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

Notch配体糖基化作为调节顺式抑制途径的机制

• 批准号: 9789686
• 负责人: SUSAN E COLE
• 金额: $ 18.66万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-20 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9789686
• 关键词: Address; Affect; Affinity; Area; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; Biological Process; Biological Testing; CISH gene; Cell Communication; Cell Culture Techniques; Cell Nucleus; Cell surface; Cells; Complex; Congenital Abnormality; Consensus Sequence; Control Locus; Data; Defect; Deformity; Development; Disease; EGF gene; Embryo; Event; Exploratory/Developmental Grant; Family; Fucose; Gene Expression; Genes; Genetic; Individual; Ligands; Link; Maintenance; Mediating; Mesoderm; Modeling; Modification; Molecular; Mus; Mutagenesis; NOTCH1 gene; Notch Signaling Pathway; Organism; Outcome; Pathway interactions; Pattern; Play; Polysaccharides; Post-Translational Protein Processing; Process; Production; Protein Family; Protein Glycosylation; Proteins; Regulation; Reporting; Research; Research Personnel; Risk; Role; Segmentation Clock Pathway; Signal Transduction; Somites; Surface; System; Testing; Time; Tissues; Work; cost; fringe protein; gene function; glycosylation; glycosyltransferase; in vivo; in vivo evaluation; interest; notch protein; novel; protein transport; receptor; rib bone structure; scoliosis; skeletal; somitogenesis; spatiotemporal; spine bone structure; sugar

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.

抽象的 Notch信号通路是一个高度保守的细胞：扮演关键的细胞通信途径 在后生发展发展的许多方面的角色。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。拟议的研究着重于调节的新型机制 需要通过糖基转移酶的边缘家族调节的条纹家族的直接配体糖基化的途径 Notch受体和配体之间的顺式相互作用。该模型将使用小鼠的体重发生来测试 探索配体糖基化和notch中CIS抑制的基本重要性的敏感模型 途径调节。两个目的将检验中心假设，即边缘家族的配体糖基化 蛋白质调节顺式中的配体相互作用，并提供了对Notch途径的时间调节 “分割时钟”的上下文，脊椎动物的物质发生。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途径和生化方法将检查配体糖基化如何调节 蛋白质：蛋白质结合词汇。在第二个目标中，对配体功能进行严格的体内评估 糖基化将完成。这些目标的完成将对功能进行首次清晰的分析 配体糖基化作为Notch信号的控制源的相关性，并将整合该模型 从蛋白质修饰和单个细胞中的运输到细胞相​​互作用的尺度 生物。 这里提出的工作将对Notch的生物学相关性进行首次严格分析 条纹糖基通过条纹蛋白质化。尽管大多数缺口配体包含保守的共识 允许通过pofut1添加聚糖的序列，然后通过边缘进行聚糖延伸 糖基转移酶，修饰的相关性未知。我们的工作将利用体积发生和 分割时钟作为敏感系统，需要条纹糖基化和抑制顺式抑制 测试配体糖基化的生物学意义，检验了以下假设 Notch配体调节顺式中蛋白质相互作用的强度，提供了一种新的机制来调节 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有广泛 对我们对槽口途径如何调节的理解的影响，允许出现的途径 表面上直接有助于跨后生动物的复杂发展决策。