RESUMEN
Influenza virus RNA polymerase has cap-dependent endonuclease activity that produces capped RNA fragments for priming viral mRNA synthesis. This enzymatic activity is essential for viral propagation, but it is not present in any host cellular enzyme, making it an attractive target for the development of anti-influenza drugs. Here, we isolated a novel inhibitor of cap-dependent endonuclease, named flupyranochromene, from the fermentation broth of the fungus Penicillium sp. f28743. Structural analysis revealed that this compound bears a putative pharmacophore that chelates divalent metal ion(s) present in the endonuclease active site in the PA subunit of the polymerase. Consistently, in vitro endonuclease assays showed that flupyranochromene exerts its inhibitory effects by blocking endonucleolytic cleavage by the PA subunit of viral RNA polymerase complex.
Asunto(s)
Antivirales/farmacología , Inhibidores Enzimáticos/farmacología , Subtipo H1N1 del Virus de la Influenza A/enzimología , Penicillium/química , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Proteínas Virales/antagonistas & inhibidores , Antivirales/química , Antivirales/aislamiento & purificación , Bioquímica/métodos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Fermentación , Técnicas Microbiológicas , Penicillium/clasificación , Penicillium/genética , Penicillium/aislamiento & purificaciónRESUMEN
Yeast capping enzymes differ greatly from those of mammalian, both structurally and mechanistically. Yeast-type capping enzyme repressors are therefore candidate antifungal drugs. The 5'-guanine-N7 cap structure of mRNAs are an essential feature of all eukaryotic organisms examined to date and is the first co-transcriptional modification of cellular pre-messenger RNA. Inhibitors of the RNA 5'-triphosphatase in yeast are likely to show fungicidal effects against pathogenic yeast such as Candida. We discovered a new RNA 5'-triphosphatase inhibitor, designated as the kribellosides, by screening metabolites from actinomycetes. Kribellosides belong to the alkyl glyceryl ethers. These novel compounds inhibit the activity of Cet1p (RNA 5'-triphosphatase) from Saccharomyces cerevisiae in vitro with IC50s of 5-8 µM and show antifungal activity with MICs ranging from 3.12 to 100 µg ml-1 against S. cerevisiae.
Asunto(s)
Ácido Anhídrido Hidrolasas/antagonistas & inhibidores , Actinobacteria/metabolismo , Antifúngicos/farmacología , Inhibidores Enzimáticos/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Antifúngicos/administración & dosificación , Antifúngicos/aislamiento & purificación , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/aislamiento & purificación , Concentración 50 Inhibidora , Pruebas de Sensibilidad Microbiana , Saccharomyces cerevisiae/enzimologíaRESUMEN
The synthesis of influenza virus mRNA is primed by capped (m(7)GpppNm-) short RNAs that are cleaved from RNA polymerase II transcripts by a virally encoded endonuclease. This cap-dependent endonuclease activity called "cap-snatching" may provide a unique target for novel anti-viral agents. To screen candidate inhibitors, it is essential to establish a method for producing efficiently a capped RNA substrate and a convenient assay for the cap-snatching activity. A 3'-biotinylated short RNA was prepared by in vitro transcription, purified by C18 reverse-phase column chromatography, and subjected to a capping reaction involving three recombinant capping enzymes. This capped RNA was shown to be an efficient substrate for the cap-snatching assay. Cap-snatching activity was then measured with the novel pull-down assay developed in this study, which is based on the streptavidin-biotin interaction. A known inhibitor for the cap-snatching reaction was evaluated by the pull-down assay, demonstrating the efficacy of the established screening system.
Asunto(s)
Antivirales/aislamiento & purificación , Evaluación Preclínica de Medicamentos/métodos , Endorribonucleasas/antagonistas & inhibidores , Inhibidores Enzimáticos/aislamiento & purificación , Orthomyxoviridae/efectos de los fármacos , Proteínas Virales/antagonistas & inhibidores , HumanosRESUMEN
mRNA capping is the first cotranscriptional modification of mRNA in the nucleus. In Saccharomyces cerevisiae, the first two steps of mRNA capping are catalyzed by the RNA triphosphatase Cet1p and the RNA guanylyltransferase Ceg1p. Cet1p and Ceg1p interact to form a mRNA capping enzyme complex and the guanylyltransferase activity of Ceg1p is stimulated by binding with Cet1p. The Cet1p-Ceg1p complex needs to be transported into the nucleus, where mRNA capping occurs. However, the molecular mechanism of nuclear transport of the Cet1p-Ceg1p complex is not known. Here, we show that Cet1p is responsible and that the Cet1p-Ceg1p interaction is essential for the nuclear localization of the Cet1p-Ceg1p complex. The results indicate that the Cet1p-Ceg1p interaction is important not only for the activation of Ceg1p, but also for nuclear import of the complex.
