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1.
ChemMedChem ; 19(11): e202300716, 2024 Jun 03.
Artículo en Inglés | MEDLINE | ID: mdl-38426720

RESUMEN

The eukaryotic initiation factor 2B (eIF2B) is a key regulator in protein-regulated signaling pathways and is closely related to the function of the central nervous system. Modulating eIF2B could retard the process of neurodegenerative diseases, including Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and vanishing white matter disease (VWM) et al. Here, we designed and synthesized a series of novel eIF2B activators containing oxadiazole fragments. The activating effects of compounds on eIF2B were investigated through testing the inhibition of ATF4 expression. Of all the targeted compounds, compounds 21 and 29 exhibited potent inhibition on ATF4 expression with IC50 values of 32.43 nM and 47.71 nM, respectively, which were stronger than that of ISRIB (IC50=67.90 nM). ATF4 mRNA assay showed that these two compounds could restore ATF4 mRNA to normal levels in thapsigargin-stimulated HeLa cells. Protein Translation assay showed that both compounds were effective in restoring protein synthesis. Compound potency assay showed that both compounds had similar potency to ISRIB with EC50 values of 5.844 and 37.70 nM. Cytotoxicity assay revealed that compounds 21 and 29 had low toxicity and were worth further investigation.


Asunto(s)
Factor de Transcripción Activador 4 , Diseño de Fármacos , Factor 2B Eucariótico de Iniciación , Humanos , Factor de Transcripción Activador 4/metabolismo , Células HeLa , Relación Estructura-Actividad , Factor 2B Eucariótico de Iniciación/metabolismo , Factor 2B Eucariótico de Iniciación/antagonistas & inhibidores , Estructura Molecular , Relación Dosis-Respuesta a Droga , Oxadiazoles/farmacología , Oxadiazoles/química , Oxadiazoles/síntesis química
2.
Nat Microbiol ; 5(11): 1361-1373, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32690955

RESUMEN

Eukaryotic cells, when exposed to environmental or internal stress, activate the integrated stress response (ISR) to restore homeostasis and promote cell survival. Specific stress stimuli prompt dedicated stress kinases to phosphorylate eukaryotic initiation factor 2 (eIF2). Phosphorylated eIF2 (p-eIF2) in turn sequesters the eIF2-specific guanine exchange factor eIF2B to block eIF2 recycling, thereby halting translation initiation and reducing global protein synthesis. To circumvent stress-induced translational shutdown, viruses encode ISR antagonists. Those identified so far prevent or reverse eIF2 phosphorylation. We now describe two viral proteins-one from a coronavirus and the other from a picornavirus-that have independently acquired the ability to counteract the ISR at its very core by acting as a competitive inhibitor of p-eIF2-eIF2B interaction. This allows continued formation of the eIF2-GTP-Met-tRNAi ternary complex and unabated global translation at high p-eIF2 levels that would otherwise cause translational arrest. We conclude that eIF2 and p-eIF2 differ in their interaction with eIF2B to such effect that p-eIF2-eIF2B association can be selectively inhibited.


Asunto(s)
Factor 2B Eucariótico de Iniciación/antagonistas & inhibidores , Factor 2 Eucariótico de Iniciación/antagonistas & inhibidores , Estrés Fisiológico/fisiología , Proteínas Virales/metabolismo , Animales , Sitios de Unión , Chlorocebus aethiops , Células Eucariotas/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Factor 2B Eucariótico de Iniciación/metabolismo , Técnicas de Inactivación de Genes , Células HEK293 , Células HeLa , Humanos , Fosforilación , Picornaviridae/metabolismo , Unión Proteica , Células Vero
3.
FEBS J ; 287(2): 239-245, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31550413

RESUMEN

The integrated stress response (ISR) regulates protein synthesis under conditions of stress. Phosphorylation of translation initiation factor eIF2 by stress-sensing kinases converts eIF2 from substrate to competitive inhibitor of its dedicated nucleotide exchange factor, eIF2B, arresting translation. A drug-like molecule called integrated stress response inhibitor (ISRIB) reverses the effects of eIF2 phosphorylation and restores translation by targeting eIF2B. When administered to mice, ISRIB enhances cognition and limits cognitive decline due to brain injury. To determine ISRIB's mechanism of action, we solved an atomic structure of ISRIB bound to the human eIF2B decamer. We found that ISRIB acts as a molecular staple, pinning together tetrameric subcomplexes of eIF2B along the assembly path to a fully active, decameric enzyme. In this Structural Snapshot, we discuss ISRIB's mechanism, its ability to rescue disease mutations in eIF2B and conservation of the enzyme and ISRIB-binding pocket.


