RESUMEN
AIMS/HYPOTHESIS: The IL-1 receptor antagonist (IL-1Ra) is an anti-inflammatory cytokine known to antagonise the actions of IL-1. We have previously shown that IL-1Ra is markedly upregulated in the serum of obese patients, is correlated with BMI and insulin resistance, and is overexpressed in the white adipose tissue (WAT) of obese humans. The aim of this study was to examine the role of IL-1Ra in the regulation of glucose homeostasis in rodents. METHODS: We assessed the expression of genes related to IL-1 signalling in the WAT of mice fed a high-fat diet, as well as the effect of Il1rn (the gene for IL-1Ra) deletion and treatment with IL-1Ra on glucose homeostasis in rodents. RESULTS: We show that the expression of Il1rn and the gene encoding the inhibitory type II IL-1 receptor was upregulated in diet-induced obesity. The blood insulin:glucose ratio was significantly lower in Il1rn ( -/- )animals, which is compatible with an increased sensitivity to insulin, reinforced by the fact that the insulin content and pancreatic islet morphology of Il1rn ( -/- ) animals were normal. In contrast, the administration of IL-1Ra to normal rats for 5 days led to a decrease in the whole-body glucose disposal due to a selective decrease in muscle-specific glucose uptake. CONCLUSIONS/INTERPRETATION: The expression of genes encoding inhibitors of IL-1 signalling is upregulated in the WAT of mice with diet-induced obesity, and IL-1Ra reduces insulin sensitivity in rats through a muscle-specific decrease in glucose uptake. These results suggest that the markedly increased levels of IL-1Ra in human obesity might contribute to the development of insulin resistance.
Asunto(s)
Grasas de la Dieta/efectos adversos , Resistencia a la Insulina/fisiología , Insulina/fisiología , Obesidad/fisiopatología , Receptores de Interleucina-1/antagonistas & inhibidores , Sialoglicoproteínas/fisiología , Tejido Adiposo/fisiopatología , Animales , Glucemia/análisis , Eliminación de Gen , Regulación de la Expresión Génica , Técnica de Clampeo de la Glucosa , Insulina/sangre , Resistencia a la Insulina/genética , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-1/fisiología , Islotes Pancreáticos/química , Islotes Pancreáticos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/metabolismo , Obesidad/etiología , Obesidad/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/fisiología , Receptores Tipo II de Interleucina-1 , Sialoglicoproteínas/genética , Sialoglicoproteínas/farmacología , Transducción de Señal , Regulación hacia ArribaRESUMEN
Alopecia areata is an inflammatory hair loss disease with a major genetic component. The presence of focal inflammatory lesions with perifollicular T-cell infiltrates reflects the importance of local cytokine production in the pathogenesis. In addition to its fundamental pro-inflammatory role, the interleukin-1 (IL-1) system has major effects on hair growth regulation in vitro, with the inhibitory actions of IL-1alpha and IL-1beta being opposed by the receptor antagonist IL-1ra. The novel interleukin-1 like molecule 1 (IL-1L1) which has greatest gene sequence homology with IL1RN, the gene encoding IL-1ra, is another potential IL-1 antagonist. In view of previous studies suggesting a significant role for IL1RN polymorphisms in the pathogenesis of autoimmune/inflammatory disease, we have analysed polymorphisms of IL-1ra (IL1RN+2018) and its homologue IL-1L1 (IL1L1+4734) in a case-control association study on 165 patients and a large number of matched controls. Homozygosity for the rare allele of IL1RN (IL1RN*2) was significantly associated with alopecia areata [odds ratio (OR) = 1.89, 95% CI (1.09, 3.28); P = 0.02], confirming our previous findings of significant association with the IL1RN variable number tandem repeat (VNTR). The results also revealed a novel association involving a polymorphism of the interleukin-1 receptor antagonist homologue IL1L1 at position + 4734, IL1RN+2018, and alopecia areata. The effect of a genotype combining three copies of the rare alleles at the IL1RN and IL1L1 loci conferred a more than additive increase in the risk of disease compared to IL1RN+2018 or IL1L1+4734 alone [OR 3.37 (1.60, 7.06); P = 0.002], suggesting possible synergy between the IL1RN and IL1L1 genes. This effect was stronger in patients with severe disease (alopecia totalis/universalis) [OR 4.62 (1.87, 11.40), P = 0.0022], and in those with early age at onset (< 20 years) [OR = 6.38 (2.64, 15.42), P = 0.0002]. Our results suggest that these polymorphisms within IL1RN and IL1L1 themselves or a gene in linkage disequilibrium with IL1RN and IL1L1 predispose to the more severe forms of alopecia areata.
