RESUMEN
Frequent outbreaks of novel coronaviruses (CoVs), highlighted by the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, necessitate the development of therapeutics that could be easily and effectively administered worldwide. The conserved mRNA-capping process enables CoVs to evade their host immune system and is a target for antiviral development. Nonstructural protein (nsp) 16 in complex with nsp10 catalyzes the final step of coronaviral mRNA capping through its 2'-O-methylation activity. Like other methyltransferases, the SARS-CoV-2 nsp10-nsp16 complex is druggable. However, the availability of an optimized assay for high-throughput screening (HTS) is an unmet need. Here, we report the development of a radioactivity-based assay for the methyltransferase activity of the nsp10-nsp16 complex in a 384-well format, kinetic characterization, and optimization of the assay for HTS (Z' factor = 0.83). Considering the high conservation of nsp16 across known CoV species, the potential inhibitors targeting the SARS-CoV-2 nsp10-nsp16 complex may also be effective against other emerging pathogenic CoVs.
Asunto(s)
Adenosina/análogos & derivados , Ensayos Analíticos de Alto Rendimiento , Caperuzas de ARN/antagonistas & inhibidores , ARN Viral/antagonistas & inhibidores , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas Reguladoras y Accesorias Virales/antagonistas & inhibidores , Adenosina/química , Adenosina/farmacología , COVID-19/virología , Clonación Molecular , Pruebas de Enzimas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Cinética , Metilación , Metiltransferasas , Modelos Moleculares , Caperuzas de ARN/genética , Caperuzas de ARN/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/enzimología , SARS-CoV-2/genética , Tritio , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Proteínas Reguladoras y Accesorias Virales/química , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Reguladoras y Accesorias Virales/metabolismoRESUMEN
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) represents a significant threat to human health. Despite its similarity to related coronaviruses, there are currently no specific treatments for COVID-19 infection, and therefore there is an urgent need to develop therapies for this and future coronavirus outbreaks. Formation of the cap at the 5' end of viral RNA has been shown to help coronaviruses evade host defenses. Nonstructural protein 14 (nsp14) is responsible for N7-methylation of the cap guanosine in coronaviruses. This enzyme is highly conserved among coronaviruses and is a bifunctional protein with both N7-methyltransferase and 3'-5' exonuclease activities that distinguish nsp14 from its human equivalent. Mutational analysis of SARS-CoV nsp14 highlighted its role in viral replication and translation efficiency of the viral genome. In this paper, we describe the characterization and development of a high-throughput assay for nsp14 utilizing RapidFire technology. The assay has been used to screen a library of 1771 Food and Drug Administration (FDA)-approved drugs. From this, we have validated nitazoxanide as a selective inhibitor of the methyltransferase activity of nsp14. Although modestly active, this compound could serve as a starting point for further optimization.
Asunto(s)
Antivirales/farmacología , Exorribonucleasas/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento , Nitrocompuestos/farmacología , Caperuzas de ARN/antagonistas & inhibidores , ARN Viral/antagonistas & inhibidores , SARS-CoV-2/efectos de los fármacos , Tiazoles/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Antiparasitarios/química , Antiparasitarios/farmacología , Antivirales/química , COVID-19/virología , Clonación Molecular , Reposicionamiento de Medicamentos , Pruebas de Enzimas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Escherichia coli/genética , Escherichia coli/metabolismo , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Cinética , Espectrometría de Masas/métodos , Metilación , Nitrocompuestos/química , Medicamentos bajo Prescripción/química , Medicamentos bajo Prescripción/farmacología , Caperuzas de ARN/genética , Caperuzas de ARN/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , SARS-CoV-2/enzimología , SARS-CoV-2/genética , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Tiazoles/química , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
SARS-CoV-2, the coronavirus that causes COVID-19, evades the human immune system by capping its RNA. This process protects the viral RNA and is essential for its replication. Multiple viral proteins are involved in this RNA capping process, including the nonstructural protein 16 (nsp16), which is an S-adenosyl-l-methionine (SAM)-dependent 2'-O-methyltransferase. Nsp16 is significantly active when in complex with another nonstructural protein, nsp10, which plays a key role in its stability and activity. Here we report the development of a fluorescence polarization (FP)-based RNA displacement assay for nsp10-nsp16 complex in a 384-well format with a Z' factor of 0.6, suitable for high-throughput screening. In this process, we purified the nsp10-nsp16 complex to higher than 95% purity and confirmed its binding to the methyl donor SAM, the product of the reaction, S-adenosyl-l-homocysteine (SAH), and a common methyltransferase inhibitor, sinefungin, using isothermal titration calorimetry (ITC). The assay was further validated by screening a library of 1124 drug-like compounds. This assay provides a cost-effective high-throughput method for screening the nsp10-nsp16 complex for RNA competitive inhibitors toward developing COVID-19 therapeutics.
