RESUMEN

Objective: To explore the antiviral activity of the small-molecule compound AM679 in hepatitis B virus (HBV) replication and infection cell models. Methods: The positive regulatory effect of AM679 on EFTUD2 expression was validated by qPCR and Western blotting. HepAD38 and HepG2-NTCP cells were treated with AM679 (0.5, 1, and 2 nmol/L). Negative control, positive control, and AM679 combined with the entecavir group were set up. HBV DNA intra-and extracellularly, as well as the expression levels of intracellular HBV total RNAs and 3.5kb-RNA changes, were detected with qPCR. Hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) levels were measured in the cell supernatant by an enzyme-linked immunosorbent assay (ELISA). The t-test method was used for the statistical analysis of the mean difference between groups. Results: EFTUD2 mRNA and protein expression levels were significantly increased in HepAD38 and HepG2-NTCP cells following AM679 treatment, with a statistically significant difference (P < 0.001). Intra-and extracellular indicators such as HBV DNA, HBV RNAs, HBV 3.5kb-RNA, HBsAg, and HBeAg were decreased to varying degrees in both cell models, and the decrease in these indicators was more pronounced with the increase in AM679 concentration and prolonged treatment duration, while the combined use of AM679 and entecavir had a more significant antiviral effect. The HBV DNA inhibition rates in the supernatant of HepAD38 cells with the use of 2 nmol/L AM679 were 21% and 48% on days three and nine, respectively. The AM679 combined with the ETV treatment group had the most significant inhibitory effect (62%), with a P < 0.01. More active HBV replication was observed after silencing EFTUD2, while the antiviral activity of AM679 was significantly weakened. Conclusion: AM679 exerts anti-HBV activity in vitro by targeting the regulation of EFTUD2 expression.

Asunto(s)

Antivirales , Virus de la Hepatitis B , Replicación Viral , Humanos , Antivirales/farmacología , ADN Viral , Guanina/análogos & derivados , Células Hep G2 , Antígenos e de la Hepatitis B/metabolismo , Antígenos de Superficie de la Hepatitis B/metabolismo , Virus de la Hepatitis B/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Indoles/química , Indoles/farmacología , Ácidos Pentanoicos/química , Ácidos Pentanoicos/farmacología , Factores de Elongación de Péptidos/antagonistas & inhibidores , Factores de Elongación de Péptidos/metabolismo , Ribonucleoproteína Nuclear Pequeña U5/antagonistas & inhibidores , Ribonucleoproteína Nuclear Pequeña U5/metabolismo

RESUMEN

Changes in mitochondrial morphology by dysregulated mitochondrial fission­fusion proteins have been increasingly recognized as a hallmark of cancer. MiD49 (mitochondrial dynamics protein of 49 kDa) is a newly identified mitochondrial fission protein involved in the dynamic regulation of mitochondrial morphology. However, the expression pattern and biological functions of MiD49 in human cancers remain largely unexplored, especially in pancreatic cancer (PC). In the present study, the expression and clinical significance of MiD49 was firstly determined by RT­qPCR and western blot analyses in PC cell lines and tumor tissues. In addition, the biologic functions of MiD49 in PC cell growth and metastasis were investigated using gain­ and loss­of­function assays both in vitro and in vivo. Moreover, the underlying mechanisms by which MiD49 regulates PC cell growth and metastasis were further explored. Our results showed that MiD49 was markedly downregulated in both PC cell lines and human PC specimens. Forced expression of MiD49 suppressed PC cell growth and metastasis both in vitro and in vivo, while knockdown of MiD49 exhibited the opposite effect. Mechanistic exploration demonstrated that the tumor­suppressive effect of MiD49 was mediated by decreased mitochondrial fission and subsequent reduced ROS production in PC cells. Our findings suggest a critical tumor­suppressive role played by MiD49 in pancreatic cancer.

Asunto(s)

Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/secundario , Dinámicas Mitocondriales , Proteínas Mitocondriales/antagonistas & inhibidores , Neoplasias Pancreáticas/patología , Factores de Elongación de Péptidos/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Biología Computacional/métodos , Bases de Datos Genéticas , Regulación hacia Abajo , Femenino , Genes Supresores de Tumor , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Factores de Elongación de Péptidos/genética , Factores de Elongación de Péptidos/metabolismo , Tasa de Supervivencia , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Excessive proliferation and apoptosis-resistance are hallmarks of cancer. Increased dynamin-related protein 1 (Drp1)-mediated mitochondrial fission is one of the mediators of this phenotype. Mitochondrial fission that accompanies the nuclear division is called mitotic fission and occurs when activated Drp1 binds partner proteins on the outer mitochondrial membrane. We examine the role of Drp1-binding partners, mitochondrial dynamics protein of 49 and 51 kDa (MiD49 and MiD51), as drivers of cell proliferation and apoptosis-resistance in non-small cell lung cancer (NSCLC) and invasive breast carcinoma (IBC). We also evaluate whether inhibiting MiDs can be therapeutically exploited to regress cancer. We show that MiD levels are pathologically elevated in NSCLC and IBC by an epigenetic mechanism (decreased microRNA-34a-3p expression). MiDs silencing causes cell cycle arrest through (a) increased expression of cell cycle inhibitors, p27Kip1 and p21Waf1 , (b) inhibition of Drp1, and (c) inhibition of the Akt-mTOR-p70S6K pathway. Silencing MiDs leads to mitochondrial fusion, cell cycle arrest, increased apoptosis, and tumor regression in a xenotransplant NSCLC model. There are positive correlations between MiD expression and tumor size and grade in breast cancer patients and inverse correlations with survival in NSCLC patients. The microRNA-34a-3p-MiDs axis is important to cancer pathogenesis and constitutes a new therapeutic target.

Asunto(s)

Biomarcadores de Tumor/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Ciclo Celular , Epigénesis Genética , Neoplasias Pulmonares/patología , Proteínas Mitocondriales/metabolismo , Factores de Elongación de Péptidos/metabolismo , Animales , Apoptosis , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/terapia , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/terapia , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Dinámicas Mitocondriales , Proteínas Mitocondriales/antagonistas & inhibidores , Proteínas Mitocondriales/genética , Factores de Elongación de Péptidos/antagonistas & inhibidores , Factores de Elongación de Péptidos/genética , Pronóstico , Tasa de Supervivencia , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Twist1, a key transcription factor regulating epithelial-mesenchymal transition and cancer metastasis, is highly expressed in invasive cancers in contrast to the loss of BTG2/TIS21 expression. Based on our observation that forced expression of BTG2/TIS21 downregulated Twist1 protein expression without altering mRNA level, we investigated molecular mechanisms of the BTG2/TIS21-inhibited Twist1 translation in the triple negative breast cancer (TNBC) cells and in vivo BTG2/TIS21-knockout (KO) mice and human breast cancer tissues. (1) C-terminal domain of Twist1 and Box B of BTG2/TIS21 interacted with each other, which abrogated Twist1 activity. (2) BTG2/TIS21 inhibited translational initiation by depleting eIF4E availability via inhibiting 4EBP1 phosphorylation. (3) Expression of BTG2/TIS21 maintained p-eIF2α that downregulates initiation of protein translation, confirmed by eIF2α-AA mutant expression and BTG2/TIS21 knockdown in MEF cells. (4) cDNA microarray analysis revealed significantly higher expression of initiation factors-eIF2A, eIF3A, and eIF4G2-in the BTG2/TIS21-KO mouse than that in the wild type. (5) BTG2/TIS21-inhibited translation initiation lead to the collapse of polysome formation and the huge peak of 80s monomer in the BTG2/TIS21 expresser, but not in the control. (6) mRNAs and protein expressions of elongation factors were also downregulated by BTG2/TIS21 expression in TNBC cells, but much higher in both TIS21-KO mice and lymph node-positive human breast cancers. (7) BTG2/TIS21-mediated Twist1 loss was not due to the protein degradation by ubiquitination and autophagy activation. (8) Twist1 protein level was significantly higher in various organs of TIS21-KO mice compared with that in the control, indicating the in vivo role of BTG2/TIS21 gene in the regulation of Twist1 protein level. Altogether, the present study support our hypothesis that BTG2/TIS21 is a promising target to combat with metastatic cancers with high level of Twist1 without BTG2/TIS21 expression.

Asunto(s)

Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Nucleares/metabolismo , Iniciación de la Cadena Peptídica Traduccional/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína 1 Relacionada con Twist/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Regulación hacia Abajo , Factor 2 Eucariótico de Iniciación/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Lisosomas/genética , Lisosomas/metabolismo , Células MCF-7 , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Factores de Elongación de Péptidos/antagonistas & inhibidores , Factores de Elongación de Péptidos/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Dominios Proteicos , Neoplasias de la Mama Triple Negativas/genética , Proteína 1 Relacionada con Twist/antagonistas & inhibidores , Proteína 1 Relacionada con Twist/genética