Asunto(s)
Alopecia Areata/genética , Interleucinas/genética , Sialoglicoproteínas/genética , Adulto , Epistasis Genética , Femenino , Predisposición Genética a la Enfermedad , Humanos , Proteína Antagonista del Receptor de Interleucina 1 , Masculino , Persona de Mediana Edad , Análisis de Secuencia de ADN , Homología de SecuenciaRESUMEN
Interleukin (IL)-1-like protein 1 (IL-1L1) is a 155-amino acid protein that shares 27% identity with IL-1beta and 47% with IL-1 receptor antagonist (IL-1ra). A 2.7-kb IL-1L1 mRNA was cloned from human placenta and is detectable in the trophoblastic cell line JEG-3, in macrophages and in endotoxin-stimulated monocytes. Expression of IL-1L1 is much less abundant and less widespread than IL-1ra. We have determined the human and mouse IL-1L1 cDNA sequences and the complete sequence of the human gene, IL1L1. IL1L1 consists of four coding exons, has two alternative non-coding first exons, lies between IL1B and IL1RN, is orientated in the same direction as IL1RN and is separated from it by approximately 53 kb. The predicted IL-1L1 protein lacks both signal sequence and glycosylation signals. A 17-kDa protein was recovered by immunoprecipitation with IL-1L1-specific antibodies from JEG-3. IL-1L1 did not stimulate IL-6 production from primary human fibroblasts or human umbilical vein endothelial cells nor did it block the IL-1alpha or IL-1beta-dependent activation of IL-6 expression. We conclude, contrary to a recent suggestion made by others, that IL-1L1 is not a functional IL-1ra. IL-1L1 also had no detectable agonistic or antagonistic effect on IFN-gamma production in response to IL-18 in KG-1 cells.
Asunto(s)
Regulación de la Expresión Génica/inmunología , Interleucina-18/inmunología , Interleucina-1/inmunología , Familia de Multigenes/inmunología , Receptores de Interleucina-1/inmunología , Receptores de Interleucina/inmunología , Sialoglicoproteínas/inmunología , Secuencia de Aminoácidos , Animales , ADN Complementario/genética , ADN Complementario/inmunología , Humanos , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-1/genética , Interleucina-18/genética , Subunidad alfa del Receptor de Interleucina-18 , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Receptores de Interleucina/antagonistas & inhibidores , Receptores de Interleucina/genética , Receptores de Interleucina-1/antagonistas & inhibidores , Receptores de Interleucina-1/genética , Receptores de Interleucina-18 , Alineación de Secuencia , Sialoglicoproteínas/genética , Transcripción GenéticaRESUMEN
Branch points and flexures in the high pressure arterial system have long been recognized as sites of unusually high turbulence and consequent stress in humans are foci for atherosclerotic lesions. We show that mice that are homozygous for a null mutation in the gene encoding an endogenous antiinflammatory cytokine, interleukin 1 receptor antagonist (IL-1ra), develop lethal arterial inflammation involving branch points and flexures of the aorta and its primary and secondary branches. We observe massive transmural infiltration of neutrophils, macrophages, and CD4(+) T cells. Animals appear to die from vessel wall collapse, stenosis, and organ infarction or from hemorrhage from ruptured aneurysms. Heterozygotes do not die from arteritis within a year of birth but do develop small lesions, which suggests that a reduced level of IL-1ra is insufficient to fully control inflammation in arteries. Our results demonstrate a surprisingly specific role for IL-1ra in the control of spontaneous inflammation in constitutively stressed artery walls, suggesting that expression of IL-1 is likely to have a significant role in signaling artery wall damage.
