Structure-Function Studies of Aquaporin 0 in Lens Development and Physiology

水通道蛋白 0 在晶状体发育和生理学中的结构功能研究

基本信息

批准号：
10334493
负责人：
Douglas J Tobias
金额：
$ 45.96万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-02-01 至 2026-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10334493
关键词：
Address Adhesions Adhesives Adverse effects Aging Amino Acids Animals Area Binding Biological Assay Biophysics Blindness CRISPR/Cas technology Calmodulin Cataract Cataract Extraction Cell Adhesion Cell Volumes Cellular Morphology Complement Computer Simulation Coupled Coupling Crystalline Lens Data Defect Development Exhibits Family Financial Hardship Fishes Future Gap Junctions Genes Genetic Genetic Engineering Health Care Costs Health system In Vitro Investigation Killifishes Knock-out Lead Lens Fiber Lens development Life Location MIP gene Maintenance Measurement Measures Medical Membrane Proteins Methodology Modeling Modification Molecular Mutation Oocytes Operative Surgical Procedures Optics Permeability Phosphoric Monoester Hydrolases Physiology Play Post-Translational Protein Processing Property Proteins Protons Refractive Indices Regulation Research Role Structure Surgical sutures Swelling Techniques Testing Variant Water Water Movements Work Xenopus oocyte Zebrafish experimental study extracellular fiber cell filensin improved in silico in vivo innovation interdisciplinary approach knock-down lens lens cortex lens transparency molecular dynamics mutant prevent regional difference therapeutic development trend water channel

项目摘要

Project Summary Cataract, the opacification of the eye lens, is the leading cause of blindness worldwide. Aquaporin 0 (AQP0), the most abundant membrane protein in the lens, functions as a water channel and as an adhesive protein. Defects in AQP0 can produce cataract, as well as have adverse effects on lens development. Despite its critical role in lens physiology, the functions of AQP0 are not fully understood. Our proposed research seeks to advance our understanding of how AQP0 water permeability (Pf), the exquisite control of which is required to maintain lens clarity, is regulated by Ca2+ and protons, whose concentrations depend on the AQP0 location within the lens. The proposed studies also seek to identify the amino acid residues that are crucial for Pf regulation and for the adhesive function of AQP0, through protein-protein and/or protein-membrane interactions, and to determine the effects of genetic modifications of AQP0 on lens physiology and development. To these ends, we will employ a tightly coupled, multi- disciplinary approach, unique within the field of aquaporin research, which employs techniques ranging in scale from the atomic/molecular to the cellular and organismal level. Specifically, in Aim 1 we propose to use in vitro Xenopus oocyte permeability measurements on a panel of mammalian and fish AQP0 mutants to assess the contribution of particular residues to AQP0 Pf and its regulation, along with adhesion assays on lens fiber cells from zebrafish containing wild-type and mutant Aqp0s. These experimental approaches will be complemented in Aim 1 with in silico multi-µs molecular dynamics simulations, validated by comparison with experimental Pf measurements, to elucidate mechanistic aspects of the influence of Ca2+ via calmodulin binding, pH, including the effects of a variety of strategically chosen mutations on the Pf of mammalian and fish AQP0s. The computer simulations proposed in Aim 1 will also address the relative role of protein-protein and protein-membrane interactions in the adhesive function of AQP0. Aim 2 will use genetically modified zebrafish to determine how AQP0 contributes to the structure and function of the lens and its development in vivo. Our work will improve understanding of lens physiology and the molecular mechanisms of water channel gating and its regulation by Ca2+ and pH, and uncover fundamental principles that could inform the future development of therapeutic strategies for delaying or eliminating cataract formation.

项目摘要白内障是眼镜镜的不透明，是全球失明的主要原因。水通道蛋白0（AQP0），镜头中最丰富的膜蛋白水通道和作为粘合蛋白。 AQP0中的缺陷会产生白内障，例如以及对晶状体发展的不利影响。尽管在镜头中起着至关重要的作用生理学，AQP0的功能尚未完全理解。我们提出的研究试图促进我们对AQP0水渗透性（PF）的理解，同等的维持晶状体清晰度所需的控制，由Ca2+和质子调节，其浓度取决于镜头内的AQP0位置。提议研究还试图确定对PF调节至关重要的氨基酸保留和通过蛋白质 - 蛋白质和/或蛋白质膜的AQP0的粘附功能相互作用，并确定AQP0遗传修饰的影响生理和发展。到这些目的，我们将采用紧密耦合，多的纪律方法，在Aquaporin Research领域独特，员工从原子/分子到细胞和生物的尺度上的技术级别。特别是，在AIM 1中，我们建议使用体外爪蟾卵母细胞的渗透性在哺乳动物和FISH AQP0突变体的面板上进行测量以评估特定残差对AQP0 PF及其法规的贡献以及广告对斑马鱼的晶状体纤维细胞进行测定，该斑马鱼含有野生型和突变体AQP0。这些实验方法将在AIM 1中完成，并在硅多µs分子中完成通过与实验PF测量值进行比较验证的动力学模拟通过钙调蛋白结合pH，阐明Ca2+影响的机械方面包括各种策略选择的突变对PF的PF的影响哺乳动物和鱼类AQP0。 AIM 1中提出的计算机模拟也将解决蛋白质 - 蛋白质和蛋白质 - 膜相互作用在该中的相对作用 AQP0的粘合功能。 AIM 2将使用一般修改的斑马鱼来确定 AQP0如何促进镜头的结构和功能及其发展体内。我们的工作将提高对晶状体生理学和分子的理解水通道门控机制及其对Ca2+和pH的调节，并发现可以告知理论未来发展的基本原则用于延迟或消除白内障形成。