RESUMEN

RfaH is required for virulence in several Gram-negative pathogens including Escherichia coli and Klebsiella pneumoniae. Through direct interactions with RNA polymerase (RNAP) and ribosome, RfaH activates the expression of capsule, cell wall and pilus biosynthesis operons by reducing transcription termination and activating translation. While E. coli RfaH has been extensively studied using structural and biochemical approaches, limited data are available for other RfaH homologs. Here we set out to identify small molecule inhibitors of E. coli and K. pneumoniae RfaHs. Results of biochemical and functional assays show that these proteins act similarly, with a notable difference between their interactions with the RNAP ß subunit gate loop. We focused on high-affinity RfaH interactions with the RNAP ß' subunit clamp helices as a shared target for inhibition. Among the top 10 leads identified by in silico docking using ZINC database, 3 ligands were able to inhibit E. coli RfaH recruitment in vitro. The most potent lead was active against both E. coli and K. pneumoniae RfaHs in vitro. Our results demonstrate the feasibility of identifying RfaH inhibitors using in silico docking and pave the way for rational design of antivirulence therapeutics against antibiotic-resistant pathogens.

Asunto(s)

ARN Polimerasas Dirigidas por ADN/química , Proteínas de Escherichia coli/química , Klebsiella pneumoniae/patogenicidad , Simulación del Acoplamiento Molecular , Factores de Elongación de Péptidos/química , Bibliotecas de Moléculas Pequeñas/química , Transactivadores/química , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Diseño de Fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/patogenicidad , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/genética , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/genética , Ligandos , Factores de Elongación de Péptidos/antagonistas & inhibidores , Factores de Elongación de Péptidos/genética , Ribosomas/química , Ribosomas/genética , Ribosomas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Transactivadores/antagonistas & inhibidores , Transactivadores/genética , Virulencia/efectos de los fármacos

RESUMEN

To maintain genome integrity, organisms employ DNA damage response, the underlying principles of which are conserved from bacteria to humans. The bacterial small RNA OxyS of Escherichia coli is induced upon oxidative stress and has been implicated in protecting cells from DNA damage; however, the mechanism by which OxyS confers genome stability remained unknown. Here, we revealed an OxyS-induced molecular checkpoint relay, leading to temporary cell cycle arrest to allow damage repair. By repressing the expression of the essential transcription termination factor nusG, OxyS enables read-through transcription into a cryptic prophage encoding kilR The KilR protein interferes with the function of the major cell division protein FtsZ, thus imposing growth arrest. This transient growth inhibition facilitates DNA damage repair, enabling cellular recovery, thereby increasing viability following stress. The OxyS-mediated growth arrest represents a novel tier of defense, introducing a new regulatory concept into bacterial stress response.

Asunto(s)

Puntos de Control del Ciclo Celular/genética , Daño del ADN/genética , Reparación del ADN/genética , Proteínas de Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/genética , Proteínas Represoras/genética , Proteínas Bacterianas/genética , División Celular/genética , Proteínas del Citoesqueleto/genética , Proteínas de Escherichia coli/antagonistas & inhibidores , Inestabilidad Genómica/genética , Estrés Oxidativo/fisiología , Factores de Elongación de Péptidos/antagonistas & inhibidores , Factores de Elongación de Péptidos/genética , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Transcripción Genética/genética

RESUMEN

BS69 (also called ZMYND11) contains tandemly arranged PHD, BROMO, and PWWP domains, which are chromatin recognition modalities. Here, we show that BS69 selectively recognizes histone variant H3.3 lysine 36 trimethylation (H3.3K36me3) via its chromatin-binding domains. We further identify BS69 association with RNA splicing regulators, including the U5 snRNP components of the spliceosome, such as EFTUD2. Remarkably, RNA sequencing shows that BS69 mainly regulates intron retention (IR), which is the least understood RNA alternative splicing event in mammalian cells. Biochemical and genetic experiments demonstrate that BS69 promotes IR by antagonizing EFTUD2 through physical interactions. We further show that regulation of IR by BS69 also depends on its binding to H3K36me3-decorated chromatin. Taken together, our study identifies an H3.3K36me3-specific reader and a regulator of IR and reveals that BS69 connects histone H3.3K36me3 to regulated RNA splicing, providing significant, important insights into chromatin regulation of pre-mRNA processing.