Asunto(s)
Acetamidas/química , Ciclohexilaminas/química , Factor 2B Eucariótico de Iniciación/antagonistas & inhibidores , Memoria/efectos de los fármacos , Fármacos Neuroprotectores/química , Acetamidas/farmacología , Animales , Sitios de Unión , Ciclohexilaminas/farmacología , Factor 2B Eucariótico de Iniciación/metabolismo , Humanos , Fármacos Neuroprotectores/farmacología , Unión Proteica
4.
Nat Cell Biol ; 21(11): 1413-1424, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31685988

RESUMEN

Tumours depend on altered rates of protein synthesis for growth and survival, which suggests that mechanisms controlling mRNA translation may be exploitable for therapy. Here, we show that loss of APC, which occurs almost universally in colorectal tumours, strongly enhances the dependence on the translation initiation factor eIF2B5. Depletion of eIF2B5 induces an integrated stress response and enhances translation of MYC via an internal ribosomal entry site. This perturbs cellular amino acid and nucleotide pools, strains energy resources and causes MYC-dependent apoptosis. eIF2B5 limits MYC expression and prevents apoptosis in APC-deficient murine and patient-derived organoids and in APC-deficient murine intestinal epithelia in vivo. Conversely, the high MYC levels present in APC-deficient cells induce phosphorylation of eIF2α via the kinases GCN2 and PKR. Pharmacological inhibition of GCN2 phenocopies eIF2B5 depletion and has therapeutic efficacy in tumour organoids, which demonstrates that a negative MYC-eIF2α feedback loop constitutes a targetable vulnerability of colorectal tumours.


Asunto(s)
Neoplasias Colorrectales/genética , Factor 2 Eucariótico de Iniciación/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-myc/genética , Proteína de la Poliposis Adenomatosa del Colon/genética , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Animales , Apoptosis/genética , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Colon/metabolismo , Colon/patología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/patología , Factor 2 Eucariótico de Iniciación/metabolismo , Factor 2B Eucariótico de Iniciación/antagonistas & inhibidores , Factor 2B Eucariótico de Iniciación/genética , Factor 2B Eucariótico de Iniciación/metabolismo , Retroalimentación Fisiológica , Femenino , Células HCT116 , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transducción de Señal , Análisis de Supervivencia , Ensayos Antitumor por Modelo de Xenoinjerto
5.
Science ; 364(6439): 495-499, 2019 05 03.
Artículo en Inglés | MEDLINE | ID: mdl-31048492

RESUMEN

A core event in the integrated stress response, an adaptive pathway common to all eukaryotic cells in response to various stress stimuli, is the phosphorylation of eukaryotic translation initiation factor 2 (eIF2). Normally, unphosphorylated eIF2 transfers the methionylated initiator tRNA to the ribosome in a guanosine 5'-triphosphate-dependent manner. By contrast, phosphorylated eIF2 inhibits its specific guanine nucleotide exchange factor, eIF2B. To elucidate how the eIF2 phosphorylation status regulates the eIF2B activity, we determined cryo-electron microscopic and crystallographic structures of eIF2B in complex with unphosphorylated or phosphorylated eIF2. The unphosphorylated and phosphorylated forms of eIF2 bind to eIF2B in completely different manners: the nucleotide exchange-active and -inactive modes, respectively. These structures explain how phosphorylated eIF2 dominantly inhibits the nucleotide exchange activity of eIF2B.


