RESUMO
Autophagy, a system for the bulk degradation of intracellular components, is essential for homeostasis and the healthy physiology and development of cells and tissues. Its deregulation is associated with human disease. Thus, methods to modulate autophagic activity are critical for analysis of its role in mammalian cells and tissues. Here we report a method to inhibit autophagy using a mutant variant of the protein ATG7, a ubiquitin E1-like enzyme essential for autophagosome formation. During autophagy, ATG7 activates the conjugation of LC3 (ATG8) with phosphatidylethanolamine (PE) and ATG12 with ATG5. Human ATG7 interactions with LC3 or ATG12 require a thioester bond involving the ATG7 cysteine residue at position 572. We generated TetOff cells expressing mutant ATG7 protein carrying a serine substitution of this critical cysteine residue (ATG7C572S). Because ATG7C572S forms stable intermediate complexes with LC3 or ATG12, its expression resulted in a strong blockage of the ATG-conjugation system and suppression of autophagosome formation. Consequently, ATG7C572S mutant protein can be used as an inhibitor of autophagy.
Assuntos
Proteína 12 Relacionada à Autofagia/química , Proteína 7 Relacionada à Autofagia/química , Família da Proteína 8 Relacionada à Autofagia/química , Autofagia/efeitos dos fármacos , Autofagossomos/efeitos dos fármacos , Proteína 5 Relacionada à Autofagia/química , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/farmacologia , Células Cultivadas , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/farmacologia , Fosfatidiletanolaminas/químicaRESUMO
Notch signaling is an evolutionarily conserved signal transduction pathway that is essential for metazoan development. Upon ligand binding, the Notch intracellular domain (NOTCH ICD) translocates into the nucleus and forms a complex with the transcription factor RBPJ (also known as CBF1 or CSL) to activate expression of Notch target genes. In the absence of a Notch signal, RBPJ acts as a transcriptional repressor. Using a proteomic approach, we identified L3MBTL3 (also known as MBT1) as a novel RBPJ interactor. L3MBTL3 competes with NOTCH ICD for binding to RBPJ In the absence of NOTCH ICD, RBPJ recruits L3MBTL3 and the histone demethylase KDM1A (also known as LSD1) to the enhancers of Notch target genes, leading to H3K4me2 demethylation and to transcriptional repression. Importantly, in vivo analyses of the homologs of RBPJ and L3MBTL3 in Drosophila melanogaster and Caenorhabditis elegans demonstrate that the functional link between RBPJ and L3MBTL3 is evolutionarily conserved, thus identifying L3MBTL3 as a universal modulator of Notch signaling in metazoans.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Histona Desmetilases/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Neuroglia/metabolismo , Receptores Notch/genética , Animais , Evolução Biológica , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Linhagem Celular Tumoral , Sequência Conservada , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica , Histona Desmetilases/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Neuroglia/citologia , Ligação Proteica , Domínios Proteicos , Receptores Notch/metabolismo , Transcrição Gênica , Técnicas do Sistema de Duplo-HíbridoRESUMO
Cell-cell interactions define a quintessential aspect of multicellular development. Metazoan morphogenesis depends on a handful of fundamental, conserved cellular interaction mechanisms, one of which is defined by the Notch signaling pathway. Signals transmitted through the Notch surface receptor have a unique developmental role: Notch signaling links the fate of one cell with that of a cellular neighbor through physical interactions between the Notch receptor and the membrane-bound ligands that are expressed in an apposing cell. The developmental outcome of Notch signals is strictly dependent on the cellular context and can influence differentiation, proliferation and apoptotic cell fates. The Notch pathway is conserved across species (Artavanis-Tsakonas et al., 1999; Bray, 2006; Kopan and Ilagan, 2009). In humans, Notch malfunction has been associated with a diverse range of diseases linked to changes in cell fate and cell proliferation including cancer (Louvi and Artavanis-Tsakonas, 2012). In this Cell Science at a Glance article and the accompanying poster we summarize the molecular biology of Notch signaling, its role in development and its relevance to disease.