Asunto(s)

Empalme Alternativo , Proteínas Portadoras/metabolismo , Cromatina/metabolismo , Histonas/metabolismo , Precursores del ARN/genética , ARN Mensajero/genética , Secuencia de Bases , Proteínas Portadoras/genética , Proteínas de Ciclo Celular , Línea Celular Tumoral , Cromatina/genética , Proteínas Co-Represoras , Metilación de ADN/genética , Proteínas de Unión al ADN , Células HeLa , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/genética , Humanos , Intrones/genética , Lisina/genética , Lisina/metabolismo , Factores de Elongación de Péptidos/antagonistas & inhibidores , Factores de Elongación de Péptidos/genética , Factores de Elongación de Péptidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Interferencia de ARN , Procesamiento Postranscripcional del ARN/genética , ARN Interferente Pequeño , Ribonucleoproteína Nuclear Pequeña U5/antagonistas & inhibidores , Ribonucleoproteína Nuclear Pequeña U5/genética , Ribonucleoproteína Nuclear Pequeña U5/metabolismo , Análisis de Secuencia de ARN , Empalmosomas/genética

RESUMEN

Ribosomes, after one round of translation, must be recycled so that the next round of translation can occur. Complete disassembly of post-termination ribosomal complex (PoTC) in yeast for the recycling consists of three reactions: release of tRNA, release of mRNA and splitting of ribosomes, catalyzed by eukaryotic elongation factor 3 (eEF3) and ATP. Here, we show that translocation inhibitors cycloheximide and lactimidomycin inhibited all three reactions. Cycloheximide is a non-competitive inhibitor of both eEF3 and ATP. The inhibition was observed regardless of the way PoTC was prepared with either release factors or puromycin. Paromomycin not only inhibited all three reactions but also re-associated yeast ribosomal subunits. On the other hand, sordarin or fusidic acid, when applied together with eEF2/GTP, specifically inhibited ribosome splitting without blocking of tRNA/mRNA release. From these inhibitor studies, we propose that, in accordance with eEF3's known function in elongation, the release of tRNA via exit site occurs first, then mRNA is released, followed by the splitting of ribosomes during the disassembly of post-termination complexes catalyzed by eEF3 and ATP.

Asunto(s)

Proteínas Fúngicas/metabolismo , Terminación de la Cadena Péptídica Traduccional/efectos de los fármacos , Factores de Elongación de Péptidos/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Saccharomyces cerevisiae/genética , Adenosina Trifosfato/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Cicloheximida/farmacología , Ácido Fusídico/farmacología , Indenos/farmacología , Macrólidos/farmacología , Paromomicina/farmacología , Factores de Elongación de Péptidos/antagonistas & inhibidores , Factores de Terminación de Péptidos/metabolismo , Piperidonas/farmacología , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Ribosomas/efectos de los fármacos , Ribosomas/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos

RESUMEN

Mitochondrial morphology is controlled by two opposing processes: fusion and fission. Drp1 (dynamin-related protein 1) and hFis1 are two key players of mitochondrial fission, but how Drp1 is recruited to mitochondria and how Drp1-mediated mitochondrial fission is regulated in mammals is poorly understood. Here, we identify the vertebrate-specific protein MIEF1 (mitochondrial elongation factor 1; independently identified as MiD51), which is anchored to the outer mitochondrial membrane. Elevated MIEF1 levels induce extensive mitochondrial fusion, whereas depletion of MIEF1 causes mitochondrial fragmentation. MIEF1 interacts with and recruits Drp1 to mitochondria in a manner independent of hFis1, Mff (mitochondrial fission factor) and Mfn2 (mitofusin 2), but inhibits Drp1 activity, thus executing a negative effect on mitochondrial fission. MIEF1 also interacts with hFis1 and elevated hFis1 levels partially reverse the MIEF1-induced fusion phenotype. In addition to inhibiting Drp1, MIEF1 also actively promotes fusion, but in a manner distinct from mitofusins. In conclusion, our findings uncover a novel mechanism which controls the mitochondrial fusion-fission machinery in vertebrates. As MIEF1 is vertebrate-specific, these data also reveal important differences between yeast and vertebrates in the regulation of mitochondrial dynamics.

Asunto(s)

GTP Fosfohidrolasas/metabolismo , Fusión de Membrana , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Factores de Elongación de Péptidos/metabolismo , Apoptosis , Western Blotting , Reactivos de Enlaces Cruzados , Citoplasma/metabolismo , Dinaminas , Técnica del Anticuerpo Fluorescente , GTP Fosfohidrolasas/genética , Glioma/genética , Glioma/metabolismo , Células HeLa , Humanos , Técnicas para Inmunoenzimas , Inmunoprecipitación , Proteínas de la Membrana/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas Mitocondriales/antagonistas & inhibidores , Proteínas Mitocondriales/genética , Neuroblastoma/genética , Neuroblastoma/metabolismo , Factores de Elongación de Péptidos/antagonistas & inhibidores , Factores de Elongación de Péptidos/genética , Unión Proteica , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fracciones Subcelulares , Células Tumorales Cultivadas

