RESUMEN
BACKGROUND: Renal cell carcinoma (RCC) comprises five major molecular and histological subtypes. The Birt-Hogg-Dubé (BHD) syndrome is a hereditary human cancer syndrome that predisposes affected individuals to develop renal carcinoma of nearly all subtypes, in addition to benign fibrofolliculomas, and pulmonary and renal cysts. BHD is caused by loss-of-function mutations in the folliculin (FLCN) protein. The molecular function of FLCN is still largely unknown; opposite and conflicting evidence of the role of FLCN in mammalian target of rapamycin signalling/phosphorylated ribosomal protein S6 (p-S6) activation had recently been reported. RESULTS AND METHODS: Here, the expression pattern of murine Flcn was described, and it was observed that homozygous disruption of Flcn results in embryonic lethality early during development. Importantly, heterozygous animals manifest early preneoplastic kidney lesions, devoid of Flcn expression, that progress towards malignancy, including cystopapillary adenomas. A bona fide tumour suppressor activity of FLCN was confirmed by nude mouse xenograft assays of two human RCC cell lines with either diminished or re-expressed FLCN. It was observed that loss of FLCN expression leads to context-dependent effects on S6 activation. Indeed, solid tumours and normal kidneys show decreased p-S6 upon diminished FLCN expression. Conversely, p-S6 is found to be elevated or absent in FLCN-negative renal cysts. CONCLUSION: In accordance with clinical data showing distinct renal malignancies arising in BHD patients, in this study FLCN is shown as a general tumour suppressor in the kidney.
Asunto(s)
Carcinoma de Células Renales/genética , Genes Supresores de Tumor/fisiología , Neoplasias Renales/genética , Proteínas Proto-Oncogénicas/fisiología , Proteínas Supresoras de Tumor/fisiología , Animales , Carcinoma de Células Renales/patología , Proliferación Celular , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Enfermedades Renales Quísticas/genética , Enfermedades Renales Quísticas/patología , Neoplasias Renales/patología , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Transgénicos , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Complejos Multiproteicos , Lesiones Precancerosas/genética , Proteínas , Proteínas Proto-Oncogénicas/genética , Proteínas Quinasas S6 Ribosómicas/metabolismo , Síndrome , Serina-Treonina Quinasas TOR , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
PHAS-I is a heat-stable protein (relative molecular mass approximately 12,400) found in many tissues. It is rapidly phosphorylated in rat adipocytes incubated with insulin or growth factors. Nonphosphorylated PHAS-I bound to initiation factor 4E (eIF-4E) and inhibited protein synthesis. Serine-64 in PHAS-I was rapidly phosphorylated by mitogen-activated (MAP) kinase, the major insulin-stimulated PHAS-I kinase in adipocyte extracts. Results obtained with antibodies, immobilized PHAS-I, and a messenger RNA cap affinity resin indicated that PHAS-I did not bind eIF-4E when serine-64 was phosphorylated. Thus, PHAS-I may be a key mediator of the stimulation of protein synthesis by the diverse group of agents and stimuli that activate MAP kinase.
Asunto(s)
Proteínas Portadoras , Insulina/farmacología , Factores de Iniciación de Péptidos/metabolismo , Fosfoproteínas/metabolismo , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Células 3T3 , Proteínas Adaptadoras Transductoras de Señales , Adipocitos/metabolismo , Animales , Proteínas de Ciclo Celular , Factores Eucarióticos de Iniciación , Péptidos y Proteínas de Señalización Intracelular , Ratones , Proteína Quinasa 1 Activada por Mitógenos , Factores de Iniciación de Péptidos/aislamiento & purificación , Fosforilación , Ratas , Proteínas Recombinantes/metabolismo , Serina/metabolismoRESUMEN
Germline mutations in the von Hippel-Lindau tumor suppressor gene (VHL) predispose individuals to a variety of tumors, including renal carcinoma, hemangioblastoma of the central nervous system, and pheochromocytoma. Here, a cellular transcription factor, Elongin (SIII), is identified as a functional target of the VHL protein. Elongin (SIII) is a heterotrimer consisting of a transcriptionally active subunit (A) and two regulatory subunits (B and C) that activate transcription elongation by RNA polymerase II. The VHL protein was shown to bind tightly and specifically to the Elongin B and C subunits and to inhibit Elongin (SIII) transcriptional activity in vitro. These findings reveal a potentially important transcriptional regulatory network in which the VHL protein may play a key role.