Asunto(s)
Ácido Anhídrido Hidrolasas/metabolismo , Núcleo Celular/enzimología , Núcleo Celular/metabolismo , Nucleotidiltransferasas/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Ácido Anhídrido Hidrolasas/genética , Nucleotidiltransferasas/genética , Unión Proteica , Transporte de Proteínas/genética , Transporte de Proteínas/fisiología , Saccharomyces cerevisiae/genéticaRESUMEN
Influenza virus RNA polymerase (RdRp) PB2 is the cap-1 binding subunit and determines host range and pathogenicity. The mutant human influenza virus RdRp containing PB2 D701N and D701N/S714R demonstrated enhanced replicon activity in mammalian cells. We investigated the influence of these mutations on RdRp activity. Cap-1-dependent transcription activities of D701N/S714R, D701N, and S714R were 348.1±6.2%, 146.4±11%, and 250.1±0.8% of that of the wild type (wt), respectively. Replication activity of these mutants for complimentary RNA to viral RNA ranged from 44% to 53% of that of the wt. Cap-1 RNA-binding activities of D701N/S714R, D701N, and S714R were 262±25%, 257±34%, and 315±9.6% of that of the wt, respectively, and their cap-dependent endonuclease activities were similar to that of the wt. These mutations did not affect template RNA-binding activities. D701N and S714R mutations enhanced transcription by enhancing cap-1 RNA-binding activity, but they may exhibit decreased efficiency of priming by the cap-1 primer. These mutations at the C-terminal domain of PB2 may affect its cap-binding domain.
Asunto(s)
Virus de la Influenza A/enzimología , Gripe Humana/virología , Nucleotidiltransferasas/genética , Proteínas de Unión a Caperuzas de ARN/genética , Caperuzas de ARN/genética , ARN Polimerasa Dependiente del ARN/genética , Proteínas Virales/genética , Regulación Viral de la Expresión Génica , Células HEK293 , Humanos , Virus de la Influenza A/patogenicidad , Gripe Humana/enzimología , Mutación , Nucleotidiltransferasas/metabolismo , Estructura Terciaria de Proteína , Proteínas de Unión a Caperuzas de ARN/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , Transcripción Genética , Proteínas Virales/metabolismoRESUMEN
The cap-dependent endonuclease activity of the influenza virus RNA-dependent RNA polymerase cleaves host mRNAs to produce capped RNA fragments for primers to initiate viral mRNA synthesis. The influenza A virus (FluA) cap-dependent endonuclease preferentially recognizes the cap1 structure (m(7)GpppNm). However, little is known about the substrate specificity of the influenza B virus (FluB) endonuclease. Here, we determined the substrate specificity of the FluB polymerase using purified viral RNPs and (32)P-labeled polyribonucleotides containing a variety of cap structures (m(7)GpppGm, m(7)GpppG, and GpppG). We found that the FluA polymerase cleaves m(7)G-capped RNAs preferentially. In contrast, the FluB polymerase could efficiently cleave not only m(7)G-capped RNAs but also unmethylated GpppG-RNAs. To identify a key amino acid(s) related to the cap recognition specificity of the PB2 subunit, the transcription activity of FluB polymerases containing mutated cap-binding domains was examined by use of a minireplicon assay system. In the case of FluA PB2, Phe323, His357, and Phe404, which stack the m(7)GTP, and Glu361 and Lys376, which make hydrogen bonds with a guanine base, were essential for the transcription activity. In contrast, in the case of FluB PB2, the stacking interaction of Trp359 with a guanine base and putative hydrogen bonds using Gln325 and Glu363 were enough for the transcription activity. Taking these results together with the result for the cap-binding activity, we propose that the cap recognition pocket of FluB PB2 does not have the specificity for m(7)G-cap structures and thus is more flexible to accept various cap structures than FluA PB2.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Virus de la Influenza A/enzimología , Virus de la Influenza B/enzimología , Caperuzas de ARN , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , ARN Polimerasas Dirigidas por ADN/química , Modelos Moleculares , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
We have established monoclonal antibodies (MoAbs) that are directed against hepatitis C virus (HCV)-expressing cells. They showed enhanced tumorigenicity after passage in culture for more than 44 days (RzM6-44d cells). To address the mechanism underlying this phenomenon, we characterized the MoAbs, and found that one of the clones recognized a molecule that was down-regulated in the RzM6-44d cells. This molecule was purified and identified as the 70-kDa thyroid autoantigen Ku70. Moreover, expression of the full-length HCV genome or HCV-core protein sequence in WRL68 human embryonic liver cells reduced the level of Ku70 protein, enhanced the ubiquitination of Ku70, and decreased the activity of DNA-activated protein kinase (DNA-PK). Therefore, it appears that the HCV-core protein facilitates the degradation of Ku70 and reduces DNA-PK activity in noncancerous liver cells.
