RESUMEN
Signaling desensitization is key to limiting signal transduction duration and intensity. Signal transducer and activator of transcription 1 (STAT1) can mediate type II interferon (IFNγ)-induced immune responses, which are enhanced and inhibited by STAT1 phosphorylation and sumoylation, respectively. Here, we identified an N-MYC interacting protein, NMI, which can enhance STAT1 phosphorylation and STAT1-mediated IFNγ immune responses by binding and sequestering the E2 SUMO conjugation enzyme, UBC9, and blocking STAT1 sumoylation. NMI facilitates UBC9 nucleus-to-cytoplasm translocation in response to IFNγ, thereby inhibiting STAT1 sumoylation. STAT1 phosphorylation at Y701 and sumoylation at K703 are mutually exclusive modifications that regulate IFNγ-dependent transcriptional responses. NMI could not alter the phosphorylation level of sumoylation-deficient STAT1 after IFNγ treatment. Thus, IFNγ signaling is modulated by NMI through sequestration of UBC9 in the cytoplasm, leading to inhibition of STAT1 sumoylation. Hence, NMI functions as a switch for STAT1 activation/inactivation cycles by modulating an IFNγ-induced desensitization mechanism.
Asunto(s)
Interferón gamma , Sumoilación , Interferón gamma/metabolismo , Transducción de Señal , Fosforilación , Factor de Transcripción STAT1/genéticaRESUMEN
Interferon-γ (IFN-γ) represents one of the most important innate immunity responses in a host to combat infections of many human viruses including human herpesviruses. Human N-myc interactor (Nmi) protein, which has been shown to interact with signal transducer and activator of transcription (STAT) proteins including STAT1, is important for the activation of IFN-γ induced STAT1-dependent transcription of many genes responsible for IFN-γ immune responses. However, no proteins encoded by herpesviruses have been reported to interact with Nmi and inhibit Nmi-mediated activation of IFN-γ immune responses to achieve immune evasion from IFN-γ responses. In this study, we show strong evidence that the UL23 protein of human cytomegalovirus (HCMV), a human herpesvirus, specifically interacts with Nmi. This interaction was identified through a yeast two-hybrid screen and co-immunoprecipitation in human cells. We observed that Nmi, when bound to UL23, was not associated with STAT1, suggesting that UL23 binding of Nmi disrupts the interaction of Nmi with STAT1. In cells overexpressing UL23, we observed (a) significantly reduced levels of Nmi and STAT1 in the nuclei, the sites where these proteins act to induce transcription of IFN-γ stimulated genes, and (b) decreased levels of the induction of the transcription of IFN-γ stimulated genes. UL23-deficient HCMV mutants induced higher transcription of IFN-γ stimulated genes and exhibited lower titers than parental and control revertant viruses expressing functional UL23 in IFN-γ treated cells. Thus, UL23 appears to interact directly with Nmi and inhibit nuclear translocation of Nmi and its associated protein STAT1, leading to a decrease of IFN-γ induced responses and an increase of viral resistance to IFN-γ. Our results further highlight the roles of UL23-Nmi interactions in facilitating viral immune escape from IFN-γ responses and enhancing viral resistance to IFN antiviral effects.
Asunto(s)
Citomegalovirus/fisiología , Evasión Inmune , Inmunidad Innata/efectos de los fármacos , Interferón gamma/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Matriz Viral/fisiología , Células Cultivadas , Citomegalovirus/inmunología , Regulación de la Expresión Génica/inmunología , Células HEK293 , Humanos , Evasión Inmune/efectos de los fármacos , Evasión Inmune/genética , Inmunidad Innata/genética , Unión Proteica , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
The present study genotyped four SNPs (rs736707, rs2229864, rs362691, and rs2073559) of the Reelin gene (RELN) in 165 autistic trios, 67 sporadic autistic children and 283 healthy controls with Chinese Han pedigree. Both case-control analysis and transmission disequilibrium test (TDT) found no evidence of significant association. The results do not support previous positive findings and suggest that the four single-nucleotide polymorphisms (SNP) of RELN are unlikely to be associated with childhood autism in Chinese Han population.