Asunto(s)
Genes Supresores de Tumor , Ligasas , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas , Enfermedad de von Hippel-Lindau/genética , Secuencia de Aminoácidos , Animales , Línea Celular , Clonación Molecular , Elonguina , Regulación de la Expresión Génica , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/genética , ARN Polimerasa II/metabolismo , Proteínas Recombinantes/metabolismo , Factores de Transcripción/química , Factores de Transcripción/aislamiento & purificación , Proteína Supresora de Tumores del Síndrome de Von Hippel-LindauRESUMEN
eIF-4A is a eukaryotic translation initiation factor that is required for mRNA binding to ribosomes. It exhibits single-stranded RNA-dependent ATPase activity, and in combination with a second initiation factor, eIF-4B, it exhibits duplex RNA helicase activity. eIF-4A is the prototype of a large family of proteins termed the DEAD box protein family, whose members share nine highly conserved amino acid regions. The functions of several of these conserved regions in eIF-4A have previously been assigned to ATP binding, ATPase, and helicase activities. To define the RNA-binding region of eIF-4A, a UV-induced cross-linking assay was used to analyze binding of mutant eIF-4A proteins to RNA. Mutants carrying mutations in the ATP-binding region (AXXXXGKT), ATPase region (DEAD), helicase region (SAT), and the most carboxy-terminal conserved region of the DEAD family, HRIGRXXR, were tested for RNA cross-linking. We show that mutations, either conservative or not, in any one of the three arginines in the HRIGRXXR sequence drastically reduced eIF-4A cross-linking to RNA. In addition, all the mutations in the HRIGRXXR region abrogate RNA helicase activity. Some but not all of these mutations affect ATP binding and ATPase activity. This is consistent with the hypothesis that the HRIGRXXR region is involved in the ATP hydrolysis reaction and would explain the coupling of ATPase and RNA-binding/helicase activities. Our results show that the HRIGRXXR region, which is QRXGRXXR or QXXGRXXR in the RNA and DNA helicases of the helicase superfamily II, is involved in ATP hydrolysis-dependent RNA interaction during unwinding. We also show that mutations in other regions of eIF-4A that abolish ATPase activity sharply decrease eIF-4A cross-linking to RNA. A model is proposed in which eIF-4A first binds ATP, resulting in a change in eIF-4A conformation which allows RNA binding that is dependent on the HRIGRXXR region. Binding of RNA induces ATP hydrolysis, leading to a more stable interaction with RNA. This process is then linked to unwinding of duplex RNA in the presence of eIF-4B.
Asunto(s)
Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/metabolismo , Factores de Iniciación de Péptidos/metabolismo , ARN Nucleotidiltransferasas/metabolismo , ARN Mensajero/metabolismo , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Secuencia Conservada , Análisis Mutacional de ADN , Factor 4A Eucariótico de Iniciación , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , ARN Helicasas , Proteínas Recombinantes/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcripción GenéticaRESUMEN
eIF-4B is a eukaryotic translation initiation factor that is required for the binding of ribosomes to mRNAs and the stimulation of the helicase activity of eIF-4A. It is an RNA-binding protein that contains a ribonucleoprotein consensus sequence (RNP-CS)/RNA recognition motif (RRM). We examined the effects of deletions and point mutations on the ability of eIF-4B to bind a random RNA, to cooperate with eIF-4A in RNA binding, and to enhance the helicase activity of eIF-4A. We report here that the RNP-CS/RRM alone is not sufficient for eIF-4B binding to RNA and that an RNA-binding region, located between amino acids 367 and 423, is the major contributor to RNA binding. Deletions which remove this region abolish the ability of eIF-4B to cooperate with eIF-4A in RNA binding and the ability to stimulate the helicase activity of eIF-4A. Point mutations in the RNP-CS/RRM had no effect on the ability of eIF-4B to cooperate with eIF-4A in RNA binding but significantly reduced the stimulation of eIF-4A helicase activity. Our results indicate that the carboxy-terminal RNA-binding region of eIF-4B is essential for eIF-4B function and is distinct from the RNP-CS/RRM.
