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

Aquaporin-0 在细胞间粘附和晶状体透明度方面的作用

基本信息

批准号：
8247061
负责人：
Kulandaiappan Varadaraj
金额：
$ 37.68万
依托单位：
STATE UNIVERSITY NEW YORK STONY BROOK
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-04-01 至 2014-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8247061
关键词：
Accounting Adhesions Aging-Related Process Amino Acids Animal Model Architecture Binding Biochemical Biological Blinded Blindness Calmodulin Cataract Categories Cell Adhesion Cell Adhesion Molecules Cell Aging Cells Charge Chimera organism Cleaved cell Computers Data Development Disease Drug Compounding Engineering Goals Homeostasis Human In Vitro Integral Membrane Protein Investigation Knock-in Mouse Knock-out Knockout Mice L Cells Lens Fiber Literature MIP gene Methods Missense Mutation Modeling Molecular Mus Mutagenesis Mutate Mutation Outcome Permeability Play Prevention Proteins Role Senile Cataract Site Site-Directed Mutagenesis Structural Protein Structure Testing Therapeutic Tissues Transgenic Organisms Tyrosine United States Vision Water Xenopus oocyte age related base cell age cytotoxicity design extracellular falls fiber cell improved in vivo insight lens lens transparency monomer mouse model mutant novel promoter protein expression protein misfolding public health relevance research study restoration simulation water channel

项目摘要

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. PUBLIC HEALTH RELEVANCE: Lens cataract is responsible for majority of the loss of sight in the United States and ranks #1 globally with over 20 million people around the world already blinded and an additional 1.25 million added to the total each year; age-related cataract by itself accounts for more than 48% of loss of vision scenarios. Lens cataract caused by AQP0 falls in the category of conformational diseases (proteopathies) as they are mainly caused by missense mutations or age related protein misfolding, aggregation and cytotoxicity and main goal of this project is to provide new insights into the structure-related functional roles of AQP0, the most abundant integral membrane protein in the lens, for maintaining transparency and homeostasis. This investigation will contribute to an improved understanding of normal lens development and aging processes, and help to design and develop therapeutic compounds/drugs for congenital and senile cataracts in humans.

描述（由申请人提供）：晶状体是一种缺乏脉管系统的透明组织。被称为水通道蛋白（AQP）的跨膜水通道在维持无血管晶状体的透明度和稳态方面发挥着重要作用。 AQP0 突变会导致人类和小鼠晶状体白内障；基因敲除会导致小鼠白内障。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。在本提案中，我们将重点放在 AQP0 上。 AQP0 至少有两个功能，即水渗透性和细胞间粘附。水渗透性已通过体外和体内研究得到真实证明，而细胞间粘附功能仍然是假设的；此外，AQP0 的完整形式和切割形式是否同时具有水孔和细胞间粘附蛋白的功能还存在争议。该提案的目标是批判性地进行实验并明确定义 AQP0 是否作为细胞间粘附蛋白发挥作用，无论是完整的还是 N 端或 C 端裂解形式都充当水孔和细胞-细胞间粘附蛋白（目标 1），细胞外环是否在细胞间粘附中发挥关键作用（目标 2），钙调蛋白结合结构域是否在粘附功能中发挥作用（目标3）以及AQP0的功能是否可以通过敲入AQP4来替代，AQP4已被证明具有水渗透性和细胞间粘附功能（目标4）。开发的一种新方法将用于研究不同形式 AQP0 的细胞间粘附功能。将使用定点诱变和基于 PCR 的结构域交换来研究细胞外环在细胞间粘附中的参与以及钙调蛋白结合结构域对 AQP0 功能的作用。将根据诱变研究的需要进行基于计算机的分子模拟。将开发敲入动物模型来测试体外研究结果的体内结果。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以验证结果。公共健康相关性：晶状体白内障是造成美国大部分失明的原因，在全球排名第一，全世界有超过 2000 万人失明，并且每年新增 125 万人；与年龄相关的白内障本身就占视力丧失情况的 48% 以上。 AQP0引起的晶状体白内障属于构象疾病（蛋白病）范畴，因为它们主要是由错义突变或年龄相关蛋白错误折叠、聚集和细胞毒性引起的，该项目的主要目标是为结构相关的功能作用提供新的见解AQP0 是晶状体中最丰富的整合膜蛋白，用于维持透明度和体内平衡。这项研究将有助于更好地了解正常晶状体发育和衰老过程，并有助于设计和开发治疗人类先天性和老年性白内障的化合物/药物。