RESUMEN

Elongation Factor P (EF-P) is an essential component of bacterial protein synthesis, enhancing the rate of translation by facilitating the addition of amino acids to the growing peptide chain. Using purified Staphylococcus aureus EF-P and a reconstituted Escherichia coli ribosomal system, an assay monitoring the addition of radiolabeled N-formyl methionine to biotinylated puromycin was developed. Reaction products were captured with streptavidin-coated scintillation proximity assay (SPA) beads and quantified by scintillation counting. Data from the assay were used to create a kinetic model of the reaction scheme. In this model, EF-P binding to the ribosome essentially doubled the rate of the ribosomal peptidyl transferase reaction. As described here, EF-P bound to the ribosomes with an apparent K(a) of 0.75 microM, and the substrates N-fMet-tRNA and biotinylated puromycin had apparent K(m)s of 19 microM and 0.5 microM, respectively. The assay was shown to be sensitive to a number of antibiotics known to target ribosomal peptide bond synthesis, such as chloramphenicol and puromycin, but not inhibitors that target other stages of protein synthesis, such as fusidic acid or thiostrepton.

Asunto(s)

Antibacterianos/análisis , Antibacterianos/farmacología , Evaluación Preclínica de Medicamentos/métodos , Pruebas de Sensibilidad Microbiana , Factores de Elongación de Péptidos/antagonistas & inhibidores , Peptidil Transferasas/antagonistas & inhibidores , Proteínas Ribosómicas/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Escherichia coli/metabolismo , Cinética , Reproducibilidad de los Resultados , Ribosomas/metabolismo , Staphylococcus aureus/metabolismo , Factores de Tiempo

RESUMEN

Bacterial elongation factor Tu (EF-Tu) and EF-Ts are interacting proteins involved in polypeptide chain elongation in protein biosynthesis. A novel scintillation proximity assay for the detection of inhibitors of EF-Tu and EF-Ts, as well as the interaction between them, was developed and used in a high-throughput screen of a chemical library. Several compounds from a variety of chemical series with inhibitory properties were identified, including certain indole dipeptides, benzimidazole amidines, 2-arylbenzimidazoles, N-substituted imidazoles, and N-substituted guanidines. The in vitro activities of these compounds were confirmed in a coupled bacterial transcription-translation assay. Several indole dipeptides were identified as inhibitors of bacterial translation, with compound 2 exhibiting a 50% inhibitory concentration of 14 microM and an MIC for S. aureus ATCC 29213 of 5.6 microg/ml. Structure-activity relationship studies around the dipeptidic indoles generated additional analogs with low micromolar MICs for both gram-negative and gram-positive bacteria. To assess the specificity of antibacterial action, these compounds were evaluated in a metabolic labeling assay with Staphylococcus aureus. Inhibition of translation, as well as limited effects on other macromolecular pathways for some of the analogs studied, indicated a possible contribution from a non-target-based antibacterial mechanism of action.

Asunto(s)

Antibacterianos/química , Dipéptidos/química , Dipéptidos/farmacología , Indoles/química , Factor Tu de Elongación Peptídica/antagonistas & inhibidores , Factores de Elongación de Péptidos/antagonistas & inhibidores , Antibacterianos/farmacología , Bencimidazoles/química , Bencimidazoles/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Cocos Grampositivos/efectos de los fármacos , Guanidinas/química , Guanidinas/farmacología , Imidazoles/química , Imidazoles/farmacología , Sustancias Macromoleculares/metabolismo , Pruebas de Sensibilidad Microbiana , Factor Tu de Elongación Peptídica/metabolismo , Factores de Elongación de Péptidos/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Relación Estructura-Actividad , Transcripción Genética/efectos de los fármacos

RESUMEN

The occurrence of fungal infection has escalated significantly in recent years and is expected to continue to increase for the foreseeable future. Unfortunately, only a limited number of antifungal drugs are currently available partially due to a lack of suitable targets. The most commonly used antifungals target the same molecule in the cell membrane and, while efficacious, are either extremely toxic or susceptible to resistance. This article examines elongation factor-3, which is unique to fungi and essential for fungal cell survival and, thus, an attractive antifungal target. A search for inhibitors of this 'perfect target' led to identification of compounds (sordarins) which inhibited elongation factor-2, a protein with a mammalian homologue. Molecular analysis demonstrated why sordarins can specifically act against fungal elongation factor-2. This data questions the validity of pursuing genes as targets only if they are unique to fungi. Proteins that are homologous to elongation factor-3 are also discussed. The advances in molecular techniques and bioinformatics will allow the re-evaluation of targets previously thought to be unattractive. In addition, molecular genetics provides new and novel information on cellular processes that can potentially introduce new targets.