Asunto(s)
Secuencia de Consenso , Factores Eucarióticos de Iniciación , Factores de Iniciación de Péptidos/química , Factores de Iniciación de Péptidos/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/química , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Escherichia coli , Glutatión Transferasa/metabolismo , Humanos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Factores de Iniciación de Péptidos/genética , Mutación Puntual , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteínas/genética , Ribosomas/metabolismo , Eliminación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Eukaryotic translation initiation factor 4E (eIF-4E), which possesses cap-binding activity, functions in the recruitment of mRNA to polysomes as part of a three-subunit complex, eIF-4F (cap-binding complex). eIF-4E is the least abundant of all translation initiation factors and a target of growth regulatory pathways. Recently, two human cDNAs encoding novel eIF-4E-binding proteins (4E-BPs) which function as repressors of cap-dependent translation have been cloned. Their interaction with eIF-4E is negatively regulated by phosphorylation in response to cell treatment with insulin or growth factors. The present study aimed to characterize the molecular interactions between eIF-4E and the other subunits of eIF-4F and to similarly characterize the molecular interactions between eIF-4E and the 4E-BPs. A 49-amino-acid region of eIF-4 gamma, located in the N-terminal side of the site of cleavage by Picornaviridae protease 2A, was found to be sufficient for interacting with eIF-4E. Analysis of deletion mutants in this region led to the identification of a 12-amino-acid sequence conserved between mammals and Saccharomyces cerevisiae that is critical for the interaction with eIF-4E. A similar motif is found in the amino acid sequence of the 4E-BPs, and point mutations in this motif abolish the interaction with eIF-4E. These results shed light on the mechanisms of eIF-4F assembly and on the translational regulation by insulin and growth factors.
Asunto(s)
Proteínas Portadoras , Iniciación de la Cadena Peptídica Traduccional , Fragmentos de Péptidos/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Sitios de Unión , Proteínas de Ciclo Celular , Clonación Molecular , Secuencia Conservada , Factor 4E Eucariótico de Iniciación , Factor 4F Eucariótico de Iniciación , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Factores de Iniciación de Péptidos/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Unión Proteica , Caperuzas de ARN/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Mutation of the von Hippel-Lindau tumor suppressor gene (vhl) causes the von Hippel-Lindau cancer syndrome as well as sporadic renal clear cell carcinoma. To pursue our study of the intracellular localization of VHL protein in relation to its function, we fused VHL to the green fluorescent protein (GFP) to produce the VHL-GFP fusion protein. Like VHL, VHL-GFP binds to elongins B and C and Cullin-2 and regulates target gene product levels, including levels of vascular endothelial growth factor and glucose transporter 1. VHL-GFP localizes predominantly to the cytoplasm, with some detectable nuclear signal. Inhibition of transcription by actinomycin D or 5,6-dichlorobenzimidazole riboside (DRB) causes VHL to be redistributed to the nucleus. A cellular fusion assay was used to demonstrate that inhibition of transcription induces a decrease in the nuclear export rate of VHL. The dependence of transcription for trafficking is lost with a deletion of exon 2, a region with a mutation causing a splice defect in the VHL gene in sporadic renal clear cell carcinoma. Addition of a strong nuclear export signal (NES) derived from the Rev protein results in complete nuclear exclusion and abrogates the redistribution of VHL-GFP-NES into the nucleus upon inhibition of transcription. Leptomycin B, which inhibits NES-mediated nuclear export, reverts the distribution of VHL-GFP-NES to that of VHL-GFP and restores sensitivity to actinomycin D and DRB. Uncoupling of VHL-GFP trafficking to transcription either by an exon 2 deletion or fusion to NES abolishes VHL function. We suggest that VHL function requires not only nuclear or cytoplasmic localization, but also exon 2-mediated transcription-dependent trafficking between these two cellular compartments.
