The Identity of the Notch Ankyrin Repeat Domain as a Determinant of Isoform-Specific Notch Signaling

项目概要/摘要： Notch信号通路是几乎所有动物发育和细胞分化的重要调节因子 器官和组织Notch受体是一种300-kDa的跨膜蛋白，由细胞外膜组成。 配体结合结构域和胞内结构域（NICD）。哺乳动物Notch通路由四个组成 受体同种型，其共享保守的结构和重叠但非冗余的功能。事实上， 每种亚型在Notch功能中起着如此关键的作用， 通过一种同种型的信号传导是极其罕见的。Notch通路中由四种受体编码的复杂性 同种型可分为两部分：1）典型Notch信号传导中的同种型特异性差异 结果，和2）与其他信号传导途径的同种型特异性串扰。在接收到信号后， 蛋白水解裂解从膜释放NICD，其在经典途径中易位到细胞膜。 细胞核，在那里它结合DNA结合蛋白CSL并募集辅激活因子Mastermind（MAML）以形成Notch 转录激活复合物（NTC）。NICD的两个结构域负责形成三元NTC， N末端无序RAM接头和锚蛋白重复结构域（ANK），其与CSL形成裂缝， MAML结合。已经表明，在典型信号传导中的异构体特异性结果由以下介导： ANK结构域中的变异，其另外负责介导特异性细胞上的NTC二聚化， 含有配对的头对头CSL结合位点的启动子。然而，迄今为止，这些报告 定性性质，并提供了不一致的结论。此外，这些研究没有调查 ANK结构域在促进与其他通路的串扰中的作用。因为ANK域无处不在 蛋白质-蛋白质相互作用基序，这是不足为奇的，许多非典型的NICD相互作用也介导 ANK域名两种最常见的Notch交叉途径是Wnt和TGFβ，尽管这两种途径都是通过免疫抑制剂来实现的。 串扰是同种型特异性的，Notch、Wnt和TGFβ信号传导的协同作用已经涉及到 许多疾病状态，特别是转移性事件和乳腺癌中肿瘤侵袭增加。 尽管有许多治疗靶向Notch信号传导的尝试，但缺乏关于调控Notch信号传导的机制的数据。 同种型特异性Notch信号传导使这些努力失败。我提出三个目标来填补这一空白： 定量每种亚型的NTC的稳定性和它们的二聚化能力，2）表征每种亚型的结合， ANK同种型与Wnt和TGFβ相互作用的关键非经典结合伴侣，以及3）定义了ANK的作用。 在乳腺癌的细胞背景下，Wnt和TGFβ串扰的ANK结构域的变化。这项工作 将体外生物物理表征与细胞功能分析相结合， 亚型特异性Notch信号传导的机制和序列基础。这些数据代表了 Notch信号传导将作为设计更有针对性的、亚型特异性Notch的关键基础 治疗学

Project Summary/Abstract: The Notch signaling pathway is a crucial regulator of development and cell differentiation in almost all animal organs and tissues. The Notch receptor is a 300-kDa transmembrane protein consisting of an extracellular ligand-binding domain and an intracellular domain (NICD). The mammalian Notch pathways consists of four receptor isoforms which share a conserved architecture and overlapping but non-redundant functions. In fact, each isoform plays such a critical role in Notch function that congenital diseases due to severely deficient signaling by one isoform are exceedingly rare. The complexity encoded in the Notch pathway by the four receptor isoforms can be divided into two components: 1) isoform-specific differences in canonical Notch signaling outcomes, and 2) isoform-specific crosstalk with other signaling pathways. Upon receiving a signal, a series of proteolytic cleavages release the NICD from the membrane which, in the canonical pathway, translocates to the nucleus where it binds DNA-binding protein CSL and recruits co-activator Mastermind (MAML) to form the Notch transcriptional activation complex (NTC). Two domains of the NICD are responsible for forming the ternary NTC, the N-terminal disordered RAM linker and the ankyrin repeat domain (ANK) which forms a cleft with CSL for MAML binding. It has been suggested that isoform-specific outcomes in canonical signaling are mediated by variations in the ANK domains which are additionally responsible for mediating NTC dimerization on specialized promoters containing paired head-to-head CSL binding sites. However, these reports, to date, have been qualitative in nature and have offered inconsistent conclusions. Additionally, these studies did not investigate the role of the ANK domains in facilitating crosstalk with other pathways. Because ANK domains are ubiquitous protein-protein interaction motifs, it is unsurprising that many non-canonical NICD interactions are also mediated by the ANK domains. Two of the most common Notch-intersecting pathways are Wnt and TGFβ, and though this crosstalk is isoform-specific, the synergistic effects of Notch, Wnt, and TGFβ signaling have been implicated in numerous disease states, particularly in metastatic events and increased tumor invasion in breast cancer. Despite numerous attempts to therapeutically target Notch signaling, a lack of data on the mechanism governing isoform-specific Notch signaling has left these efforts unsuccessful. I propose three Aims to fill this void: I will 1) quantify the stability of each isoform’s NTC and their dimerization capacities, 2) characterize the binding of each ANK isoform with key non-canonical binding partners that crosstalk with Wnt and TGFβ, and 3) define the role of variations in the ANK domains on Wnt and TGFβ crosstalk in the cellular context of breast cancer. This work will integrate in vitro biophysical characterizations with functional analyses in cells to define the biophysical mechanism(s) and sequence basis of isoform-specific Notch signaling. These data represent a holistic view of Notch signaling that will serve as a critical foundation for designing more targeted, isoform-specific Notch therapeutics.