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

摘要：白内障是世界范围内致盲的主要原因，给美国医疗保健系统造成了巨大的损失 每年数十亿美元用于外科治疗。透镜混浊与主要眼功能障碍有关， 膜蛋白，水通道蛋白-0（AQP 0）和Lim 2。我的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，微循环系统输送营养物质 并将废物移到内部纤维细胞中，并且对于在几十年的寿命中保持透镜的透明度是必不可少的; 然而，这种微循环系统是如何建立和维持的，作为年龄的函数， 明白使用先进的质谱技术，如氢氘交换-MS， 天然MS和化学交联研究，我打算阐明如何具体的蛋白质和脂质相互作用 影响AQP 0和Lim 2的结构和功能;膜蛋白是基础的 透镜的微循环系统。最丰富的透镜膜蛋白，AQP 0，在晶状体的形成中起重要作用。 透镜纤维细胞粘附和透水性，透水性通过与晶状体纤维细胞的相互作用来调节。 钙结合蛋白，钙调蛋白。我的实验室和其他实验室的数据表明，钙调素与 AQP 0的C-末端尾;然而，分子动力学（MD）模拟表明非典型相互作用 有一个水通道蛋白0的胞浆环这一MD预测尚未得到实验验证。除了AQP 0- 蛋白质相互作用（目的1），我假设AQP 0-脂质相互作用（目的2）调节AQP 0渗透性， 作为透镜透明度基础的粘附特性;然而，关于AQP 0相互作用的报告有限 与天然脂质。鉴于AQP 0在维持透镜透明度方面的重要作用及其与白内障的联系， 了解与蛋白质如钙调素和天然脂质的调节相互作用将阐明其在细胞凋亡中的作用。 微循环系统透镜中第二丰富的膜蛋白是Lim 2，与AQP 0一样， 突变与小鼠白内障的发生有关;然而，对Lim 2蛋白知之甚少。 相互作用（目标1）。已经报道了Lim 2的结合配偶体，即钙调蛋白和半乳糖凝集素-3，但是如何结合Lim 2呢？ 这些相互作用调节Lim 2的结构和功能尚不清楚。此外，天然透镜脂质已经被 据报道，影响Lim 2亚基组装，但这一现象背后的细节还没有 探讨了由于缺乏关于Lim 2-天然脂质相互作用的实验研究（目标2），我将使用 天然MS鉴定特定的脂质相互作用，并确定它们如何影响Lim 2结构。我的发现将 研究人员开发了可以预防、逆转或延迟白内障的治疗目标和/或实践， 阵

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.