Asunto(s)
Ligasas , Proteínas/metabolismo , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas , Animales , Secuencia de Bases , Transporte Biológico Activo/efectos de los fármacos , Células COS , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/metabolismo , Fusión Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Cartilla de ADN/genética , Dactinomicina/farmacología , Genes Supresores de Tumor , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Mutación , Proteínas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transcripción Genética , Células Tumorales Cultivadas , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau , Enfermedad de von Hippel-Lindau/genética , Enfermedad de von Hippel-Lindau/metabolismoRESUMEN
Initiation in eukaryotes is the rate limiting step of translation. The binding of the mRNA to the 40S ribosomal subunit, which is mediated by the mRNA cap structure, is a key target for control of protein synthesis. The cap binding protein, eIF4E, is the most limiting of all initiation factors and its overexpression in NIH3T3 cells causes malignant transformation. 4E-binding protein 1 (BP1) and 4E-BP2 are small proteins that bind to eIF4E and inhibit translation. Here, 4E-BPs were expressed in cells transformed by eIF4E or by v-src to determine the effect of 4E-BPs on cell growth and tumorigenicity. We show that 4E-BPs cause a significant reversion of the transformed phenotype. Thus, we demonstrate that the eIF4E-binding proteins act as negative regulators of cell growth. We propose that 4E-BPs are members of a class of negative regulators of cell growth acting on the translation machinery of the cell.
Asunto(s)
División Celular/genética , Factores de Iniciación de Péptidos/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Células 3T3/metabolismo , Células 3T3/patología , Animales , Ensayo de Unidades Formadoras de Colonias , Ciclinas/metabolismo , Ratones , Ratones Desnudos , Ornitina Descarboxilasa/metabolismo , Fenotipo , Proteínas de Unión a Caperuzas de ARNRESUMEN
Under conditions of reduced tissue oxygenation, hypoxia-inducible factor (HIF) controls many processes, including angiogenesis and cellular metabolism, and also influences cell proliferation and survival decisions. HIF is centrally involved in tumour growth in inherited diseases that give rise to renal cell carcinoma (RCC), such as Von Hippel-Lindau syndrome and tuberous sclerosis complex. In this study, we examined whether HIF is involved in tumour formation of RCC in Birt-Hogg-Dubé syndrome. For this, we analysed a Birt-Hogg-Dubé patient-derived renal tumour cell line (UOK257) that is devoid of the Birt-Hogg-Dubé protein (BHD) and observed high levels of HIF activity. Knockdown of BHD expression also caused a threefold activation of HIF, which was not as a consequence of more HIF1α or HIF2α protein. Transcription of HIF target genes VEGF, BNIP3 and CCND1 was also increased. We found nuclear localization of HIF1α and increased expression of VEGF, BNIP3 and GLUT1 in a chromophobe carcinoma from a Birt-Hogg-Dubé patient. Our data also reveal that UOK257 cells have high lactate dehydrogenase, pyruvate kinase and 3-hydroxyacyl-CoA dehydrogenase activity. We observed increased expression of pyruvate dehydrogenase kinase 1 (a HIF gene target), which in turn leads to increased phosphorylation and inhibition of pyruvate dehydrogenase. Together with increased protein levels of GLUT1, our data reveal that UOK257 cells favour glycolytic rather than lipid metabolism (a cancer phenomenon termed the 'Warburg effect'). UOK257 cells also possessed a higher expression level of the L-lactate influx monocarboxylate transporter 1 and consequently utilized L-lactate as a metabolic fuel. As a result of their higher dependency on glycolysis, we were able to selectively inhibit the growth of these UOK257 cells by treatment with 2-deoxyglucose. This work suggests that targeting glycolytic metabolism may be used therapeutically to treat Birt-Hogg-Dubé-associated renal lesions.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Regulación de la Expresión Génica/genética , Glucólisis/fisiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Síndrome de Birt-Hogg-Dubé/genética , Síndrome de Birt-Hogg-Dubé/metabolismo , Western Blotting , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/metabolismo , Línea Celular Tumoral , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Inmunohistoquímica , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , Proteínas Supresoras de Tumor/genéticaAsunto(s)
