Aggregation of Deamidated Crystallins as a Major Cause of Cataracts

项目总结： 我们的实验室专注于我们已确定的最普遍的化学修饰 与存在于晶状体细胞核中的不溶蛋白质有关--脱酰胺和氧化。这些 修饰与年龄相关的核性白内障最相关，这是迄今为止最常见的白内障类型。在……里面 在这项拟议的工作中，我们将研究脱酰胺和氧化之间的相互作用，以模拟 晶状体中与年龄有关的过程。尽管晶状体环境通常处于还原状态，但晶状体的氧化 晶体蛋白中的巯基长期以来被认为与老年性白内障有关，因为晶状体中含有谷胱甘肽。 随着晶状体中心年龄的增长而减少。非天然二硫化物交联结晶的形成 因此，通过氧化半胱氨酸残基的亚基预计是导致 晶状体蛋白的聚集和不溶解。在目标1中，我们将确定具体的、与年龄相关的 γ中的脱氨基S通过鉴定反应形成的非天然二硫键促进其聚集 联合脱酰亚胺和氧化。在目标2中，我们将检验非天然二硫键的假设 形成较高有序的γS齐聚物，而AIMS 1和2的焦点是氧化 疯狂的γS，Aim 3探索了γS中的这些与年龄相关的修改是否会导致非本地交叉链接 γ-和β-晶体蛋白亚基之间的相互作用，从而打乱晶体蛋白的天然四元排列。 总体而言，这些发现将阐明脱酰胺，一种自发的修饰，如何有助于 氧化驱动的聚集级联反应是老年性核性白内障的基础。确立这一点 脱酰亚胺主要通过加强晶体蛋白的氧化来调节其作用，从而提供 旨在延缓年龄相关核发病的治疗策略发展的稳健模型 通过恢复晶状体的抗氧化剂水平来防治白内障。这项工作也将对 长寿命蛋白的脱酰胺和氧化与淀粉样原纤维相关的其他几种疾病 队形。

Project Summary: Our laboratories have focused on the most prevalent chemical modifications that we have identified to be associated with the insoluble proteins present in the nucleus of the lens- deamidation and oxidation. These modifications are most relevant to age-related nuclear cataract, by far the most common type of cataract. In this proposed work, we will examine the interplay between deamidation and oxidation in order to mimic the age-related processes in the lens. Although the lens environment is normally in a reduced state, oxidation of sulfhydryls in crystallins has long been associated with age-related cataract as the pool of lens glutathione diminishes with aging in the center of the lens. The formation of non-native, disulfide crosslinked crystallin subunits via the oxidation of Cys residues is therefore anticipated to be a key process leading to the aggregation and insolubilization of lens proteins. In Aim 1, we will determine how specific, age-related deamidations in γS promote its aggregation by identifying non-native disulfide bond formed in response to combined deamidation and oxidation. In Aim 2, we will test the hypothesis that non-native disulfide bond formation leads to higher ordered oligomers of γS. While the focus of Aims 1 and 2 is the oxidation of deamidated γS, Aim 3 explores whether these age-related modifications in γS lead to non-native crosslinks between γ- and β-crystallin subunits and thereby disrupt the native quaternary arrangement of the crystallins. Overall, these findings will elucidate how deamidation, a spontaneous modification, contributes to the oxidation-driven aggregation cascade that underlies age-related nuclear cataract. Establishing that deamidation mediates its effects predominantly via augmenting the oxidation of crystallin proteins will provide a robust model for the development of therapeutic strategies aiming to delay the onset of age-related nuclear cataract by restoring the antioxidant levels of the lens. The work will also have significant implications for several other diseases where deamidation and oxidation of long-lived proteins is associated with amyloid fibril formation.