Structural analysis of protein-protein and protein-lipid interactions of lens membrane proteins.

晶状体膜蛋白的蛋白质-蛋白质和蛋白质-脂质相互作用的结构分析。

• 批准号: 10508511
• 负责人: Carla O'Neale
• 金额: $ 3.5万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10508511
• 关键词: Adhesions; Adopted; Affect; Age; Binding; Biological Assay; Blindness; C-terminal; Calcium-Binding Proteins; Calmodulin; Cataract; Cell Adhesion; Chemicals; Complement; Crystalline Lens; Data; Deuterium; Development; Environment; Functional disorder; Galectin 3; Generations; Goals; Healthcare Systems; Homeostasis; Human; Hydrogen; Integral Membrane Protein; Lens Fiber; Life; Link; Lipid Binding; Lipids; MIP gene; Mass Spectrum Analysis; Membrane; Membrane Proteins; Microcirculation; Mus; Mutation; Nutrient; Operative Surgical Procedures; Permeability; Physiological; Play; Property; Protein Analysis; Protein Dynamics; Proteins; Regulation; Reporting; Research Personnel; Role; Site; Structure; System; Tail; Techniques; Testing; Tissues; Vascular blood supply; Vision; Water; cell water; common treatment; cost; crosslink; experimental study; fiber cell; insight; lens; lens transparency; molecular dynamics; prevent; protein function; protein structure; reconstitution; therapeutic development; therapeutic target; wasting; water channel

Summary/abstract: Cataract is the leading cause of blindness worldwide, costing the US Healthcare system billions of dollars annually for surgical treatment. Lens opacification has been linked to dysfunction in major membrane proteins, Aquaporin-0 (AQP0) and Lim2. The overall goal of my project is to develop a comprehensive understanding of the structure and function of protein-protein and protein-lipid interactions of these membrane proteins. In the absence of a blood supply, the microcirculation system transports nutrients and removes wastes to inner fiber cells and is essential for maintaining lens transparency over decades of life; however, how this microcirculation system is established and maintained as a function of age is not well understood. Using advanced mass spectrometry techniques such as hydrogen-deuterium exchange-MS, native-MS and chemical crosslinking studies, I intend to elucidate how specific protein and lipid interactions impact the structure and function of AQP0 and Lim2; membrane proteins that are fundamental to the microcirculation system of the lens. The most abundant lens membrane protein, AQP0, plays important roles in lens fiber cell adhesion and water permeability with water permeability regulated by interaction with the calcium-binding protein, calmodulin. Data from my lab and others demonstrated that calmodulin interacts with the C-terminal tail of AQP0; however, molecular dynamic (MD) simulations suggest a non-canonical interaction with a cytosolic loop of AQP0. This MD prediction has not been experimentally validated. In addition to AQP0- protein interactions (Aim 1), I hypothesize that AQP0-lipid interactions (Aim 2) regulate AQP0 permeability and adhesion properties that underlie lens transparency; however, there are limited reports on AQP0 interactions with native lipids. Given the vital role of AQP0 in maintaining lens transparency and its connection to cataract, understanding regulatory interactions with proteins such as calmodulin and native lipids will clarify its role in the microcirculation system. The second most abundant membrane protein in the lens is Lim2 and, like AQP0, its mutation has been associated with cataractogenesis in mice; however, little is known about Lim2-protein interactions (Aim 1). Binding partners to Lim2 have been reported, i.e. calmodulin and galectin-3, but how these interactions modulate Lim2 structure and function are not clear. Additionally, native lens lipids have been reported to impact Lim2 subunit assembly, but the details underlying this phenomenon have not been explored. As a result of the scarcity of experimental research on Lim2-native lipid interactions (Aim 2), I will use native MS to identify specific lipid interactions and determine how they affect Lim2 structure. My findings will aid researchers develop therapeutic targets and/or practices that can prevent, reverse or delay cataract formation.

摘要/摘要：白内障是全球失明的主要原因，使美国医疗保健系统造成了损失 每年数十亿美元用于手术治疗。镜头无情已与主要功能障碍有关 膜蛋白，Aquaporin-0（AQP0）和LIM2。我项目的总体目标是开发 对蛋白质 - 蛋白质蛋白和蛋白质脂质相互作用的结构和功能的全面理解 这些膜蛋白。在没有血液供应的情况下，微循环系统运输营养 并去除对内部纤维细胞的废物，对于维持晶状体透明度数十年是至关重要的。 但是，如何建立和维持随着年龄的函数建立和维护该微循环系统 理解。使用高级质谱技术，例如氢 - 偏见交换-MS， 我打算阐明特定蛋白质和脂质相互作用的天然MS和化学交联研究 影响AQP0和LIM2的结构和功能；膜蛋白是至关重要的 镜头的微循环系统。最丰富的镜头膜蛋白AQP0在 晶状体纤维细胞的粘附和水的渗透性，与与水的渗透率相互作用 钙结合蛋白，钙调蛋白。我实验室和其他人的数据表明，钙调蛋白与 AQP0的C末端尾部；但是，分子动力学（MD）模拟表明非经典相互作用 带有AQP0的胞质环。该MD预测尚未经过实验验证。除了AQP0- 蛋白质相互作用（AIM 1），我假设AQP0-脂质相互作用（AIM 2）调节AQP0渗透性和 透镜透明度的粘附特性；但是，关于AQP0交互的报告有限 与天然脂质。鉴于AQP0在维持晶状体透明度及其与白内障的联系中的重要作用， 了解与蛋白质（例如钙调蛋白和本地脂质）的调节性相互作用将阐明其在 微循环系统。镜头中第二大最丰富的膜蛋白是LIM2，就像AQP0一样， 突变与小鼠的白内施术相关。但是，关于lim2-蛋白的知之甚少 互动（目标1）。据报道，与LIM2的结合伙伴，即钙调蛋白和Galectin-3，但如何 这些相互作用调节LIM2结构和功能尚不清楚。此外，本地透镜脂质已经 据报道会影响LIM2亚基组装，但这种现象的细节尚未 探索。由于缺乏针对LIM2本地脂质相互作用的实验研究（AIM 2），我将使用 天然MS确定特定的脂质相互作用并确定它们如何影响LIM2结构。我的发现会 援助研究人员开发可以预防，逆转或延迟白内障的治疗目标和/或实践 形成。