Interaction of alpha-crystallin with cholesterol bilayer domains in cataract formation

α-晶状体蛋白与胆固醇双层结构域在白内障形成中的相互作用

• 批准号: 10186757
• 负责人: Laxman Mainali
• 金额: $ 29.26万
• 依托单位: BOISE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10186757
• 关键词: Affect; Affinity; Age; Animals; Binding; Biological; Cataract; Cell Nucleus; Cell membrane; Cholesterol; Crystalline Lens; Crystallins; Cytoplasm; Development; Electron Spin Resonance Spectroscopy; Eye; Goals; Health; Homeostasis; Human; Individual; Knowledge; Lead; Lecithin; Lipids; Measurement; Membrane; Membrane Lipids; Methods; Modeling; Mole the mammal; Molecular; Molecular Chaperones; Nuclear; Phosphatidylethanolamine; Phosphatidylserines; Phospholipids; Physiological; Play; Property; Proteins; Race; Recording of previous events; Reporting; Research; Role; Sampling; Sphingomyelins; Structural Protein; Techniques; Testing; Work; age group; age related; alpha-Crystallins; aqueous; base; experience; fiber cell; fluidity; insight; lens; lens transparency; light scattering; membrane model; oxygen transport; physical property; prevent; sex

PROJECT SUMMARY/ABSTRACT The objective of this proposal is to understand the interaction of α-crystallin with membrane cholesterol (Chol) and cholesterol bilayer domains (CBDs) in the fiber-cell plasma membranes of the human eye lens. CBDs are formed in the fiber-cell plasma membrane of the eye lens and have positive physiological functions, helping to maintain lens transparency and possibly protect against cataract formation. The soluble lens protein, α-crystallin, is a major structural protein that, under healthy conditions, forms a transparent lattice in the lens and plays a major role in maintaining lens transparency. Several discoveries report that the level of α-crystallin in the lens cytoplasm declines with age and cataract progression, accompanied by a corresponding increase in the amount of membrane-bound α-crystallin. However, the mechanism by which α-crystallin associates with fiber-cell plasma membrane and how the age-related change in membrane lipid composition affects the α-crystallin binding is unclear. I hypothesize that the binding of α-crystallin to membrane is inhibited by CBDs, which decreases the light scattering and helps maintain lens transparency. In their proposed role, CBDs should increase the level of α-crystallin in the lens cytoplasm favoring its chaperone function and maintaining lens cytoplasm homeostasis. I discovered that the properties of CBDs change significantly with age and are related to the size of the CBD, which increases with age and is greater in nuclear than in cortical membranes. Based on my extensive experience working with CBDs in model and human lens membranes, I will (i) determine the lipid composition in fiber-cell plasma membranes that promotes or inhibits the binding of α-crystallin to membranes, (ii) test the hypothesis that CBDs inhibit the binding of α-crystallin to membranes, and finally (iii) determine the effects of CBD on the binding of α-crystallin in clear and cataractous human lens membranes of different age groups. The analysis will include donor's health history, sex, and race. I developed electron paramagnetic resonance (EPR) methods to study small-volume aqueous biological samples (3 µL at X-band or 150 nL at W-band), particularly for studies of lens membranes obtained from the eyes of a single donor. This technique provides a major advantage when studying the binding of α-crystallin in membranes of age-matched clear and cataractous lenses from human donors. In addition, the EPR approach has the unique ability to simultaneously provide information about the CBDs and the binding of α-crystallin. For the last eight years, my research has focused on understanding the molecular organization of lipids and proteins in plasma membrane of intact fiber cells of human eye lenses. Building upon the knowledge I acquired during these studies, here I propose moving my research in a new direction to focus on the interaction of CBDs with α-crystallin. There is a clear need for a more in-depth understanding of the roles of CBDs in the binding of α-crystallin in the fiber cell plasma membrane. The findings from this study will help fill this gap and produce valuable insights in maintaining lens transparency.

项目概要/摘要 该提案的目的是了解 α-晶状体蛋白与膜胆固醇 (Chol) 的相互作用 人眼晶状体纤维细胞质膜中的胆固醇双层结构域（CBD）是。 在眼晶状体的纤维细胞质膜中形成，具有积极的生理功能，有助于 保持晶状体透明度并可能防止白内障形成可溶性晶状体蛋白，α-晶状体蛋白， 是一种主要的结构蛋白，在健康条件下，它在晶状体中形成透明晶格，并起到 一些发现表明，晶状体中 α-晶状体蛋白的水平在维持晶状体透明度方面发挥着重要作用。 细胞质随着年龄和白内障进展而减少，并伴随着数量的相应增加 然而，α-晶状体蛋白与纤维细胞血浆结合的机制。 膜以及膜脂质成分与年龄相关的变化如何影响 α-晶状体蛋白结合 不清楚。我认为 CBD 会抑制 α-晶状体蛋白与膜的结合，从而降低 光散射并有助于保持晶状体透明度 在其提议的作用中，CBD 应该提高光的水平。 晶状体细胞质中的α-晶状体蛋白有利于其伴侣功能并维持晶状体细胞质稳态I。 发现CBD的特性随着年龄的增长而显着变化，并且与CBD的大小有关， 根据我的广泛研究，它随着年龄的增长而增加，并且在核中比在皮质膜中更大。 凭借在模型和人类晶状体膜中使用 CBD 的经验，我将 (i) 确定 促进或抑制 α-晶状体蛋白与膜结合的纤维细胞质膜，(ii) 测试 假设 CBD 抑制 α-晶状体蛋白与膜的结合，最后 (iii) 确定 CBD 对不同年龄组透明和白内障人晶状体膜中 α-晶状体蛋白结合的影响。 分析将包括捐赠者的健康史、性别和种族。我开发了电子顺磁共振（EPR）。 研究小体积水性生物样品（X 波段 3 µL 或 W 波段 150 nL）的方法，特别是 为研究从单个捐赠者的眼睛获得的晶状体膜提供了重要的技术。 研究年龄匹配的透明晶状体和白内障晶状体膜中 α-晶状体蛋白的结合时的优势 此外，EPR 方法具有同时提供信息的独特能力。 关于 CBD 和 α-晶状体蛋白的结合 在过去的八年里，我的研究重点是 了解人类完整纤维细胞质膜中脂质和蛋白质的分子组织 基于我在这些研究中获得的知识，我建议将我的研究转移到眼睛镜片上。 关注 CBD 与 α-晶状体蛋白相互作用的新方向显然需要更深入的研究。 了解 CBD 在纤维细胞质膜中 α-晶状体蛋白结合中的作用。 这项研究将有助于填补这一空白，并在保持镜片透明度方面产生有价值的见解。