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：细胞通讯通路， 在后生动物发育的许多方面发挥作用。该途径的紧密空间和时间调节是 在许多发展决策中至关重要，并了解有助于此的分子机制 优雅控制是一个广泛关注的领域。拟议的研究集中在一种新的机制，以调节 该途径需要糖基转移酶边缘家族的直接配体糖基化来调节 Notch受体和配体之间的顺式相互作用。该模型将使用小鼠体节发生进行测试， 一个敏感的模型，探索配体糖基化和顺式抑制在Notch中的重要性 途径调节两个目标将测试中心假设，即配体糖基化的边缘家庭的 蛋白质调节顺式配体相互作用，并提供Notch途径的时间调节。 这是脊椎动物体节发生的“分段时钟”的背景。首先，细胞培养分析和 诱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途径和生物化学方法将研究配体糖基化如何调节 蛋白质：蛋白质结合亲和体。在第二个目标中，对配体的功能进行严格的体内评估， 糖基化将完成。这些目标的完成将产生第一个明确的功能分析， 作为Notch信号传导的控制位点的配体糖基化的相关性，并将整合该模型 从单个细胞中的蛋白质修饰和运输到细胞间的相互作用和模式化， 有机体 本文提出的工作将首次对Notch的生物学相关性进行严格的分析。 边缘蛋白的配体糖基化。尽管大多数Notch配体含有保守的共有序列， 允许通过Pofut 1进行聚糖添加，然后通过fringe进行聚糖延伸的序列 在糖基转移酶中，修饰的相关性是未知的。我们的工作将利用体节发生， 所述分段时钟作为需要边缘糖基化和顺式抑制以严格 测试配体糖基化的生物学意义，检验边缘修饰的假设， Notch配体调节顺式蛋白质相互作用的强度，提供了一种新的机制来调节蛋白质相互作用。 Notch信号传导的空间和时间激活。我们预计，这项工作的结果将具有广泛的意义。 这对我们理解Notch通路是如何调节的有着重要意义， 表面上很简单，有助于后生动物复杂的发育决定。

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.