Asunto(s)

Antifúngicos/uso terapéutico , Proteínas Fúngicas/antagonistas & inhibidores , Micosis/tratamiento farmacológico , Factores de Elongación de Péptidos/antagonistas & inhibidores , Antifúngicos/farmacología , Candida/efectos de los fármacos , Candida/genética , Candida/metabolismo , Farmacorresistencia Fúngica , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Marcación de Gen , Humanos , Factor 1 de Elongación Peptídica/fisiología , Factores de Elongación de Péptidos/genética , Fenotipo , Priones/efectos de los fármacos , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae , Especificidad de la Especie

RESUMEN

Sordarin derivatives are remarkably selective inhibitors of fungal protein synthesis. Available evidence points to a binding site for these inhibitors on elongation factor 2, but high affinity binding requires the presence of ribosomes. The gene mutated in one of the two isolated complementation groups of Saccharomyces cerevisiae mutants resistant to the sordarin derivative GM193663 has now been identified. It is RPP0, encoding the essential protein of the large ribosomal subunit stalk rpP0. Resistant mutants are found to retain most of the binding capacity for the drug, indicating that mutations in rpP0 endow the ribosome with the capacity to perform translation elongation in the presence of the inhibitor. Other proteins of the ribosomal stalk influence the expression of resistance, pointing to a wealth of interactions between stalk components and elongation factors. The involvement of multiple elements of the translation machinery in the mode of action of sordarin antifungals may explain the large selectivity of these compounds, even though the individual target components are highly conserved proteins.

Asunto(s)

Antifúngicos/metabolismo , Proteínas Protozoarias , Proteínas Ribosómicas/metabolismo , Secuencia de Aminoácidos , Animales , Prueba de Complementación Genética , Humanos , Indenos , Datos de Secuencia Molecular , Mutación , Factor 2 de Elongación Peptídica , Factores de Elongación de Péptidos/antagonistas & inhibidores , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido

RESUMEN

Elongation factor 3 (EF-3) is an essential requirement for translation in fungi. We previously reported activation of EF-3-ATPase by yeast ribosomes. EF-3 interacts with both ribosomal subunits and shows high affinity for 60S subparticles. Translational inhibitors alpha-sarcin, ricin and auto-immune antibodies to GTPase-activation center inhibit binding of EF-2 but not of EF-3 to yeast ribosomes. EF-2 competes with EF-3 for the ribosomal binding sites and inhibits EF-3-ATPase activity. Neomycin relieves the inhibitory effect of EF-2 on EF-3 function. The apparent competition between EF-2 and EF-3 may represent binding of these two proteins to specific conformational states of the ribosome. EF-3 stimulates ternary complex binding to yeast ribosomes. Neither the binding of EF-3 to ribosomes, nor the ribosome-dependent EF-3-ATPase activity are influenced by EF-1 alpha. Three lines of experimental evidence suggest a direct interaction between EF-1 alpha and EF-3. A polyclonal antibody to EF-3 immunoprecipitates EF-1 alpha along with EF-3. EF-1 alpha co-migrates with GST-EF-3 on glutathione-Sepharose columns. ELISA tests demonstrate an interference of EF-3/anti-EF-3 interaction by EF-1 alpha but not by EF-2. These results strongly suggest that the stimulatory effect of EF-3 on the ternary complex binding to yeast ribosomes involves a direct interaction between EF-1 alpha and EF-3.

Asunto(s)

Proteínas Fúngicas/fisiología , Factores de Elongación de Péptidos/fisiología , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Unión Competitiva , Factor 1 de Elongación Peptídica , Factor 2 de Elongación Peptídica , Factores de Elongación de Péptidos/antagonistas & inhibidores , Factores de Elongación de Péptidos/genética , Factores de Elongación de Péptidos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae

RESUMEN

Some plants were used for a long time in folk medicine as sources of anti-tumour remedies. Their effects on protein biosynthesis in vitro have been examined and described. The separate features of the peptide elongation system, isolated from tumoural cells, have been demonstrated. Some elongation factors or ribosomes have been shown to be a target site for the inhibition of protein biosynthesis caused by the substances isolated from various sources. The glycoside and caffeic acid, isolated from Melissa officinalis leaves, inhibited protein biosynthesis by direct influence the elongation factor eEF-2. The activity of this factor was also inhibited by aloin and aloeemodin. Saponin glycoside and its aglycon, isolated from Verbascum thapsiforme flowers, as well as digoxin, emetine and cepheline directly inactivated ribosomes. "Chagi" fraction, isolated from Inonotus obliquus, is responsible for the inhibitory effect caused by the aqueous tannin--less extract from this fungus. The target site for quercetin has been found to be the subunit form EF-1 alpha. It may be supposed that, the plant inhibitors of protein biosynthesis could be utilized for searching specific antitumoural preparations.