Asunto(s)
Antígenos Nucleares/metabolismo , Proteína Quinasa Activada por ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Hepacivirus/fisiología , Hepatocitos/virología , Proteínas Nucleares/antagonistas & inhibidores , Proteínas del Núcleo Viral/fisiología , Línea Celular , Células Cultivadas , Femenino , Humanos , Autoantígeno Ku , Masculino , UbiquitinaciónRESUMEN
We have established four monoclonal antibodies (MAbs) against the nucleocapsid protein (NP) of canine distemper virus (CDV). A competitive binding assay has revealed that the MAbs are directed against two antigenic domains. An immunofluorescence assay using a series of deletion clones of the NP and an immunoprecipitation assay using the NP have revealed that two of the MAbs recognize the C-terminal region of the NP while the other two recognize the tertiary structure of the N-terminal domain. These MAbs reacted with all eight strains of CDV used in this study, but showed different reactivities against measles virus and rinderpest virus.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Virus del Moquillo Canino/inmunología , Proteínas de la Nucleocápside/inmunología , Animales , Anticuerpos Monoclonales/genética , Especificidad de Anticuerpos , Variación Antigénica , Unión Competitiva , Células COS , Chlorocebus aethiops , Moquillo/virología , Perros , Mapeo Epitopo , Técnica del Anticuerpo Fluorescente Indirecta , Ratones , Ratones Endogámicos BALB C , Mutagénesis Insercional , Proteínas de la Nucleocápside/genética , ARN Viral/química , ARN Viral/genética , Ensayo de Radioinmunoprecipitación , Células VeroRESUMEN
Hepatitis C virus (HCV) core protein is a putative nucleocapsid protein with a number of regulatory functions. In tissue culture cells, HCV core protein is mainly located at the endoplasmic reticulum as well as mitochondria and lipid droplets within the cytoplasm. However, it is also detected in the nucleus in some cells. To elucidate the mechanisms by which cellular trafficking of the protein is controlled, we performed subcellular fractionation experiments and used confocal microscopy to examine the distribution of heterologously expressed fusion proteins involving various deletions and point mutations of the HCV core combined with green fluorescent proteins. We demonstrated that a region spanning amino acids 112 to 152 can mediate association of the core protein not only with the ER but also with the mitochondrial outer membrane. This region contains an 18-amino-acid motif which is predicted to form an amphipathic alpha-helix structure. With regard to the nuclear targeting of the core protein, we identified a novel bipartite nuclear localization signal, which requires two out of three basic-residue clusters for efficient nuclear translocation, possibly by occupying binding sites on importin-alpha. Differences in the cellular trafficking of HCV core protein, achieved and maintained by multiple targeting functions as mentioned above, may in part regulate the diverse range of biological roles of the core protein.