Asunto(s)
Trastorno Autístico/genética , Moléculas de Adhesión Celular Neuronal/genética , Proteínas de la Matriz Extracelular/genética , Salud de la Familia , Proteínas del Tejido Nervioso/genética , Polimorfismo Genético/genética , Serina Endopeptidasas/genética , Adulto , Trastorno Autístico/etnología , Estudios de Casos y Controles , Distribución de Chi-Cuadrado , China/etnología , Femenino , Frecuencia de los Genes , Estudio de Asociación del Genoma Completo/métodos , Genotipo , Humanos , Masculino , Proteína Reelina , Adulto JovenRESUMEN
BACKGROUND: A well-characterized single nucleotide polymorphism (472G/A-Val/Met-SNP8) in the coding sequence of the catechol-O-methyltransferase (COMT) gene leads to a three- to fourfold difference in enzymatic activity and clinical and animal studies suggest a role in anxiety states like panic disorder. METHODS: Subjects from 70 panic disorder pedigrees, and 83 "triads", were genotyped at seven single nucleotide polymorphisms (SNPs), polymorphic microsatellites in the first intron of COMT and approximately 339kb upstream of COMT (D22S944) and analyzed for genetic association and linkage. RESULTS: Linkage analysis showed elevated LOD scores for 472G/A (SNP 8), silent exon 3 substitution (186C/T-SNP 5), and the marker D22S944 (2.88, 2.62, and 2.93, respectively), using a variety of diagnostic and genetic models. Association tests were not significant for the SNPs, but were highly significant for D22S944 (p =.0001-.0003). One three-marker haplotype formed from the above three polymorphisms was significantly associated with panic disorder (p =.0001), as was the "global" p value for this combination (p =.005). In addition, numerous haplotypes with combinations of D22S944 and COMT SNPs were found to be significantly associated with panic disorder. CONCLUSIONS: Our findings provide strong evidence for a susceptibility locus for panic disorder either within the COMT gene or in a nearby region of chromosome 22.
Asunto(s)
Catecol O-Metiltransferasa/genética , Cromosomas Humanos Par 22 , Trastorno de Pánico/genética , Adulto , Femenino , Marcadores Genéticos , Predisposición Genética a la Enfermedad/genética , Haplotipos , Humanos , Desequilibrio de Ligamiento , Escala de Lod , Masculino , Trastorno de Pánico/diagnóstico , Trastorno de Pánico/psicología , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
DNA templates harboring specific single nucleotide polymorphism (SNP) sites are largely needed as positive controls in practical SNP analysis and in determination of the reliability of newly developed methods in high-throughput screening assays. Here we report a one-step method to produce SNP templates by amplifying a wild-type sequence with primers having single nucleotide mismatches at or near their 3' ends. A short amplicon harboring an EcoRI site was used to evaluate the feasibility of our strategy. Perfectly matched primers and primers with a single base mismatch occurring from the first base to the sixth base of the EcoRI site were used for primer extension. By using polymerase without a proofreading function, we kept mismatched nucleotides from occurring in extended primer products, as confirmed by EcoRI digestion and sequencing analysis. The strategy of using primers with a single mismatched base and exo- polymerase was shown to be an efficient one-step method for preparing SNP templates, either for application in the development of SNP screening assays or as positive controls in practical SNP assays.