Clonación Molecular , Genes Supresores de Tumor , Neoplasias Renales/genética , Ligasas , Proteínas/genética , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas , Enfermedad de von Hippel-Lindau/genética , Secuencia de Aminoácidos , Animales , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Proteínas/química , Proteína Supresora de Tumores del Síndrome de Von Hippel-LindauAsunto(s)
Genes Supresores de Tumor , Ligasas , Proteínas/metabolismo , ARN Polimerasa II/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas , Clonación Molecular , Elonguina , Escherichia coli , Histidina , Humanos , Oligodesoxirribonucleótidos , Biosíntesis de Proteínas , Proteínas/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Lugares Marcados de Secuencia , Moldes Genéticos , Factores de Transcripción/biosíntesis , Factores de Transcripción/aislamiento & purificación , Proteína Supresora de Tumores del Síndrome de Von Hippel-LindauRESUMEN
Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene predisposes to vascular tumor formation in several organs. VHL regulates two evolutionary conserved pathways: the targeting of hydroxylated hypoxia-inducible factor-alpha (HIF-alpha) for proteasomal degradation and the remodeling of extracellular matrix (ECM). The biochemical mechanisms of the ECM assembly pathway remain poorly defined. Here, we provide evidence supporting a biochemical role for VHL in ECM assembly. We show that VHL directly binds to the collagen IV alpha 2 (COL4A2) chain and that this interaction is necessary for its assembly into the ECM. The VHL-COL4A2 interaction is dependent on endoplasmic reticulum (ER)-mediated COL4A2 hydroxylation and independent of cytosolic, hypoxia regulated HIF-alpha-modifying enzymes. We find that the N-terminal tail of COL4A2 protrudes from the ER lumen into the cytosol where it is bound by VHL. Failure of VHL to interact with COL4A2 correlates with loss of collagen IV network formation in vitro and collagen IV remodeling in vivo. Our data suggest a HIF-alpha-independent role for the VHL-COL4A2 interaction in suppression of angiogenic tumor formation through collagen IV network assembly.
Asunto(s)
Carcinoma de Células Renales/metabolismo , Colágeno Tipo IV/metabolismo , Matriz Extracelular/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Hipoxia de la Célula , Membrana Celular/metabolismo , Colágeno Tipo IV/genética , Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Hidroxilación , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Inmunoprecipitación , Neoplasias Renales/metabolismo , Oxigenasas de Función Mixta/metabolismo , Unión Proteica , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genéticaRESUMEN
eIF-4A is a translation initiation factor that exhibits bidirectional RNA unwinding activity in vitro in the presence of another translation initiation factor, eIF-4B and ATP. This activity is thought to be responsible for the melting of secondary structure in the 5' untranslated region of eukaryotic mRNAs to facilitate ribosome binding. eIF-4A is a member of a fast growing family of proteins termed the DEAD family. These proteins are believed to be RNA helicases, based on the demonstrated in vitro RNA helicase activity of two members (eIF-4A and p68) and their homology in eight amino acid regions. Several related biochemical activities were attributed to eIF-4A: (i) ATP binding, (ii) RNA-dependent ATPase and (iii) RNA helicase. To determine the contribution of the highly conserved regions to these activities, we performed site-directed mutagenesis. First we show that recombinant eIF-4A, together with recombinant eIF-4B, exhibit RNA helicase activity in vitro. Mutations in the ATPase A motif (AXXXXGKT) affect ATP binding, whereas mutations in the predicted ATPase B motif (DEAD) affect ATP hydrolysis. We report here that the DEAD region couples the ATPase with the RNA helicase activity. Furthermore, two other regions, whose functions were unknown, have also been characterized. We report that the first residue in the HRIGRXXR region is involved in ATP hydrolysis and that the SAT region is essential for RNA unwinding. Our results suggest that the highly conserved regions in the DEAD box family are critical for RNA helicase activity.
