Study of Intracellular Regulators of Notch Signaling

我们在果蝇中发现了一个与Mastermind同源的基因（hMam-1），该基因被认为是Notch信号通路的重要正调控因子。在本研究项目中，我们进一步鉴定了人类Man家族的两个新成员（hMam-2和hMam-3），它们保留了与hMam-1和果蝇Mam相似的特征。hMam-2和hMam-3均稳定并参与DNA结合复合物RBP-J蛋白和Notch细胞内结构域，其充当信号传导的中间体。hMam-2和hMam-3都通过Notch信号增强了从靶启动子的转录激活。然而，我们还获得了证据，即Notch 3和Notch 4对靶启动子的激活被hMam-2比被hMam-1或-3更有效地增强。因此，在哺乳动物系统中的Mam蛋白的多样性可能有助于提供分歧的强度的Notch信号在不同的细胞type.We还研究了一个新的母体基因，神经质，这被认为是一个重要组成部分的Notch信号，神经质编码的哺乳动物GDP-岩藻糖蛋白O-岩藻糖基转移酶，增加岩藻糖的表皮生长因子样重复果蝇同源物的功能。我们已经发现Neurotic对于Notch与其配体Delta的物理相互作用以及Fringe在果蝇培养细胞中调节这种相互作用的能力是必需的。我们的研究结果表明，O-岩藻糖基化催化的神经也参与了条纹的非依赖性活动的Notch，并可能提供一种新的开关机制，调节配体-受体相互作用。这是配体-受体相互作用绝对需要蛋白质糖基化事件的前所未有的例子。

We had identified a human homolog of Mastermind (Man), which had been implicated as an important positive regulator of Notch signaling pathway by genetic analyses in Drosophila (hMam-1). During this research project, we have further identified two new members of human Man family (hMam-2 and hMam-3), which retain characteristics similar to hMam-1 and Drosophila Mam. Both hMam-2 and hMam-3 stabilize and participate in the DNA-binding complex RBP-J protein and the Notch intracellular domains that serve as intermediates of the signaling. Both hMam-2 and hMam-3 enhanced the activation of transcription from a target promoter by Notch signaling. However, we also obtained evidence that the activation of the target promoter by Notch3 and Notch4 is more efficiently potentiated by hMam-2 than by hMam-1 or -3. Thus the multiplicity of Mam proteins in the mammalian system may help provide divergence to the strength of the Notch signals in different cell types.We have also investigated function of a novel maternal gene, neurotic, which is suggested to be an essential component for Notch signaling, neurotic encodes a Drosophila homolog of mammalian GDP-fucose protein O-fucosyltransferase, which adds fucose sugar to epidermal growth factor-like repeats. We have found Neurotic is essential for the physical interaction of Notch with its ligand Delta, and for the ability of Fringe to modulate this interaction in Drosophila cultured cells. Our results suggest that O-fucosylation catalyzed by Neurotic is also involved in the Fringe-independent activities of Notch and may provide a novel on-off mechanism that regulates ligand-receptor interactions. This is an unprecedented example of an absolute requirement of a protein glycosylation event for a ligand-receptor interaction.