Asunto(s)
Factor 2B Eucariótico de Iniciación/antagonistas & inhibidores , Factor 2B Eucariótico de Iniciación/química , Factor 2 Eucariótico de Iniciación/química , Estrés Fisiológico , Secuencias de Aminoácidos , Microscopía por Crioelectrón , Factor 2B Eucariótico de Iniciación/metabolismo , Humanos , Fosforilación
6.
Biochemistry ; 57(9): 1426-1435, 2018 03 06.
Artículo en Inglés | MEDLINE | ID: mdl-29425030

RESUMEN

Eukaryotic translation initiation factor 2B (eIF2B) is the guanine nucleotide exchange factor of the GTPase eIF2, which brings the initiator Met-tRNAi to the ribosome in the form of the eIF2-GTP·Met-tRNAi ternary complex (TC). The activity of eIF2B is inhibited by phosphorylation of its substrate eIF2 by several stress-induced kinases, which triggers the integrated stress response (ISR). The ISR plays a central role in maintaining homeostasis in the cell under various stress conditions, and its dysregulation is a causative factor in the pathology of a number of neurodegenerative disorders. Over the past three decades, virtually every aspect of eIF2B function has been the subject of uncertainty or controversy: from the catalytic mechanism of nucleotide exchange, to whether eIF2B only catalyzes nucleotide exchange on eIF2 or also promotes binding of Met-tRNAi to eIF2-GTP to form the TC. Here, we provide the first complete thermodynamic analysis of the process of recycling of eIF2-GDP to the TC. The available evidence leads to the conclusion that eIF2 is channeled from the ribosome (as an eIF5·eIF2-GDP complex) to eIF2B, converted by eIF2B to the TC, which is then channeled back to eIF5 and the ribosome. The system has evolved to be regulated by multiple factors, including post-translational modifications of eIF2, eIF2B, and eIF5, as well as directly by the energy balance in the cell, through the GTP:GDP ratio.


Asunto(s)
Factor 2B Eucariótico de Iniciación/química , Factor 2B Eucariótico de Iniciación/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Factor 2B Eucariótico de Iniciación/antagonistas & inhibidores , Humanos , Fosforilación , Schizosaccharomyces/metabolismo , Termodinámica
7.
Elife ; 4: e07314, 2015 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-25875391

RESUMEN

The general translation initiation factor eIF2 is a major translational control point. Multiple signaling pathways in the integrated stress response phosphorylate eIF2 serine-51, inhibiting nucleotide exchange by eIF2B. ISRIB, a potent drug-like small molecule, renders cells insensitive to eIF2α phosphorylation and enhances cognitive function in rodents by blocking long-term depression. ISRIB was identified in a phenotypic cell-based screen, and its mechanism of action remained unknown. We now report that ISRIB is an activator of eIF2B. Our reporter-based shRNA screen revealed an eIF2B requirement for ISRIB activity. Our results define ISRIB as a symmetric molecule, show ISRIB-mediated stabilization of activated eIF2B dimers, and suggest that eIF2B4 (δ-subunit) contributes to the ISRIB binding site. We also developed new ISRIB analogs, improving its EC50 to 600 pM in cell culture. By modulating eIF2B function, ISRIB promises to be an invaluable tool in proof-of-principle studies aiming to ameliorate cognitive defects resulting from neurodegenerative diseases.


Asunto(s)
Acetamidas/química , Ciclohexilaminas/química , Factor 2B Eucariótico de Iniciación/genética , Fármacos Neuroprotectores/química , Nootrópicos/química , Subunidades de Proteína/genética , Acetamidas/síntesis química , Acetamidas/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Ciclohexilaminas/síntesis química , Ciclohexilaminas/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Factor 2B Eucariótico de Iniciación/antagonistas & inhibidores , Factor 2B Eucariótico de Iniciación/metabolismo , Expresión Génica , Genes Reporteros , Células HEK293 , Células HeLa , Ensayos Analíticos de Alto Rendimiento , Humanos , Células K562 , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/farmacología , Nootrópicos/síntesis química , Nootrópicos/farmacología , Fosforilación , Unión Proteica , Multimerización de Proteína/efectos de los fármacos , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Relación Estructura-Actividad , Tapsigargina/antagonistas & inhibidores , Tapsigargina/farmacología
8.
J Neurosci ; 33(17): 7165-74, 2013 Apr 24.
Artículo en Inglés | MEDLINE | ID: mdl-23616526

