RESUMEN
BACKGROUND: Severe physical and environmental stress seems to have a suppressive effect on the hypothalamic-pituitary-gonadal (HPG) axis in men. Examining hormonal responses to an extreme 160-km competition across frozen Alaska provides a unique opportunity to study this intense stress. OBJECTIVE: To examine hormonal responses to an ultra-endurance race. METHODS: Blood samples were obtained from 16 men before and after racing and analyzed for testosterone, interleukin-6 (IL-6), growth hormone (GH) and cortisol. Six subjects (mean (SD) age 42 (7) years; body mass 78.9 (7.1) kg; height 1.78 (0.05) m raced by bicycle (cyclists) and 10 subjects (age 35 (9) years; body mass 77.9 (10.6) kg; height, 1.82 (0.05) m) raced by foot (runners). Mean (SD) finish times were 21.83 (6.27) and 33.98 (6.12) h, respectively. RESULTS: In cyclists there were significant (p< or =0.05) mean (SD) pre-race to post-race increases in cortisol (254.83 (135.26) to 535.99 (232.22) nmol/l), GH (0.12 (0.23) to 3.21 (3.33) microg/ml) and IL-6 (2.36 (0.42) to 10.15 (3.28) pg/ml), and a significant decrease in testosterone (13.81 (3.19) to 5.59 (3.74) nmol/l). Similarly, in runners there were significant pre-race to post-race increases in cortisol (142.09 (50.74) to 452.21 (163.40) ng/ml), GH (0.12 (0.23) to 3.21 (3.33) microg/ml) and IL-6 (2.42 (0.68) to 12.25 (1.78) pg/ml), and a significant decrease in testosterone (12.32 (4.47) to 6.96 (3.19) nmol/l). There were no significant differences in the hormonal levels between cyclists and runners (p>0.05). CONCLUSIONS: These data suggest a suppression of the hypopituitary-gonadal axis potentially mediated by amplification of adrenal stress responses to such an ultra-endurance race in environmentally stressful conditions.
Asunto(s)
Ciclismo/fisiología , Frío/efectos adversos , Sistema Hipotálamo-Hipofisario/metabolismo , Resistencia Física/fisiología , Sistema Hipófiso-Suprarrenal/metabolismo , Carrera/fisiología , Adulto , Alaska , Hormona del Crecimiento/sangre , Humanos , Hidrocortisona/sangre , Interleucina-6/sangre , Masculino , Persona de Mediana Edad , Testosterona/sangreRESUMEN
The mechanism of selenocysteine incorporation in eukaryotes has been assumed for almost a decade to be inherently different from that in prokaryotes, due to differences in the architecture of selenoprotein mRNAs in the two kingdoms. After extensive efforts in a number of laboratories spanning the same time frame, some of the essential differences between these mechanisms are finally being revealed, through identification of the factors catalyzing cotranslational selenocysteine insertion in eukaryotes. A single factor in prokaryotes recognizes both the selenoprotein mRNA, via sequences in the coding region, and the unique selenocysteyl-tRNA, via both its secondary structure and amino acid. The corresponding functions in eukaryotes are conferred by two distinct but interacting factors, one recognizing the mRNA, via structures in the 3' untranslated region, and the second recognizing the tRNA. Now, with these factors in hand, crucial questions about the mechanistic details and efficiency of this intriguing process can begin to be addressed.
Asunto(s)
Regiones no Traducidas 3'/genética , Factores de Elongación de Péptidos/metabolismo , Proteínas/genética , ARN de Transferencia Aminoácido-Específico/metabolismo , Selenocisteína/metabolismo , Regiones no Traducidas 3'/metabolismo , Animales , Células Eucariotas/metabolismo , Methanococcus/genética , Methanococcus/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo , SelenoproteínasRESUMEN
A case of primary nasal meningioma in a 69-year-old women is described. The pathologic, radiologic and clinical characteristics are described. A summary of previously published articles on the subject is given.