Asunto(s)
Arteritis/inmunología , Receptores de Interleucina-1/antagonistas & inhibidores , Sialoglicoproteínas/genética , Edad de Inicio , Alelos , Animales , Arteritis/genética , Arteritis/patología , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Femenino , Humanos , Proteína Antagonista del Receptor de Interleucina 1 , Longevidad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Interleucina-1/análisis , Sialoglicoproteínas/inmunologíaRESUMEN
The family of interleukin-1 receptor-like genes currently has six known members. We have constructed a contig of 10 overlapping human PAC clones that covers 530 kb and includes five of the six family members. The termini of the contig were mapped to the interval between D2S373 and D2S176 (chromosome 2q12) by radiation hybrid mapping. The contig contains the genes (cen --> tel), in the order given, for the type II interleukin-1 (IL-1) receptor (IL1R2), the type I IL-1 receptor (IL1R1), the IL-1 receptor-related protein 2 (IL1RL2), T1/ST2/fit-1 (IL1RL1), and the IL-1 receptor-related protein 1, which has recently been shown to be a component of the IL-18 receptor (IL18R1). We show that all the genes are transcribed in the same direction, with IL1R2 being transcribed toward the cluster. The only known family member that is absent from the human contig is the IL-1 receptor accessory protein gene (IL1RAP), which maps to 3q28.
Asunto(s)
Cromosomas Humanos Par 2 , Familia de Multigenes , Receptores de Interleucina-1/genética , Southern Blotting , Mapeo Contig , Biblioteca de Genes , Humanos , Hibridación in Situ , Repeticiones de Microsatélite , Modelos Genéticos , Sondas de Oligonucleótidos , Mapeo RestrictivoRESUMEN
In population- and family-based association studies, it is useful to have some knowledge of the patterns of linkage disequilibrium that exist between markers in candidate regions. When such studies are carried out with multiallelic markers, it is often convenient to group the alleles into a biallelic system, for analysis. In this study, we specifically examined the interleukin-1 (IL-1) gene cluster on chromosome 2, a region containing candidates for many inflammatory and autoimmune disorders. Data were collected on eight markers, four of which were multiallelic. Using these data, we investigated the effect of three allele-grouping strategies, including a novel method, on the detection of linkage disequilibrium. The novel approach, termed the "delta method," measures the deviation from the expected haplotype frequencies under linkage equilibrium, for each allelic combination. This information is then used to group the alleles, in an attempt to avoid the grouping together of alleles at one locus that are in opposite disequilibrium with the same allele at the second locus. The estimate haplotype frequencies (EH) program was used to estimate haplotype frequencies and the disequilibrium measure. In our data it was found that the delta method compared well with the other two strategies. Using this method, we found that there was a reasonable correlation between disequilibrium and physical distance in the region (r=-.540, P=.001, one-tailed). We also identified a common, eight-locus haplotype of the IL-1 gene cluster.
Asunto(s)
Alelos , Interleucina-1/genética , Desequilibrio de Ligamiento , Familia de Multigenes , Marcadores Genéticos , HumanosRESUMEN
Genes of the interleukin-1 (IL-1) gene cluster localized on chromosome 2q13 are implicated in many physiological and pathophysiological processes. We present here a high-resolution physical map of this region between markers D2S2008 and D2S4/PAX8. An integrated YAC/PAC contig and a partial transcriptional map were constructed by STS-constent mapping using the CEPH YAC library and three PAC libraries. A total of 3 YACs, 34 PACs, and 56 STSs were integrated: 33 newly generated probes to PAC end sequences, 9 polymorphic and 4 nonpolymorphic markers, 5 known genes, 4 expressed sequence tags, and 1 pseudogene. Within the map, a complete PAC contig of > 1 Mb encompasses the IL-1 gene cluster and PAX8, a paired-box-containing gene. This allowed us to define the transcriptional orientation of GLVR1, IL1B, and IL1RN and to show that PAX8 is localized outside the IL-1 gene cluster. FISH analysis localized PAC clones containing the IL-1 gene cluster to 2q12-q13. The data provide the basis for further characterization of the IL-1 gene cluster and for the construction of a sequence-ready PAC contig of this region.
Asunto(s)
Cromosomas Humanos Par 2/genética , Interleucina-1/genética , Familia de Multigenes , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Artificiales de Levadura/genética , Clonación Molecular , Cartilla de ADN/genética , Expresión Génica , Marcadores Genéticos , Humanos , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Lugares Marcados de SecuenciaRESUMEN
AP-1-binding elements from promoter proximal DNA (the small HpaII-digested fraction of mouse genomic DNA) were affinity-selected with recombinant AP-1 complexes. One of the selected AP-1-binding elements originated from 1 kb 3' of the transcription start site of SEZ-6. We show that the mouse SEZ-6 gene extends over 49 kbp and contains 17 exons. SEZ-6 has been reported as a mouse brain-specific transcript encoding an integral membrane protein with a short cytoplasmic tail which we note may have a signalling function. We show that SEZ-6 mRNA expression in rat brain is specific to neurons but shows sharp regional differences, unconnected with the localization of major neurotransmitters. Full-length and a 3' truncated transcript are also abundant in testis. We define the origins of all reported sequence variants. The hypothetical domain structure of the protein is in excellent agreement with the exonic structure of the gene. The SEZ-6 promoter is a CpG island. In transient transfections, even the smallest promoter fragment tested (157 bp) was extremely selective towards a mouse neuronal cell line, Neuro 2a, compared with NIH-3T3, a non-expressing line.
