Role of Aquaporin-0 for cell-to-cell adhesion and lens transparency

描述(申请人提供)：晶状体是一种透明的组织，缺乏血管系统。跨膜水通道，称为水通道蛋白(AQPs)，在维持无血管晶状体的透明度和动态平衡方面发挥着重要作用。AQP0基因突变导致人类和小鼠晶状体白内障；基因敲除导致小鼠白内障。我们的长期目标是为晶状体白内障的治疗和预防做出贡献。在这项提案中，我们将重点放在AQP0上。AQP0至少有两个功能，即透水性和细胞间黏附。体外和体内研究已经证实了AQP0的透水性，但细胞与细胞间的黏附功能仍是一个假说；此外，AQP0的完整和裂解形式是否同时具有水孔和细胞间黏附蛋白的功能仍存在争议。这项建议的目标是进行关键实验并明确确定AQP0是否作为细胞间黏附蛋白发挥功能，是否完整以及N-或和C-末端裂解形式是否同时作为水孔和细胞间黏附蛋白(目标1)，细胞外环是否在细胞间黏附中发挥关键作用(目标2)。钙调蛋白结合域是否具有与黏附功能有关的作用(目标3)以及是否可以通过敲打既具有透水性又具有细胞间黏附功能的AQP4来取代AQP0的功能(AQP4)(目标4)。一种新开发的方法将被用来研究不同形式的AQP0的细胞间黏附功能。细胞外环参与细胞间黏附以及钙调蛋白结合域在AQP0功能中的作用将通过定点突变和基于PCR的结构域交换来研究。根据诱变研究的需要，将进行基于计算机的分子模拟。将建立敲入动物模型来测试体外发现的体内结果。将使用结构-功能方法来实现这些目标，并酌情进行细胞学、生化和分子生物学实验来验证结果。

DESCRIPTION (provided by applicant): Lens is a transparent tissue that lacks vasculature. Transmembrane water channels known as aquaporins (AQPs) play a significant role in maintaining transparency and homeostasis in the avascular lens. Mutations in AQP0 result in lens cataract in both human and mouse; knockout leads to cataract in mouse. Our long term goal is to contribute to the treatment and prevention of lens cataract. In this proposal, we are directing our focus on AQP0. At least two functions have been attributed for AQP0 viz., water permeability and cell-to-cell adhesion. Water permeability has been proven authentically through in vitro and in vivo studies while cell-to-cell adhesion function remains hypothetical; moreover, it is controversial whether intact as well as cleaved forms of AQP0 function both as a water pore and a cell-to-cell adhesion protein. The goals of this proposal is to critically experiment and clearly define whether AQP0 functions as a cell-to-cell adhesion protein, whether intact as well as the N- or and C- terminus cleaved forms function both as a water pore and a cell-to-cell adhesion protein (Aim 1), whether the extracellular loops play a critical role in cell-to-cell adhesion (Aim 2), whether the calmodulin-binding domain has a role with regard to adhesion function (Aim 3) and whether the function/s of AQP0 can be replaced by knocking in AQP4 which is proven to have both water permeability and cell-to-cell adhesion function (Aim 4). A novel method developed will be used to study the cell-to-cell adhesion function of the different forms of AQP0. Involvement of extracellular loop in cell-to-cell adhesion and the role of calmodulin binding domain for the functions of AQP0 will be studied using site-directed mutagenesis and PCR-based domain swapping. Computer based molecular simulation will be performed as necessary for mutagenesis studies. Knock-in animal models will be developed to test the in vivo outcome of the in vitro findings. The objectives will be pursued using structure-function approach and performing cytological, biochemical and molecular biological experiments as appropriate to verify the results.