Asunto(s)
Hepacivirus/química , Proteínas del Núcleo Viral/análisis , Secuencia de Aminoácidos , Línea Celular , Núcleo Celular/química , Retículo Endoplásmico/química , Humanos , Carioferinas/fisiología , Mitocondrias/química , Datos de Secuencia Molecular , Señales de Localización Nuclear , Proteínas del Núcleo Viral/químicaRESUMEN
The Sendai virus (SeV) RNA-dependent RNA polymerase complex, which consists of L and P proteins, participates in the synthesis of viral mRNAs that possess a methylated cap structure. To identify the SeV protein(s) involved in mRNA cap methylation, we developed an in vitro assay system to detect mRNA (guanine-7-)methyltransferase (G-7-MTase) activity. Viral ribonucleoprotein complexes and purified recombinant L protein but not P protein exhibited G-7-MTase activity. On the other hand, mRNA synthesis in a reconstituted transcription system using purified N-RNA (N protein-genomic RNA) complex as a template required both the L and P proteins. The enzymatic properties of SeV G-7-MTase were different from those of cellular G-7-MTase. In particular, unlike cellular G-7-MTase, the SeV enzyme preferentially methylated capped RNA containing the viral mRNA 5'-end sequences (GpppApGpG-). The C-terminal part (amino acid residues 1,756-2,228) of the L protein catalyzed cap methylation, whereas the N-terminal half (residues 1-1,120) containing putative RNA polymerase subdomains did not. This is to our knowledge the first direct biochemical evidence that supports the idea that mononegavirus L protein catalyzes cap methylation as well as RNA synthesis.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/fisiología , ARN Mensajero/metabolismo , ARN Viral , ARN Polimerasa Dependiente del ARN/fisiología , Virus Sendai/enzimología , Proteínas Virales/fisiología , Baculoviridae/metabolismo , Western Blotting , ADN Complementario/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Humanos , Metilación , Metiltransferasas/metabolismo , Modelos Genéticos , Estructura Terciaria de Proteína , ARN/química , Caperuzas de ARN , ARN Viral/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , Cloruro de Sodio/farmacología , Especificidad por Sustrato , Proteínas Virales/metabolismoRESUMEN
The ARF tumor suppressor gene antagonizes generation of various tumors. ARF-mediated tumor suppression occurs in a p53-independent manner as well as in a p53-dependent manner. We here demonstrate that BCL6 is a target of the ARF tumor suppressor. Either mouse p19(ARF) or human p14(ARF) binds to BCL6 and downregulates BCL6-induced transcriptional repression. ARF-mediated downregulation of the BCL6 activity may account in part for ARF-mediated tumor suppression.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Osteosarcoma/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Proteína p14ARF Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Sitios de Unión , Células COS , Línea Celular , Chlorocebus aethiops , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Humanos , Ratones , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-bcl-6RESUMEN
Cdc7 expression repressor (CR)/periphilin has been originally cloned as an interactor with periplakin, a precursor of the cornified cell envelope, and suggested to constitute a new type of nuclear matrix. We here show that CR/periphilin is a ubiquitously expressed nuclear protein with speckled distribution. Overexpression of CR/periphilin induces S-phase arrest. Analysis of expression of regulators involved in DNA replication has revealed that both mRNA and protein expression of Cdc7, a regulator of the initiation and continuation of DNA replication, are markedly downregulated by overexpression of CR/periphilin. However, co-expression of Cdc7 only marginally rescues S-phase arrest induced by CR, indicating that CR retards S-phase progression by modifying expression of some genes including Cdc7, which are involved in progression of DNA replication or coordination of DNA replication and S-phase progression.
