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

当今生物学研究中的重大挑战。对Notch基因突变的研究就是一个例证，Notch基因是一种重要的信号受体，调节着发育和干细胞活动的许多方面。Notch是通过配体与胞外结构域的结合而激活的，胞外结构域由许多与表皮生长因子(EGF)相关的串联重复组成。与EGF区域的配体结合启动蛋白水解性切割，释放Notch胞内域(NICD)转位到细胞核，在那里NICD与转录因子和辅助激活蛋白相互作用，启动靶基因的表达。NICD内的Anankyrin结构域区起着重要的作用，为核复合体的不同成分提供结合位点。Ankyrin区域还形成了一个调节平台，用于结合参与胞内运输和分选的泛素连接酶和接头蛋白复合体等成分，它们在调节Notch信号水平方面发挥着重要作用。果蝇遗传/发育分析和癌症基因组测序表明，Notch蛋白不同位置的点突变，包括不同的EGF和Ankyrin重复序列，会产生不同的表型结果。其中许多突变的机制基础，即序列变化如何通过改变Notch功能导致表型结果，人们对此知之甚少，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.