Notch ligand glycosylation as a mechanism to regulate pathway cis-inhibition

摘要 Notch信号通路是一种高度保守的细胞：起关键作用的细胞通讯通路 在后生动物发展的许多方面发挥作用。对这一途径的严格时空调节是 在许多发育决策中至关重要，并了解导致这一现象的分子机制 优雅的控制是一个广受关注的领域。拟议中的研究集中在一种新的监管机制上 需要糖基转移酶边缘家族直接配体糖基化才能调节的途径 Notch受体与配体的顺式相互作用。这一模型将用小鼠的体细胞发生来测试 探讨配体糖基化和顺式抑制在Notch中的重要作用的灵敏模型 途径调控。两个AIMS将检验中心假设，即边缘家族的配体糖基化 蛋白质调节顺式反应中的配基相互作用，并提供对Notch途径的时间调节 脊椎动物体细胞发生的“分段时钟”的背景。首先，细胞培养分析和 突变将直接评估配体糖基化如何影响蛋白质相互作用和配体呈递。 Notch途径和生化方法将检测配体糖基化是如何调节的 蛋白质：蛋白质结合的亲和基团。在第二个目标中，对配体的功能进行了严格的体内评估 糖基化将完成。完成这些目标将产生第一个明确的功能分析 配体糖基化作为Notch信号的控制点的相关性，并将这一模型整合到 从单个细胞的蛋白质修饰和运输到细胞相互作用和图案形成 有机体。 这里提出的工作将为Notch的生物学相关性提供第一个严格的分析 边缘蛋白的配体糖基化作用。尽管大多数Notch配体包含保守的共识 允许通过Pofut1加成多聚糖，然后通过边缘延伸多聚糖的序列 糖基转移酶的修饰的相关性尚不清楚。我们的工作将利用体细胞发生和 分段时钟作为一个敏感的系统，需要边缘糖基化和顺式抑制来严格 检验配体糖基化的生物学意义，检验边缘修饰的假说 缺口配体调节顺式蛋白相互作用的强度，提供了一种新的机制来调节 Notch信号的空间和时间激活。我们预计，这项工作的结果将具有广泛的 对我们理解Notch途径是如何调节的，从而允许出现一条途径的暗示 在表面上是直截了当的，有助于后生动物复杂的发展决策。

Abstract The Notch signaling pathway is a highly conserved cell:cell communication pathway that plays critical roles in many aspects of metazoan development. Tight spatial and temporal regulation of this pathway is critical in many developmental decisions, and understanding the molecular mechanisms contributing to this elegant control is an area of broad interest. The proposed research focuses on a novel mechanism to regulate the pathway that requires direct ligand glycosylation by the fringe family of glycosyltransferases to modulate cis-interactions between Notch receptors and ligands. This model will be tested using mouse somitogenesis as a sensitive model to explore the fundamental importance of ligand glycosylation and cis-inhibition in Notch pathway regulation. Two aims will test the central hypothesis that ligand glycosylation by the fringe family of proteins modulates ligand interactions in cis and provides temporal regulation of the Notch pathway in the context of the "segmentation clock" that times vertebrate somitogenesis. First, cell culture analyses and mutagenesis will directly assess how ligand glycosylation affects protein interactions and ligand presentation in the Notch pathway, and biochemical approaches will examine how ligand glycosylation modulates protein:protein binding affinitites. In the second aim, a rigorous in vivo assessment of the function of ligand glycosylation will be completed. Completion of these aims will produce the first clear analysis of the functional relevance of ligand glycosylation as a locus of control for Notch signaling, and will integrate this model across scales from protein modification and trafficking in individual cells to cellular interactions and patterning in an organism. The work proposed here will provide the first rigorous analysis of the biological relevance of Notch ligand glycosylation by fringe proteins. Although the majority of Notch ligands contain conserved consensus sequences that would allow glycan addition by Pofut1 followed by glycan extension by fringe glycosyltransferases, the relevance of the modifications are unknown. Our work will exploit somitogenesis and the segmentation clock as a sensitive system that requires fringe glycosylation and cis-inhibition to rigorously test the biological significance of ligand glycosylation, examining the hypothesis that fringe modification of Notch ligands modulate the strength of protein interactions in cis, providing a novel mechanism to regulate the spatial and temporal activation of Notch signaling. We anticipate that the results from this work will have broad implications for our understanding of how the Notch pathway is regulated, allowing a pathway that appears straightforward on the surface to contribute to complex developmental decisions across metazoans.