Asunto(s)
Proteínas de Ciclo Celular/biosíntesis , Regulación hacia Abajo , Proteínas Nucleares/biosíntesis , Proteínas Serina-Treonina Quinasas/biosíntesis , Fase S , Adenoviridae/genética , Animales , Antígenos de Neoplasias , Northern Blotting , Células COS , Ciclo Celular , Línea Celular , Línea Celular Tumoral , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Clonación Molecular , ADN/metabolismo , ADN Complementario/metabolismo , Etiquetas de Secuencia Expresada , Células HeLa , Humanos , Immunoblotting , Inmunohistoquímica , Inmunoprecipitación , Queratinocitos/metabolismo , Modelos Genéticos , Plásmidos/metabolismo , Reacción en Cadena de la Polimerasa , Unión Proteica , Isoformas de Proteínas , ARN Mensajero/metabolismoRESUMEN
Combined disruption of the ARF gene and the p53 gene causes mouse predisposition to tumors of a wider variety and at a higher frequency than disruption of the p53 gene, indicating that the ARF gene has p53-independent anti-tumor function in addition to p53-dependent function. Coincidentally with this notion, ectopic expression of the p19(ARF) induces apoptosis for wild-type mouse embryo fibroblasts which have been immortalized by introduction of the SV40 virus genome (SV40-MEFs). The protein expression levels of p53, p21(Cip1), and Bax were not upregulated by ectopic expression of p19(ARF) in SV40-MEFs, indicating that expression of p19(ARF) induced apoptosis through p53-independent pathways in this system. Ectopic expression of p19(ARF) induced prominent apoptosis even in SV40-Bak-/-MEFs. In contrast, expression of p19(ARF) induced only a very low grade of apoptosis in Bax-/- or Bax-/-/Bak-/-SV40-MEFs. Remarkable attenuation of p19(ARF)-induced apoptosis by disruption of the Bax gene thus leads to the conclusion that Bax plays a major role in p53-independent apoptosis induced by p19(ARF).
Asunto(s)
Apoptosis/fisiología , Fibroblastos/citología , Fibroblastos/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Proto-Oncogénicas/metabolismo , Proteína p14ARF Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis/efectos de los fármacos , Ciclo Celular/fisiología , Células Cultivadas , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Relación Dosis-Respuesta a Droga , Fibroblastos/efectos de los fármacos , Humanos , Riñón/embriología , Riñón/metabolismo , Ratones , Estaurosporina/farmacología , Proteína Destructora del Antagonista Homólogo bcl-2 , Proteína X Asociada a bcl-2RESUMEN
Cellular tubulin has been shown to activate in vitro transcription with Sendai virus (SeV) particles. In this study, the molecular basis for the transcriptional activation by tubulin was investigated. We showed that tubulin dissociates viral matrix (M) protein, which acts as a negative regulator for transcription, from viral ribonucleoprotein (RNP) consisting of L, P, N proteins, and the genome RNA. Both alpha and beta subunits of human tubulin, which were expressed as GST fusion proteins, were found to stimulate viral mRNA synthesis similar to native alpha/beta-heterodimer tubulin. Pull-down assay using GST-tubulin subunits demonstrated that M protein is released from the RNP as a complex with each tubulin subunit. In vitro-binding analyses revealed that M protein directly interacts with tubulin as well as microtubules. These findings suggest that interaction of M protein with tubulin may have an important role in the regulation of SeV transcription.
Asunto(s)
Regulación Viral de la Expresión Génica/fisiología , Genoma Viral , Virus Sendai/genética , Virus Sendai/metabolismo , Activación Transcripcional/fisiología , Tubulina (Proteína)/metabolismo , Proteínas de la Matriz Viral/metabolismo , Sitios de Unión , Células Cultivadas , Humanos , Microtúbulos/química , Microtúbulos/genética , Microtúbulos/metabolismo , Unión Proteica , Subunidades de Proteína , Virus Sendai/química , Relación Estructura-Actividad , Tubulina (Proteína)/química , Proteínas de la Matriz Viral/químicaRESUMEN
The ARF (p19ARF for the mouse ARF consisting of 169 amino acids and p14ARF for the human ARF consisting of 132 amino acids) genes upregulate p53 activities to induce cell cycle arrest and sensitize cells to apoptosis by inhibiting Mdm2 activity. p53-independent apoptosis also is induced by ectopic expression of p19ARF. We constructed various deletion mutants of p19ARF with a cre/loxP-regulated adenoviral vector to determine the regions of p19ARF which are responsible for p53-independent apoptosis. Ectopic expression of the C-terminal region (named C40) of p19ARF whose primary sequence is unique to the rodent ARF induced prominent apoptosis in p53-deficient mouse embryo fibroblasts. Relatively low-grade but significant apoptosis also was induced in p53-deficient mouse embryo fibroblasts by ectopic expression of p19ARF1-129, a p19ARF deletion mutant deficient in the C40 region. In contrast, ectopic expression of the wild-type p14ARF did not induce significant apoptosis in human cells. Taken together, we concluded that p53-independent apoptosis was mediated through multiple regions of the mouse ARF including C40, and the ability of the ARF gene to mediate p53-independent apoptosis has been not well conserved during mammalian evolution.