Asunto(s)
Antivirales/farmacología , Ensayos Analíticos de Alto Rendimiento , ARN Viral/antagonistas & inhibidores , SARS-CoV-2/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas Reguladoras y Accesorias Virales/antagonistas & inhibidores , Adenosina/análogos & derivados , Adenosina/farmacología , Unión Competitiva , COVID-19/virología , Inhibidores Enzimáticos/farmacología , Polarización de Fluorescencia , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Metiltransferasas , Unión Proteica , Caperuzas de ARN/antagonistas & inhibidores , Caperuzas de ARN/genética , Caperuzas de ARN/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Transducción de Señal , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Reguladoras y Accesorias Virales/metabolismo , Replicación Viral , Tratamiento Farmacológico de COVID-19RESUMEN
We report the chemical synthesis of a set of nicotinamide adenine dinucleotide (NAD) cap analogues containing chemical modifications that reduce their susceptibility to NAD-RNA-degrading enzymes. These analogues can be incorporated into transcripts in a similar way as NAD. Biochemical characterization of RNAs carrying these caps with DXO, NudC, and Nudt12 enzymes led to the identification of compounds that can be instrumental in unraveling so far unaddressed biological aspects of NAD-RNAs.
Asunto(s)
Adenina/farmacología , NAD/antagonistas & inhibidores , Niacinamida/farmacología , Caperuzas de ARN/antagonistas & inhibidores , Adenina/análogos & derivados , Adenina/química , Conformación Molecular , NAD/metabolismo , Niacinamida/análogos & derivados , Niacinamida/química , Caperuzas de ARN/metabolismoRESUMEN
The p53 tumor suppressor plays a critical role in protecting normal cells from malignant transformation. Development of small molecules to reactivate p53 in cancer cells has been an area of intense research. We previously identified an internal ribosomal entry site (IRES) within the 5' untranslated region of p53 mRNA that mediates translation of the p53 mRNA independent of cap-dependent translation. Our results also show that in response to DNA damage, cells switch from cap-dependent translation to cap-independent translation of p53 mRNA. In the present study, we discovered a specific inhibitor of cap-dependent translation, 4EGI-1, that is capable of inducing the accumulation of p53 in cancer cells retaining wild-type p53. Our results show that 4EGI-1 causes an increase in p53 IRES activity, leading to increased translation of p53 mRNA. We also observed that 4EGI-1 induces cancer cell apoptosis in a p53-dependent manner. Furthermore, 4EGI-1 induces p53 in cancer cells without causing DNA double-strand breaks. In conclusion, we discovered a mechanistic link between inhibition of cap-dependent translation and enhanced p53 accumulation. This leads to apoptosis of cancer cells without causing collateral damage to normal cells, thus providing a novel and effective therapeutic strategy for cancer.
Asunto(s)
Caperuzas de ARN/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/biosíntesis , Regiones no Traducidas 5' , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Daño del ADN/genética , Células HCT116 , Humanos , Hidrazonas/farmacología , Sitios Internos de Entrada al Ribosoma/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/genética , Caperuzas de ARN/efectos de los fármacos , ARN Mensajero/genética , Ribosomas , Tiazoles/farmacología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Eukaryotic gene expression is dependent on the modification of the first transcribed nucleotide of pre-mRNA by the addition of the 7-methylguanosine cap. The cap protects transcripts from exonucleases and recruits complexes which mediate transcription elongation, processing and translation initiation. The cap is synthesized by a series of reactions which link 7-methylguanosine to the first transcribed nucleotide via a 5' to 5' triphosphate bridge. In mammals, cap synthesis is catalysed by the sequential action of RNGTT (RNA guanylyltransferase and 5'-phosphatase) and RNMT (RNA guanine-7 methyltransferase), enzymes recruited to RNA pol II (polymerase II) during the early stages of transcription. We recently discovered that the mammalian cap methyltransferase is a heterodimer consisting of RNMT and the RNMT-activating subunit RAM (RNMT-activating mini-protein). RAM activates and stabilizes RNMT and thus is critical for cellular cap methylation and cell viability. In the present study we report that RNMT interacts with the N-terminal 45 amino acids of RAM, a domain necessary and sufficient for maximal RNMT activation. In contrast, smaller components of this RAM domain are sufficient to stabilize RNMT. RAM functions in the nucleus and we report that nuclear import of RAM is dependent on PY nuclear localization signals and Kapß2 (karyopherin ß2) nuclear transport protein.
