Structural and functional analysis of the repressor complex in the Notch signaling pathway of Drosophila melanogaster.
Structural and functional analysis of the repressor complex in the Notch signaling pathway of Drosophila melanogaster.
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DOI：
10.1091/mbc.e11-05-0420
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发表时间：
2011-09
影响因子：
3.3
通讯作者：
Preiss A
中科院分区：
文献类型：
--
作者：
Maier D;Kurth P;Schulz A;Russell A;Yuan Z;Gruber K;Kovall RA;Preiss A
尽管Notch途径高度保守，但其抑制在生物体之间似乎是不同的。在果蝇中，CSL直系同源物Su（H）和Hairless之间形成高亲和力复合物，其在体外和体内进行了非常详细的分析。在哺乳动物细胞中，无毛果蝇显示出结合CBF 1并抑制Notch转录输出。在后生动物中，高度保守的Notch途径驱动细胞特化。在受体激活时，Notch的胞内结构域组装转录激活因子复合物，该转录激活因子复合物包括DNA结合蛋白CSL、人C启动子结合因子1、黑腹果蝇的无毛抑制因子[Su（H）]以及秀丽隐杆线虫的lin-12和Glp-1表型的复合物。在不存在配体的情况下，CSL抑制Notch靶基因。然而，尽管CSL直向同源物的结构相似性，但阻遏在生物体之间表现出很大的差异。在这里，我们分析了果蝇中的Notch阻遏复合物，由果蝇CSL蛋白Su（H）和辅助阻遏物Hairless组成，后者招募一般阻遏蛋白。我们证明了Su（H）的C-末端结构域是与Hairless形成高亲和力复合物的必要和充分条件。影响与Notch和Mastermind相互作用的Su（H）突变对Hairless结合没有影响。尽管如此，我们证明了Notch和Hairless在体外和细胞培养中竞争CSL。此外，我们确定了一个网站的无毛，这是至关重要的结合苏（H），随后表明，这种无毛突变体是强烈受损，未能正确组装的阻遏物复合物在体内。最后，我们证明了无毛介导的抑制Notch信号在细胞培养试验中，这暗示了一个潜在的类似的抑制机制，在哺乳动物中，可能被利用为治疗目的。
Despite high conservation of the Notch pathway, its repression appears diverse between organisms. In Drosophila, a high-affinity complex forms between the CSL orthologue Su(H) and Hairless, which is analyzed in great detail in vitro and in vivo. Drosophila Hairless is shown to bind CBF1 and inhibit Notch transcriptional output in mammalian cells. In metazoans, the highly conserved Notch pathway drives cellular specification. On receptor activation, the intracellular domain of Notch assembles a transcriptional activator complex that includes the DNA-binding protein CSL, a composite of human C-promoter binding factor 1, Suppressor of Hairless of Drosophila melanogaster [Su(H)], and lin-12 and Glp-1 phenotype of Caenorhabditis elegans. In the absence of ligand, CSL represses Notch target genes. However, despite the structural similarity of CSL orthologues, repression appears largely diverse between organisms. Here we analyze the Notch repressor complex in Drosophila, consisting of the fly CSL protein, Su(H), and the corepressor Hairless, which recruits general repressor proteins. We show that the C-terminal domain of Su(H) is necessary and sufficient for forming a high-affinity complex with Hairless. Mutations in Su(H) that affect interactions with Notch and Mastermind have no effect on Hairless binding. Nonetheless, we demonstrate that Notch and Hairless compete for CSL in vitro and in cell culture. In addition, we identify a site in Hairless that is crucial for binding Su(H) and subsequently show that this Hairless mutant is strongly impaired, failing to properly assemble the repressor complex in vivo. Finally, we demonstrate Hairless-mediated inhibition of Notch signaling in a cell culture assay, which hints at a potentially similar repression mechanism in mammals that might be exploited for therapeutic purposes.