Asunto(s)
Meningioma/diagnóstico , Meningioma/cirugía , Neoplasias Nasales/diagnóstico , Neoplasias Nasales/cirugía , Anciano , Femenino , Humanos , Pronóstico , Tomografía Computarizada por Rayos X , Resultado del TratamientoRESUMEN
Selenocysteine is a rare amino acid in protein that is encoded by UGA with the requirement of a downstream mRNA stem-loop structure, the selenocysteine insertion sequence element. To detect selenoproteins in Drosophila, the entire genome was analyzed with a novel program that searches for selenocysteine insertion sequence elements, followed by selenoprotein gene signature analyses. This computational screen and subsequent metabolic labeling with (75)Se and characterization of selenoprotein mRNA expression resulted in identification of three selenoproteins: selenophosphate synthetase 2 and novel G-rich and BthD selenoproteins that had no homology to known proteins. To assess a biological role for these proteins, a simple chemically defined medium that supports growth of adult Drosophila and requires selenium supplementation for optimal survival was devised. Flies survived on this medium supplemented with 10(-8) to 10(-6) m selenium or on the commonly used yeast-based complete medium at about twice the rate as those on a medium without selenium or with >10(-6) m selenium. This effect correlated with changes in selenoprotein mRNA expression. The number of eggs laid by Drosophila was reduced approximately in half in the chemically defined medium compared with the same medium supplemented with selenium. The data provide evidence that dietary selenium deficiency shortens, while supplementation of the diet with selenium normalizes the Drosophila life span by a process that may involve the newly identified selenoproteins.
Asunto(s)
Proteínas de Drosophila , Drosophila/metabolismo , Fertilidad , Proteínas/metabolismo , ARN Mensajero/metabolismo , Selenio/metabolismo , Selenocisteína/metabolismo , Algoritmos , Secuencia de Aminoácidos , Animales , Northern Blotting , ADN Complementario/metabolismo , Etiquetas de Secuencia Expresada , Fertilidad/efectos de los fármacos , Genoma , Esperanza de Vida , Datos de Secuencia Molecular , Fosfotransferasas/biosíntesis , Unión Proteica , ARN de Transferencia/metabolismo , Selenio/farmacología , Selenoproteínas , Homología de Secuencia de Aminoácido , Programas Informáticos , Factores de TiempoRESUMEN
Selenocysteine (Sec) tRNA (tRNA([Ser]Sec)) serves as both the site of Sec biosynthesis and the adapter molecule for donation of this amino acid to protein. The consequences on selenoprotein biosynthesis of overexpressing either the wild type or a mutant tRNA([Ser]Sec) lacking the modified base, isopentenyladenosine, in its anticodon loop were examined by introducing multiple copies of the corresponding tRNA([Ser]Sec) genes into the mouse genome. Overexpression of wild-type tRNA([Ser]Sec) did not affect selenoprotein synthesis. In contrast, the levels of numerous selenoproteins decreased in mice expressing isopentenyladenosine-deficient (i(6)A(-)) tRNA([Ser]Sec) in a protein- and tissue-specific manner. Cytosolic glutathione peroxidase and mitochondrial thioredoxin reductase 3 were the most and least affected selenoproteins, while selenoprotein expression was most and least affected in the liver and testes, respectively. The defect in selenoprotein expression occurred at translation, since selenoprotein mRNA levels were largely unaffected. Analysis of the tRNA([Ser]Sec) population showed that expression of i(6)A(-) tRNA([Ser]Sec) altered the distribution of the two major isoforms, whereby the maturation of tRNA([Ser]Sec) by methylation of the nucleoside in the wobble position was repressed. The data suggest that the levels of i(6)A(-) tRNA([Ser]Sec) and wild-type tRNA([Ser]Sec) are regulated independently and that the amount of wild-type tRNA([Ser]Sec) is determined, at least in part, by a feedback mechanism governed by the level of the tRNA([Ser]Sec) population. This study marks the first example of transgenic mice engineered to contain functional tRNA transgenes and suggests that i(6)A(-) tRNA([Ser]Sec) transgenic mice will be useful in assessing the biological roles of selenoproteins.
