Combining structure and genetic data to probe cis and trans regulation of Notch by ligands.

生物体的健康取决于成人健康所依赖的组织和器官的适当发育。了解导致发育复杂性的信号的调节将在BBSRC的优先领域“整个生命过程中的健康老龄化”中产生相当大的影响。这不仅仅是因为同样的发育信号在成人身上继续被使用，以确保器官和组织的适当的动态平衡、更新和修复。了解这些过程是如何控制的，也将有助于推进组织工程和干细胞开发领域，以改善老龄化人口的健康和生活，因为这将促进操纵细胞命运的方法。进化保守的Notch受体可能是最重要的发育信号之一，因为它在发育和成人组织动态平衡的许多方面发挥着重要作用，因此参与了许多细胞类型的调节。这些关键功能是通过影响Notch受体结构/功能的突变来揭示的，这些突变与许多不同的发育障碍和癌症有关。只有在少数情况下，我们才能充分了解Notch不同部分的这些错义突变是如何导致其活性上调或下调的，这与影响健康的不同结果有关。这是因为Notch的调节是复杂的，涉及到受体不同部分的许多不同的细胞外和细胞内相互作用。Notch定位于细胞膜，并被两类膜结合配体中的一种激活。相邻细胞上的配体(反式提呈的配体)激活Notch信号。然而，如果配体在与Notch(顺式配体)相同的细胞中表达，它反而会阻止信号转导。配体与Notch相互作用的这些不同结果是如何产生的，以及这些相反的调控机制如何映射到Notch的结构和功能是本项目将解决的重要问题。我们基于之前的初步研究假设，不同的突变体将选择性地干扰不同模式的调控相互作用，从而对活性产生不同的结果。了解基因与表型之间的联系很重要，因为了解Notch突变导致功能丧失或功能获得的不同方式将促进我们对这一重要信号如何正常调控的了解，并将为补偿这些缺陷提供新的策略，从长远来看，这将最终为改善健康和福祉的治疗或干预提供新的途径。我们处理这个问题的方法将把果蝇诺奇作为一个模型系统来使用。以最近发表的Notch配体复合体的结构为指导，我们将通过细胞培养和体内实验，比较Notch与两种不同类型的配体(锯齿/锯齿类和Delta类)的顺式和反式相互作用的结构/功能/表型关系。果蝇在对细胞信号调节提供基本和保守的新见解方面有着良好的记录。苍蝇是我们研究的一个很好的模型系统，因为Notch的序列、结构和信号机制在苍蝇和人类之间是高度保守的，并且没有任何生物体能够更好地理解Notch信号，并且可以轻松地将新的突变体引入苍蝇基因组中，这使得在体内探测结构/功能成为一种可行的方法。将果蝇作为模式生物，有助于BBSRC的优先领域“替代、改进和减少使用动物的研究”。这是因为用脊椎动物模型进行类似的研究需要使用大量的动物，特别是因为哺乳动物的基因组包含四个Notch副本，这使得实验结果更难控制和解释。

The well being of the organism depends on the proper development of the tissues and organs on which adult health depends. Understanding the regulation of the signals that contribute to developmental complexity will have considerable impact within the BBSRC priority area of "healthy aging across the lifecourse". This is not in the least because the same developmental signals continue to be used in the adult to ensure proper homeostasis, renewal and repair of organs and tissues. Understanding how these processes are controlled will also help advance the field of tissue engineering and exploitation of stem cells to improve the health and life of the aging population because it will facilitate ways to manipulate cell fates. The evolutionary conserved Notch receptor mediates arguably one of the most important developmental signals because it plays numerous critical roles in many aspects of development and adult tissue homeostasis, and hence is involved in regulating many cell types. These critical functions are revealed by mutations that affect the structure/function of the Notch receptor and which are associated with many different developmental disorders and with cancer. Only in a small number of cases do we fully understand how these missense mutations in different parts of Notch lead to up or down-regulation of its activity that is linked to different outcomes affecting health. This is because the regulation of Notch is complex involving many different extracellular and intracellular interactions in different parts of the receptor. Notch is localised to the cell membrane and activated there by one of two classes of membrane bound ligand. Ligands on adjacent cells (trans-presented ligands) activate the Notch signal. However, if the ligand is expressed in the same cell as Notch (cis-ligand) it instead blocks signalling. How these different outcomes of ligands interacting with Notch arise, and how these opposing regulatory mechanisms map onto the structure and function of Notch are important questions that will be addressed in this project. We hypothesise based on previous pilot studies that different mutants will selectively interfere with different modes of regulatory interactions and hence have different outcomes on activity. Understanding the genotype to phenotype connection is important because understanding the different ways in which Notch mutants lead to loss or gain of function will advance our knowledge of how this important signal is normally regulated, and it will inform new strategies to allow compensation for these defects which in the longer term will ultimately provide new routes to therapies or interventions to improve health and well being. Our approach to this question will exploit Drosophila Notch as a model system. Guided by recent published structures of Notch ligand complexes, we will compare the structure/function/phenotype relationships of cis and trans-interactions of Notch with two different classes of ligand, the Serrate/Jagged class and the Delta class, using cell culture and in vivo assays. Drosophila has a strong track record of delivering fundamental and conserved new insights into cell signalling regulation. The fly excellent is an excellent model system for our study since Notch sequence, structure and signalling mechanisms are highly conserved between flies and humans and there is no organism in which Notch signalling is better understood and the ease by which novel mutants can be introduced into the fly genome makes probing structure/function in vivo a viable approach. Using the fruitfly Drosophila as a model organism contributes to the BBSRC priority area of "replacement, refinement and reduction in research using animals". This is because doing similar research with vertebrate models would require the use of a large number of animals particular because of the fact that mammalian genomes contain four copies of Notch, making experimental results much harder to control and interpret.