Characterisation of the co-adapter function of the E3 ligase Neuralized (Neur) during DSL-ligand induced Notch signalling

Notch信号在所有后生动物的许多发育过程中起着至关重要的作用。此外，通过参与成体干细胞的维持，它是包括人类在内的几种成体组织的稳态所必需的。内吞作用是整个配体诱导的通路激活所必需的。它由膜相关E3连接酶Neuralized （Neur）和Mindbomb1 （Mib1）启动。它们结合到DSL配体的胞内结构域（ICDs）上，介导赖氨酸(K)侧链上的泛素化（ubi）。泛素化配体通过其泛素结合域被内吞适配器Liquid Facets (Lqf)/Epsin识别，从而连接到内吞核心机制。在果蝇中，这两个E3连接酶在翅膀发育过程中可以在功能上相互替代，这表明它们可以执行相似的功能，尽管它们除了ubi所需的RING （Really Interesting New Gene）手指结构域（RF）之外没有序列相似性。然而，在神经发生过程中，Mib1不能取代Neur的功能，这表明Neur启动Notch信号的方式与Mib1不同。我们最近的工作通过揭示不能被泛素化的配体可以被Neur激活而不能被Mib1激活来支持这一观点。因此，Neur除了具有E3连接酶活性外，还具有Mib1无法提供的另一种迄今未知的功能。在本应用中，我们将进一步描述Neur的功能，并假设Neur是一种新型的内吞共适配器，它与Lqf/Epsin形成复合物，以一种额外的方式将配体连接到核心内吞机制。

Notch signalling plays a crucial role in many developmental processes in all metazoans. Moreover, it is required for homeostasis of several adult tissues, including in humans, through its involvement in maintenance of adult stem cells. Endocytosis is required for the full ligand induced activation of the pathway. It is initiated by the membrane-associated E3 ligases Neuralized (Neur) and Mindbomb1 (Mib1). They bind to the intracellular domains (ICDs) of DSL ligands to mediate the ubiquitylation (ubi) on lysine (K) side chains. The ubiquitylated ligands are recognised by the endocytic adapter Liquid Facets (Lqf)/Epsin via its ubiquitin binding domains and thereby connected to the endocytic core machinery. In Drosophila, the two E3 ligases can functionally substitute for one another during wing development, indicating that they can perform similar functions, even though they share no sequence similarity apart from a RING (Really Interesting New Gene) Finger domain (RF), which is required for ubi. However, Mib1 cannot replace the function of Neur during neurogenesis, suggesting that Neur can initiate Notch signalling in a manner different from Mib1. Our recent work supports this notion by revealing that ligands that cannot be ubiquitylated can be activated by Neur, but not Mib1. Hence, Neur has another so far unknown function despite its E3 ligase activity, which cannot be provided by Mib1. In this application, we will further characterise the function of Neur and follow the hypothesis that Neur is a novel type of endocytic co-adapter that forms a complex with Lqf/Epsin to connect the ligands in an additional way to the core endocytic machinery.