with KI-67 labeled clusters. Taken together, in the adult human testis, FGFR3 expression is a feature of small clones of rarely dividing type A Spg which resemble "undifferentiated" Spg, including the spermatogonial stem cells.
Asunto(s)
División Celular , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/biosíntesis , Espermatogonias/metabolismo , Membrana Celular/metabolismo , Vesículas Citoplasmáticas/metabolismo , Proteínas de Unión al ADN/biosíntesis , Técnica del Anticuerpo Fluorescente , Humanos , Antígeno Ki-67/biosíntesis , Masculino , Microscopía Confocal , Proteínas Nucleares/biosíntesis , Proteínas Proto-Oncogénicas c-kit/análisis , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/análisis , Túbulos Seminíferos/citología , Túbulos Seminíferos/metabolismo , Espermatogénesis/fisiología , Testículo/citología , Testículo/metabolismo , Transactivadores/biosíntesis , Factores de Transcripción/análisis , Factores de Transcripción/biosíntesisRESUMEN
It is unclear whether the distinct nuclear morphologies of human A(dark) (Ad) and A(pale) (Ap) spermatogonia are manifestations of different stages of germ cell development or phases of the mitotic cycle, or whether they may reflect still unknown molecular differences. According to the classical description by Clermont, human dark type A spermatogonium (Ad) may contain one, sometimes two or three nuclear 'vacuolar spaces' representing chromatin rarefaction zones. These structures were readily discerned in paraffin sections of human testis tissue during immunohistochemical and immunofluorescence analyses and thus represented robust morphological markers for our study. While a majority of the marker proteins tested did not discriminate between spermatogonia with and without chromatin rarefaction zones, doublesex- and mab-3-related transcription factor (DMRT1), tyrosine kinase receptor c-Kit/CD117 (KIT) and proliferation-associated antigen Ki-67 (KI-67) appeared to be restricted to subtypes which lacked the rarefaction zones. Conversely, exosome component 10 (EXOSC10) was found to accumulate within the rarefaction zones, which points to a possible role of this nuclear domain in RNA processing.
Asunto(s)
Espermatogonias/citología , Espermatogonias/metabolismo , Biomarcadores/metabolismo , Diferenciación Celular , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Cromatina/metabolismo , Cromatina/ultraestructura , Proteínas de Unión al ADN/metabolismo , Exorribonucleasas/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Humanos , Inmunohistoquímica , Antígeno Ki-67/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Masculino , Microscopía Confocal , Microscopía Fluorescente , Proteínas Nucleares/metabolismo , Proteína de la Leucemia Promielocítica con Dedos de Zinc , Proteínas Proto-Oncogénicas c-kit/metabolismo , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/metabolismo , Espermatogénesis , Espermatogonias/clasificación , Transactivadores/metabolismo , Factores de Transcripción/metabolismoRESUMEN
SPOC1 (PHF13) is a recently identified protein that has been shown to dynamically associate with somatic chromatin, to modulate chromatin compaction and to be important for proper cell division. Here, we report on the expression of SPOC1 in promyelocytic leukaemia zinc finger (PLZF)-positive undifferentiated spermatogonial stem cells (SSCs) of the mouse testis. To investigate further the biological function of SPOC1 in germ cells we generated Spoc1 mutant mice from a gene-trap embryonic stem cell clone. Postpubertal homozygous Spoc1(-/-) animals displayed a pronounced progressive loss of germ cells from an initially normal germ epithelium of the testis tubules leading to testis hypoplasia. This loss first affected non-SSC stages of germ cells and then, at a later time point, the undifferentiated spermatogonia. Remarkably, successive loss of all germ cells (at >20 weeks of age) was preceded by a transient increase in the number of undifferentiated A(aligned) (A(al)) spermatogonia in younger mice (at >10 weeks of age). The number of primary Spoc1(-/-) gonocytes, the proliferation of germ cells, and the initiation and progression of meiosis was normal, but we noted a significantly elevated level of apoptosis in the Spoc1(-/-) testis. Taken together, the data argue that SPOC1 is indispensable for stem cell differentiation in the testis and for sustained spermatogenesis.