Asunto(s)
Biosíntesis de Proteínas , Proteínas , ARN de Transferencia Aminoácido-Específico/biosíntesis , Animales , Secuencia de Bases , Northern Blotting/métodos , Expresión Génica , Isopenteniladenosina/genética , Isopenteniladenosina/metabolismo , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Selenio/metabolismo , SelenoproteínasRESUMEN
Selenium has been shown to prevent cancer in a variety of animal model systems. Both epidemiological studies and supplementation trials have supported its efficacy in humans. However, the mechanism by which selenium suppresses tumor development remains unknown. Selenium is present in known human selenoproteins as the amino acid selenocysteine (Sec). Sec is inserted cotranslationally in response to UGA codons within selenoprotein mRNAs in a process requiring a sequence within the 3'-untranslated region (UTR), referred to as a Sec insertion sequence (SECIS) element. Recently, a human Mr 15,000 selenoprotein (Sep15) was identified that contains an in-frame UGA codon and a SECIS element in the 3'-UTR. Examination of the available cDNA sequences for this protein revealed two polymorphisms located at position 811 (C/T) and at position 1125 (G/A) located within the 3'-UTR. Here, we demonstrate significant differences in Sep15 allele frequencies by ethnicity and that the identity of the nucleotides at the polymorphic sites influences SECIS function in a selenium-dependent manner. This, together with genetic data indicating loss of heterozygosity at the Sep15 locus in certain human tumor types, suggests that Sep15 may be involved in cancer development, risk, or both.
Asunto(s)
Regiones no Traducidas 3'/genética , Polimorfismo de Nucleótido Simple/fisiología , Proteínas/genética , Adulto , Población Negra/genética , ADN/sangre , ADN/genética , ADN de Neoplasias/genética , Femenino , Genotipo , Humanos , Pérdida de Heterocigocidad , Masculino , Neoplasias/genética , Selenoproteínas , Población Blanca/genéticaRESUMEN
Mammalian selenocysteine-containing proteins characterized with respect to function are involved in redox processes and exhibit distinct expression patterns and cellular locations. A recently identified 15-kDa selenoprotein (Sep15) has no homology to previously characterized proteins, and its function is not known. Here we report the intracellular localization and identification of a binding partner for this selenoprotein which implicate Sep15 in the regulation of protein folding. The native Sep15 isolated from rat prostate and mouse liver occurred in a complex with a 150-kDa protein. The latter protein was identified as UDP-glucose:glycoprotein glucosyltransferase (UGTR), the endoplasmic reticulum (ER)-resident protein, which was previously shown to be involved in the quality control of protein folding. UGTR functions by glucosylating misfolded proteins, retaining them in the ER until they are correctly folded or transferring them to degradation pathways. To determine the intracellular localization of Sep15, we expressed a green fluorescent protein-Sep15 fusion protein in CV-1 cells, and this protein was localized to the ER and possibly other perinuclear compartments. We determined that Sep15 contained the N-terminal signal peptide that was essential for translocation and that it was cleaved in the mature protein. However, C-terminal sequences of Sep15 were not involved in trafficking and retention of Sep15. The data suggest that the association between Sep15 and UGTR is responsible for maintaining the selenoprotein in the ER. This report provides the first example of the ER-resident selenoprotein and suggests a possible role of the trace element selenium in the quality control of protein folding.
Asunto(s)
Glucosiltransferasas/metabolismo , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN , Glucosiltransferasas/química , Masculino , Ratones , Datos de Secuencia Molecular , Próstata/metabolismo , Unión Proteica , Ratas , Selenoproteínas , Fracciones Subcelulares/metabolismoRESUMEN
Many mammalian retroviruses express their protease and polymerase by ribosomal frameshifting. It was originally proposed that a specialized shifty tRNA promotes the frameshift event. We previously observed that phenylalanine tRNA(Phe) lacking the highly modified wybutoxosine (Y) base on the 3' side of its anticodon stimulated frameshifting, demonstrating that this tRNA is shifty. We now report the shifty tRNA(Phe) contains 1-methylguanosine (m(1)G) in place of Y and that the m(1)G form from rabbit reticulocytes stimulates frameshifting more efficiently than its m(1)G-containing counterpart from mouse neuroblastoma cells. The latter tRNA contains unmodified C and G nucleosides at positions 32 and 34, respectively, while the former tRNA contains the analogous 2'-O-methylated nucleosides at these positions. The data suggest that not only does the loss of a highly modified base from the 3' side of the anticodon render tRNA(Phe) shifty, but the modification status of the entire anticodon loop contributes to the degree of shiftiness. Possible biological consequences of these findings are discussed.
