Understanding the function of microtubule-associated signalling networks in health and disease: role of adaptor proteins in network assembly

微管对真核细胞中的关键过程，特别是货物的运输、细胞分裂和迁移的调控是必不可少的。这些细胞过程的核心是MT+末端跟踪蛋白(+TIPS)的动态组装。它们将MT精确地引导到细胞内的不同区域，然后有选择地触发特定的反应。+末端-网络组装由适配蛋白控制，这些适配蛋白包含一小组短蛋白质相互作用基序(Sxip，Eey和Basic-S/P-)，嵌入到固有的无序区域，然而，这个故事远未完成。大多数研究都集中在单个成分上，很少进行定量分析。因此，当前模型的预测是不可靠的。该项目是正在进行的开发+TIP信令网络的全面、多组件模型的研究的组成部分。癌症中接头蛋白的突变有助于疾病的进展和转移，证明了它们的关键作用，并证明了进行更全面研究的理由。利用核磁共振、X射线结晶学和ITC，我们将解决适配蛋白复合体的结构，并定义目前尚未表征的正端网络中主要相互作用的热力学特征。我们将主要关注连接网络核心的SXIP、EEY和BASIC-S/P-主题。我们还将使用高分辨率荧光显微镜监测活细胞中的网络，并使用基于细胞渗透结构的多肽抑制剂通过破坏特定的相互作用来调节它们的特征。这些变化将与对正常细胞和癌细胞迁移和增殖的影响相关联，以确定特定蛋白质的贡献。利用工程细胞底物，我们将调整细胞环境，以模拟健康和病理的肿瘤和老化组织，从而定义细胞对环境变化的反应能力。将所有信息结合在一起，我们希望确定信号网络的关键组件，并测试它们作为药物靶标的潜力。

Microtubules (MTs) are essential for the regulation of key processes in eukaryotic cells, in particular cargo transport, cell division and migration. At the heart of these cellular processes are dynamic assemblies of MT plus-end tracking proteins (+TIPs). They guide MTs precisely to different areas in cells, and then selectively trigger specific responses "on arrival". +TIP-network assembly is governed by adaptor proteins that contain a small set of short protein interaction motifs (SxIP, EEY and basic-S/P-), embedded into intrinsically disordered regions however, this story is far from complete. Most of the research is focused on individual components, and little quantitative analysis is conducted. As a result, predictions from current models are unreliable. This project is an integral part of an on-going study to develop a comprehensive, multi-component model of +TIP signalling networks. Mutations of adaptor proteins in cancer contributes to disease progression and metastasis, demonstrating their critical role and the justification for more comprehensive research. Using NMR, X-ray crystallography and ITC we will solve structures of the adaptor protein complexes and define thermodynamic characteristics of the main interactions within the plus-end networks that are currently uncharacterised. Primarily we will focus on the SxIP, EEY and basic-S/P-motifs that interconnect the core of the networks. We will also monitor the networks in live cells with high-resolution fluorescent microscopy and use cell-permeable structure-based peptide inhibitors to modulate their characteristics by disrupting specific interactions. These changes will be correlated with the effects on migration and proliferation of normal and cancer cells to define the contributions of specific proteins. With engineered cell substrates we will modulate cell environment to emulate healthy and pathological tumour and ageing tissues, thus defining the ability of cells to react to the environmental changes. Joining all the information together we hope to identify critical components of the signalling networks and test their potential as drug targets.