Asunto(s)
Células Madre Adultas/metabolismo , Proteínas de Unión al ADN/metabolismo , Espermatogénesis , Espermatogonias/metabolismo , Testículo/metabolismo , Factores de Transcripción/metabolismo , Células Madre Adultas/patología , Animales , Apoptosis/genética , Diferenciación Celular/genética , Supervivencia Celular/genética , Ensamble y Desensamble de Cromatina , Proteínas de Unión al ADN/genética , Humanos , Masculino , Ratones , Ratones Noqueados , Mutación/genética , Espermatogénesis/genética , Espermatogonias/patología , Testículo/patología , Factores de Transcripción/genéticaRESUMEN
In this study, we characterize the molecular and functional features of a novel protein called SPOC1. SPOC1 RNA expression was previously reported to be highest in highly proliferating tissues and increased in a subset of ovarian carcinoma patients, which statistically correlated with poor prognosis and residual disease. These observations implied that SPOC1 might play a role in cellular proliferation and oncogenesis. Here we show that the endogenous SPOC1 protein is labile, primarily chromatin associated and its expression as well as localization are regulated throughout the cell cycle. SPOC1 is dynamically regulated during mitosis with increased expression levels and biphasic localization to mitotic chromosomes indicating a functional role of SPOC1 in mitotic processes. Consistent with this postulate, SPOC1 siRNA knockdown experiments resulted in defects in mitotic chromosome condensation, alignment and aberrant sister chromatid segregation. Finally, we have been able to show, using micrococcal nuclease (MNase) chromatin-digestion assays that SPOC1 expression levels proportionally influence the degree of chromatin compaction. Collectively, our findings show that SPOC1 modulates chromatin structure and that tight regulation of its expression levels and subcellular localization during mitosis are crucial for proper chromosome condensation and cell division.
Asunto(s)
Cromatina/metabolismo , Cromosomas Humanos/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Mitosis , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Animales , Línea Celular Tumoral , Células Eucariotas , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Metafase , Profase , Complejo de la Endopetidasa Proteasomal/metabolismo , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Transporte de Proteínas , ARN Interferente Pequeño/metabolismo , Fracciones Subcelulares/metabolismoAsunto(s)
Virus de la Hepatitis B , Hepatitis B/epidemiología , Hepatitis B/transmisión , Carcinoma Hepatocelular/etiología , ADN Viral/genética , Hepatitis B/complicaciones , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/inmunología , Humanos , Italia , Neoplasias Hepáticas/etiología , Factores de RiesgoRESUMEN
UNLABELLED: Formation of enveloped viruses involves assembly and budding at cellular membranes. In this study, we elucidated the morphogenesis of hepadnaviruses on the ultrastructural and biochemical level using duck hepatitis B virus (DHBV) as a model system. Formation of virus progeny initiates at the endoplasmic reticulum (ER) and is conserved both in vitro and in vivo. The morphogenesis proceeds via membrane-surrounded vesicles containing both virions and subviral particles, indicating a common morphogenetic pathway. The virus particle-containing vesicles (VCVs) are generated and maintained by reorganization of endomembranes accompanied by a striking disorganization of the rough ER (rER). VCVs are novel organelles with unique identity and properties of ER, intermediate compartment, endosomes, and multivesicular bodies. VCVs are dynamic structures whose size and shape are regulated by both membrane fusion and fission. CONCLUSION: Our data indicate a strong reorganization of endomembranes during DHBV infection, resulting in the biogenesis of novel organelles serving as multifunctional platforms for assembly and budding of virus progeny.
Asunto(s)
Virus de la Hepatitis B del Pato/genética , Virus de la Hepatitis B/fisiología , Hepatocitos/virología , Animales , Fraccionamiento Celular , Membrana Celular/virología , Patos , Retículo Endoplásmico/virología , Virus de la Hepatitis B del Pato/crecimiento & desarrollo , Virus de la Hepatitis B del Pato/aislamiento & purificación , Virus de la Hepatitis B/crecimiento & desarrollo , Virus de la Hepatitis B/aislamiento & purificación , Hepatocitos/ultraestructura , Orgánulos/virología , Plásmidos , Replicación ViralRESUMEN
The human hepatitis B virus (HBV) and the duck hepatitis B virus (DHBV) share several fundamental features. Both viruses have a partially double-stranded DNA genome that is replicated via a RNA intermediate and the coding open reading frames (ORFs) overlap extensively. In addition, the genomic and structural organization, as well as replication and biological characteristics, are very similar in both viruses. Most of the key features of hepadnaviral infection were first discovered in the DHBV model system and subsequently confirmed for HBV. There are, however, several differences between human HBV and DHBV. This review will focus on the molecular and cellular biology, evolution, and host adaptation of the avian hepatitis B viruses with particular emphasis on DHBV as a model system.
