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: 10542473
• 负责人: Carla O'Neale
• 金额: $ 0.25万
• 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/10542473
• 关键词: AQP1 gene; 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; Molecular; Mus; Mutation; Nutrient; Operative Surgical Procedures; Permeability; Physiological; Play; Property; Protein Analysis; 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 function; reconstitution; simulation; 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：白内障是世界范围内导致失明的主要原因，使美国医疗系统蒙受损失 每年数十亿美元用于外科治疗。晶状体混浊与主要的 膜蛋白，水通道蛋白-0(AQP0)和LIM2。我这个项目的总体目标是开发一个 全面了解蛋白质-蛋白质和蛋白质-脂质相互作用的结构和功能 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物质 并清除内部纤维细胞的废物，对于几十年的使用寿命保持镜片的透明度是必不可少的； 然而，这种微循环系统是如何随着年龄的变化而建立和维持的还不是很好。 明白了。使用先进的质谱学技术，如氢-氚交换-质谱仪， 通过对天然-MS和化学交联的研究，我打算阐明特定的蛋白质和脂肪是如何相互作用的 影响AQP0和LIM2的结构和功能；膜蛋白是 晶状体的微循环系统。最丰富的晶状体膜蛋白AQP0在 晶状体纤维细胞粘附性和透水性通过与水的相互作用来调节 钙结合蛋白，钙调素。来自我的实验室和其他人的数据表明，钙调蛋白与 AQP0的C-末端；然而，分子动力学(MD)模拟表明存在非正则相互作用 与AQP0的胞浆环路。这一MD预测尚未得到实验验证。除了AQP0- 蛋白质相互作用(目标1)，我假设AQP0-脂质相互作用(目标2)调节AQP0通透性和 作为晶状体透明度基础的粘附性；然而，关于AQP0相互作用的报道有限 用天然的脂肪。鉴于AQP0在维持晶状体透明度及其与白内障的联系中起着至关重要的作用， 了解与钙调蛋白和天然脂类等蛋白质的调节相互作用将澄清它在 微循环系统。晶状体中第二丰富的膜蛋白是LIM2，和AQP0一样，它的 突变与小鼠白内障的发生有关；然而，对Lim2蛋白知之甚少。 互动(目标1)。已经报道了与Lim2结合的伙伴，即钙调蛋白和Galectin-3，但如何结合 这些相互作用对LIM2的结构和功能的调控尚不清楚。此外，天然的晶状体脂质已经被 据报道影响Lim2亚基组装，但这一现象背后的细节尚未得到 探索过了。由于缺乏对Lim2-天然脂质相互作用的实验研究(目标2)，我将使用 天然MS用于识别特定的脂类相互作用并确定它们如何影响LIM2结构。我的发现将 帮助研究人员开发可以预防、逆转或延缓白内障的治疗目标和/或做法 队形。