Asunto(s)
Apoptosis/fisiología , Proteína p14ARF Supresora de Tumor/química , Proteína p14ARF Supresora de Tumor/fisiología , Secuencia de Aminoácidos , Animales , Línea Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Expresión Génica , Humanos , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Proteína p14ARF Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
Hashimoto's encephalopathy (HE) is a rare autoimmune disease associated with Hashimoto's thyroiditis (HT). To identify the HE-related autoantigens, we developed a human brain proteome map using two-dimensional electrophoresis and applied it to the immuno-screening of brain proteins that react with autoantibodies in HE patients. After sequential MALDI-TOF-MASS analysis, immuno-positive spots of 48 kDa (pI 7.3-7.8) detected from HE patient sera were identified as a novel autoimmuno-antigen, alpha-enolase, harboring several modifications. Specific high reactivities against human alpha-enolase were significant in HE patients with excellent corticosteroid sensitivity, whereas the patients with fair or poor sensitivity to the corticosteroid treatment showed less reactivities than cut-off level. Although a few HT patients showed faint reactions to alpha-enolase, 95% of HT patients, patients with other neurological disorders, and healthy subjects tested were all negative. These results suggest that the detection of anti-alpha-enolase antibody is useful for defining HE-related pathology, and this proteomic strategy is a powerful method for identifying autoantigens of various central nervous system diseases with unknown autoimmune etiologies.
Asunto(s)
Autoantígenos/genética , Encefalopatías/enzimología , Encefalopatías/inmunología , Encéfalo/enzimología , Encéfalo/inmunología , Fosfopiruvato Hidratasa/genética , Fosfopiruvato Hidratasa/inmunología , Tiroiditis Autoinmune/enzimología , Tiroiditis Autoinmune/inmunología , Secuencia de Aminoácidos , Especificidad de Anticuerpos , Autoanticuerpos/sangre , Electroforesis en Gel Bidimensional , Humanos , Datos de Secuencia Molecular , Mapeo Peptídico , Proteoma , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The RNA-dependent RNA polymerase of influenza virus is composed of three viral P proteins (PB1, PB2, and PA) and involved in both transcription and replication of the RNA genome. The PB1 subunit plays a key role in both the assembly of three P protein subunits and the catalytic function of RNA polymerization. We have established a simultaneous expression system of three P proteins in various combinations using recombinant baculoviruses, and isolated the PA-PB1-PB2 ternary (3P) complex and two kinds of the binary (2P) complex, PA-PB1 and PB1-PB2. The affinity-purified 3P complex showed all of the catalytic properties characteristic of the transcriptase, including capped RNA-binding, capped RNA cleavage, model viral RNA binding, model viral RNA-directed RNA synthesis, and polyadenylation of newly synthesized RNA. The PB1-PB2 binary complex showed essentially the same catalytic properties as does the 3P complex, whereas the PA-PB1 complex catalyzed de novo initiation of RNA synthesis in the absence of primers. Taken together we propose that the catalytic specificity of PB1 subunit is modulated to the transcriptase by binding PB2 or the replicase by interaction with PA.
Asunto(s)
Orthomyxoviridae/genética , Orthomyxoviridae/fisiología , ARN/ultraestructura , Transcripción Genética , Replicación Viral , Secuencia de Bases , Catálisis , ARN Polimerasas Dirigidas por ADN/metabolismo , Genoma Viral , Immunoblotting , Modelos Biológicos , Datos de Secuencia Molecular , Unión Proteica , ARN/metabolismo , ARN Viral , ARN Polimerasa Dependiente del ARN , Ribonucleasa H/metabolismoRESUMEN
p19(ARF) is a potent tumor suppressor. By inactivating Mdm2, p19(ARF) upregulates p53 activities to induce cell cycle arrest and sensitize cells to apoptosis in the presence of collateral signals. It has also been demonstrated that cell cycle arrest is induced by overexpressed p19(ARF) in p53-deficient mouse embryonic fibroblasts, only in the absence of the Mdm2 gene. Here, we show that apoptosis can be induced without additional apoptosis signals by expression of p19(ARF) using an adenovirus-mediated expression system in p53-intact cell lines as well as p53-deficient cell lines. Also, in primary mouse embryonic fibroblasts (MEFs) lacking p53/ARF, p53-independent apoptosis is induced irrespective of Mdm2 status by expression of p19(ARF). In agreement, p19(ARF)-mediated apoptosis in U2OS cells, but not in Saos2 cells, was attenuated by coexpression of Mdm2. We thus conclude that there is a p53-independent pathway for p19(ARF)-induced apoptosis that is insensitive to inhibition by Mdm2.
