Understanding receptor clustering via multivalent ligand display

背景细胞外信号传导允许细胞与外界环境进行交流和反应。细胞表面受体的配体结合触发下游细胞内过程，最终决定细胞命运。重要的是，这些信号传导事件是浓度依赖性的，受体聚集和寡聚化与增加的信号效力和持久性相关。然而，激活所需的配体组织/价态知之甚少。因此，在这个项目中，我们将创建一个模块化的聚合物-蛋白质阵列平台，能够向细胞呈现精确定义的多配体结构，使我们能够将下游细胞命运与受体取向和寡聚化首次联系起来。ObjectivesTo allow us to achieve this goal，we have three key objectives：i）开发新的化学物质，可以选择性地靶向蛋白质的N-末端进行修饰; ii）合成具有常见细胞信号传导蛋白的精确定义的聚合物-蛋白质缀合物阵列;和iii）将聚合物-蛋白质缀合物结构与细胞内信号传导激活和持久性相关联。影响虽然受体的二聚化或更高阶聚集的重要性已被广泛证明，但我们的机制理解主要限于证明单个分离的受体-配体基序的活化增加-对下游信号传导所需的配体呈递的最佳方向，结构和价态知之甚少。这里描述的项目通过提供一种精确的手段来探测蛋白质-配体展示，从而研究关键的“生命规则”之一，直接解决了这一挑战。因此，我们将使前所未有的了解受体-配体相互作用，通过提供深入了解受体聚集的浓度依赖性。重要的是，异常受体激活广泛涉及疾病，迫切需要研究受体聚集的新工具。高纯度的生长因子制剂和低容量的聚合物阵列只是最近才出现，而N-末端蛋白质靶向仍然处于起步阶段。通过提供一种简便的方法，以可控的方式将任何感兴趣的蛋白质与聚合物阵列库偶联，我们还将为高通量多价配体筛选提供一个强大的平台。因此，该项目的产出将创造更多的机会，“将生物科学与工程和物理科学的创新相结合”，正如BBSRC的“转型技术”优先领域所描述的那样。

BackgroundExtracellular signalling allows cells to communicate and respond to their environment. Ligand binding by cell surface receptors triggers downstream intracellular processes that ultimately dictate cell fate. Critically, these signalling events are concentration dependent with receptor clustering and oligomerisation being correlated to increased signal potency and persistence. However, the ligand organisation/valency necessary for activation is poorly understood. In this project, we will therefore create a modular polymer-protein array platform able to present precisely defined multi-ligand architectures to cells, enabling us to correlate downstream cell fate to receptor orientation and oligomerisation for the first time.ObjectivesTo allow us to achieve this goal, we have three key objectives: i) To develop novel chemistries that can selectively target the N-termini of proteins for modification; ii) To synthesise precisely defined polymer-protein conjugate arrays with common cell signalling proteins; and iii) To correlate polymer-protein conjugate architecture to intracellular signalling activation and persistence. ImpactWhile the importance of dimerisation or higher order clustering of receptors has been widely demonstrated, our mechanistic understanding is mostly limited to demonstrations of increased activation over single isolated receptor-ligand motifs - the optimal orientation, architecture, and valency of ligand presentation necessary for downstream signalling is poorly understood. The project described here directly addresses this challenge by providing a precise means to probe protein-ligand display, and thus study one of the key 'rules of life'. We will therefore enable unprecedented understanding of receptor-ligand interactions by providing insight into the concentration dependent nature of receptor clustering. Importantly, abnormal receptor activation is widely implicated in disease, and there is an urgent need for new tools for studying receptor clustering. High-purity growth factor preparations and low volume polymer arrays have only recently emerged while N-terminal protein targeting remains in its infancy. The novel combination of technologies described here is therefore timely.By providing a facile means to conjugate any protein of interest to a polymer array library in a controlled manner, we will also deliver a powerful platform for high throughput multi-valent ligand screening. The output of this project will therefore create additional opportunities to 'integrate bioscience with innovation in the engineering and physical sciences', as described by the 'Transformative technologies' priority area of the BBSRC.