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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.

项目总结