RESUMEN

Axonal protein synthesis is a complex process involving selective mRNA localization and translational regulation. In this study, using in situ hybridization and metabolic labeling, we show that the mRNAs encoding eukaryotic translation initiation factors eIF2B2 and eIF4G2 are present in the axons of rat sympathetic neurons and are locally translated. We also report that a noncoding microRNA, miR16, modulates the axonal expression of eIF2B2 and eIF4G2. Transfection of axons with precursor miR16 and anti-miR16 showed that local miR16 levels modulated axonal eIF2B2 and eIF4G2 mRNA and protein levels, as well as axon outgrowth. siRNA-mediated knock-down of axonal eIF2B2 and eIF4G2 mRNA also resulted in a significant decrease in axonal eIF2B2 and eIF4G2 protein. Moreover, results of metabolic labeling studies showed that downregulation of axonal eIF2B2 and eIF4G2 expression also inhibited local protein synthesis and axon growth. Together, these data provide evidence that miR16 mediates axonal growth, at least in part, by regulating the local protein synthesis of eukaryotic translation initiation factors eIF2B2 and eIF4G2 in the axon.


Asunto(s)
Fibras Adrenérgicas/metabolismo , Axones/metabolismo , Factor 2B Eucariótico de Iniciación/biosíntesis , Factor 4G Eucariótico de Iniciación/biosíntesis , Biosíntesis de Proteínas/fisiología , Fibras Adrenérgicas/fisiología , Animales , Axones/fisiología , Células Cultivadas , Regulación hacia Abajo/fisiología , Factor 2B Eucariótico de Iniciación/antagonistas & inhibidores , Factor 2B Eucariótico de Iniciación/fisiología , Factor 4G Eucariótico de Iniciación/antagonistas & inhibidores , Factor 4G Eucariótico de Iniciación/fisiología , Femenino , Masculino , Ratas , Ratas Sprague-Dawley , Ganglio Cervical Superior/metabolismo , Ganglio Cervical Superior/fisiología
9.
Cancer Lett ; 313(2): 235-48, 2011 Dec 27.
Artículo en Inglés | MEDLINE | ID: mdl-22056078

RESUMEN

Salubrinal is a specific eIF2α phosphatase inhibitor that inhibits ER stress-mediated apoptosis. However, maintaining hyper-phosphorylated eIF2α state with high doses of salubrinal treatment promotes apoptosis in some cancer cells. In this report, we found that salubrinal induced apoptosis of EBV-transformed B cells. Notably, salubrinal induced ROS generation and p38 MPAK activation, which then induced expression of FasL. Moreover, salubrinal subsequently led to activation of caspases, calcium redistribution, Bax translocation, cytochrome c release, and apoptosis. These findings suggest that salubrinal may be a novel therapeutic approach for EBV-associated malignant diseases.


Asunto(s)
Apoptosis/efectos de los fármacos , Linfocitos B/metabolismo , Cinamatos/farmacología , Herpesvirus Humano 4/patogenicidad , Especies Reactivas de Oxígeno/metabolismo , Tiourea/análogos & derivados , Proteína X Asociada a bcl-2/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/patología , Linfocitos B/virología , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Calcio/metabolismo , Caspasas/metabolismo , Línea Celular Transformada , Citocromos c/metabolismo , Retículo Endoplásmico/metabolismo , Factor 2B Eucariótico de Iniciación/antagonistas & inhibidores , Factor 2B Eucariótico de Iniciación/metabolismo , Proteína Ligando Fas/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Transporte de Proteínas , Tiourea/farmacología , Proteína Letal Asociada a bcl/metabolismo , Proteína bcl-X/metabolismo
10.
Am J Physiol Endocrinol Metab ; 300(3): E564-70, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21224482

