Binding and regulation of the activity of the DSL ligand Serrate by E3 ligases

Notch通路在所有后生动物的许多发育和稳态过程中都是必需的。此外，它还参与越来越多疾病的发病机制（例如，G.癌症）。因此，重要的是要详细了解其活动的调节。Notch受体通过与D1/Ser/Lag 2（DSL）蛋白家族的配体结合而被激活。在果蝇的基因组中存在两种DSL配体：Delta（D1）和Serrate（Ser）。为了激活Notch受体，信号发送细胞中的配体必须被内吞。到目前为止的工作表明，膜相关的E3连接酶Neuralized（Neur）和Mindbomb 1（Mib 1）在配体的激活及其内吞作用中起着至关重要的作用。这些酶介导赖氨酸（K）侧链上DSL配体的胞内结构域（ICD）的泛素化（ubi）。配体活化所需的另一个组分并且在胞吞作用中具有作为货物衔接子的记录作用是Epsin同系物液体小面（Lqf）4。基于这些发现，提出了一个简单的模型，其中连接酶通过泛在化其ICD来启动DSL配体的内吞作用。这种标记被Lqf识别，将配体与内吞核心机制物理连接。在果蝇中，大多数DSL信号由Mib 1介导。我们最近发现，MIB 1结合到两个独立的小的结合盒在细胞内结构域ICD的人丝氨酸直系同源物JAGGED 1（JAG 1），称为N-和C-盒。这两种相互作用都是Notch信号转导正常刺激所必需的。我们还可以表明Ks对于D1信号传导是重要的。然而，目前尚不清楚MIB 1依赖性JAG 1结合的规则是否适用于其他配体。此外，还不知道Ser或Dl的ICD中哪些Ks被Mib 1泛素化，以及是否存在共同的ubi规则。通过专注于果蝇配体Serrate，将在本资助期内回答这些问题。

The Notch pathway is required in many developmental and homeostatic processes in all metazoans. In addition, it is involved in the pathogenesis of an increasing number of diseases (e. g. cancer) in humans. Hence, it is important to understand the regulation of its activity in detail. The Notch receptor is activated through binding to ligands of the Dl/Ser/Lag2 (DSL) protein family, In the genome of Drosophila two DSL ligands are present: Delta (Dl) and Serrate (Ser). To activate the Notch receptor, the ligands in the signal-sending cells must be endocytosed. The work so far indicates that the membrane-associated E3 ligases Neuralized (Neur) and Mindbomb1 (Mib1) play crucial roles during activation of the ligands and their endocytosis 3. These enzymes mediate the ubiquitylation (ubi) of the intracellular domains (ICDs) of DSL ligands on lysine (K) side chains. A further component required for ligand activation and with a documented role as a cargo adapter in endocytosis is the Epsin homolog Liquid facets (Lqf) 4. Based on these findings, a simple model has been suggested in which the ligases initiate endocytosis of the DSL ligands by ubiquitylating their ICDs. This label is recognised by Lqf to physically link the ligands to the endocytic core machinery.In Drosophila most DSL signalling is mediated by Mib1. We recently found that MIB1 binds to two separate small binding boxes in the intracellular domain ICD of the human Ser ortholog JAGGED1 (JAG1), termed N- and C-box. Both interactions are needed for normal stimulation of Notch signal transduction. We could also show that the Ks are important for Dl signalling. However, it is not clear whether the rules found for MIB1 dependent JAG1 binding apply to other ligands. Moreover, it is not known which Ks in the ICD of Ser or Dl are ubiquitylated by Mib1 and whether there is a common rule underlying ubi. By focussing on the Drosophila ligand Serrate, will answer these questions in this funding period.