Asunto(s)
Sistema de Lectura Ribosómico , Guanina/análogos & derivados , Guanina/química , Guanosina/análogos & derivados , Guanosina/química , ARN de Transferencia de Fenilalanina/química , ARN de Transferencia de Fenilalanina/genética , Retroviridae/genética , Animales , Secuencia de Bases , Hígado/química , Ratones , Datos de Secuencia Molecular , Neuroblastoma/química , Conformación de Ácido Nucleico , ARN de Transferencia de Fenilalanina/metabolismo , Conejos , Reticulocitos/químicaRESUMEN
Representatives of three primary life domains--bacteria, archaea, and eukaryotes--possess specific selenium-containing proteins. The majority of naturally occurring selenoproteins contain an amino acid, selenocysteine, that is incorporated into protein in response to the code word UGA. The presence of selenium in natural selenoproteins and in proteins in which this element is introduced by chemical or biological manipulations provides additional opportunities for characterizing structure, function, and mechanism of action. This unit provides an overview of known selenocysteine-containing proteins, examples of targeted incorporation of selenium into proteins, and methods specific for selenoprotein identification and characterization.
Asunto(s)
Selenocisteína/análisis , Selenoproteínas/química , Secuencia de Bases , Código Genético/genética , Espectroscopía de Resonancia Magnética/métodos , Espectrometría de Masas/métodos , ARN de Transferencia Aminoácido-Específico/química , ARN de Transferencia Aminoácido-Específico/genética , Radioisótopos de Selenio/química , Selenocisteína/química , Selenoproteínas/genéticaRESUMEN
Animal thioredoxin reductases (TRs) are selenocysteine-containing flavoenzymes that utilize NADPH for reduction of thioredoxins and other protein and nonprotein substrates. Three types of mammalian TRs are known, with TR1 being a cytosolic enzyme, and TR3, a mitochondrial enzyme. Previously characterized TR1 and TR3 occurred as homodimers of 55-57-kDa subunits. We report here that TR1 isolated from mouse liver, mouse liver tumor, and a human T-cell line exhibited extensive heterogeneity as detected by electrophoretic, immunoblot, and mass spectrometry analyses. In particular, a 67-kDa band of TR1 was detected. Furthermore, a novel form of mouse TR1 cDNA encoding a 67-kDa selenoprotein subunit with an additional N-terminal sequence was identified. Subsequent homology analyses revealed three distinct isoforms of mouse and rat TR1 mRNA. These forms differed in 5' sequences that resulted from the alternative use of the first three exons but had common downstream sequences. Similarly, expression of multiple mRNA forms was observed for human TR3 and Drosophila TR. In these genes, alternative first exon splicing resulted in the formation of predicted mitochondrial and cytosolic proteins. In addition, a human TR3 gene overlapped with the gene for catechol-O-methyltransferase (COMT) on a complementary DNA strand, such that mitochondrial TR3 and membrane-bound COMT mRNAs had common first exon sequences; however, transcription start sites for predicted cytosolic TR3 and soluble COMT forms were separated by approximately 30 kilobases. Thus, this study demonstrates a remarkable heterogeneity within TRs, which, at least in part, results from evolutionary conserved genetic mechanisms employing alternative first exon splicing. Multiple transcription start sites within TR genes may be relevant to complex regulation of expression and/or organelle- and cell type-specific location of animal thioredoxin reductases.