Asunto(s)
Mapeo Encefálico , Encéfalo/metabolismo , Regulación de la Expresión Génica/fisiología , Genoma , Proteínas del Tejido Nervioso/genética , Regiones Promotoras Genéticas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/citología , Células Cultivadas , Fragmentación del ADN , Proteínas de Unión al ADN/metabolismo , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Ratas , Factor de Transcripción AP-1/metabolismoRESUMEN
We have cloned a cDNA from a mouse gene, Pso (peroxisomal sarcosine oxidase). Pso appears to encode a homolog of the single-subunit (40 kDa) bacterial sarcosine oxidases. The mouse Pso gene product would contain a peroxisomal localization sequence, like that of the recently reported rabbit enzyme, Mouse Pso lies between 20 and 50 kb upstream of the promoter of the Sez6 gene, close to Crybal on chromosome 11. Pso is expressed very strongly and specifically in liver and kidney. The gene appears to be present widely in eutherian mammals.
Asunto(s)
Mapeo Cromosómico , Cristalinas/genética , Oxidorreductasas N-Desmetilantes/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario , Mamíferos , Ratones , Datos de Secuencia Molecular , Conejos , Sarcosina-Oxidasa , Homología de Secuencia de Aminoácido , Cadena A de beta-CristalinaRESUMEN
A variable number of tandem repeats (VNTR) polymorphism has been described in intron 2 of the interleukin-1 receptor antagonist gene. Allele 2 of this polymorphism is associated with many chronic inflammatory diseases. Using direct sequencing of polymerase chain reaction products from individuals of known genotype for the VNTR, we have identified four single base change polymorphisms in exons 1ic and 2 and one upstream of exon 1ic, all of which are probably in linkage disequilibrium with the intron 2 VNTR. The exonic polymorphisms do not alter the encoded amino acid sequence. Using the exon 2 polymorphism as a marker for the intron 2 disease-associated allele, we have been able to analyse allele-specific mRNA in heterozygotic keratinocyte cell lines. The disease-associated allele shows no difference from other alleles in this cell type with respect to mRNA accumulation.
Asunto(s)
Alelos , Interleucina-1 , Liquen Escleroso y Atrófico/genética , Polimorfismo Genético , ARN Mensajero/genética , Sialoglicoproteínas/genética , Secuencia de Bases , Células Cultivadas , Análisis Mutacional de ADN , Exones/genética , Marcadores Genéticos , Humanos , Proteína Antagonista del Receptor de Interleucina 1 , Intrones/genética , Queratinocitos , Desequilibrio de Ligamiento , Repeticiones de Minisatélite , Datos de Secuencia Molecular , Mutación PuntualRESUMEN
We have constructed a restriction map of the human genomic region containing the genes encoding the three members of the interleukin-1 (IL-1) family, IL-1 alpha, IL-1 beta, and IL-1 receptor antagonist (IL-1 ra). For this purpose, pulsed-field gel electrophoresis blots were hybridized with probes derived from the three genes. The genes (IL1A, IL1B, and IL1RN, respectively) were found to map to a common restriction fragment of approximately 430 kb that is flanked by two clusters of sites for methylation-sensitive rare-cutter restriction enzymes (putative CpG islands). A likely third internal CpG island was marked by two rare-cutter sites. CpG and non-CpG-specific enzymes were used to map the three genes. Relative to one terminal CpG island, the three genes were mapped to the following intervals: IL1A was between +0 and +35 kb, IL1B was between +70 and +110 kb, and IL1RN was between +330 and +430 kb.