RESUMEN

Statins are a widely prescribed class of cholesterol lowering drugs whose use is frequently associated with muscle-related ailments. A number of mechanisms have been implicated in statin-induced myotoxicity including alterations in both protein synthesis and protein degradation. The objective of the present study was to explore the mechanism(s) contributing to the statin-induced reduction in protein synthesis in the muscle-derived C2C12 cell line. Cells were treated with 10 µM simvastatin or vehicle alone for 24 h in 1% serum. Cells exposed to simvastatin exhibited reduced rates of protein synthesis, as evidenced by [(35)S]methionine and [(35)S]cysteine incorporation into protein. The reduction in protein synthesis occurred with a concomitant decrease in expression and activity of eukaryotic initiation factor 2B (eIF2B), a regulated and rate-controlling guanine nucleotide exchange factor known to affect global rates of protein synthesis. The reductions in protein synthesis and eIF2B expression were prevented by coincubation with mevalonate. Simvastatin treatment also resulted in a proteasome-sensitive reduction in the protein expression of all the subunits of the eIF2B heteropentameric complex. Finally, increased phosphorylation of the catalytic ε-subunit at Ser(535) was observed, an event consistent with an observed reduction in eIF2B activity. These results suggest that repression of eIF2B expression and activity may contribute, at least in part, to the statin-induced reduction in protein synthesis.


Asunto(s)
Factor 2B Eucariótico de Iniciación/antagonistas & inhibidores , Factor 2B Eucariótico de Iniciación/biosíntesis , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Células Musculares/metabolismo , Proteínas Musculares/biosíntesis , Inhibidores de la Síntesis de la Proteína , Simvastatina/farmacología , Western Blotting , Línea Celular , Cisteína/metabolismo , Factor 2B Eucariótico de Iniciación/genética , Nucleótidos de Guanina/metabolismo , Humanos , Metionina/metabolismo , Ácido Mevalónico/farmacología , Células Musculares/efectos de los fármacos
11.
Mol Biol Cell ; 21(13): 2202-16, 2010 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-20444979

RESUMEN

Recycling of eIF2-GDP to the GTP-bound form constitutes a core essential, regulated step in eukaryotic translation. This reaction is mediated by eIF2B, a heteropentameric factor with important links to human disease. eIF2 in the GTP-bound form binds to methionyl initiator tRNA to form a ternary complex, and the levels of this ternary complex can be a critical determinant of the rate of protein synthesis. Here we show that eIF2B serves as the target for translation inhibition by various fusel alcohols in yeast. Fusel alcohols are endpoint metabolites from amino acid catabolism, which signal nitrogen scarcity. We show that the inhibition of eIF2B leads to reduced ternary complex levels and that different eIF2B subunit mutants alter fusel alcohol sensitivity. A DNA tiling array strategy was developed that overcame difficulties in the identification of these mutants where the phenotypic distinctions were too subtle for classical complementation cloning. Fusel alcohols also lead to eIF2alpha dephosphorylation in a Sit4p-dependent manner. In yeast, eIF2B occupies a large cytoplasmic body where guanine nucleotide exchange on eIF2 can occur and be regulated. Fusel alcohols impact on both the movement and dynamics of this 2B body. Overall, these results confirm that the guanine nucleotide exchange factor, eIF2B, is targeted by fusel alcohols. Moreover, they highlight a potential connection between the movement or integrity of the 2B body and eIF2B regulation.


Asunto(s)
Alcoholes/farmacología , Factor 2B Eucariótico de Iniciación/antagonistas & inhibidores , Factor 2B Eucariótico de Iniciación/metabolismo , Complejos Multiproteicos , Biosíntesis de Proteínas/efectos de los fármacos , Subunidades de Proteína/metabolismo , Alcoholes/metabolismo , Secuencia de Bases , Factor 2B Eucariótico de Iniciación/química , Factor 2B Eucariótico de Iniciación/genética , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Datos de Secuencia Molecular , Mutación , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
12.
FASEB J ; 22(12): 4327-37, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-18728216