Asunto(s)
Empalme Alternativo , Variación Genética , Reductasa de Tiorredoxina-Disulfuro/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario/análisis , Drosophila/enzimología , Drosophila/genética , Exones , Humanos , Intrones , Masculino , Ratones , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Ratas , Homología de Secuencia de Aminoácido , Reductasa de Tiorredoxina-Disulfuro/aislamiento & purificaciónRESUMEN
The selenocysteine (Sec) tRNA[Ser]Sec population in higher vertebrates consists of two major isoacceptors that differ from each other by a single nucleoside modification in the wobble position of the anticodon (position 34). One isoacceptor contains 5-methylcarboxymethyluridine (mcmU) in this position, whereas the other contains 5-methylcarboxymethyluridine-2'-O-methylribose (mcmUm). The other modifications in these tRNAs are N6-isopentenyladenosine (i6A), pseudouridine (psi), and 1-methyladenosine (m1A) at positions 37, 55, and 58, respectively. As methylation of the ribose at position 34 is influenced by the intracellular selenium status and the presence of this methyl group dramatically alters tertiary structure, we investigated the effect of the modifications at other positions as well as tertiary structure on its formation. Mutations were introduced within a synthetic gene encoded in an expression vector, transcripts generated and microinjected into Xenopus oocytes, and the resulting tRNA products analyzed for the presence of modified bases. The results suggest that efficient methylation of mcmU to yield mcmUm requires the prior formation of each modified base and an intact tertiary structure, whereas formation of modified bases at other positions, including mcmU, is not as stringently connected to precise primary and tertiary structure. These results, along with the observations that methylation of mcmU is enhanced in the presence of selenium and that this methyl group affects tertiary structure, further suggest that the mcmUm isoacceptor must have a role in selenoprotein synthesis different from that of the mcmU isoacceptor.
Asunto(s)
Aminoacil-ARN de Transferencia/química , Animales , Femenino , Humanos , Metilación , Mutación , Conformación de Ácido Nucleico , Oocitos , Aminoacil-ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/metabolismo , Ribosa/metabolismo , Transfección , Xenopus laevisRESUMEN
Selenium has been implicated in cancer prevention, but the mechanism and possible involvement of selenoproteins in this process are not understood. To elucidate whether the 15-kDa selenoprotein may play a role in cancer etiology, the complete sequence of the human 15-kDa protein gene was determined, and various characteristics associated with expression of the protein were examined in normal and malignant cells and tissues. The 51-kilobase pair gene for the 15-kDa selenoprotein consisted of five exons and four introns and was localized on chromosome 1p31, a genetic locus commonly mutated or deleted in human cancers. Two stem-loop structures resembling selenocysteine insertion sequence elements were identified in the 3'-untranslated region of the gene, and only one of these was functional. Two alleles in the human 15-kDa protein gene were identified that differed by two single nucleotide polymorphic sites that occurred within the selenocysteine insertion sequence-like structures. These 3'-untranslated region polymorphisms resulted in changes in selenocysteine incorporation into protein and responded differently to selenium supplementation. Human and mouse 15-kDa selenoprotein genes manifested the highest level of expression in prostate, liver, kidney, testis, and brain, and the level of the selenoprotein was reduced substantially in a malignant prostate cell line and in hepatocarcinoma. The expression pattern of the 15-kDa protein in normal and malignant tissues, the occurrence of polymorphisms associated with protein expression, the role of selenium in differential regulation of polymorphisms, and the chromosomal location of the gene may be relevant to a role of this protein in cancer.