Asunto(s)
Genes , Interleucina-1/genética , Receptores de Interleucina-1/genética , Mapeo Cromosómico , Cromosomas Humanos Par 2 , Enzimas de Restricción del ADN , Electroforesis en Gel de Campo Pulsado , HumanosRESUMEN
Fos protein heterodimerizes through one surface of an alpha-helical domain called the leucine zipper. We have investigated the effect of destabilizing this domain by multiply substituting small residues of its non-interacting surface with glycine. Ternary complex formation between mutated Fos, Jun and DNA was determined in vitro in the presence of denaturant. We also tested the ability of constitutively expressed, mutated Fos proteins to support anchorage independent growth of the cell line Rat1A. Combinations of two substitutions are tolerated in both assays of Fos function, while four substitutions resulted in attenuation in both functions. Rat1A expressing one of the quadruple mutants also showed temperature sensitivity in anchorage independent growth. In dense monolayers of these cells, stromelysin (a Fos-responsive gene product) decreased in abundance as a function of temperature and was less abundant even at 34 degrees C than in cells that overexpressed the wild-type c-fos mRNA. However the mutant transgene itself appeared to show temperature sensitive expression. We suggest that creating a range of glycine substitutions for small residues in the non-interacting face of a leucine zipper might be of general use as a strategy to produce attenuated mutants of other transcription factors.
Asunto(s)
Leucina Zippers , Estructura Secundaria de Proteína , Proteínas Proto-Oncogénicas c-fos/química , Animales , Secuencia de Bases , División Celular , Línea Celular , Expresión Génica , Genes fos , Datos de Secuencia Molecular , Mutación , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/fisiología , Proteínas Proto-Oncogénicas c-jun/química , Ratas , Relación Estructura-Actividad , TemperaturaRESUMEN
A cDNA clone encoding the 3CD proteinase (3CDpro) of poliovirus type 2 (Sabin), the precursor to proteinase 3Cpro and RNA polymerase 3Dpol, was expressed in bacteria by using a T7 expression system. Site-specific mutagenesis of the 3C/3D cleavage site was performed to generate active proteolytic precursors impaired in their ability to process themselves to 3Cpro and 3Dpol. Of these mutations, the exchange of the Thr residue at the P4 position of the 3C/3D cleavage site for a Lys residue (3CDpro T181K) resulted in a mutant polypeptide exhibiting the smallest amount of autoprocessing. This mutant was purified to 86% homogeneity and used for subsequent proteolytic studies. Purified 3CDproM (M designates the cleavage site mutant 3CDpro T181K) was capable of cleaving the P1 capsid precursor, a peptide representing the 2BC cleavage site, and the 2BC precursor polypeptide. Purified 3CDproM demonstrated the same detergent sensitivity in processing experiments with the capsid precursor as was observed by using P1 and crude extracts of poliovirus-infected HeLa cell lysates. Purified 3CDproM did not have any detectable RNA polymerase activity, whereas 3Dpol, separated from 3CDproM by gel filtration in the last step of purification, did. We conclude that 3CDproM can process both structural and nonstructural precursors of the poliovirus polyprotein and that it is active against a synthetic peptide substrate. Moreover, cleavage of 3CD to 3Dpol is needed to activate the 3D RNA polymerase.
Asunto(s)
Cisteína Endopeptidasas/aislamiento & purificación , Cisteína Endopeptidasas/metabolismo , ARN Polimerasas Dirigidas por ADN/aislamiento & purificación , ARN Polimerasas Dirigidas por ADN/metabolismo , Endopeptidasas/aislamiento & purificación , Endopeptidasas/metabolismo , Precursores Enzimáticos/aislamiento & purificación , Precursores Enzimáticos/metabolismo , Poliovirus/enzimología , Proteínas Virales , Proteasas Virales 3C , Cápside/metabolismo , Cromatografía DEAE-Celulosa , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Clonación Molecular , Cisteína Endopeptidasas/genética , ADN Viral/genética , ADN Viral/aislamiento & purificación , ARN Polimerasas Dirigidas por ADN/genética , Electroforesis en Gel de Poliacrilamida , Endopeptidasas/genética , Precursores Enzimáticos/genética , Escherichia coli/genética , Cinética , Peso Molecular , Mutagénesis Sitio-Dirigida , Plásmidos , Poliovirus/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
The Fos and Jun proteins, which are components of the transcription factor AP1, associate through the interaction of their so-called leucine zipper domains and bind strongly and specifically to DNA at phorbol ester-responsive elements. Jun also homodimerizes and binds the same element whereas Fos seems to have no specific affinity for DNA. We show that a single amino-acid change in the leucine zipper of Fos is sufficient to allow a truncated Fos protein to homodimerize and thus form a complex with DNA, even in the absence of Jun. This Fos-derived homodimer recognizes the consensus phorbol-ester responsive element specifically, in vitro. We conclude that the structural requirements for specific DNA binding are present in the Fos protein itself, with the exception of its lack of self-affinity.