Asunto(s)

Factores de Elongación de Péptidos/antagonistas & inhibidores , Plantas/química , Inhibidores de la Síntesis de la Proteína/aislamiento & purificación , Animales , Ácidos Cafeicos/aislamiento & purificación , Emodina/análogos & derivados , Emodina/aislamiento & purificación , Glicósidos/aislamiento & purificación , Inhibidores de la Síntesis de la Proteína/farmacología , Quercetina/aislamiento & purificación , Células Tumorales Cultivadas/efectos de los fármacos

RESUMEN

The structural and functional characteristics of the elongation system (ribosomes and elongation factors) are presented. The immunochemical and diagnostic meaning of the ribosome investigations is considered. Evidence of the participation of ribosomes in the first step of protein glycosylation is presented. The heterogeneous elongation factor eEF-1, isolated from Guerin epithelioma, can be separated into three fractions: one of them functionally corresponds to EF-1 alpha, the second on to EF-1 beta gamma, and the third is an unidentified, active aggregate named EF-1B, which contains the subunit forms EF-1 alpha and EF-1 beta gamma, and other polypeptides showing protein kinase activity. The aggregate EF-1B can be autophosphorylated, while the subunit forms EF-1 alpha and EF-1 beta gamma can neither become autophosphorylated nor phosphorylate other polypeptides. The subunit form EF-beta gamma consists from two polypeptides of 32 and 51 kDa, corresponding to other eukaryotic beta and gamma polypeptides, respectively. EF-1 beta gamma is thermostable and protects against thermal inactivation of EF-1 alpha in the EF-1 alpha-EF-1 beta gamma complex. Pure eEF-2 preparations isolated from normal and neoplastic tissues show different structural features. The existence of eEF-2 in multiple forms, differing in molecular mass, have been found. The eEF-2 with molecular weight of about 100 kDa can be phosphorylated, while eEF-2 of about 65 kDa was not phosphorylated by protein kinase eEF-2. The phosphorylated eEF-2 lost its activity, and this effect was reversed by dephosphorylation. The eEF-2 (65 kDa) was isolated from the active polyribosomes, and it may directly participate in the translocation step of the peptide elongation. It was noted that the components of elongation system can be inhibited, in separate steps, by the substances isolated from various sources of plant origin. Alkaloids emetine and cepheline, cardiac remedy digoxin, saponin glycoside, and its aglycon directly inactivated ribosomes. Quercetin inhibited eEF-1 activity by directly influencing its subunit form EF-1 alpha. eEF-2 was shown to be a target site of the inhibitory action of the glycoside isolated from Melissa officinalis leaves.

Asunto(s)

Células Eucariotas/metabolismo , Extensión de la Cadena Peptídica de Translación/efectos de los fármacos , Factores de Elongación de Péptidos/antagonistas & inhibidores , Extractos Vegetales/farmacología , Animales , Factor 1 de Elongación Peptídica , Factor 2 de Elongación Peptídica , Factores de Elongación de Péptidos/química , Factores de Elongación de Péptidos/metabolismo , Ribosomas/efectos de los fármacos , Ribosomas/fisiología , Ribosomas/ultraestructura

RESUMEN

The effect of quercetin (3,3',4',5,7-pentahydroxyflavone) on the polypeptide elongation system isolated from rat liver cells, was investigated. Quercetin inhibited [14C]leucine incorporation into proteins in vitro and the inhibitory effect is being directed towards the elongation factor eEF-1, but not to eEF-2 and ribosomes. Quercetin was found to form a complex with EF-1 alpha, which was inactive in GTP-dependent binding to ribosomes. It can be suggested that quercetin can block the total or the part of the domain of EF-1 alpha structure that is responsible for formation of the ternary complex EF-1 alpha-GTP-[14C]Phe-tRNA and therefore preventing formation of the quaternary complex with ribosomes.