Asunto(s)
Avihepadnavirus/genética , Avihepadnavirus/fisiología , Infecciones por Hepadnaviridae/patología , Secuencia de Aminoácidos , Animales , Avihepadnavirus/crecimiento & desarrollo , Avihepadnavirus/patogenicidad , ADN Viral/genética , Modelos Animales de Enfermedad , Patos , Infecciones por Hepadnaviridae/tratamiento farmacológico , Infecciones por Hepadnaviridae/fisiopatología , Virus de la Hepatitis B del Pato/genética , Virus de la Hepatitis B del Pato/crecimiento & desarrollo , Virus de la Hepatitis B del Pato/patogenicidad , Virus de la Hepatitis B del Pato/fisiología , Datos de Secuencia Molecular , Morfogénesis/fisiología , Tropismo/fisiología , Proteínas Virales/análisis , Proteínas Virales/fisiología , Vacunas Virales/genética , Vacunas Virales/uso terapéutico , Internalización del Virus , Replicación Viral/fisiologíaRESUMEN
Caspase-8-binding protein FLICE-associated huge protein (FLASH) has been proposed to regulate death receptor CD95-induced apoptosis through facilitating caspase-8 activation at the death-inducing signaling complex. Here, we found that FLASH interacts with the PML nuclear body component Sp100 and predominantly resides in the nucleus and nuclear bodies (NBs). In response to CD95 activation, FLASH leaves the NBs and translocates into the cytoplasm where it accumulates at mitochondria. The nucleo-cytoplasmic translocation of FLASH requires CD95-induced caspase activation and is facilitated by the Crm1-dependent nuclear export pathway. Downregulation of FLASH by RNA interference or inhibition of its nucleo-cytoplasmic shuttling reduced CD95-induced apoptosis. Furthermore, we show that the adenoviral anti-apoptotic Bcl-2 family member E1B19K traps FLASH and procaspase-8 in a ternary complex at mitochondria, thereby blocking CD95-induced caspase-8 activation. Knock-down of Sp100 potentiated CD95-activated apoptosis through enhancing nucleo-cytoplasmic FLASH translocation. In summary, our findings suggest that CD95 signals via a previously unrecognized nuclear pathway mediated by nucleo-cytoplasmic translocation of FLASH.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/fisiología , Proteínas de Unión al Calcio/fisiología , Núcleo Celular , Cuerpos de Inclusión Intranucleares , Receptor fas/fisiología , Animales , Antígenos Nucleares/metabolismo , Apoptosis , Proteínas Reguladoras de la Apoptosis/metabolismo , Autoantígenos/metabolismo , Proteínas de Unión al Calcio/metabolismo , Caspasa 8/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Humanos , Cuerpos de Inclusión Intranucleares/metabolismo , Carioferinas/fisiología , Modelos Biológicos , Proteínas Nucleares/metabolismo , Unión Proteica , Transporte de Proteínas , Receptores Citoplasmáticos y Nucleares/fisiología , Transducción de Señal , Distribución Tisular , Proteína Exportina 1RESUMEN
Entry and intracellular transport of hepatitis B viruses have several unusual, largely unknown aspects. In this study, we explored the mode of virus entry using the duck hepatitis B virus (DHBV) and the primary hepatocyte infection model. Upon internalization, viral particles were enriched in an endosomal compartment, as revealed by biochemical and ultrastructural analysis. Virus-containing vesicles harbored early endosome markers. Kinetic analysis revealed time-dependent partial translocation of viral DNA from endosomes into the cytosol. This was strongly reduced by inhibition of vacuolar ATPase; (vATPase) activity with bafilomycin A1 and resulted in abortive infection and prevention of cccDNA formation. Inactivation of vATPase induced accumulation and stabilization of incoming viral particles in endosomes, presumably by blocking endosomal carrier vesicle-mediated cargo transport and sorting. Although neutralization of the endomembrane organelles alone led to stabilization of incoming viral particles, it did not inhibit virus infection. In line with this, a pH-dependent ectopic virus fusion at the plasma membrane could not be artificially induced. This provided further evidence for a pH-neutral translocation mechanism. Endosomal membrane potential was required for viral infection because cotreatment of cells with monensin partially overcame the inhibitory effect of bafilomycin A1. In conclusion, hepatitis B viral infection is mediated by a novel cellular entry mechanism with features different from that of all other known viruses.