RESUMEN

The critical role of protein synthesis in regulating lifespan has been evidenced. This study shows that adult-onset RNAi inactivation of eukaryotic initiation factor 2Bdelta (eIF2Bdelta/F11A3.2), a subunit of eIF2B, extends the mean lifespan of Caenorhabditis elegans. eIF2B is a GDP-GTP exchange factor for eIF2--a rate-limiting factor for protein synthesis initiation. (35)S-methionine incorporation assay showed that global protein synthesis is reduced by eIF2Bdelta/F11A3.2 RNAi. Inhibition of eIF2Bdelta/F11A3.2 during adulthood conferred thermal and oxidative stress resistance and reduced the fecundity and fat storage, suggesting the possible trade-offs of resources between reproduction and somatic maintenance. Lifespan extension by adult-onset eIF2Bdelta/F11A3.2 RNAi is suppressed in FOXO transcription factor daf-16 deletion mutants. Adult-onset eIF2Bdelta/F11A3.2 RNAi increases the expression of stress-resistant genes, including hsp-16.2, hsp-70, hsp90, and sod-3, some of which are reported to be targets of DAF-16. Adult-onset eIF2Bdelta/F11A3.2 RNAi in daf-16 mutants reduced fecundity, but did not extend lifespan. Furthermore, adult-onset eIF2Bdelta/F11A3.2 RNAi did not extend the lifespan of germline-defective glp-4 organisms. Thus, it is possible that eIF2Bdelta/F11A3.2 RNAi during adulthood prolongs lifespan via daf-16, which induces stress resistance in organisms. This might be the mechanism, at least in part, for trade-offs of resources between reproduction and somatic maintenance.


Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Factor 2B Eucariótico de Iniciación/antagonistas & inhibidores , Longevidad/efectos de los fármacos , Interferencia de ARN , Envejecimiento/fisiología , Animales , Proteínas de Caenorhabditis elegans/genética , Fertilidad/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica , Longevidad/fisiología , Estrés Oxidativo/efectos de los fármacos
13.
Curr Biol ; 18(14): 1025-33, 2008 Jul 22.
Artículo en Inglés | MEDLINE | ID: mdl-18635357

RESUMEN

BACKGROUND: Cell-size-control systems, coupled with apoptotic- and cell-proliferation-regulatory mechanisms, determine the overall dimensions of organs and organisms, and their dysregulation can lead to tumor formation. The interrelationship between cell-growth-regulatory mechanisms and apoptosis during normal development and cancer is not understood. The TRK-fused gene (TFG) promotes tumorigenesis when present in chromosomal rearrangements from various human-cancer types by unknown mechanisms. Apaf1/CED-4 is essential for apoptosis but has not been shown to function in cell-growth control. RESULTS: We found that loss of TFG-1, the TFG ortholog in Caenorhabditis elegans, results in supernumerary apoptotic corpses, whereas its overexpression is sufficient to inhibit developmentally programmed cell death. TFG-1 is also required for cells and nuclei to grow to normal size. Furthermore, we found that CED-4 is required for cell-growth inhibition in animals lacking TFG-1. However, caspases, the downstream effectors of CED-4-mediated apoptosis, are not required in TFG-1- or CED-4-regulated cell-size control. CED-4 acts to inhibit cell growth by antagonizing the effects of other conserved cell-size-regulating proteins, including cAMP response element binding (CREB) protein, translation-initiation factor eIF2B, and the nucleolar p53-interacting protein nucleostemin. CONCLUSIONS: These findings show that TFG-1 suppresses apoptosis and is essential for normal cell-size control, suggesting that abnormalities in the cell-growth-promoting and apoptosis-inhibiting functions of TFG might be responsible for its action in tumorigenesis. Also, they reveal that CED-4 plays a pivotal role in activating apoptosis and restricting cell and nuclear size, thereby determining the appropriate overall size of an animal. Thus, these findings reveal links between the control mechanisms for apoptosis and cell growth.


Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Proteínas de Unión al Calcio/genética , Animales , Apoptosis/genética , Apoptosis/fisiología , Tamaño Corporal/genética , Tamaño Corporal/fisiología , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/antagonistas & inhibidores , Proteínas de Caenorhabditis elegans/fisiología , Proteínas de Unión al Calcio/antagonistas & inhibidores , Proteínas de Unión al Calcio/fisiología , Tamaño de la Célula , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/antagonistas & inhibidores , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Factor 2B Eucariótico de Iniciación/antagonistas & inhibidores , Factor 2B Eucariótico de Iniciación/genética , Factor 2B Eucariótico de Iniciación/fisiología , Genes de Helminto , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Proto-Oncogenes Mas , Proto-Oncogenes , Interferencia de ARN
14.
Am J Physiol Gastrointest Liver Physiol ; 285(3): G517-28, 2003 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-12773302

RESUMEN

Acute pancreatitis (AP) has been shown in some studies to inhibit total protein synthesis in the pancreas, whereas in other studies, protein synthesis was not affected. Previous in vitro work has shown that high concentrations of cholecystokinin both inhibit protein synthesis and inhibit the activity of the guanine nucleotide exchange factor eukaryotic initiation factor (eIF)2B by increasing the phosphorylation of eIF2alpha. We therefore evaluated in C57BL/6 mice the effects of caerulein-induced AP on pancreatic protein synthesis, eIF2B activity and other protein translation regulatory mechanisms. Repetitive hourly injections of caerulein were administered at 50 microg/kg ip. Pancreatic protein synthesis was reduced 10 min after the initial caerulein administration and was further inhibited after three and five hourly injections. Caerulein inhibited the two major regulatory points of translation initiation: the activity of the guanine nucleotide exchange factor eIF2B (with an increase of eIF2alpha phosphorylation) and the formation of the eIF4F complex due, in part, to degradation of eIF4G. This inhibition was not accounted for by changes in the upstream stimulatory pathway, because caerulein activated Akt as well as phosphorylating the downstream effectors of mTOR, 4E-BP1, and ribosomal protein S6. Caerulein also decreased the phosphorylation of the eukaryotic elongation factor 2, implying that this translation factor was not inhibited in AP. Thus the inhibition of pancreatic protein synthesis in this model of AP most likely results from the inhibition of translation initiation as a result of increased eIF2alpha phosphorylation, reduction of eIF2B activity, and the inhibition of eIF4F complex formation.


Asunto(s)
Ceruletida , Factor 2B Eucariótico de Iniciación/metabolismo , Factor 4F Eucariótico de Iniciación/metabolismo , Pancreatitis/metabolismo , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas , Enfermedad Aguda , Animales , Factor 2B Eucariótico de Iniciación/antagonistas & inhibidores , Factor 4E Eucariótico de Iniciación/metabolismo , Ratones , Pancreatitis/inducido químicamente , Factor 2 de Elongación Peptídica/metabolismo , Fosforilación , Proteínas Quinasas/metabolismo , Proteínas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Serina-Treonina Quinasas TOR
16.
Biochemistry ; 39(42): 12929-38, 2000 Oct 24.
Artículo en Inglés | MEDLINE | ID: mdl-11041858

RESUMEN

Phosphorylation of serine 51 residue on the alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha) inhibits the guanine nucleotide exchange (GNE) activity of eIF2B, presumably, by forming a tight complex with eIF2B. Inhibition of the GNE activity of eIF2B leads to impairment in eIF2 recycling and protein synthesis. We have partially purified the wild-type (wt) and mutants of eIF2alpha in which the serine 51 residue was replaced with alanine (51A mutant) or aspartic acid (51D mutant) in the baculovirus system. Analysis of these mutants has provided novel insight into the role of 51 serine in the interaction between eIF2 and eIF2B. Neither mutant was phosphorylated in vitro. Both mutants decreased eIF2alpha phosphorylation occurring in hemin and poly(IC)-treated reticulocyte lysates due to the activation of double-stranded RNA-dependent protein kinase (PKR). However, addition of 51D, but not 51A mutant eIF2alpha protein promoted inhibition of the GNE activity of eIF2B in hemin-supplemented rabbit reticulocyte lysates in which relatively little or no endogenous eIF2alpha phosphorylation occurred. The 51D mutant enhanced the inhibition in GNE activity of eIF2B that occurred in hemin and poly(IC)-treated reticulocyte lysates where PKR is active. Our results show that the increased interaction between eIF2 and eIF2B protein, occurring in reticulocyte lysates due to increased eIF2alpha phosphorylation, is decreased significantly by the addition of mutant 51A protein but not 51D. Consistent with the idea that mutant 51D protein behaves like a phosphorylated eIF2alpha, addition of this partially purified recombinant subunit, but not 51A or wt eIF2alpha, increases the interaction between eIF2 and 2B proteins in actively translating hemin-supplemented lysates. These findings support the idea that phosphorylation of the serine 51 residue in eIF2alpha promotes complex formation between eIF2alpha(P) and eIF2B and thereby inhibits the GNE activity of eIF2B.