Asunto(s)

Extensión de la Cadena Peptídica de Translación/efectos de los fármacos , Factores de Elongación de Péptidos/antagonistas & inhibidores , Quercetina/farmacología , Animales , Guanosina Trifosfato/metabolismo , Técnicas In Vitro , Leucina/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Extensión de la Cadena Peptídica de Translación/fisiología , Factor 1 de Elongación Peptídica , Factores de Elongación de Péptidos/metabolismo , ARN de Transferencia de Fenilalanina/metabolismo , Ratas , Ratas Wistar , Ribosomas/efectos de los fármacos , Ribosomas/metabolismo

RESUMEN

EF-3 is a translational elongation factor specific to yeasts and fungi. Its carboxy-terminal region contains three lysine-clusters and is very basic. The region has been reported to be responsible for the interaction with ribosomes [Ishiyama, A., Ogawa, K., & Miyazaki, M. (1992) in Abstracts of the 15th Annual Meeting of the Molecular Biology Society of Japan, p.190]. To find specific inhibitors for the interaction of EF-3 with ribosomes, the effects of two basic polyamino acids, poly-L-(Lys) and poly-L-(Arg), and two acidic polyamino acids, poly-L-(Asp) and poly-L-(Glu), were examined using two assay systems for ATPase of EF-3. One was for the ribosome-activated ATPase and the other for the intrinsic (ribosome-independent) ATPase of EF-3. Basic polyamino acids were expected to act as analogues of the carboxy-terminal region of EF-3, and acidic ones to interact with EF-3. The basic polyamino acids inhibited the ribosome-activated ATPase, but they also inhibited the intrinsic one more effectively. Acidic polyamino acids, poly-L-(Asp) and poly-L-(Glu), inhibited the ribosome-activated ATPase but not the intrinsic one. Thus, acidic polyamino acids could be specific inhibitors of the interaction between EF-3 and ribosomes. Furthermore, a system for detecting the binding of EF-3 to ribosomes was constructed. That is, ribosome-bound EF-3 was detected by measuring the ATPase on precipitated ribosomes after a mixture of EF-3 and ribosomes had been ultracentrifuged. Using this system, poly-L-(Asp) was shown to inhibit the binding of EF-3 to ribosomes directly.

Asunto(s)

Proteínas Fúngicas/antagonistas & inhibidores , Factores de Elongación de Péptidos/antagonistas & inhibidores , Péptidos/farmacología , Biosíntesis de Proteínas , Ribosomas/efectos de los fármacos , Saccharomyces/química , Adenosina Trifosfatasas/antagonistas & inhibidores , Ácido Poliglutámico/farmacología , Polilisina/farmacología , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae

RESUMEN

As it has been found, the incubation of [gamma-32P]ATP with elongation factor--1 alpha purified from rabbit reticulocytes resulted in the phosphorylation of several substrate proteins /Tuhácková, Z. (1992) In: Rec. Adv. Cell. Mol. Biol. 4, 79-86, Peeters Press, Leuven/ (1). In the present paper chromatofocusing of the purified eEF-1 alpha demonstrates that the ATP-dependent protein kinase activity is associated with a single protein catalyzing the GTP-dependent binding of aminoacyl-tRNA to ribosomes. Both of these activities are inhibited by staurosporine and gossypol. The inhibition by GDP but not by GTP indicates a possible involvement of conformation changes also in the modulation of the protein kinase activity displayed by eEF-1 alpha.

Asunto(s)

Alcaloides/farmacología , Gosipol/farmacología , Factores de Elongación de Péptidos/antagonistas & inhibidores , Reticulocitos/metabolismo , Animales , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Guanosina Difosfato/farmacología , Histonas/metabolismo , Cinética , Peso Molecular , Factor 1 de Elongación Peptídica , Factores de Elongación de Péptidos/sangre , Factores de Elongación de Péptidos/aislamiento & purificación , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Conejos , Ribosomas/metabolismo , Estaurosporina

RESUMEN

Plant ribosome sensitivity to either pokeweed antiviral protein (PAP) or fusidic acid has been investigated using a highly coupled translation system obtained from Vicia sativa L., a plant that lacks ribosome-inactivating protein activity. Protein synthesis directed by endogenous messengers was more sensitive to PAP than synthesis of polyphenylalanine directed by polyuridylic acid. Conversely, the effects of fusidic acid were the highest in polyphenylalanine synthesis. Additionally, the concentration of magnesium ions during translation also played a determinant role in the extent of inhibition by both PAP and fusidic acid. The effects seem to be exerted on the interaction of the translational elongation factor 2 with the ribosome.

Asunto(s)

Fabaceae/efectos de los fármacos , Ácido Fusídico/farmacología , N-Glicosil Hidrolasas , Péptidos , Proteínas de Plantas/farmacología , Plantas Medicinales , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mensajero/genética , Ribosomas/efectos de los fármacos , Sistema Libre de Células , Fabaceae/genética , Magnesio/farmacología , Biosíntesis de Péptidos , Factor 2 de Elongación Peptídica , Factores de Elongación de Péptidos/antagonistas & inhibidores , Proteínas de Plantas/biosíntesis , Poli U/genética , Proteínas Inactivadoras de Ribosomas Tipo 1