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: 9906932
• 负责人: Robert S. Haltiwanger
• 金额: $ 47.52万
• 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/9906932
• 关键词: Acute Lymphocytic Leukemia; Acute T Cell Leukemia; Address; Adult; Affect; Affinity; Binding; Biological Assay; Biophysics; Blood; C57BL/6 Mouse; Cancer Cell Growth; Cancer Etiology; Carbon; Cell Maturation; Cells; Congenital Abnormality; Congenital Heart Defects; Consensus Sequence; Data; Defect; Development; Dimethyl Sulfoxide; Endocytosis; Enzymes; Epidermal Growth Factor; Event; Extracellular Domain; Family; Female; Fucose; Fucosyltransferase 1; Grant; Human; In Vitro; Individual; Injections; Knowledge; Laws; Lead; Ligand Binding; Ligands; Link; Malignant Neoplasms; Mammals; Manic; Mediating; Methods; Modification; Molecular; Monitor; Mus; Mutation; N-Acetylglucosaminyltransferases; NOTCH1 gene; Notch Signaling Pathway; Nucleic Acid Regulatory Sequences; Oncogenes; Oral; Pain; Pathway interactions; Patients; Peptide Hydrolases; Polysaccharides; Procedures; Proteins; PubMed; Publishing; Receptor Activation; Receptor Signaling; Regulation; Signal Pathway; Signal Transduction; Site; Specificity; Spleen; Squamous cell carcinoma; System; T cell differentiation; T-Cell Development; T-Lymphocyte; Tandem Repeat Sequences; Testing; Thymus Gland; Time; Tumor Suppressor Proteins; Wild Type Mouse; Work; analog; base; design; developmental disease; experimental study; glycosylation; human disease; in vitro Assay; in vitro activity; in vivo; inhibitor/antagonist; intraperitoneal; male; mutant; notch protein; novel therapeutics; receptor; receptor binding; small molecule; stem cells

Modified Project Summary/Abstract Section 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 Ofucose 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. Analogs which affect Notch activity in vitro will be evaluated in vivo in mice to monitor effects on the Notch-dependent maturation of T cells.

修改后的项目摘要/摘要部分 Notch 受体启动对所有后生动物发育至关重要的信号通路，该通路的缺陷会导致许多先天性出生缺陷和癌症，因此，可以增加或减少 Notch 活性的疗法。 Notch 在多个层面上受到调节，但 Notch 胞外结构域 (ECD) 的糖基化已成为一种主要调节因子，可根据具体情况增加或减少 Notch 活性。 36 个串联表皮生长因子样 (EGF) 重复序列，其中许多包含 O-连接岩藻糖的共有序列，通过蛋白 O-岩藻糖基转移酶 1 (POFUT1) 将 O-岩藻糖添加到 EGF 重复序列中，并且对所有的 Notch 功能至关重要。根据检查的背景，而 β3-N-乙酰氨基葡萄糖转移酶的 Fringe 家族对 Ofucose 的延伸具有调节作用。 Notch 已成为通过改变信号受体糖基化状态来调节信号受体的范例。Fringe 修饰通常会增强 Delta 样配体家族（DLL1、DLL4）对 Notch1 (N1) 的激活，但会抑制 Jagged 配体（J1、J2）的激活。在过去的资助周期中，我们通过识别 N1 的哪些 EGF 重复序列被每种 Fringe 酶修饰，并确定哪些 EGF 被修饰，从而在 Fringe 修饰区分配体的分子机制方面取得了重大进展。我们的结果表明，Fringes“标记”Notch ECD，其中一些标记激活 DLL1-N1 激活，而不同的标记抑制 J1-N1 激活。碳 6 的修饰）以配体特异性方式抑制 Notch 激活，这为我们可以使用小分子改变 Notch 糖基化并调节其功能提供了概念证明。我们的结果得出了总体假设。对于此应用：Notch 上特定位点的 O-聚糖通过直接调节初始 Notch-配体结合，或通过调节配体结合之后但在蛋白水解受体激活之前的事件来调节其活性。旨在解决边缘修饰如何抑制 J1-N1 激活的问题，我们发表的研究表明边缘增强了 DLL1 与 N1 的结合，为增强 DLL1-N1 激活提供了分子解释，但我们也表明了这一点。边缘增强 J1 与 N1 的结合。因此，边缘修饰必须通过影响配体结合之后但在蛋白水解受体激活之前的步骤来抑制 J1-N1 激活，目标 2 中的实验将检查这种效应的几种可能机制。 O-岩藻糖在Notch EGF重复序列上通过边缘进行显着位点特异性延伸的基础。这一知识将使我们能够消除特定位点的边缘修饰，而不影响O-岩藻糖的添加。最后，目标 3 研究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这些类似物来抑制依赖于 Notch 活性进行分裂的癌细胞的生长。体外试验将在小鼠体内进行评估，以监测对 T 细胞的 Notch 依赖性成熟的影响。