Vüllings Nicole, Airich Alina, Seib Ekaterina, Troost Tobias, Klein Thomas
Institute of Genetics, Heinrich-Heine-Universitaet Duesseldorf, Universitaetsstr. 1, Duesseldorf, 40225, Germany.
BMC Biol. 2025 Mar 6;23(1):71. doi: 10.1186/s12915-025-02162-6.
Notch signalling plays a crucial role in many developmental, homoeostatic and pathological processes in metazoans. The pathway is activated by binding of the ligand to the Notch receptor, which changes the conformation of the receptor by exerting a pulling force. The pulling force is generated by the endocytosis of the interacting ligand into the signal-sending cell. Endocytosis of ligands requires the action of the E3 ligases Mindbomb1 (Mib1) and Neuralized (Neur) that ubiquitylate lysines (Ks) of their intracellular domains. It has been shown that human MIB1 binds JAGGED1 (JAG1) via a bipartite binding motif in its ICD. This interaction is required for the activation of JAG1. However, it is not known whether this bipartite binding mode is of general importance. It is also not rigorously tested whether it occurs in vivo. Moreover, it is not known whether Mib1 ubiquitylates specific Ks in the ICD of ligands, or is rather non-selective.
We therefore investigated how Mib1 interacts with the Notch ligand Delta of Drosophila in an in vivo trans-activation assay and determined the Ks which are required for signalling. We show that the activation of Dl by Mib1 follows similar rules as has been found for mammalian MIB1 and JAG1. We present evidence that a combination of six Ks of the ICD is required for the full signalling activity of Dl by Mib1, with K742 being the most important one.
Altogether, our analysis further reveals the rules of Mib1-mediated DSL-ligand-dependent Notch-signalling.
Notch信号通路在多细胞动物的许多发育、稳态和病理过程中起着至关重要的作用。该信号通路通过配体与Notch受体结合而被激活，配体与受体结合通过施加拉力改变受体的构象。这种拉力是由相互作用的配体被信号发送细胞内吞作用产生的。配体的内吞作用需要E3连接酶Mindbomb1（Mib1）和Neuralized（Neur）的作用，它们使配体细胞内结构域的赖氨酸（Ks）泛素化。已表明人类MIB1通过其胞内结构域中的双组分结合基序与JAGGED1（JAG1）结合。这种相互作用是JAG1激活所必需的。然而，尚不清楚这种双组分结合模式是否具有普遍重要性。也未严格测试其是否在体内发生。此外，尚不清楚Mib1是否使配体细胞内结构域中的特定赖氨酸泛素化，或者是否更具非选择性。
因此，我们在体内反式激活试验中研究了Mib1如何与果蝇的Notch配体Delta相互作用，并确定了信号传导所需的赖氨酸。我们表明，Mib1对Dl的激活遵循与哺乳动物MIB1和JAG1类似的规则。我们提供的证据表明，Mib1对Dl的完全信号传导活性需要胞内结构域六个赖氨酸的组合，其中K742是最重要的一个。
总之，我们的分析进一步揭示了Mib1介导的依赖DSL配体Notch信号传导的规则。
