Mapping Notch endocytic signalling pathways via quantitative proteomics in D. melanogaster

细胞外信号传导允许细胞在组织环境中相互通信并影响其相邻细胞的命运，将信号从外部环境转导到细胞核以影响靶基因的表达。这些过程在发育和细胞分化过程中非常活跃。这个过程中的关键参与者之一是无处不在的信号受体Notch，它支撑着细胞中的许多生理过程。蛋白质在细胞膜上的蛋白水解裂解导致其细胞内结构域的释放，该结构域可以通过几种信号传导途径（通常涉及内体）找到其进入细胞核的途径。这些后一种途径可以以网格蛋白依赖性和非依赖性方式发生，并导致不同基因组的激活/抑制。然而，在这些内体信号传导途径中，各种Notch相互作用伴侣究竟如何影响它尚不清楚。使用果蝇S2细胞作为模型系统，我们进行了两次RNAi筛选，以确定一组高质量的Notch相互作用物，这些相互作用物影响两种内体信号通路的Notch表达，并希望将这些数据与更广泛的生物信息学数据整合，以更好地了解信号网络是如何构成的。然后，我们将进行全面的全球定量蛋白质组学实验，以确定两种不同的信号通路如何影响蛋白质组，定义差异表达的下游Notch靶点。这将加强生物信息学网络分析产生的假设，我们随后将使用定制蛋白质标准品的靶向蛋白质组学实验以及伴随的细胞生物学和生物化学来验证这些假设。我们还旨在研究在体内对最具信息性和有效的Notch相互作用物的敲低效应。因此，我们的总体目标是研究两种Notch信号通路促进的不同调控机制，在蛋白质水平上定义靶基因的伴随变化，并将其置于果蝇的表型背景中。该项目将定量蛋白质组学方法与生物信息学相结合，以在蛋白质水平上描绘关键的细胞信号传导组分，并体现了系统生物学方法，以提供对生命规则的新理解。我们的合作是新颖的，将分子和细胞生物学与尖端的分析科学结合在一起，因此很好地利用了BBSRC所支持的变革性技术。通过关注后生动物生活中无处不在的信号通路，我们试图揭示与理解整个生命过程中的健康相关的发展和细胞分化的基本原则。

Extracellular signalling allows cells to communicate with one another in the context of tissues and influence the fate of their neighbouring cells, transducing signals from the external environment to the nucleus to affect the expression of target genes. Such processes are critically active during development and cell differentiation. One of the key players in this process is the ubiquitous signalling receptor Notch, which underpins many physiological processes in the cell. Proteolytic cleavage of the protein at the cell membrane leads to release of its intracellular domain which can find its way to the nucleus via several signalling routes, often involving endosomes. These latter routes can occur in a Clathrin-dependent and independent manner, and lead to activation/suppression of different sets of genes. Exactly how the various Notch interacting partners influence it during these endosomal signalling pathways is, however, not known. Using Drosophila S2 cells as a model system, we have carried out two RNAi screens to determine a high-quality set of Notch interactors that influence Notch expression for the two endosomal signalling routes, and wish to integrate this data with wider bioinformatics data to better understand how the signalling networks are constituted. We will then carry out a comprehensive global quantitative proteomics experiment to determine how the two different signalling pathways affect the proteome, defining the downstream Notch targets that are differentially expressed. This will reinforce hypotheses generated by the bioinformatics network analyses, which we will subsequently validate using a combination of targeted proteomics experiments using bespoke protein standards, and attendant cell biology and biochemistry. We also aim to examine the knockdown effects on the most informative and potent Notch interactors in vivo. Our overall aim is therefore the characterise the different regulatory mechanisms promoted by the two Notch signalling routes, defining attendant changes in the target genes at the protein level, and placing this into a phenotypic context in the Drosophila. The project integrates quantitative proteomics methods with bioinformatics to delineate key cell signalling components at the protein level, and embodies a systems biology approach to deliver new understanding of the rules of life. Our collaboration is novel, bringing together molecular and cellular biology with cutting-edge analytical science, and consequently makes good use of the transformative technologies BBSRC espouses. By focusing on a ubiquitous signalling pathway in metazoan life, we seek to uncover fundamental principles that govern development and cell differentiation that are relevant to understanding health across the life course.