Asunto(s)
Neoplasias/genética , Proteínas/genética , Selenio/metabolismo , Regiones no Traducidas 3' , Adolescente , Adulto , Anciano , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , Western Blotting , Línea Celular , Mapeo Cromosómico , Cromosomas Humanos Par 1 , Elementos Transponibles de ADN , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Exones , Femenino , Genes Reporteros , Humanos , Intrones , Yoduro Peroxidasa/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Modelos Genéticos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Polimorfismo Genético , Polimorfismo de Nucleótido Simple , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Ratas , Selenoproteínas , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Distribución Tisular , Transcripción Genética , Transfección , Células Tumorales CultivadasRESUMEN
To gain a better understanding of the biological consequences of the exposure of tumor cells to selenium, we evaluated the selenium-dependent responses of two selenoproteins (glutathione peroxidase and the recently characterized 15-kDa selenoprotein) in three human glioma cell lines. Protein levels, mRNA levels, and the relative distribution of the two selenocysteine tRNA isoacceptors (designated mcm(5)U and mcm(5)Um) were determined for standard as well as selenium-supplemented conditions. The human malignant glioma cell lines D54, U251, and U87 were maintained in normal or selenium-supplemented (30 nM sodium selenite) conditions. Northern blot analysis demonstrated only minor increases in steady-state GSHPx-1 mRNA in response to selenium addition. Baseline glutathione peroxidase activity was 10.7 +/- 0.7, 7.6 +/- 0.7, and 4.3 +/- 0.7 nmol NADPH oxidized/min/mg protein for D54, U251, and U87, respectively, as determined by the standard coupled spectrophotometric assay. Glutathione peroxidase activity increased in a cell line-specific manner to 19.7 +/- 1.4, 15.6 +/- 2.1, and 6. 7 +/- 0.5 nmol NADPH oxidized/min/mg protein, respectively, as did a proportional increase in cellular resistance to H(2)O(2), in response to added selenium. The 15-kDa selenoprotein mRNA levels likewise remained constant despite selenium supplementation. The selenium-dependent change in distribution between the two selenocysteine tRNA isoacceptors also occurred in a cell line-specific manner. The percentage of the methylated isoacceptor, mcm(5)Um, changed from 35.5 to 47.2 for D54, from 38.1 to 47.3 for U251, and from 49.0 to 47.6 for U87. These data represent the first time that selenium-dependent changes in selenoprotein mRNA and protein levels, as well as selenocysteine tRNA distribution, were examined in human glioma cell lines.
Asunto(s)
Glioma/metabolismo , Proteínas/metabolismo , Selenio/farmacología , Regulación Neoplásica de la Expresión Génica , Glioma/patología , Glutatión Peroxidasa/metabolismo , Humanos , Peso Molecular , Estrés Oxidativo/efectos de los fármacos , Biosíntesis de Proteínas , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Aminoacil-ARN de Transferencia/efectos de los fármacos , Aminoacil-ARN de Transferencia/metabolismo , Selenoproteínas , Células Tumorales Cultivadas , Glutatión Peroxidasa GPX1RESUMEN
A common posttranscriptional modification of tRNA is the isopentenylation of adenosine at position 37, creating isopentenyladenosine (i(6)A). The role of this modified nucleoside in protein synthesis of higher eukaryotes is not well understood. Selenocysteyl (Sec) tRNA (tRNA([Ser]Sec)) decodes specific UGA codons and contains i(6)A. To address the role of the modified nucleoside in this tRNA, we constructed a site-specific mutation, which eliminates the site of isopentenylation, in the Xenopus tRNA([Ser]Sec) gene. Transfection of the mutant tRNA([Ser]Sec) gene resulted in 80% and 95% reduction in the expression of co-transfected selenoprotein genes encoding type I and II iodothyronine deiodinases, respectively. A similar decrease in type I deiodinase synthesis was observed when transfected cells were treated with lovastatin, an inhibitor of the biosynthesis of the isopentenyl moiety. Neither co-transfection with the mutant tRNA gene nor lovastatin treatment reduced type I deiodinase mRNA levels. Also, mutant tRNA expression did not alter initiation of translation or degradation of the type I deiodinase protein. Furthermore, isopentenylation of tRNA([Ser]Sec) was not required for synthesis of Sec on the tRNA. We conclude that isopentenylation of tRNA([Ser]Sec) is required for efficient translational decoding of UGA and synthesis of selenoproteins.