Asunto(s)
Núcleo Celular/metabolismo , Metiltransferasas/metabolismo , Señales de Localización Nuclear/metabolismo , Proteínas de Unión al ARN/metabolismo , beta Carioferinas/metabolismo , Línea Celular , Núcleo Celular/enzimología , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Humanos , Metilación , Metiltransferasas/antagonistas & inhibidores , Metiltransferasas/genética , Proteínas Mutantes/antagonistas & inhibidores , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Señales de Localización Nuclear/antagonistas & inhibidores , Señales de Localización Nuclear/química , Señales de Localización Nuclear/genética , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Transporte de Proteínas , Caperuzas de ARN/antagonistas & inhibidores , Caperuzas de ARN/metabolismo , Precursores del ARN/antagonistas & inhibidores , Precursores del ARN/metabolismo , Estabilidad del ARN , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , beta Carioferinas/antagonistas & inhibidores , beta Carioferinas/genéticaRESUMEN
The RNA guanylyltransferase (GTase) is involved in the synthesis of the (m7)Gppp-RNA cap structure found at the 5' end of eukaryotic mRNAs. GTases are members of the covalent nucleotidyl transferase superfamily, which also includes DNA and RNA ligases. GTases catalyze a two-step reaction in which they initially utilize GTP as a substrate to form a covalent enzyme-GMP intermediate. The GMP moiety is then transferred to the diphosphate end of the RNA transcript in the second step of the reaction to form the Gppp-RNA structure. In the current study, we used a combination of virtual database screening, homology modeling, and biochemical assays to search for novel GTase inhibitors. Using this approach, we demonstrate that mycophenolic acid (MPA) can inhibit the GTase reaction by preventing the catalytic transfer of the GMP moiety onto an acceptor RNA. As such, MPA represents a novel type of inhibitor against RNA guanylyltransferases that inhibits the second step of the catalytic reaction. Moreover, we show that the addition of MPA to S. cerevisiae cells leads to a reduction of capped mRNAs. Finally, biochemical assays also demonstrate that MPA can inhibit DNA ligases through inhibition of the second step of the reaction. The biological implications of these findings for the MPA-mediated inhibition of members of the covalent nucleotidyl superfamily are discussed.
Asunto(s)
Ácido Micofenólico/farmacología , Caperuzas de ARN/antagonistas & inhibidores , Caperuzas de ARN/metabolismo , ADN Ligasas/metabolismo , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Sistema de la Enzima Desramificadora del Glucógeno/química , Sistema de la Enzima Desramificadora del Glucógeno/metabolismo , Guanosina Monofosfato/metabolismo , Humanos , Nucleotidiltransferasas/química , Nucleotidiltransferasas/metabolismo , Estructura Secundaria de Proteína , ARN/metabolismo , Saccharomyces cerevisiae/enzimologíaRESUMEN
A number of different human pathogens code for their own enzymes involved in the synthesis of the RNA cap structure. Although the RNA cap structures originating from human and microbial enzymes are often identical, the subunit composition, structure and catalytic mechanisms of the microbial-encoded enzymes involved in the synthesis of the RNA cap structure are often significantly different from those of host cells. As a consequence, these pathogenic cap-forming enzymes are potential targets for antimicrobial drugs. During the past few years, experimental studies have started to demonstrate that inhibition of the RNA capping activity is a reasonable approach for the development of antimicrobial agents. The combination of structural, biochemical, and molecular modeling studies are starting to reveal novel molecules that can serve as starting blocks for the design of more potent and specific antimicrobial agents. Here, we examine various strategies that have been developed to inhibit microbial enzymes involved in the synthesis of the RNA cap structure, emphasizing the challenges remaining to design potent and selective drugs.
