O-Glycosylation of Epidermal Growth Factor-like Motifs

修改项目摘要/摘要部分 Notch受体启动所有后生动物发育所必需的信号通路。该途径的缺陷导致许多先天性出生缺陷和癌症。Notch在癌症中可以作为致癌基因或肿瘤抑制基因发挥作用，因此需要可以增加或减少Notch活性的疗法。Notch在许多水平上受到调节，但Notch细胞外结构域（ECD）的糖基化已经成为主要的调节剂，其可以根据环境增加或降低Notch活性。Notch ECD含有多达36个串联表皮生长因子样（EGF）重复序列，其中许多含有O-连接岩藻糖的共有序列。O-岩藻糖被添加到EGF重复的蛋白O-岩藻糖基转移酶1（POFUT 1），是必不可少的Notch功能在所有的情况下检查，而扩展的Ofucose边缘家庭？3-N-乙酰葡糖胺转移酶是调节性的。Notch的边缘调节已经成为通过改变其糖基化状态来调节信号传导受体的范例。边缘修饰通常通过配体的Delta样家族（DLL 1，DLL 4）增强Notch 1（N1）活化，但抑制Jagged配体（J1，J2）的活化。在过去的资助周期中，我们在分子机制上取得了重大进展，通过这种机制，我们可以通过识别每个条纹酶修饰的N1的EGF重复序列来区分配体，并确定哪些EGF重复序列负责调节作用。我们的研究结果表明，条纹“标记”Notch ECD，一些标记激活DLL 1-N1激活，而不同的标记抑制J1-N1激活。此外，我们最近已经证明岩藻糖类似物（碳6修饰的岩藻糖）以配体特异性方式抑制Notch活化，提供了我们可以使用小分子改变Notch糖基化并调节其功能的概念证据。我们的研究结果导致了这一应用的总体假设：在特定位点上的Notch的O-聚糖调节其活性，直接调节初始Notch配体结合，或通过调节配体结合后，但之前的蛋白水解受体活化的事件。我们将从三个方面来检验这一假设。目的1试图解决边缘修饰如何抑制J1-N1激活。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受体活化之前的步骤来抑制J1-N1活化。目标1中的实验将研究这种效应的几种可能机制。目的2研究了O-岩藻糖对Notch EGF重复序列的显著位点特异性延伸的基础。这些知识将使我们能够消除特定位点的条纹修饰，而不影响O-岩藻糖的添加。最后，目标3检查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类似物来抑制依赖于Notch活性的癌细胞的生长。将在小鼠体内评估影响Notch体外活性的类似物，以监测对Notch依赖性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.