Asunto(s)
Factores Eucarióticos de Iniciación , Factores de Iniciación de Péptidos/metabolismo , ARN Nucleotidiltransferasas/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Autorradiografía , Reactivos de Enlaces Cruzados , Electroforesis en Gel de Poliacrilamida , Factor 4A Eucariótico de Iniciación , Hidrólisis , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Factores de Iniciación de Péptidos/genética , ARN/metabolismo , ARN Helicasas , ARN Nucleotidiltransferasas/genéticaRESUMEN
The trans-activation response element (TAR) at the 5' end of the human immunodeficiency virus type 1 (HIV-1) mRNAs forms a stable hairpin structure which is a target for binding of the virally encoded protein Tat, which activates viral gene expression, as well as several cellular factors. TAR is also inhibitory to translation. One of several host factors that binds to TAR RNA is the La autoantigen, an RNA-binding protein which functions in RNA polymerase III transcription termination and has also been implicated in cap-independent internal translation initiation on poliovirus RNA. Here we show that La autoantigen alleviates translational repression by the HIV-1 leader RNA. In rabbit reticulocyte lysate, La relieves the cis-inhibitory effect of the TAR RNA on translation of bacterial chloramphenicol acetyltransferase (CAT) mRNA but not inhibition that is mediated by an artificial secondary structure element. Canonical translation factors exhibited slight (eIF-2 and GEF) or no (eIF-4A, eIF-4B, eIF-4E, eIF-4F, eIF-3, and eEF-1 alpha) stimulatory activity on translation of TAR-containing CAT mRNA. In addition, we show that poliovirus RNA, in spite of being an inefficient template in rabbit reticulocyte lysate, is a strong competitive inhibitor of translation of TAR-containing CAT mRNA but not CAT mRNA. This inhibition can be relieved by La but not by any other translation factor. The results suggest a possible involvement of the La autoantigen in HIV-1 gene expression.
Asunto(s)
Autoantígenos/fisiología , VIH-1/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Viral/genética , Ribonucleoproteínas/fisiología , Activación Transcripcional , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Poliovirus/genética , Antígeno SS-BRESUMEN
An important aspect of the regulation of gene expression is the modulation of translation rates in response to growth factors, hormones and mitogens. Most of this control is at the level of translation initiation. Recent studies have implicated the MAP kinase pathway in the regulation of translation by insulin and growth factors. MAP kinase phosphorylates a repressor of translation initiation [4E-binding protein (BP) 1] that binds to the mRNA 5' cap binding protein eukaryotic initiation factor (eIF)-4E and inhibits cap-dependent translation. Phosphorylation of the repressor decreases its affinity for eIF-4E, and thus relieves translational inhibition. eIF-4E forms a complex with two other polypeptides, eIF-4A and p220, that promote 40S ribosome binding to mRNA. Here, we have studied the mechanism by which 4E-BP1 inhibits translation. We show that 4E-BP1 inhibits 48S pre-initiation complex formation. Furthermore, we demonstrate that 4E-BP1 competes with p220 for binding to eIF-4E. Mutants of 4E-BP1 that are deficient in their binding to eIF-4E do not inhibit the interaction between p220 and eIF-4E, and do not repress translation. Thus, translational control by growth factors, insulin and mitogens is affected by changes in the relative affinities of 4E-BP1 and p220 for eIF-4E.
Asunto(s)
Proteínas Portadoras , Fragmentos de Péptidos/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Fosfoproteínas/metabolismo , Biosíntesis de Proteínas , Caperuzas de ARN/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Sitios de Unión , Unión Competitiva , Proteínas de Ciclo Celular , Línea Celular , Factor 4E Eucariótico de Iniciación , Vectores Genéticos , Células HeLa , Humanos , Datos de Secuencia Molecular , Nucleopoliedrovirus/genética , Nucleopoliedrovirus/aislamiento & purificación , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/química , Factores de Iniciación de Péptidos/antagonistas & inhibidores , Factores de Iniciación de Péptidos/química , Fosfoproteínas/química , ARN Mensajero/metabolismo , ARN Ribosómico/metabolismo , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The p46 subunit of eukaryotic initiation factor (eIF)-4F purified from rabbit reticulocyte lysate has previously been found to be composed of eIF-4AI and eIF-4AII in a 4:1 ratio, respectively, whereas the free form of rabbit eIF-4A is composed solely of eIF-4AI. Using sucrose gradient centrifugation and an m7GTP-Sepharose 4B assay, it was shown that eIF-4A exchanges with the p46 subunit of eIF-4F. Incubation of [14C]eIF-4A and eIF-4F resulted in the incorporation of [14C] eIF-4A into the eIF-4F complex. Conversely, the [14C] p46 subunit of [14C]eIF-4F was shown to dissociate from the [14C]eIF-4F complex in the presence of eIF-4A, presumably due to the incorporation of unlabeled eIF-4A. Similar experiments were conducted in which 14C-labeled initiation factors were incubated with rabbit reticulocyte lysate. When [14C]eIF-4A was incubated with lysate, [14C]eIF-4A became incorporated into the eIF-4F complex present in the lysate. Additionally, when [14C]eIF-4F was incubated with lysate, the [14C]p46 subunit of [14C]eIF-4F dissociated from the [14C]eIF-4F complex, most likely due to the exchange of unlabeled eIF-4A (present in the lysate) with the [14C]p46 subunit. The exchange of mouse eIF-4AI and eIF-4AII expressed in Escherichia coli was also investigated in the presence of eIF-4F and rabbit reticulocyte lysate. Both the sucrose gradient experiments and m7GTP-Sepharose 4B assays demonstrated that the [14C]p46 subunit of [14C]eIF-4F was displaced in the presence of eIF-4AI or eIF-4AII and that mouse [14C]eIF-4AI or [14C]eIF-4AII became incorporated into the eIF-4F complex in the same manner as rabbit reticulocyte eIF-4A.
