RESUMEN
BACKGROUND: NR2E1 (Tlx) is an orphan nuclear receptor that regulates the maintenance and self-renewal of neural stem cells, and promotes tumourigenesis. Nr2e1-null mice exhibit reduced cortical and limbic structures and pronounced retinal dystrophy. NR2E1 functions mainly as a repressor of gene transcription in association with the co-repressors atrophin-1, LSD1, HDAC and BCL11A. Recent evidence suggests that NR2E1 also acts as an activator of gene transcription. However, co-activator complexes that interact with NR2E1 have not yet been identified. In order to find potential novel co-regulators for NR2E1, we used a microarray assay for real-time analysis of co-regulator-nuclear receptor interaction (MARCoNI) that contains peptides representing interaction motifs from potential co-regulatory proteins, including known co-activator nuclear receptor box sequences (LxxLL motif). RESULTS: We found that NR2E1 binds strongly to an atrophin-1 peptide (Atro box) used as positive control and to 19 other peptides that constitute candidate NR2E1 partners. Two of these proteins, p300 and androgen receptor (AR), were further validated by reciprocal pull-down assays. The specificity of NR2E1 binding to peptides in the array was evaluated using two single amino acid variants, R274G and R276Q, which disrupted the majority of the binding interactions observed with wild-type NR2E1. The decreased binding affinity of these variants to co-regulators was further validated by pull-down assays using atrophin1 as bait. Despite the high conservation of arginine 274 in vertebrates, its reduced interactions with co-regulators were not significant in vivo as determined by retinal phenotype analysis in single-copy Nr2e1-null mice carrying the variant R274G. CONCLUSIONS: We showed that MARCoNI is a specific assay to test interactions of NR2E1 with candidate co-regulators. In this way, we unveiled 19 potential co-regulator partners for NR2E1, including eight co-activators. All the candidates here identified need to be further validated using in vitro and in vivo models. This assay was sensitive to point mutations in NR2E1 ligand binding domain making it useful to identify mutations and/or small molecules that alter binding of NR2E1 to protein partners.
Asunto(s)
Ligandos , Receptores Nucleares Huérfanos/agonistas , Receptores Citoplasmáticos y Nucleares/agonistas , Secuencia de Aminoácidos , Animales , Proteínas Portadoras , Descubrimiento de Drogas , Variación Genética , Humanos , Ratones , Ratones Noqueados , Receptores Nucleares Huérfanos/química , Receptores Nucleares Huérfanos/metabolismo , Fenotipo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/metabolismoRESUMEN
BACKGROUND: Prostate cancer consists of multifactorial and multifocal events, generating differential gene expression in tumor cells. METHODS: The molecular profile of 14 gene expression was analyzed through cDNA array in blood samples of patients with prostate cancer (PCa) and benign prostatic hyperplasia (BPH). RESULTS: Messenger RNA from patient's blood showed significant differences between PCa and BPH groups only for the NOS3 gene, with an occurrence chance for PCa5.8-fold higher than BPH disease. CONCLUSION: The NOS3 gene expression in the patient's blood may be used as a putative biomarker for prostate cancer.
Asunto(s)
Perfilación de la Expresión Génica , Hiperplasia Prostática/sangre , Hiperplasia Prostática/genética , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/genética , Adulto , Expresión Génica , Genes Relacionados con las Neoplasias/genética , Humanos , Masculino , Óxido Nítrico Sintasa de Tipo III/sangre , Óxido Nítrico Sintasa de Tipo III/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/sangre , ARN Mensajero/genéticaRESUMEN
O aumento da produtividade e qualidade dos produtos animais vem se tornando de grande interesse econômico. A prolactina (PRL) é um hormônio essencial para o sucesso reprodutivo e seu receptor (RPRL) tem sido detectado em vários tecidos². O gene RPRL foi recentemente mapeado em suínos no cromossomo 16(6). Este trabalho teve como objetivo analisar a frequência genotípica do RPRL em três diferentes raças de suíno, Landrace, Large White e Pietrain e correlacionar os genótipos com características de interesse. Foram analisados um total de 124 animais. O DNA foi extraído de sangue total suíno e submetido a técnica de PCR-RFLP, para determinação do genótipo do gene do receptor da prolactina. As análises estatísticas mostraram que o genótipo RPRL teve efeito sobre peso médio diário na raça Landrace (p<0,0135). As médias de DEPGMD na raça Landrace também foram diferentes em relação ao genótipo (p< 0,0610), confirmando a análise dos dados reais de Ganho de Peso Médio Diário. Métodos de seleção assistida por marcadores, juntamente com métodos de seleção tradicional poderão ser utilizados para potencializar e acelerar o melhoramento de características de interesse econômico em suínos, onde o gene do receptor de prolactina (RPRL) poderá ser utilizado como um marcador molecular para o ganho de peso médio diário real e sua DEP.