Aquaporin Regulation: A New Translational Route to Cytotoxic CNS Oedema Therapies

水通道蛋白(AQP)控制所有组织中的水流。我们发现，AQP的功能是通过调节质膜上AQP4水孔的数量来响应紧张度或低氧的变化来控制的。可逆性囊泡到质膜的再定位是由机械敏感通道激活导致细胞内钙离子水平升高和cAMP积聚，导致钙调蛋白依赖的磷酸化和细胞骨架重组而触发的。为了推动这一领域向前发展，我们现在需要充分了解所涉及的机制，这是本项目的总体目标。这一新的认识将为水失衡疾病提供新的靶点，并为指导寻找新的抑制剂提供一个平台。我们已经建立了一种基于细胞表面生物素化和细胞表面AQP的ELISA检测的内源性AQP质膜定位的方法，目前采用24孔板模式。将其扩展到96孔和384孔格式将提供一个使用高含量化合物文库的AQP重定位抑制剂的高通量筛选系统。本项研究的目标是：目标1：使用高含量筛选来鉴定AQP抑制剂。我们的96孔化验方法测量细胞肿胀。高含量筛选方面的进展将被用来使这一分析方法适应于基于图像的384孔跨荧光格式，该格式量化了细胞质到膜的转运和细胞表面分子的循环。通过精心挑选的经过批准、注释和多样化的化学集合的小组进行筛选，将识别专门调节AQP4亚细胞定位的新分子。确认的HITS将进入我们的细胞表面生物素化分析。药物动力学上合适的药物将被选择用于体内的概念验证测试。优先HITS将使用一套细胞药理学工具深入分析，以在表型、蛋白质组和转录水平阐明作用机制。目标2：定义AQP4调节的完整机制。为了获得一个完整的机制框架，我们将详细研究钙离子内流如何触发AQP4重新定位。通过分子和细胞生物学工具、我们的抑制物小组和蛋白质组/脂体学分析，将AQP4转移到质膜和从质膜转移出AQP4以响应星形胶质细胞紧张性变化的囊泡和调节蛋白的分子同一性目前尚不清楚。将调查AQP4同源寡聚和贩运之间的联系。AQP4和调节其膜丰度的蛋白质之间的相互作用将得到表征。全长AQP4和调节AQP4复合体的结构表征将提供一个可以直接用于寻找新抑制剂的平台。

Aquaporins (AQP) control water flow in all tissues. We have discovered that AQP function is controlled by regulating the number of AQP4 water pores in the plasma membrane in response to changes in tonicity or hypoxia. Reversible vesicle-to-plasma-membrane relocalization is triggered by activation of mechanosensitive channels leading to increased intracellular Ca2+ levels and cAMP accumulation, resulting in calmodulin-dependent phosphorylation and cytoskeletal reorganization. To move the field forward, we now need to fully understand the mechanisms involved, which is the overall aim of this project. This new understanding will provide novel targets for diseases of water imbalance and a platform to guide the search for new inhibitors. We have developed an assay for endogenous AQP plasma membrane localization based on cell-surface biotinylation and ELISA detection of cell-surface AQP, currently in 24-well plate format. Scaling this to 96- and 384-well format will provide a high-throughput screening system for inhibitors of AQP relocalization using high content compound libraries.The objectives of this studentship are:Objective 1: Identify AQP inhibitors using high-content screening. Our 96-well assay measures cell swelling. Advances in high-content screening will be used to adapt this assay into a robust image-based 384-well transfluor format that quantifies cytoplasmic-to-membrane translocation and recycling of cell-surface molecules. Screening across a carefully-selected panel of approved, annotated and diverse chemical sets will identify new molecules that specifically modulate AQP4 sub-cellular localization. Confirmed hits will progress to our cell-surface biotinylation assay. Pharmacokinetically-appropriate hits will be selected for proof-of-concept testing in vivo. Prioritized hits will be profiled in-depth using a suite of cellular pharmacology tools to elucidate mechanism-of-action at phenotypic, proteomic and transcriptomic levels.Objective 2: Define the complete mechanism of AQP4 regulation. To obtain a complete mechanistic framework, we will examine in molecular detail how calcium influx triggers AQP4 relocalization. The currently-unknown molecular identity of vesicles and regulatory proteins transferring AQP4 to and from the plasma membrane in response to tonicity changes in astrocytes will be identified using molecular- and cell-biology tools, our inhibitor panel and proteomic/lipidomic analyses. The link between AQP4 homo-oligomerisation and trafficking will be investigated. Interactions between AQP4 and proteins that regulate its membrane abundance will be characterized. Structural characterization of full-length AQP4 and regulatory AQP4 complexes will provide a platform that can be directly exploited in the search for new inhibitors.