Asunto(s)
Factores de Iniciación de Péptidos/metabolismo , Animales , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Escherichia coli , Factor 4A Eucariótico de Iniciación , Factor 4F Eucariótico de Iniciación , Ratones , Fragmentos de Péptidos/metabolismo , Factores de Iniciación de Péptidos/genética , Conejos , ReticulocitosRESUMEN
Eukaryotic translation initiation factor-4A (eIF-4A) plays a critical role in binding of eukaryotic mRNAs to ribosomes. It has been biochemically characterized as an RNA-dependent ATPase and RNA helicase and is a prototype for a growing family of putative RNA helicases termed the DEAD box family. It is required for mRNA-ribosome binding both in its free form and as a subunit of the cap binding protein complex, eIF-4F. To gain further understanding into the mechanism of action of eIF-4A in mRNA-ribosome binding, defective eIF-4A mutants were tested for their abilities to function in a dominant negative manner in a rabbit reticulocyte translation system. Several mutants were demonstrated to be potent inhibitors of translation. Addition of mutant eIF-4A to a rabbit reticulocyte translation system strongly inhibited translation of all mRNAs studied including those translated by a cap-independent internal initiation mechanism. Addition of eIF-4A or eIF-4F relieved inhibition of translation, but eIF-4F was six times more effective than eIF-4A, whereas eIF-4B or other translation factors failed to relieve the inhibition. Kinetic experiments demonstrated that mutant eIF-4A is defective in recycling through eIF-4F, thus explaining the dramatic inhibition of translation. Mutant eIF-4A proteins also inhibited eIF-4F-dependent, but not eIF-4A-dependent RNA helicase activity. Taken together these results suggest that eIF-4A functions primarily as a subunit of eIF-4F, and that singular eIF-4A is required to recycle through the complex during translation. Surprisingly, eIF-4F, which binds to the cap structure, appears to be also required for the translation of naturally uncapped mRNAs.
Asunto(s)
Iniciación de la Cadena Peptídica Traduccional , Factores de Iniciación de Péptidos/metabolismo , Caperuzas de ARN/metabolismo , ARN Mensajero/metabolismo , Secuencia de Aminoácidos , Animales , Cloranfenicol O-Acetiltransferasa/biosíntesis , Cloranfenicol O-Acetiltransferasa/metabolismo , Factor 4A Eucariótico de Iniciación , Factor 4F Eucariótico de Iniciación , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Factores de Iniciación de Péptidos/genética , Mutación Puntual , Biosíntesis de Proteínas , ARN Helicasas , ARN Nucleotidiltransferasas/metabolismo , Conejos , Reticulocitos/metabolismo , Ribosomas/metabolismo , Transcripción GenéticaRESUMEN
The cleavage of the hepatitis C virus polyprotein between the nonstructural proteins NS2 and NS3 is mediated by the NS2/3 protease, whereas the NS3 protease is responsible for the cleavage of the downstream proteins. Purification and in vitro characterization of the NS2/3 protease has been hampered by its hydrophobic nature. NS2/3 protease activity could only be detected in cells or in in vitro translation assays with the addition of microsomal membranes or detergent. To facilitate purification of this poorly characterized protease, we truncated the N-terminal hydrophobic domain, resulting in an active enzyme with improved biophysical properties. We define a minimal catalytic region of NS2/3 protease retaining autocleavage activity that spans residues 904-1206 and includes the C-terminal half of NS2 and the N-terminal NS3 protease domain. The NS2/3 (904-1206) variant was purified from Escherichia coli inclusion bodies and refolded by gel filtration chromatography. The purified inactive form of NS2/3 (904-1206) was activated by the addition of glycerol and detergent to induce autocleavage at the predicted site between Leu(1026) and Ala(1027). NS2/3 (904-1206) activity was dependent on zinc ions and could be inhibited by NS4A peptides, peptides that span the cleavage site, or an N-terminal peptidic cleavage product. This NS2/3 variant will facilitate the development of an assay suitable for identifying inhibitors of HCV replication.