Asunto(s)
Desoxirribonucleasa EcoRI/genética , Mutagénesis Sitio-Dirigida/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Oligonucleótidos/genética , Polimorfismo de Nucleótido Simple/genética , Moldes Genéticos , Secuencia de Bases , Escherichia coli/genética , Ingeniería Genética/métodos , Datos de Secuencia Molecular , Mutación , Reacción en Cadena de la Polimerasa/métodos , Análisis de Secuencia de ADNRESUMEN
A novel coronavirus was identified as the cause for severe acute respiratory syndrome (SARS). The complete sequence of SARS genome has provided an opportunity for the development of molecular diagnostic assays. To restrain further outbreak of SARS, the World Health Organization has posted several pairs of polymerase chain reaction (PCR) primers for early diagnosis and urged more research to be done on PCR protocols. Here we report a strategy for the de novo synthesis of PCR templates complimentary to the SARS virus genome, which has the advantage of working on PCR templates without concern about viral infection and also has the advantage that it can be used by those who do not have access to the SARS virus. This highly efficient and safe strategy for obtaining SARS gene fragments is useful for the development of PCR assays, as well as for the preparation of reliable positive controls for PCR testing kits.
Asunto(s)
Pruebas Diagnósticas de Rutina/métodos , Reacción en Cadena de la Polimerasa/métodos , Síndrome Respiratorio Agudo Grave/diagnóstico , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Moldes Genéticos , Secuencia de Bases , ADN Complementario/genética , Datos de Secuencia Molecular , Síndrome Respiratorio Agudo Grave/virologíaRESUMEN
OBJECTIVE: To determine whether mutations in the bone morphogenetic protein receptor 2 gene (BMPR2), initially reported in primary pulmonary hypertension, were present in patients with pulmonary arterial hypertension and scleroderma spectrum of disease. Methods. BMPR2 gene mutations were determined using nucleic acid sequencing in 24 patients with pulmonary arterial hypertension and scleroderma spectrum of disease and in 2 control groups, 96 healthy North American individuals and 100 Israeli Ashkenazi Jews. The patients also had antinuclear antibody determinations and underwent right heart catheterization. RESULTS: One BMPR2 guanine to adenine (G to A) mutation in exon 13 was found in a 59-year-old Ashkenazi Jewish woman with the limited cutaneous variant, a normal chest radiograph, and positive anticentromere and rheumatoid factor autoantibodies. However, this mutation is thought to be a polymorphism because the same mutation was also found in an ethnically matched healthy Ashkenazi Jew. CONCLUSION: Pulmonary arterial hypertension in scleroderma spectrum of disease was not associated with heterogeneous germline mutations of BMPR2.
Asunto(s)
Hipertensión Pulmonar/genética , Hipertensión Pulmonar/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Esclerodermia Sistémica/complicaciones , Esclerodermia Sistémica/genética , Adulto , Anciano , Receptores de Proteínas Morfogenéticas Óseas de Tipo II , Análisis Mutacional de ADN , Femenino , Pruebas Genéticas , Humanos , Judíos/genética , Masculino , Persona de Mediana Edad , Mutación/genética , Polimorfismo Genético/genética , Esclerodermia Sistémica/fisiopatologíaRESUMEN
We report a large monocentric case series of 82 patients with human immunodeficiency virus-associated pulmonary arterial hypertension (PAH). No germline mutations of the PPH1 gene (bone morphogenetic protein receptor-II) were found in any of the 19 patients tested. PAH was the direct cause of death in 72% of cases. Survival rates of the overall population at 1, 2, and 3 years were 73, 60, and 47%, respectively. Survival was significantly poorer in patients in New York Heart Association functional class III-IV at the time of diagnosis, as compared with those in functional class I-II with respective rates of 60, 45, and 28% versus 100, 90, 84% at 1, 2, and 3 years (p < 0.0001). Subsequently, we analyzed prognostic factors in patients in functional class III-IV. Univariate analysis indicated that CD4 lymphocyte count of more than 212 cells mm(-3), the use of combination antiretroviral therapy (CART), and epoprostenol infusion were related with a better survival. On multivariate analysis only CD4 lymphocyte count was an independent predictor of survival, presumably because CART and epoprostenol infusion were strongly linked in our patient population. These results suggest that patients with severe human immunodeficiency virus-associated PAH should be considered for long-term epoprostenol infusion in association with CART.