Asunto(s)
Yoduro Peroxidasa/genética , Isopenteniladenosina/metabolismo , Proteínas/genética , Aminoacil-ARN de Transferencia/química , Aminoacil-ARN de Transferencia/metabolismo , Animales , Células CHO , Codón/genética , Cricetinae , Cinética , Lovastatina/farmacología , Mutagénesis Sitio-Dirigida , Biosíntesis de Proteínas , Aminoacil-ARN de Transferencia/genética , Proteínas Recombinantes/biosíntesis , Selenoproteínas , Transcripción Genética/efectos de los fármacos , Transfección , XenopusRESUMEN
In eukaryotes, the decoding of the UGA codon as selenocysteine (Sec) requires a Sec insertion sequence (SECIS) element in the 3' untranslated region of the mRNA. We purified a SECIS binding protein, SBP2, and obtained a cDNA clone that encodes this activity. SBP2 is a novel protein containing a putative RNA binding domain found in ribosomal proteins and a yeast suppressor of translation termination. By UV cross-linking and immunoprecipitation, we show that SBP2 specifically binds selenoprotein mRNAs both in vitro and in vivo. Using (75)Se-labeled Sec-tRNA(Sec), we developed an in vitro system for analyzing Sec incorporation in which the translation of a selenoprotein mRNA was both SBP2 and SECIS element dependent. Immunodepletion of SBP2 from the lysates abolished Sec insertion, which was restored when recombinant SBP2 was added to the reaction. These results establish that SBP2 is essential for the co-translational insertion of Sec into selenoproteins. We hypothesize that the binding activity of SBP2 may be involved in preventing termination at the UGA/Sec codon.
Asunto(s)
Biosíntesis de Proteínas , Proteínas/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Codón/genética , Secuencia Conservada , Humanos , Neoplasias Hepáticas Experimentales , Mamíferos , Datos de Secuencia Molecular , ARN Mensajero/genética , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Ratas , Proteínas Recombinantes/metabolismo , Selenocisteína/genética , Selenocisteína/metabolismo , Selenoproteínas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Transfección , Células Tumorales CultivadasRESUMEN
Decoding UGA as selenocysteine requires a unique tRNA, a specialized elongation factor, and specific secondary structures in the mRNA, termed SECIS elements. Eukaryotic SECIS elements are found in the 3' untranslated region of selenoprotein mRNAs while those in prokaryotes occur immediately downstream of UGA. Consequently, a single eukaryotic SECIS element can serve multiple UGA codons, whereas prokaryotic SECIS elements only function for the adjacent UGA, suggesting distinct mechanisms for recoding in the two kingdoms. We have identified and characterized the first eukaryotic selenocysteyl-tRNA-specific elongation factor. This factor forms a complex with mammalian SECIS binding protein 2, and these two components function together in selenocysteine incorporation in mammalian cells. Expression of the two functional domains of the bacterial elongation factor-SECIS binding protein as two separate proteins in eukaryotes suggests a mechanism for rapid exchange of charged for uncharged selenocysteyl-tRNA-elongation factor complex, allowing a single SECIS element to serve multiple UGA codons.
Asunto(s)
Regiones no Traducidas 3'/genética , Conformación de Ácido Nucleico , Factores de Elongación de Péptidos/metabolismo , Proteínas , Aminoacil-ARN de Transferencia/genética , Secuencias Reguladoras de Ácidos Nucleicos , Selenocisteína/genética , Secuencia de Aminoácidos , Animales , Línea Celular , Humanos , Ratones , Datos de Secuencia Molecular , Factores de Elongación de Péptidos/química , Factores de Elongación de Péptidos/genética , Biosíntesis de Proteínas , Aminoacil-ARN de Transferencia/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ratas , Selenocisteína/metabolismo , Selenoproteínas , Alineación de Secuencia , TransfecciónRESUMEN
Several recent observations have indicated that the primary structure of the Chinese hamster selenocysteine tRNA([Ser]sec) is different than those of other mammalian species. These reports prompted us to investigate the gene sequence for this tRNA in Chinese hamsters. Southern blotting of Chinese hamster ovary (CHO) genomic DNA derived from cultured cells with a tRNA([Ser]sec) probe indicated several hybridizing bands, and each of the corresponding genetic loci was isolated from a recombinant CHO library by molecular cloning. Sequence analysis of these regions indicated three likely pseudogenes and a single functional gene whose sequence differed from those of other mammals. Of these, only one pseudogene and the putative functional gene are actively transcribed following their microinjection into Xenopus oocytes. The possibility that the functional CHO tRNA([Ser]sec) evolved from an edited transcript is discussed.
