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

• 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
• 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

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.

项目概要 白内障，即眼睛晶状体混浊，是全世界失明的主要原因。 水通道蛋白 0 (AQP0) 是晶状体中最丰富的膜蛋白，充当 水通道并作为粘附蛋白。 AQP0 缺陷可导致白内障，如 并对晶状体发育产生不良影响。尽管它在镜头中发挥着至关重要的作用 生理学上，AQP0 的功能尚不完全清楚。我们提出的研究 旨在加深我们对 AQP0 透水性 (Pf) 的理解， 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 AQP0 在晶状体内的位置。拟议的 研究还试图确定对 Pf 调节至关重要的氨基酸残基 通过蛋白质-蛋白质和/或蛋白质-膜实现 AQP0 的粘附功能 相互作用，并确定 AQP0 基因修饰对晶状体的影响 生理学和发育。为此，我们将采用紧密耦合的、多 学科方法，在水通道蛋白研究领域是独一无二的，它采用 技术范围从原子/分子到细胞和有机体 等级。具体来说，在目标 1 中，我们建议使用体外非洲爪蟾卵母细胞通透性 对一组哺乳动物和鱼类 AQP0 突变体进行测量，以评估 特定残基对 AQP0 Pf 的贡献及其调节以及粘附 对含有野生型和突变型 Aqp0 的斑马鱼晶状体纤维细胞进行测定。这些 实验方法将在目标 1 中通过计算机多微秒分子进行补充 动力学模拟，通过与实验 Pf 测量值的比较进行验证，以 阐明 Ca2+ 通过钙调蛋白结合、pH、 包括各种策略性选择的突变对 Pf 的影响 哺乳动物和鱼类 AQP0。目标 1 中提出的计算机模拟也将 解决蛋白质-蛋白质和蛋白质-膜相互作用的相对作用 AQP0的粘附功能。目标 2 将使用转基因斑马鱼来确定 AQP0 对晶状体结构和功能的贡献及其在晶状体中的发展 体内。我们的工作将增进对晶状体生理学和分子生物学的理解 水通道门控机制及其 Ca2+ 和 pH 的调节，并揭示 可以为治疗策略的未来发展提供信息的基本原则 用于延迟或消除白内障的形成。