Aggregation of Deamidated Crystallins as a Major Cause of Cataracts

项目摘要： 我们的实验室专注于我们已经确定的最普遍的化学修饰， 与存在于透镜核中的不溶性蛋白质有关-脱酰胺和氧化。这些 这些修饰与年龄相关的核性白内障最相关，核性白内障是目前最常见的白内障类型。在 这项拟议的工作，我们将研究脱酰胺和氧化之间的相互作用，以模拟 透镜中与年龄相关的过程。虽然透镜环境通常处于还原状态，但是， 晶状体蛋白中的巯基作为透镜谷胱甘肽的库，长期以来与年龄相关性白内障有关 随着透镜中心的老化而减小。非天然二硫键交联晶状体蛋白的形成 因此，预期通过Cys残基的氧化来降解亚基是导致细胞凋亡的关键过程。 透镜蛋白的聚集和不溶解。在目标1中，我们将确定如何具体，年龄相关 γS中的脱酰胺化通过识别响应于 结合脱酰胺和氧化。在目标2中，我们将检验非天然二硫键 形成导致γS的更高有序的低聚物。虽然目标1和2的重点是 脱酰胺的γS，目的3探讨了γS中这些与年龄相关的修饰是否会导致非天然交联 在γ-和β-晶状体蛋白亚基之间，从而破坏晶状体蛋白的天然四级排列。 总的来说，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，是年龄相关性核性白内障的基础。确立 脱酰胺作用主要通过增加晶状体蛋白的氧化来介导其作用， 用于开发治疗策略的稳健模型，旨在延迟与年龄相关的核 通过恢复透镜的抗氧化剂水平来治疗白内障。这项工作还将对以下方面产生重大影响： 其中长寿命蛋白质的脱酰胺和氧化与淀粉样蛋白原纤维相关的几种其它疾病 阵

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.