Asunto(s)
Apoptosis , Proteínas Nucleares , Proteína p14ARF Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Adenoviridae/genética , Ciclo Celular , Muerte Celular , Línea Celular , Separación Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Fibroblastos/metabolismo , Citometría de Flujo , Humanos , Immunoblotting , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Operón Lac , Plásmidos/metabolismo , Pruebas de Precipitina , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Transducción de Señal , Factores de Tiempo , Transfección , Regulación hacia ArribaRESUMEN
S-adenosylhomocysteine hydrolase (SAHH) is the only enzyme known to cleave S-adenosylhomocysteine (SAH), a product and an inhibitor of all S-adenosylmethionine-dependent transmethylation reactions. Xenopus SAHH is a nuclear enzyme in transcriptionally active cells and inhibition of xSAHH prevents cap methylation of hnRNA [Mol. Biol. Cell 10 (1999) 4283]. Here, we demonstrate that inhibition of xSAHH in Xenopus XTC cells results in a cytoplasmic accumulation of the shuttling hnRNPs, while xSAHH itself remains in the nucleus. The functional link between xSAHH and mRNA cap methylation is further supported by a physical association between xSAHH and mRNA(guanine-7-)methyltransferase (CMT). We show by co-immunoprecipitation of tagged proteins that both enzymes interact in vivo. Direct interaction in vitro is shown by pull-down experiments that further demonstrate that the N-terminal 55 amino acids of xSAHH are sufficient for binding to CMT. Since CMT is known to bind to the hyperphoshorylated C-terminal domain (CTD) of its large subunit of RNA polymerase II, we have studied the co-localisation of RNA polymerase II and xSAHH in oocyte nuclei. Immunolocalisation on spreads of lampbrush chromosomes shows xSAHH on the loops of the transcriptionally active lampbrush chromosomes, in Cajal bodies and in B-snurposomes, the nuclear compartments that are most likely engaged in storage and recycling of RNA polymerase II and its cofactors. We therefore suggest that a subfraction of the nuclear xSAHH remains associated with the RNA polymerase holoenzyme complexes, also while these are not actively engaged in transcription.
Asunto(s)
Hidrolasas/metabolismo , Metiltransferasas/metabolismo , Caperuzas de ARN/metabolismo , Adenosilhomocisteinasa , Animales , Compartimento Celular , Línea Celular , Núcleo Celular/enzimología , Inhibidores Enzimáticos/farmacología , Ribonucleoproteínas Nucleares Heterogéneas , Hidrolasas/antagonistas & inhibidores , Hidrolasas/genética , Metilación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteínas/metabolismo , Transfección , Tubercidina/farmacología , Xenopus laevisRESUMEN
ik3-1/Cables is associated with cdk3 in self-replicating cells. In postmitotic neurons, it may serve as an adaptor molecule, functionally connecting c-abl and cdk5, and supporting neurite growth. Here we report that ik3-1 binds to p53 and p73 in vivo. Ectopically expressed ik3-1 potentiates p53-induced cell death but not p73-induced cell death in U2OS cells. On the contrary, coexpression of ik3-1-DeltaC, an ik3-1 deletion mutant lacking the C-terminal 139 [corrected] amino acids (corresponding to the cyclin box-homologous region), inhibits p73-induced cell death but not p53-induced cell death. ik3-1-DeltaC-mediated inhibition of p73-induced cell death are partially attenuated by overexpression of ik3-1. These data indicate that ik3-1 is not only a regulator for p53-induced cell death but also an essential regulator for p73-induced cell death, and ik3-1-DeltaC competes with ik3-1 only in p73-induced cell death. Furthermore, functional domains of p53 responsible for its interaction with ik3-1 are partially different from those of p73. In conclusion, we found that ik3-1, a putative component of cell cycle regulation, is functionally connected with p53 and p73, but in distinct fashions.