Asunto(s)
Antiinfecciosos/síntesis química , Diseño de Fármacos , Terapia Molecular Dirigida/métodos , Caperuzas de ARN/antagonistas & inhibidores , Caperuzas de ARN/metabolismo , Animales , Antiinfecciosos/química , Humanos , Modelos Biológicos , Modelos Moleculares , Nucleotidiltransferasas/antagonistas & inhibidores , Nucleotidiltransferasas/metabolismo , Fosfatos/síntesis química , Fosfatos/química , Análogos de Caperuza de ARN/síntesis química , Análogos de Caperuza de ARN/química , Vanadatos/síntesis química , Vanadatos/químicaRESUMEN
The Hippo tumour suppressor pathway is a conserved signalling pathway that controls organ size. The core of the Hpo pathway is a kinase cascade, which in Drosophila involves the Hpo and Warts kinases that negatively regulate the activity of the transcriptional coactivator Yorkie. Although several additional components of the Hippo pathway have been discovered, the inputs that regulate Hippo signalling are not fully understood. Here, we report that induction of extra F-actin formation, by loss of Capping proteins A or B, or caused by overexpression of an activated version of the formin Diaphanous, induced strong overgrowth in Drosophila imaginal discs through modulating the activity of the Hippo pathway. Importantly, loss of Capping proteins and Diaphanous overexpression did not significantly affect cell polarity and other signalling pathways, including Hedgehog and Decapentaplegic signalling. The interaction between F-actin and Hpo signalling is evolutionarily conserved, as the activity of the mammalian Yorkie-orthologue Yap is modulated by changes in F-actin. Thus, regulators of F-actin, and in particular Capping proteins, are essential for proper growth control by affecting Hippo signalling.
Asunto(s)
Actinas/genética , Proteínas de Drosophila/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/genética , Alas de Animales/citología , Alas de Animales/crecimiento & desarrollo , Actinas/biosíntesis , Actinas/química , Animales , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Proliferación Celular , Células Cultivadas , Citoesqueleto/química , Citoesqueleto/genética , Proteínas de Drosophila/biosíntesis , Proteínas de Drosophila/química , Drosophila melanogaster/química , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Forminas , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Especificidad de Órganos/genética , Fenotipo , Proteínas Serina-Treonina Quinasas/química , Caperuzas de ARN/antagonistas & inhibidores , Caperuzas de ARN/química , Caperuzas de ARN/genética , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Alas de Animales/químicaAsunto(s)
Bencenosulfonatos/farmacología , Citarabina/farmacología , Factor 4E Eucariótico de Iniciación/antagonistas & inhibidores , Idarrubicina/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mielomonocítica Aguda/tratamiento farmacológico , Piridinas/farmacología , Ribavirina/farmacología , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Unión Competitiva , Ensayos Clínicos Fase II como Asunto , Sinergismo Farmacológico , Humanos , Leucemia Mieloide Aguda/patología , Leucemia Mielomonocítica Aguda/patología , Persona de Mediana Edad , Niacinamida/análogos & derivados , Compuestos de Fenilurea , Caperuzas de ARN/antagonistas & inhibidores , Caperuzas de ARN/metabolismo , Ribavirina/metabolismo , Ribavirina/uso terapéutico , Sorafenib , Células Tumorales Cultivadas/efectos de los fármacos , Ensayo de Tumor de Célula MadreRESUMEN
PURPOSE: The growth and survival of acute myeloid leukemia (AML) cells are enhanced by the deregulation of signaling pathways such as phosphoinositide 3-kinase (PI3K)/Akt and mammalian target of rapamycin (mTOR). Major efforts have thus been made to develop molecules targeting these activated pathways. The mTOR serine/threonine kinase belongs to two separate complexes: mTORC1 and mTORC2. The mTORC1 pathway is rapamycin sensitive and controls protein translation through the phosphorylation of 4E-BP1 in most models. In AML, however, the translation process is deregulated and rapamycin resistant. Furthermore, the activity of PI3K/Akt and mTOR is closely related, as mTORC2 activates the oncogenic kinase Akt. We therefore tested, in this study, the antileukemic activity of the dual PI3K/mTOR ATP-competitive inhibitor NVP-BEZ235 compound (Novartis). EXPERIMENTAL DESIGN: The activity of NVP-BEZ235 was tested in primary AML samples (n = 21) and human leukemic cell lines. The different signaling pathways were analyzed by Western blotting. The cap-dependent mRNA translation was studied by 7-methyl-GTP pull-down experiments, polysomal analysis, and [(3)H]leucine incorporation assays. The antileukemic activity of NVP-BEZ235 was tested by analyzing its effects on leukemic progenitor clonogenicity, blast cell proliferation, and survival. RESULTS: The NVP-BEZ235 compound was found to inhibit PI3K and mTORC1 signaling and also mTORC2 activity. Furthermore, NVP-BEZ235 fully inhibits the rapamycin-resistant phosphorylation of 4E-BP1, resulting in a marked inhibition of protein translation in AML cells. Hence, NVP-BEZ235 reduces the proliferation rate and induces an important apoptotic response in AML cells without affecting normal CD34(+) survival. CONCLUSIONS: Our results clearly show the antileukemic efficiency of the NVP-BEZ235 compound, which therefore represents a promising option for future AML therapies.
