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

项目摘要/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 配体结合区和胞内区(NICD)。哺乳动物的Notch通路由四个部分组成 受体亚型具有保守的结构和重叠但非冗余的功能。事实上, 每一种亚型在Notch功能中都起着至关重要的作用，以至于由于严重缺乏而导致的先天性疾病 通过一种异构体来传递信号是极其罕见的。四种受体在Notch通路中编码的复杂性 异构体可以分为两个部分：1)典范Notch信号的异构体特异性差异 结果，以及2)与其他信号通路的异构体特异性串扰。在接收到信号时，一系列 蛋白水解酶将NICD从膜上释放出来，在规范的途径中，NICD被转移到 与DNA结合蛋白CSL结合的细胞核，并招募共激活因子MasterMind(MAML)形成Notch 转录激活复合体(NTC)。NICD的两个结构域负责形成三元NTC， N-末端无序的RAM接头和与CSL形成裂隙的Ankyrin重复结构域(ANK) MAML绑定。已有研究表明，典型信号转导中的异构体特异性结果是由 ANK结构域的变异，这些变异还负责调节NTC在专门化上的二聚化 含有成对的CSL结合位点的启动子。然而，迄今为止，这些报告一直是 在性质上是定性的，得出的结论也不一致。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，难怪许多非规范的NICD相互作用也是介导的 通过ANK域。两个最常见的缺口交叉通路是WNT和转化生长因子β，尽管这 串扰是异构体特有的，Notch、Wnt和转化生长因子β信号的协同效应已被牵涉到 多种疾病状态，特别是在乳腺癌的转移事件和肿瘤侵袭增加的情况下。 尽管多次尝试以Notch信号为治疗靶点，但缺乏有关其调控机制的数据 同型异构体特异性的Notch信号使这些努力没有成功。我提出了三个目标来填补这一空白：我将 量化每个异构体的NTC的稳定性和它们的二聚化能力；2)表征每个异构体的结合 与WNT和转化生长因子β串扰的关键非规范结合伙伴的ANK异构体，以及3)定义角色 在乳腺癌的细胞环境中，Wnt和转化生长因子β串扰的ANK结构域的变异。这部作品 将把体外生物物理特征与细胞内的功能分析结合起来，以定义生物物理 异构体特异性Notch信号的机制(S)和序列基础。这些数据代表了对 Notch信号转导，将成为设计更有针对性的、异构体特异的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.