O-Glycosylation of Epidermal Growth Factor-like Motifs

修改后的项目摘要/摘要部分 Notch受体启动了所有后生动物发育所必需的信号通路。该通路的缺陷会导致许多先天出生缺陷和癌症。Notch在癌症中既可以作为癌基因发挥作用，也可以作为肿瘤抑制因子发挥作用，因此需要提高或降低Notch活性的治疗方法。Notch在许多水平上受到调节，但Notch胞外区(ECD)的糖基化已成为一种主要的调节因子，可以根据上下文增加或降低Notch的活性。Notch ECD包含多达36个串联表皮生长因子样(EGF)重复序列，其中许多重复序列包含O-连接岩藻糖的共同序列。O-岩藻糖通过蛋白O-岩藻糖基转移酶1(POFUT1)被添加到EGF重复序列中，在所有被研究的环境中，O-岩藻糖是Notch功能所必需的，而3-N-乙酰氨基葡萄糖转移酶的边缘家族对O-岩藻糖的延伸是调节的。Notch的边缘调节已经成为通过改变信号受体的糖基化状态来调节信号受体的范例。边缘修饰通常增强Delta样配体家族(DLL1，DL4)对Notch1(N1)的激活，但抑制锯齿状配体(J1，J2)的激活。在过去的拨款周期中，我们在条纹修饰区分配体的分子机制方面取得了重大进展，方法是确定N1的哪些EGF重复序列被每个条纹酶修饰，并确定这些EGF重复序列中的哪些负责调节效应。我们的结果表明，条纹“标记”了Notch ECD，一些标记激活了DLL1-N1的激活，而明显的标记抑制了J1-N1的激活。此外，我们最近发现岩藻糖类似物(岩藻糖与碳6的修饰)以配体特异性的方式抑制Notch的激活，为我们可以使用小分子改变Notch糖基化并调节其功能的概念提供了证据。我们的结果导致了这一应用的总体假设：Notch上特定位置的O-葡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蛋白水解性受体激活之前的事件来调节其活性。我们将在三个目标上检验这一假设。目标1试图解决条纹修饰如何抑制J1-N1激活。我们发表的工作表明，条纹增强了DLL1与N1的结合，为增强DLL1-N1的激活提供了分子解释，但我们也表明条纹增强了J1与N1的结合。因此，条纹修饰必须通过影响配体结合之后但蛋白水解性受体激活之前的步骤来抑制J1-N1的激活。目标1中的实验将检验这种效应的几种可能机制。目的2研究O-岩藻糖在Notch EGF重复序列上的显著位置特异性延长的基础。这一知识将使我们能够消除特定部位的条纹修饰，而不会影响O-岩藻糖的添加。最后，目标3研究岩藻糖类似物如何影响Notch活性，我们是否可以产生更有效的具有不同活性的类似物，以及我们是否可以使用这些类似物来抑制依赖Notch活性进行分裂的癌细胞的生长。在体外影响Notch活性的类似物将在小鼠体内进行评估，以监测对依赖Notch的T细胞成熟的影响。

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.