RESUMO
Control of stability of beta-catenin is central in the wnt signaling pathway. Here, the protein conductin was found to form a complex with both beta-catenin and the tumor suppressor gene product adenomatous polyposis coli (APC). Conductin induced beta-catenin degradation, whereas mutants of conductin that were deficient in complex formation stabilized beta-catenin. Fragments of APC that contained a conductin-binding domain also blocked beta-catenin degradation. Thus, conductin is a component of the multiprotein complex that directs beta-catenin to degradation and is located downstream of APC. In Xenopus embryos, conductin interfered with wnt-induced axis formation.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas Repressoras , Transativadores , Proteína da Polipose Adenomatosa do Colo , Sequência de Aminoácidos , Animais , Proteína Axina , Sítios de Ligação , Padronização Corporal , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Quinase 3 da Glicogênio Sintase , Humanos , Camundongos , Dados de Sequência Molecular , Mutação , Fosforilação , Proteínas/química , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Xenopus/embriologia , Proteínas de Xenopus , beta CateninaRESUMO
The mouse M-lysozyme downstream enhancer has been previously characterized on several levels of gene regulation. The enhancer was co-localized with a DNase I hypersensitive site in the chromatin of mature macrophages, the in vivo interaction of transcription factor GABP with the enhancer core (MLDE) demonstrated binding being restricted to mature macrophage cells, and analysis of the MLDE methylation state revealed a correlation between demethylation of CpG dinucleotides and the in vivo GABP binding. Here, we analyzed in detail the full-length enhancer in addition to the core element. We identified a total of nine binding sites for nuclear factors. Most of these factors are found ubiquitously in all cell types tested. These factors include several unknown proteins as well as the transcription factor NF-Y. In addition, three binding sites for a new single-stranded DNA binding protein were found. The presence of this factor in mature macrophages correlates with the in vivo DNA melting of one of the binding sites and with the enhancer strength.
Assuntos
DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Muramidase/genética , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Cromatina/fisiologia , Pegada de DNA , Desoxirribonuclease I , Fosfatos de Dinucleosídeos , Fator de Transcrição de Proteínas de Ligação GA , Regulação Enzimológica da Expressão Gênica , Isoenzimas/biossíntese , Isoenzimas/genética , Macrófagos/enzimologia , Camundongos , Dados de Sequência Molecular , Muramidase/biossíntese , Oligodesoxirribonucleotídeos/química , Proteínas Recombinantes/biossíntese , Deleção de Sequência , TransfecçãoRESUMO
An international task force of academic and industry leaders in sarcopenia research met on December 5, 2012 in Orlando, Florida to develop guidelines for designing and executing randomized clinical trials of sarcopenia treatments. The Task Force reviewed results from previous trials in related disease areas to extract lessons relevant to future sarcopenia trials, including practical issues regarding the design and conduct of trials in elderly populations, the definition of appropriate target populations, and the selection of screening tools, outcome measures, and biomarkers. They discussed regulatory issues, the challenges posed by trials of different types of interventions, and the need for standardization and harmonization. The Task Force concluded with recommendations for advancing the field toward better clinical trials.
Assuntos
Idoso Fragilizado , Ensaios Clínicos Controlados Aleatórios como Assunto , Projetos de Pesquisa , Sarcopenia/tratamento farmacológico , Comitês Consultivos , Idoso , Idoso de 80 Anos ou mais , Congressos como Assunto , União Europeia , Humanos , Estados UnidosRESUMO
The mouse lysozyme downstream enhancer was previously colocalized with the DNase I-hypersensitive site in the chromatin of mature macrophages. This hypersensitive site was shown to be macrophage differentiation-dependent. Demethylation of CpG sequences within the enhancer is correlated with lysozyme expression in mature macrophages. Binding of the GABP heterotetrameric transcription factor to the enhancer core element (MLDE), only seen in vivo on the demethylated MLDE element in macrophages, is inhibited by DNA methylation. Here, we analyzed the DNA sequences required for demethylation. In electrophoretic mobility shift experiments we found that in addition to the complete methylated MLDE the hemimethylated form of the lower strand inhibits GABP binding as well. Therefore, GABP is unlikely to be the mediator of demethylation. In addition, we show by stable DNA transfections of methylated mouse lysozyme enhancer sequences that MLDE-flanking sequences are required for demethylation. We narrowed down these DNA elements to two short regions of 163 and 79 base pairs on either side of the MLDE, each of which is sufficient to mediate demethylation of the GABP site.
Assuntos
DNA/metabolismo , Elementos Facilitadores Genéticos , Muramidase/genética , Animais , Sequência de Bases , Linhagem Celular , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Remoção de Radical Alquila , Fator de Transcrição de Proteínas de Ligação GA , Macrófagos/metabolismo , Camundongos , Dados de Sequência Molecular , Fatores de Transcrição/metabolismoRESUMO
Interactions between beta-catenin and LEF-1/TCF, APC and conductin/axin are essential for wnt-controlled stabilization of beta-catenin and transcriptional activation. The wnt signal transduction pathway is important in both embryonic development and tumor progression. We identify here amino acid residues in beta-catenin that distinctly affect its binding to LEF-1/TCF, APC and conductin. These residues form separate surface clusters, termed hot spots, along the armadillo superhelix of beta-catenin. We also show that complementary charged and hydrophobic amino acids are required for formation of the bipartite beta-catenin-LEF-1 transcription factor. Moreover, we demonstrate that conductin/axin binding to beta-catenin is essential for beta-catenin degradation, and that APC acts as a cofactor of conductin/axin in this process. Binding of APC to conductin/axin activates the latter and occurs between their SAMP and RGS domains, respectively.