Combining structural, biophysical and in vivo techniques to probe structure/function of stem cell signalling receptor, Notch

今天生物学研究的重要挑战。Notch基因是一种重要的信号受体，调节发育和干细胞活性的许多方面。Notch是由配体与细胞外结构域结合而激活的，细胞外结构域由许多与表皮生长因子（EGF）相关的串联重复序列组成。配体与EGF区域结合启动蛋白水解裂解，释放Notch细胞内结构域（NICD）转移到细胞核，NICD与转录因子和辅激活蛋白相互作用，开启靶基因表达。NICD内的锚定结构域区域起着重要的作用，为核复合物的不同组分提供结合位点。锚蛋白区域还形成了一个结合成分的调节平台，如泛素连接酶和适配器蛋白复合物，参与内吞运输和分选，在调节Notch信号水平中起重要作用。果蝇遗传/发育分析和癌症基因组测序表明，Notch蛋白不同位置（包括不同的EGF和Ankyrin重复序列）的点突变会产生多样性的表型结果。机制基础，即序列改变如何通过改变Notch功能导致表型结果，对许多这些突变知之甚少，但结果的多样性表明不同的突变探测不同的调控相互作用。

important challenge in biological research today. This is exemplified by studies of mutations in the Notch gene, an important signalling receptor regulating many aspects of development and stem cell activity. Notch is activated by binding of ligand to the extracellular domain which is comprised of numerous tandem repeats related to Epidermal Growth Factor (EGF). Ligand binding to the EGF region initiates proteolytic cleavages that release the Notch intracellular domain (NICD) to translocate to the nucleus, where NICD interacts with transcription factors and coactivator proteins to switch on target gene expression. The ankyrin domain region within the NICD plays an important role, providing binding sites for different components of the nuclear complex. The ankyrin region also forms a regulatory platform for binding components such as ubiquitin ligases and Adapter protein complexes involved in the endocytic trafficking and sorting which play an important part in modulating Notch signalling levels. Drosophila genetic/developmental analysis and cancer genome sequencing has shown that point mutations in different locations of the Notch protein including different EGF and Ankyrin repeats produce a diversity of phenotypic outcomes. The mechanistic basis, i.e. how the sequence change leads to phenotypic outcome through altered Notch function, is poorly understood for many of these mutations but the diversity of outcomes suggests different mutations probe distinct regulatory interactions.