Asunto(s)
ARN de Transferencia Aminoácido-Específico/genética , Animales , Secuencia de Bases , Células CHO , Clonación Molecular , Cricetinae , ADN/química , ADN/genética , Evolución Molecular , Femenino , Genes/genética , Datos de Secuencia Molecular , Oocitos/metabolismo , Seudogenes/genética , Procesamiento Postranscripcional del ARN , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Transcripción Genética , Xenopus/genéticaRESUMEN
We propose the existence of a relationship of stereochemical complementarity between gene sequences that code for interacting components: nucleic acid-nucleic acid, protein-protein and protein-nucleic acid. Such a relationship would impose evolutionary constraints on the DNA sequences themselves, thus retaining these sequences and governing the direction of the evolutionary process. Therefore, we propose that prebiotic, template-directed autocatalytic synthesis of mutally cognate peptides and polynucleotides resulted in their amplification and evolutionary conservation in contemporary prokaryotic and eukaryotic organisms as a genetic regulatory apparatus. If this proposal is correct, then the relationships between the sequences in DNA coding for these interactions constitute a life code of which the genetic code is only one aspect of the many related interactions encoded in DNA.
Asunto(s)
Evolución Molecular Dirigida , Secuencia de Bases , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , EstereoisomerismoRESUMEN
The intracellular generation of reactive oxygen species, together with the thioredoxin and glutathione systems, is thought to participate in redox signaling in mammalian cells. The activity of thioredoxin is dependent on the redox status of thioredoxin reductase (TR), the activity of which in turn is dependent on a selenocysteine residue. Two mammalian TR isozymes (TR2 and TR3), in addition to that previously characterized (TR1), have now been identified in humans and mice. All three TR isozymes contain a selenocysteine residue that is located in the penultimate position at the carboxyl terminus and which is encoded by a UGA codon. The generation of reactive oxygen species in a human carcinoma cell line was shown to result in both the oxidation of the selenocysteine in TR1 and a subsequent increase in the expression of this enzyme. These observations identify the carboxyl-terminal selenocysteine of TR1 as a cellular redox sensor and support an essential role for mammalian TR isozymes in redox-regulated cell signaling.
Asunto(s)
Selenocisteína/metabolismo , Transducción de Señal , Reductasa de Tiorredoxina-Disulfuro/genética , Secuencia de Aminoácidos , Animales , Dinitroclorobenceno/química , Factor de Crecimiento Epidérmico/farmacología , Fluoresceínas/química , Regulación Enzimológica de la Expresión Génica/genética , Humanos , Peróxido de Hidrógeno/farmacología , Isoenzimas/química , Isoenzimas/genética , Hígado/enzimología , Masculino , Espectrometría de Masas , Ratones , Datos de Secuencia Molecular , Oxidación-Reducción , Fragmentos de Péptidos/análisis , Peroxidasas , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/genética , Testículo/enzimología , Reductasa de Tiorredoxina-Disulfuro/química , Células Tumorales CultivadasRESUMEN
The zebrafish is widely used as a model system for studying mammalian developmental genetics and more recently, as a model system for carcinogenesis. Since there is mounting evidence that selenium can prevent cancer in mammals, including humans, we characterized the selenocysteine tRNA[Ser]sec gene and its product in zebrafish. Two genes for this tRNA were isolated and sequenced and were found to map at different loci within the zebrafish genome. The encoding sequences of both are identical and their flanking sequences are highly homologous for several hundred bases in both directions. The two genes likely arose from gene duplication which is a common phenomenon among many genes in this species. In addition, zebrafish tRNA[Ser]sec was isolated from the total tRNA population and shown to decode UGA in a ribosomal binding assay.