Asunto(s)
Antineoplásicos/farmacología , Imidazoles/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Proteínas/antagonistas & inhibidores , Quinolinas/farmacología , Factores de Transcripción/antagonistas & inhibidores , Antineoplásicos/uso terapéutico , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Imidazoles/uso terapéutico , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas/metabolismo , Quinolinas/uso terapéutico , Caperuzas de ARN/antagonistas & inhibidores , Caperuzas de ARN/genética , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Serina-Treonina Quinasas TOR , Factores de Transcripción/metabolismo , Células Tumorales CultivadasRESUMEN
In this study, we demonstrate the identification of an internal ribosome entry site (IRES) within the 5'-untranslated region (5'-UTR) of the mouse mammary tumor virus (MMTV). The 5'-UTR of the full-length mRNA derived from the infectious, complete MMTV genome was cloned into a dual luciferase reporter construct containing an upstream Renilla luciferase gene (RLuc) and a downstream firefly luciferase gene (FLuc). In rabbit reticulocyte lysate, the MMTV 5'-UTR was capable of driving translation of the second cistron. In vitro translational activity from the MMTV 5'-UTR was resistant to the addition of m(7)GpppG cap-analog and cleavage of eIF4G by foot-and-mouth disease virus (FMDV) L-protease. IRES activity was also demonstrated in the Xenopus laevis oocyte by micro-injection of capped and polyadenylated bicistronic RNAs harboring the MMTV-5'-UTR. Finally, transfection assays showed that the MMTV-IRES exhibits cell type-dependent translational activity, suggesting a requirement for as yet unidentified cellular factors for its optimal function.
Asunto(s)
Regiones no Traducidas 5' , Virus del Tumor Mamario del Ratón/genética , Iniciación de la Cadena Peptídica Traduccional , ARN Viral/química , Animales , Línea Celular , Humanos , Luciferasas de Luciérnaga/análisis , Luciferasas de Luciérnaga/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Oocitos/metabolismo , Plásmidos/genética , Regiones Promotoras Genéticas , Caperuzas de ARN/antagonistas & inhibidores , ARN Mensajero/química , Conejos , Xenopus laevis , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
We report synthesis and properties of a pair of new potent inhibitors of translation, namely two diastereomers of 7-methylguanosine 5'-(1-thiotriphosphate). These new analogs of mRNA 5'cap (referred to as m(7)GTPalphaS (D1) and (D2)) are recognized by translational factor eIF4E with high affinity and are not susceptible to hydrolysis by Decapping Scavenger pyrophosphatase (DcpS). The more potent of diastereomers, m(7)GTPalphaS (D1), inhibited cap-dependent translation in rabbit reticulocyte lysate approximately 8-fold and approximately 15-fold more efficiently than m(7)GTP and m(7)GpppG, respectively. Both analogs were also significantly more stable in RRL than unmodified ones.
