O-Glycosylation of Epidermal Growth Factor-like Motifs

表皮生长因子样基序的 O-糖基化

• 批准号: 9769051
• 负责人: Robert S. Haltiwanger
• 金额: $ 47.66万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9769051
• 关键词: Acute Lymphocytic Leukemia; Acute T Cell Leukemia; Address; Affect; Affinity; Binding; Biological Assay; Biophysics; Cancer Cell Growth; Cancer Etiology; Carbon; Cells; Congenital Abnormality; Congenital Heart Defects; Consensus Sequence; Data; Defect; Development; Endocytosis; Enzymes; Epidermal Growth Factor; Event; Extracellular Domain; Family; Fucose; Fucosyltransferase 1; Grant; Human; In Vitro; Individual; Knowledge; Lead; Ligand Binding; Ligands; Link; Malignant Neoplasms; Mammals; Manic; Mediating; Methods; Modification; Molecular; Mutation; N-Acetylglucosaminyltransferases; NOTCH1 gene; Notch Signaling Pathway; Nucleic Acid Regulatory Sequences; Oncogenes; Pathway interactions; Patients; Peptide Hydrolases; Polysaccharides; Proteins; Publishing; Receptor Activation; Receptor Signaling; Regulation; Signal Pathway; Signal Transduction; Site; Specificity; Squamous cell carcinoma; Stem cells; System; T-Cell Development; T-Lymphocyte; Tandem Repeat Sequences; Testing; Tumor Suppressor Proteins; Work; analog; base; design; developmental disease; experimental study; glycosylation; human disease; in vitro Assay; inhibitor/antagonist; mutant; notch protein; novel therapeutics; receptor; receptor binding; small molecule

Notch receptors initiate a signaling pathway essential for development in all metazoans. Defects in the pathway cause a number of congenital birth defects and cancers. Notch can function as either an oncogene or tumor suppressor in cancers, so therapies that can increase or decrease Notch activity are needed. Notch is regulated at numerous levels, but glycosylation of the Notch extracellular domain (ECD) has emerged as a major regulator that can increase or decrease Notch activity depending on context. The Notch ECD contains up to 36 tandem Epidermal Growth Factor-like (EGF) repeats, many of which contain consensus sequences for O-linked fucose. O-Fucose is added to EGF repeats by Protein O-fucosyltransferase 1 (POFUT1) and is essential for Notch function in all contexts examined, while extension of O-fucose by the Fringe family of 3-N- acetylglucosaminyltransferases is modulatory. Fringe regulation of Notch has become a paradigm for regulation of a signaling receptor by altering its glycosylation status. Fringe modifications typically enhance Notch1 (N1) activation by the Delta-like family of ligands (DLL1, DLL4), but inhibit activation by Jagged ligands (J1, J2). In the past grant cycle we made significant progress on the molecular mechanisms by which Fringe modifications differentiate between ligands by identifying which EGF repeats of N1 are modified by each Fringe enzyme, and determining which of those EGF repeats are responsible for the modulatory effect. Our results demonstrate that Fringes “mark” the Notch ECD, with some marks activating DLL1-N1 activation, and distinct marks inhibiting J1-N1 activation. In addition, we have recently shown that fucose analogs (fucose with modifications to carbon 6) inhibit Notch activation in a ligand-specific manner, providing proof of concept that we can use small molecules to alter Notch glycosylation and regulate its function. Our results have led to the overall hypothesis for this application: O-glycans at specific sites on Notch regulate its activity by directly modulating initial Notch-ligand binding, or by modulating events subsequent to ligand binding but prior to proteolytic receptor activation. We will test this hypothesis in three aims. Aim 1 seeks to address how Fringe modifications inhibit J1-N1 activation. Our published work shows that Fringes enhance binding of DLL1 to N1, providing a molecular explanation for enhanced DLL1-N1 activation, but we also showed that Fringes enhance J1 binding to N1. Thus, Fringe modification must inhibit J1-N1 activation by affecting a step subsequent to ligand binding but prior to proteolytic receptor activation. The experiments in Aim 1 will examine several possible mechanisms for this effect. Aim 2 examines the basis for the striking site-specific elongation of O- fucose on Notch EGF repeats by the Fringes. This knowledge will allow us to eliminate Fringe modification at specific sites without affecting addition of O-fucose. Finally, Aim 3 examines how the fucose analogs affect Notch activity, whether we can generate more potent analogs with distinct activities, and whether we can use the analogs to inhibit growth of cancer cells dependent on Notch activity for division.

Notch受体启动所有后生动物发育所必需的信号通路。缺陷 途径导致许多先天性出生缺陷和癌症。Notch可以作为致癌基因发挥作用， Notch是癌症中的肿瘤抑制因子，因此需要可以增加或减少Notch活性的疗法。缺口 在许多水平调节，但糖基化的Notch细胞外结构域（ECD）已出现作为一个重要的功能。 主要的调节因子，可以增加或减少Notch的活动，这取决于上下文。Notch ECD包含 多达36个串联的表皮生长因子样（EGF）重复序列，其中许多包含共有序列 O-连接的岩藻糖。通过蛋白质O-岩藻糖基转移酶1（POFUT 1）将O-岩藻糖添加到EGF重复序列中， 在所有检查的情况下，Notch功能都是必不可少的，而O-岩藻糖的延伸是由α 3-N- 乙酰葡糖胺转移酶是调节性的。Notch的边缘调控已经成为一种范式， 通过改变其糖基化状态来调节信号受体。边缘修改通常会增强 Notch 1（N1）被Delta样配体家族（DLL 1，DLL 4）激活，但抑制Jagged配体的激活 (J1，J2）。在过去的资助周期中，我们在Fringe的分子机制方面取得了重大进展。 通过识别N1的哪些EGF重复被每个Fringe修饰来区分配体之间的修饰 酶，并确定哪些EGF重复序列负责调节作用。我们的结果 证明条纹“标记”Notch ECD，一些标记激活DLL 1-N1激活， 抑制J1-N1激活的标志物。此外，我们最近表明，岩藻糖类似物（岩藻糖与 对碳6的修饰）以配体特异性方式抑制Notch活化，提供了概念证明， 我们可以使用小分子来改变Notch糖基化并调节其功能。我们的研究结果导致了 本申请的总体假设：Notch上特定位点的O-聚糖通过直接调节其活性 调节初始Notch-配体结合，或通过调节配体结合之后但在Notch-配体结合之前的事件， 蛋白水解受体激活。我们将从三个方面来检验这一假设。目标1旨在解决边缘如何 修饰抑制J1-N1活化。我们发表的工作表明，条纹增强DLL 1与N1的结合， 为增强DLL 1-N1激活提供了分子解释，但我们也表明，条纹增强了DLL 1-N1的激活。 J1与N1结合。因此，Fringe修饰必须通过影响J1-N1活化后的步骤来抑制J1-N1活化。 配体结合但在蛋白水解受体活化之前。目标1中的实验将检查几个 这种影响的可能机制。目的2研究了O- Notch EGF的重复序列。这些知识将使我们能够消除边缘修改， 特定位点，而不影响O-岩藻糖的添加。最后，目标3考察了岩藻糖类似物如何影响 Notch活性，我们是否可以产生具有不同活性的更有效的类似物，以及我们是否可以使用 抑制依赖于Notch分裂活性的癌细胞生长的类似物。