Asunto(s)
Proteínas Proto-Oncogénicas/genética , Secuencia de Aminoácidos , Southern Blotting , Proteínas de Unión al ADN/genética , Técnicas In Vitro , Leucina Zippers/genética , Leucina Zippers/fisiología , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Biosíntesis de Proteínas , Conformación Proteica , Proteínas Proto-Oncogénicas c-fos , Proteínas Proto-Oncogénicas c-jun , Mapeo Restrictivo , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Synthetic peptides, 14-16 residues in length, were used as substrates for purified recombinant poliovirus proteinase 3C. The sequences of the substrates correspond to the sequences of authentic cleavage sites in the poliovirus polyprotein, all of which contain Gln-Gly at the scissile bond. Specificity of cleavages was demonstrated by analysis of 3C digests of synthetic peptides. Relative rate constants for the cleavages were derived by competition experiments. The rate constants roughly correlated with the estimated half-life of the homologous precursor proteins detected in poliovirus-infected cells. The peptide most resistant to cleavage corresponded to the 3C/3D junction, a site known to be cleaved very slowly by 3C in vivo. Substitution of threonine for alanine in P4 position of this peptide, however, resulted in significant cleavage. This observation supports the hypothesis that the residue in P4 position, in addition to the Gln-Gly in P1 and P1', respectively, contributes to substrate recognition. Ac-Gln-Gly-NH2 was not a substrate for 3C.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Poliovirus/enzimología , Proteínas Virales , Proteasas Virales 3C , Secuencia de Aminoácidos , Cinética , Oligopéptidos/síntesis química , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Proteinase 3C of poliovirus type 2 (Sabin) was expressed at 4% total protein in Escherichia coli. The protein was soluble and could be purified by a simple scheme. It was weakly active on the capsid precursor P1 (expressed in vitro), which contains two cleavage sites. The products of processing P1 were 1ABC and 1D (VP1). The activity was insensitive to Triton X-100. Crude extracts of cells infected with poliovirus type 1 (Mahoney) gave strong processing and yielded 1AB (VP0), 1C (VP3), and 1D in the same assay system but were sensitive to detergent. 3C from cell extracts that was separated from its precursors resembled the recombinant proteinase in its activity. Recombinant 3C cleaved the peptide dansyl-Glu-Glu-Glu-Ala-Met-Glu-Gln-Gly-Ile-Thr-Asn-Lys-NH2 at the Gln-Gly bond. We conclude that 3C is merely the core of the Gln-Gly-cleaving activity which processes P1 in vivo and that there is probably a hydrophobic contact between a larger 3C precursor and its P1 substrate which allows the second processing reaction: 1ABC, 1D----1AB, 1C, 1D.
Asunto(s)
Cisteína Endopeptidasas/genética , Poliovirus/enzimología , Proteínas Virales , Proteasas Virales 3C , Cápside/metabolismo , Cisteína Endopeptidasas/biosíntesis , Cisteína Endopeptidasas/aislamiento & purificación , Cisteína Endopeptidasas/metabolismo , Electroforesis en Gel de Poliacrilamida , Precursores Enzimáticos/metabolismo , Escherichia coli/genética , Regulación de la Expresión Génica , Células HeLa , Humanos , Péptidos/metabolismo , Plásmidos , Poliovirus/genética , Precursores de Proteínas/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Picornavirus RNAs are uncapped messengers and have unusually long 5' nontranslated regions (5'NTRs) which contain many noninitiating AUG triplets. The translational efficiency of different picornavirus RNAs varies between different cell-free extracts and even in the same extract, such as micrococcal nuclease-treated rabbit reticulocyte lysates. The effect of the poliovirus 5'NTR on in vitro translation was compared with that of the 5'NTR of encephalomyocarditis virus by the use of synthetic mRNAs, micrococcal nuclease-treated HeLa cell extracts, and rabbit reticulocyte lysates. Artificial mono- and dicistronic mRNAs synthesized with T7 RNA polymerase were used to investigate whether the 5'NTR of encephalomyocarditis virus RNA contains a potential internal ribosomal entry site. The sequence between nucleotides 260 and 484 in the 5'NTR of encephalomyocarditis RNA was found to play a critical role in the efficient translation in both mono- and dicistronic mRNAs. Our data suggest that an internal ribosomal entry site resides in this region.