Asunto(s)
Guanosina Trifosfato/análogos & derivados , Inhibidores de la Síntesis de la Proteína/química , Inhibidores de la Síntesis de la Proteína/farmacología , Análogos de Caperuza de ARN/química , Caperuzas de ARN/antagonistas & inhibidores , Animales , Endorribonucleasas/metabolismo , Factor 4E Eucariótico de Iniciación/antagonistas & inhibidores , Factor 4E Eucariótico de Iniciación/metabolismo , Guanosina Trifosfato/síntesis química , Guanosina Trifosfato/química , Guanosina Trifosfato/farmacología , Humanos , Ratones , Biosíntesis de Proteínas , Inhibidores de la Síntesis de la Proteína/síntesis química , Análogos de Caperuza de ARN/síntesis química , Análogos de Caperuza de ARN/farmacología , Caperuzas de ARN/metabolismo , ARN Mensajero/metabolismo , Estereoisomerismo , Tionucleótidos/síntesis química , Tionucleótidos/química , Tionucleótidos/farmacologíaRESUMEN
Eukaryotic cells utilize a cycle of ribosome trafficking on the endoplasmic reticulum (ER) to partition mRNAs between the cytosol and ER compartments. In this process, ribosomes engaged in the synthesis of signal sequence-bearing proteins are trafficked to the endoplasmic reticulum via the signal-recognition particle pathway and are released from the ER upon translation termination. Though the processes governing ribosome trafficking to the ER are well understood, little is known regarding the complementary ribosome release process. In this study, Coxsackie B virus (CBV) infection was used to inactivate the initiation stage of protein synthesis, thereby limiting translation to the elongation and termination stages. Ribosome partitioning between the cytosol and ER compartments was examined to determine the role of termination in ribosome release from the ER. CBV infection resulted in efficient cleavage of eIF4G and PABP, coincident with polyribosome breakdown in the cytosol and ER compartments. Termination resulted in the continued association of ribosomes with the ER compartment, rather than the expected process of ribosome release. Analyses of ribosome/mRNA loading patterns in the cytosol and ER revealed that CBV infection was accompanied by a suppression of mRNA translation in the cytosol and the sustained, although reduced, translation in the ER compartment. Direct biosynthetic labeling experiments demonstrated that protein synthesis on the ER was enhanced relative to the cytosol following CBV infection. In total, these data demonstrate that ribosome and mRNA release from the ER is regulated independent of translation termination and identify the ER as a privileged site for protein synthesis.
Asunto(s)
Retículo Endoplásmico/metabolismo , Biosíntesis de Proteínas , Inhibidores de la Síntesis de la Proteína/metabolismo , Caperuzas de ARN/antagonistas & inhibidores , ARN Mensajero/metabolismo , Northern Blotting , Western Blotting , Proteínas Portadoras/metabolismo , Compartimento Celular , Fraccionamiento Celular , Infecciones por Coxsackievirus/genética , Infecciones por Coxsackievirus/metabolismo , Citosol/metabolismo , Enterovirus Humano B/genética , Enterovirus Humano B/metabolismo , Factor 4G Eucariótico de Iniciación/metabolismo , Células HeLa , Humanos , Caperuzas de ARN/genética , Ribosomas/metabolismoRESUMEN
Translation is downregulated in response to a variety of moderate stresses, including serum deprivation, hyperosmolarity and ionizing radiation. The cytostatic p21-activated protein kinase 2 (Pak2)/gamma-PAK is activated under the same stress conditions. Expression of wild-type Pak2 in cells and addition of Pak2 to reticulocyte lysate inhibit translation, while kinase-inactive mutants have no effect. Pak2 binds to and phosphorylates initiation factor (eIF)4G, which inhibits association of eIF4E with m(7)GTP, reducing initiation. The Pak2-binding site maps to the region on eIF4G that contains the eIF4E-binding site; Pak2 and eIF4E compete for binding to this site. Using an eIF4G-depleted reticulocyte lysate, reconstitution with mock-phosphorylated eIF4G fully restores translation, while phosphorylated eIF4G reduces translation to 37%. RNA interference releases Pak2-induced inhibition of translation in contact-inhibited cells by 2.7-fold. eIF4G mutants of the Pak2 site show that S896D inhibits translation, while S896A has no effect. Activation of Pak2 in response to hyperosmotic stress inhibits cap-dependent, but not IRES-driven, initiation. Thus, a novel pathway for mammalian cell stress signaling is identified, wherein activation of Pak2 leads to inhibition of cap-dependent translation through phosphorylation of eIF4G.
