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

项目总结/摘要 本研究旨在了解α-晶状体蛋白与膜胆固醇（Chol）的相互作用 和人眼透镜的纤维细胞质膜中的胆固醇双层结构域（CBD）。CBD是 形成于眼透镜的纤维细胞质膜中，具有积极的生理功能，有助于 保持透镜的透明度并可能防止白内障的形成。可溶性透镜蛋白，α-晶状体蛋白， 是一种主要的结构蛋白，在健康条件下，在透镜中形成透明晶格， 主要作用是保持透镜的透明度。有几项发现报告说，透镜中的α-晶状体蛋白水平 细胞质随着年龄和白内障进展而下降，同时数量相应增加 膜结合的α-晶体蛋白。然而，α-晶状体蛋白与纤维细胞浆结合的机制 膜以及与年龄相关的膜脂质组成变化如何影响α-晶状体蛋白结合， 不清楚我推测α-晶状体蛋白与膜的结合被CBD抑制，这降低了α-晶状体蛋白与膜的结合。 光散射并有助于保持透镜的透明度。在其拟议的作用中，CBD应提高 透镜细胞质中的α-晶状体蛋白有利于其分子伴侣功能并维持透镜细胞质的稳态。我 发现CBD的性质随着年龄的增长而发生显着变化，并且与CBD的大小有关， 随着年龄的增长而增加，并且在细胞核中比在皮质膜中更大。基于我对 根据在模型和人类透镜膜中使用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）检测 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透镜膜中α-晶状体蛋白结合的影响。的 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 用于研究从单个供体的眼睛获得的透镜膜。这项技术提供了一个主要的 研究年龄匹配的透明晶状体和白内障晶状体膜中α-晶状体蛋白结合时的优势 从人类捐赠者。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，我的研究主要集中在 了解人类完整纤维细胞质膜中脂质和蛋白质的分子结构 眼镜片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 为研究CBD与α-晶体蛋白的相互作用提供了新的方向。显然需要更深入地 了解CBD在纤维细胞质膜α-晶体蛋白结合中的作用。这些发现 从这项研究将有助于填补这一空白，并产生宝贵的见解，在保持透镜透明度。

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.