The role of lysines of the intracellular domain of Notch during regulation of the Notch pathway

通过底物特异性E3连接酶将多肽泛素转移到细胞内结构域的赖氨酸侧链上，是溶酶体内吞作用和随后几种跨膜蛋白（TMPs）降解的重要信号。此外，据推测，通过泛素化启动的内吞作用对于通过配体激活进化保守的Notch信号受体是必不可少的。这一假设是基于Notch完全激活所需的几个E3连接酶的鉴定。Notch是一个在其胞内结构域中含有27种赖氨酸的TMP。为了测试赖氨酸/泛素化对Notch受体活性的重要性，我们用天冬酰胺（Rs）交换了所有赖氨酸（Ks），并测试了由此产生的NK2R变体在果蝇中的活性。我们发现该变体比相应的对照更活跃，并且它可能以不依赖于配体的方式激活Notch通路。在本应用程序中，我们将进一步描述NK2R变体，并询问它是如何激活的。我们将使用这种变体来确定哪些赖氨酸对受体活性的调节是重要的。我们将进一步确定所鉴定的E3连接酶在受体调控中的作用。我们将通过显微镜分析（电子和共聚焦显微镜，实时成像），生化分析以及分子和遗传实验的结合来实现这些目标。该分析将揭示Lysines of Notch和E3连接酶在Notch信号通路活性调控中的作用，对哺乳动物Notch信号传导也有重要意义。

Transfer of the polypeptide ubiquitin to the lysine side chains of intracellular domains performed by substrate specific E3 Ligases is an important signal for endocytosis and subsequent degradation of several transmembrane proteins (TMPs) in the lysosome. Moreover, it is assumed that endocytosis, initiated through ubiquitylation, is essential for the activation of the evolutionary conserved Notch signalling receptor by its ligands. This assumption is based on the identification of several E3 ligase required for the full activity of Notch. Notch is a TMP that contains 27 lysines in its intracellular domain. In order to test the importance of lysines/ ubiquitylation for the Notch receptor activity, we have exchanged all lysines (Ks) by asparagine (Rs) and tested the activity of the resulting NK2R variant in Drosophila melanogaster. We found that the variant is more active than the corresponding control and that it can activate the Notch pathway probably in a ligand-independent manner. In this application, we will characterise the NK2R variant further and ask how it is activated. We will use this variant to determine which lysines are important for the regulation of the activity of the receptor. We will further determine the role of the identified E3 ligases in the regulation of the receptor. We will achieve these goals by a combination of microscope analysis (electron and confocal microscope, Live imaging), biochemical assays and molecular and genetic experiments.The analysis will reveal the meaning of Lysines of Notch and the function of E3 ligases during the regulation of the activity of the Notch signalling pathway and is of importance also for Notch signalling in mammals.