Asunto(s)
Regulación hacia Abajo/fisiología , Factor 4G Eucariótico de Iniciación/metabolismo , Iniciación de la Cadena Peptídica Traduccional/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Caperuzas de ARN/antagonistas & inhibidores , Caperuzas de ARN/fisiología , Sitios de Unión , Línea Celular , Humanos , Fosforilación , Interferencia de ARN , Reticulocitos/enzimología , Ribosomas/fisiología , Quinasas p21 ActivadasRESUMEN
MicroRNAs (miRNAs) repress translation of target mRNAs by interaction with partially mismatched sequences in their 3' UTR. The mechanism by which they act on translation has remained largely obscure. We examined the translation of mRNAs containing four partially mismatched miRNA-binding sites in the 3' UTR in HeLa cells cotransfected with a cognate miRNA. The mRNAs were prepared by in vitro transcription and were engineered to employ different modes of translation initiation. We find that the 5' cap structure and the 3' poly(A) tail are each necessary but not sufficient for full miRNA-mediated repression of mRNA translation. Replacing the cap structure with an internal ribosome entry site from either the cricket paralysis virus or the encephalomyocarditis virus impairs miRNA-mediated repression. Collectively, these results demonstrate that miRNAs interfere with the initiation step of translation and implicate the cap-binding protein eukaryotic initiation factor 4E as a molecular target.
Asunto(s)
Factor 4E Eucariótico de Iniciación/antagonistas & inhibidores , MicroARNs/fisiología , Proteínas de Unión a Poli(A)/antagonistas & inhibidores , Caperuzas de ARN/antagonistas & inhibidores , Células HeLa , Humanos , ARN Mensajero/metabolismo , Receptores CXCR4/genética , Ribosomas/fisiologíaRESUMEN
Pokeweed antiviral protein (PAP) is a single-chain ribosome inactivating protein (RIP) that binds to ribosomes and depurinates the highly conserved alpha-sarcin/ricin loop (SRL) of the large subunit rRNA. Catalytic depurination of a specific adenine has been proposed to result in translation arrest and cytotoxicity. Here, we show that both precursor and mature forms of PAP are localized in the endoplasmic reticulum (ER) in yeast. The mature form is retro-translocated from the ER into the cytosol where it escapes degradation unlike the other substrates of the retro-translocation pathway. A mutation of a highly conserved asparagine residue at position 70 (N70A) delays ribosome depurination and the onset of translation arrest. The ribosomes are eventually depurinated, yet cytotoxicity and loss of viability are markedly absent. Analysis of the variant protein, N70A, does not reveal any decrease in the rate of synthesis, subcellular localization, or the rate of transport into the cytosol. N70A destabilizes its own mRNA, binds to cap, and blocks cap dependent translation, as previously reported for the wild-type PAP. However, it cannot depurinate ribosomes in a translation-independent manner. These results demonstrate that N70 near the active-site pocket is required for depurination of cytosolic ribosomes but not for cap binding or mRNA destabilization, indicating that the activity of PAP on capped RNA can be uncoupled from its activity on rRNA. These findings suggest that the altered active site of PAP might accommodate a narrower range of substrates, thus reducing ribotoxicity while maintaining potential therapeutic benefits.
Asunto(s)
Citosol/metabolismo , Retículo Endoplásmico/metabolismo , N-Glicosil Hidrolasas/metabolismo , Phytolacca , Proteínas de Plantas/metabolismo , Inhibidores de la Síntesis de la Proteína/metabolismo , Caperuzas de ARN/antagonistas & inhibidores , Ribosomas/metabolismo , Alanina/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Asparagina/genética , Sitios de Unión/genética , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Secuencia Conservada , Citosol/química , Retículo Endoplásmico/química , Retículo Endoplásmico/genética , Datos de Secuencia Molecular , N-Glicosil Hidrolasas/química , N-Glicosil Hidrolasas/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Inhibidores de la Síntesis de la Proteína/química , Transporte de Proteínas/genética , Caperuzas de ARN/química , Caperuzas de ARN/genética , Proteínas Inactivadoras de Ribosomas Tipo 1 , Ribosomas/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Adenovirus (Ad) infection results in a marked inhibition of cellular protein synthesis that initiates during the late phase of the viral infectious cycle. We show that the mechanism used for suppression of cellular protein synthesis during cell cycle progression is exploited by Ad to repress host and enhance late viral mRNA translation. Discrimination between cellular and late Ad mRNAs and inhibition of host protein synthesis are shown to involve viral-mediated underphosphorylation of cap-binding protein (CBP) and subsequent inactivation of CBP complex, a large enzymatic complex required for cap-dependent mRNA translation. Late Ad mRNAs, like those of poliovirus, possess the unique ability to translate independent of a normal cap recognition process and do not require the activity of CBP complex. Inhibition of cellular translation by these two viruses is quite similar, except that whereas CBP complex is proteolytically degraded by poliovirus, it is functionally inactivated by Ad.