Asunto(s)
Factor 2B Eucariótico de Iniciación/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Serina/metabolismo , Alanina/genética , Animales , Ácido Aspártico/genética , Baculoviridae/genética , Sistema Libre de Células/metabolismo , Factor 2 Eucariótico de Iniciación/biosíntesis , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/inmunología , Factor 2B Eucariótico de Iniciación/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido/metabolismo , Hemina/metabolismo , Humanos , Masculino , Nucleopoliedrovirus/genética , Nucleopoliedrovirus/metabolismo , Fosforilación , Poli I-C/metabolismo , Conejos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Reticulocitos/metabolismo , Spodoptera/genética , Spodoptera/metabolismo
17.
Biochemistry ; 38(46): 15398-405, 1999 Nov 16.
Artículo en Inglés | MEDLINE | ID: mdl-10563826

RESUMEN

Phosphorylation of the serine 51 residue in the alpha-subunit of translational initiation factor 2 in eukaryotes (eIF2 alpha) impairs protein synthesis presumably by sequestering eIF2B, a rate-limiting pentameric guanine nucleotide exchange protein which catalyzes the exchange of GTP for GDP in the eIF2-GDP binary complex. To further understand the importance of eIF2 alpha phosphorylation in the interaction between eIF2 alpha(P) and eIF2B proteins and thereby the regulation of eIF2B activity, we expressed the wild type (wt) and a mutant eIF2 alpha in which the serine 48 residue was replaced with alanine (48A mutant) in the baculovirus system. The findings reveal that the expression of both of these recombinant subunits was very efficient (15-20% of the total protein) and both proteins were recognized by an eIF2 alpha monoclonal antibody and were phosphorylated to the same extent by reticulocyte eIF2 alpha kinases. However, partially purified recombinant subunits (wt or 48A mutant) were not phosphorylated as efficiently as the eIF2 alpha subunit present in the purified reticulocyte trimeric eIF2 complex and were also found to inhibit the phosphorylation of eIF2 alpha of the trimeric complex. Furthermore, the extents of inhibition of eIF2B activity and formation of the eIF2 alpha(P)-eIF2B complex that occurs due to eIF2 alpha phosphorylation in poly(IC)-treated rabbit reticulocyte lysates were decreased significantly in the presence of insect cell extracts expressing the 48A mutant eIF2 alpha compared to those for wt. These findings support the hypothesis that the serine 48 residue is required for high-affinity interaction between eIF2 alpha(P) and eIF2B.


Asunto(s)
Factor 2B Eucariótico de Iniciación/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Serina/metabolismo , Sustitución de Aminoácidos/genética , Animales , Anticuerpos Monoclonales/metabolismo , Sitios de Unión , Western Blotting , Fraccionamiento Celular , Factor 2 Eucariótico de Iniciación/química , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/inmunología , Factor 2B Eucariótico de Iniciación/antagonistas & inhibidores , Factor 2B Eucariótico de Iniciación/química , Vectores Genéticos/síntesis química , Humanos , Sustancias Macromoleculares , Nucleopoliedrovirus/genética , Fosforilación , Poli I-C/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reticulocitos/efectos de los fármacos , Reticulocitos/metabolismo , Serina/genética , Spodoptera/genética , Spodoptera/metabolismo , Spodoptera/virología , Transfección
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