Asunto(s)
Hepacivirus/enzimología , Proteínas no Estructurales Virales/metabolismo , Secuencia de Aminoácidos , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Técnicas In Vitro , Datos de Secuencia Molecular , Pliegue de Proteína , Proteínas no Estructurales Virales/aislamiento & purificaciónRESUMEN
Elongin C is a 112-amino acid protein that is found in mammalian cells as a positive regulatory subunit of heterotrimeric RNA polymerase II elongation factor Elongin (SIII) and as a component of a multiprotein complex containing the von Hippel-Lindau (VHL) tumor suppressor protein. As a subunit of the Elongin complex, Elongin C interacts directly with the transcriptionally active Elongin A subunit and potently induces its elongation activity; in addition, Elongin C interacts with the ubiquitin-like Elongin B subunit, which regulates the interaction of Elongin C with Elongin A. As a component of the VHL complex, Elongin C interacts directly with both Elongin B and the VHL protein. Binding of the VHL protein to Elongin C was found to prevent Elongin C from interacting with and activating Elongin A in vitro, leading to the proposal that one function of the VHL protein may be to regulate RNA polymerase II elongation by negatively regulating the Elongin complex. In this report, we identify Elongin C sequences required for its interaction with the VHL protein. We previously demonstrated that the ability of Elongin C to bind and activate Elongin A is sensitive to mutations in the C-terminal half of Elongin C, as well as to mutations in an N-terminal Elongin C region needed for formation of the Elongin BC complex. Here we show that interaction of Elongin C with the VHL tumor suppressor protein depends strongly on sequences in the C terminus of Elongin C but is independent of the N-terminal Elongin C region required for binding to Elongin B and for binding and activation of Elongin A. Taken together, our results are consistent with the proposal that the VHL protein negatively regulates Elongin C activation of the Elongin complex by sterically blocking the interaction of C-terminal Elongin C sequences with Elongin A. In addition, our finding that only a subset of Elongin C sequences required for its interaction with Elongin A are critical for binding to VHL may offer the opportunity to develop reagents that selectively interfere with Elongin and VHL function.
Asunto(s)
Ligasas , Proteínas/química , Proteínas/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas , Secuencia de Aminoácidos , Animales , Sitios de Unión , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans , Elonguina , Genes Supresores de Tumor , Humanos , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Proteínas/aislamiento & purificación , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Alineación de Secuencia , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Lugares Marcados de Secuencia , Factores de Transcripción/aislamiento & purificación , Proteína Supresora de Tumores del Síndrome de Von Hippel-LindauRESUMEN
The eukaryotic translation initiation factor 4E (eIF4E) plays an important role in the control of cell growth. eIF4E binds to the mRNA 5' cap structure m(7)GpppN (where N is any nucleotide), and promotes ribosome binding to the mRNA in the cytoplasm. However, a fraction of eIF4E localizes to the nucleus. Here we describe the cloning and functional characterization of a new eIF4E-binding protein, referred to as 4E-T (eIF4E-Transporter). We demonstrate that 4E-T is a nucleocytoplasmic shuttling protein that contains an eIF4E-binding site, one bipartite nuclear localization signal and two leucine-rich nuclear export signals. eIF4E forms a complex with the importin alphabeta heterodimer only in the presence of 4E-T. Overexpression of wild-type 4E-T, but not of a mutant defective for eIF4E binding, causes the nuclear accumulation of HA-eIF4E in cells treated with leptomycin B. Taken together, these results demonstrate that the novel nucleocytoplasmic shuttling protein 4E-T mediates the nuclear import of eIF4E via the importin alphabeta pathway by a piggy-back mechanism.