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：
8494216
负责人：
Kulandaiappan Varadaraj
金额：
$ 7.5万
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：
2012
资助国家：
美国
起止时间：
2012-04-01 至 2014-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8494216
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）：镜头是缺乏脉管系统的透明组织。称为水通道蛋白（AQP）的跨膜水通道在保持透明度和稳态中起着重要作用。 AQP0中的突变导致人和小鼠的镜头白内障。敲除导致鼠标白内障。我们的长期目标是为透镜白内障的治疗和预防做出贡献。在此提案中，我们将关注AQP0。至少有两个功能归因于AQP0，即水的渗透性和细胞对细胞粘附。水的渗透性已通过体外和体内研究得到了真实的证明，而细胞间粘附功能仍然假设。此外，有争议的是完整以及AQP0的分裂形式既可以作为水孔和细胞到细胞粘附蛋白。 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关于粘附函数（AIM 3）以及是否可以通过敲击AQP4来代替AQP0的功能/S，该aqp4被证明具有水的渗透性和细胞间粘附函数（AIM 4）。开发的新方法将用于研究不同形式的AQP0的细胞间粘附功能。将使用位置定向的诱变和基于PCR的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在AQP0功能中的作用。对于诱变研究，将必要进行基于计算机的分子模拟。将开发敲入动物模型以测试体外发现的体内结果。将使用结构功能方法和进行细胞学，生化和分子生物学实验来实现目标，以验证结果。公共卫生相关性：镜头白内障负责美国失去视力的大部分，全球排名第一，全球超过2000万人已经蒙蔽了双眼，每年总数增加了125万人；与年龄相关的白内障本身占视力情景丧失的48％以上。 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稳态。这项调查将有助于对正常的晶状体发展和衰老过程的了解，并有助于设计和开发人类先天性和老年性白内障的治疗化合物/药物。

项目成果

期刊论文数量（11）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Intact and N- or C-terminal end truncated AQP0 function as open water channels and cell-to-cell adhesion proteins: end truncation could be a prelude for adjusting the refractive index of the lens to prevent spherical aberration.

DOI：
10.1016/j.bbagen.2014.05.002
发表时间：
2014-09
期刊：
BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS
影响因子：
3
作者：
Kumari, S. Sindhu;Varadaraj, Kulandaiappan
通讯作者：
Varadaraj, Kulandaiappan

Deletion of beaded filament proteins or the C-terminal end of Aquaporin 0 causes analogous abnormal distortion aberrations in mouse lens.

DOI：
10.1016/j.exer.2021.108645
发表时间：
2021-08
期刊：
EXPERIMENTAL EYE RESEARCH
影响因子：
3.4
作者：
Varadaraj, Kulandaiappan;FitzGerald, Paul G.;Kumari, S. Sindhu
通讯作者：
Kumari, S. Sindhu

Functional characterization of an AQP0 missense mutation, R33C, that causes dominant congenital lens cataract, reveals impaired cell-to-cell adhesion.

DOI：
10.1016/j.exer.2013.09.019
发表时间：
2013-11
期刊：
EXPERIMENTAL EYE RESEARCH
影响因子：
3.4
作者：
Kumari, Sindhu S.;Gandhi, Jason;Mustehsan, Mohammed H.;Eren, Semih;Varadaraj, Kulandaiappan
通讯作者：
Varadaraj, Kulandaiappan

Positively charged amino acid residues in the extracellular loops A and C of lens aquaporin 0 interact with the negative charges in the plasma membrane to facilitate cell-to-cell adhesion.

晶状体水通道蛋白 0 的细胞外环 A 和 C 中带正电荷的氨基酸残基与质膜中的负电荷相互作用，以促进细胞间粘附。