Elucidating divergent regulation of mammalian Notch ligands by Mindbomb1 using humanized Drosophila

Notch信号通路在许多发育过程中调节短程细胞通讯，在可能所有真核生物中调节动态平衡。因此，Notch通路被发现在越来越多的疾病(如癌症)和衰老的发病机制中发挥重要作用也就不足为奇了。在信号传递过程中，信号发送细胞中属于DSL家族的配体结合并激活信号接收端的Notch受体。在哺乳动物中，有5种Notch配体和4种Notch受体，它们都可以相互作用。虽然已经证明不同的配体在体内具有不同的功能作用，但目前还不清楚它们的不同活性是如何在分子上编码的。Notch配体的活性依赖于E3连接酶Mindomb1(Mib1)启动的内吞作用，它催化配体的胞内域(ICD)的泛素化(UBI)，并产生一种拉力，诱导受体的激活构象变化。我们假设，配体活性的差异是由MIB1和哺乳动物配体的ICD之间的相互作用以及由此产生的ICD的UBI模式编码的。我们将通过回答以下问题来验证这一假设：1.哺乳动物配体的ICD激活Notch途径的能力是否不同？2.ICD与MIB1是如何相互作用的？3.这些相互作用对功能的影响是什么？4.配体ICD中的哪些氨基酸是泛素化的，对信号和内吞作用是重要的？5.配体是否也能像已发现的果蝇配体和Delta那样以一种普遍独立的方式发挥作用？为了回答这些问题，我们将使用人性化的果蝇，用所有哺乳动物配体的ICD取代果蝇Delta配体ICD，用哺乳动物MIB1取代果蝇Mib1。这将允许利用果蝇可用的大量技术(例如遗传学、成像-在TK实验室进行)，并在活体环境中测试功能差异。同时，我们将使用最先进的体外和细胞培养分析，包括FRET和实时成像内吞分析，以提供哺乳动物细胞配体尾巴活性的补充分析(DS LAB)。这些结果将揭示Mib1与DSL配体相互作用的基本规律，以及这些相互作用如何在不同的环境中促进可变Notch活性。

The Notch signalling pathway mediates short-range cell communication in many developmental processes and homeostasis in probably all eukaryotes. It is therefore not surprising that the Notch pathway was found to play an important role in the pathogenesis of an increasing number of diseases (e. g. cancer) and in ageing. During signalling, the ligands, belonging to the DSL family, in the signal-sending cells bind and activate Notch receptors on the signal-receiving side. In mammals, there are 5 Notch ligands and 4 Notch receptors that can all interact with each other. While it has been shown that different ligands have different functional roles in vivo, it is unclear how their different activities are encoded molecularly. Notch ligand activity has been shown to depend on endocytosis initiated by the E3 ligases Mindbomb1 (Mib1), which catalyses the ubiquitylation (ubi) of the intracellular domains (ICDs) of the ligands, and creates a pulling force that induces an activating conformational change of the receptor. We hypothesize that differences in ligand activities are encoded by the interaction between MIB1 and the mammalian ligands’ ICDs, as well as by the resulting ubi patterns of the ICDs. We will test this hypothesis by answering the following questions: 1. Do the ICDs of mammalian ligands differ in their ability to activate the Notch pathway? 2. How do the ligands ICDs interact with MIB1? 3. What is the consequence of these interactions for function? 4. Which amino acids in the ICDs of the ligands are ubiquitylated and important for signalling and endocytosis? 5. Can the ligands also act in a ubi-independent manner, as has been found for Drosophila ligand Delta? To answer these questions, we will use ‘humanized’ flies by replacing the Drosophila Delta ligand ICD with the ICDs of all mammalian ligands, and the Drosophila Mib1 with the mammalian MIB1. This will allow exploiting the vast array of techniques available in Drosophila (e.g. genetics, imaging – performed in the TK lab) and test the functional differences in an in vivo settings. In parallel, we will use state of the art in vitro and cell culture assays including FRET and live imaging endocytosis assays to provide a complementary analysis of ligand tail activity in mammalian cells (DS lab). The results will reveal the fundamental rules for the interaction of Mib1 with the DSL ligands and how these interactions promote variable Notch activity in different contexts.