RESUMO
Human mutations in the cardiac transcription factor gene TBX5 cause congenital heart disease (CHD), although the underlying mechanism is unknown. We report characterization of the endogenous TBX5 cardiac interactome and demonstrate that TBX5, long considered a transcriptional activator, interacts biochemically and genetically with the nucleosome remodeling and deacetylase (NuRD) repressor complex. Incompatible gene programs are repressed by TBX5 in the developing heart. CHD mis-sense mutations that disrupt the TBX5-NuRD interaction cause depression of a subset of repressed genes. Furthermore, the TBX5-NuRD interaction is required for heart development. Phylogenetic analysis showed that the TBX5-NuRD interaction domain evolved during early diversification of vertebrates, simultaneous with the evolution of cardiac septation. Collectively, this work defines a TBX5-NuRD interaction essential to cardiac development and the evolution of the mammalian heart, and when altered may contribute to human CHD.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Coração/embriologia , Miocárdio/metabolismo , Proteínas com Domínio T/genética , Animais , Humanos , Camundongos Transgênicos , Organogênese/genética , Organogênese/fisiologia , Proteínas com Domínio T/metabolismo , Transcrição Gênica/genéticaRESUMO
The immunoaffinity isolation of protein complexes is an essential technique for the purification and -concentration of protein complexes from cells and tissues. In this chapter we present the methodologies for the purification of proteins and protein complexes from Xenopus laevis and Xenopus tropicalis. Specific to this protocol are the techniques for the cryolysis of Xenopus cells and tissues, a procedure that limits contamination from yolk proteins while preserving endogenous protein complexes, the methodologies for immunoaffinity purification of proteins using magnetic beads, and the protocols for western blot analysis. In addition, the procedures in this chapter can be extended to use with proteomic analysis of protein complexes as presented in the following chapter.
Assuntos
Imunoprecipitação/métodos , Complexos Multiproteicos/isolamento & purificação , Proteínas de Xenopus/isolamento & purificação , Xenopus laevis/metabolismo , Animais , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Afinidade de Anticorpos , Western Blotting , Soluções Tampão , Eletroforese em Gel de Poliacrilamida , Embrião não Mamífero/metabolismo , Imãs/química , Microesferas , Complexos Multiproteicos/imunologia , Proteínas de Xenopus/imunologiaRESUMO
Congenital heart defects affect nearly 1% of all newborns and are a significant cause of infant death. Clinical studies have identified a number of congenital heart syndromes associated with mutations in genes that are involved in the complex process of cardiogenesis. The African clawed frog, Xenopus, has been instrumental in studies of vertebrate heart development and provides a valuable tool to investigate the molecular mechanisms underlying human congenital heart diseases. In this review, we discuss the methodologies that make Xenopus an ideal model system to investigate heart development and disease. We also outline congenital heart conditions linked to cardiac genes that have been well studied in Xenopus and describe some emerging technologies that will further aid in the study of these complex syndromes.
Assuntos
Modelos Animais de Doenças , Cardiopatias Congênitas/embriologia , Cardiopatias Congênitas/genética , Xenopus/embriologia , Xenopus/metabolismo , Animais , Coração/embriologia , Morfogênese , Xenopus/genéticaRESUMO
Protein ubiquitination regulates numerous cellular functions in eukaryotes. The prevailing view about the role of RING or U-box ubiquitin ligases (E3) is to provide precise positioning between the attached substrate and the ubiquitin-conjugating enzyme (E2). However, the mechanism of ubiquitin transfer remains obscure. Using the carboxyl terminus of Hsc70-interacting protein as a model E3, we show herein that although U-box binding is required, it is not sufficient to trigger the transfer of ubiquitin onto target substrates. Furthermore, additional regions of the E3 protein that have no direct contact with E2 play critical roles in mediating ubiquitin transfer from E2 to attached substrates. By combining computational structure modeling and protein engineering approaches, we uncovered a conformational flexibility of E3 that is required for substrate ubiquitination. Using an engineered version of the carboxyl terminus of Hsc70-interacting protein ubiquitin ligase as a research tool, we demonstrate a striking flexibility of ubiquitin conjugation that does not affect substrate specificity. Our results not only reveal conformational changes of E3 during ubiquitin transfer but also provide a promising approach to custom-made E3 for targeted proteolysis.
