Crystallin-Derived Anti-Chaperones in the Lens

描述(申请人提供)：晶状体蛋白结构的变化，是晶状体的主要蛋白质，被认为是导致晶状体透明度随年龄增长而丧失的原因。与年轻的非白内障晶状体相比，老年性和白内障患者的晶状体蛋白降解和聚集增加。然而，与年龄相关的蛋白质聚集的分子机制还不完全清楚。我们假设，老化晶状体(40岁)中的蛋白质聚集是由新型蛋白酶的作用启动的，这种酶识别晶体蛋白中的特定序列，并负责切割晶体蛋白并产生具有抗伴侣活性和毒性特性的晶体蛋白片段。具有抗伴侣活性的晶体蛋白片段与位于伴侣位点的1A和1B-晶体蛋白相互作用，导致已知在老化、不透明的晶状体和白内障晶体中发生的1-晶体蛋白伴侣活性的丧失。此外，晶状体蛋白片段与完整、修饰或截断的晶状体蛋白之间的相互作用导致与年龄相关的蛋白质聚集和1-晶状体蛋白在晶状体核区的不溶解，导致透明度丧失和白内障的形成。为了支持我们的假设，即晶体蛋白衍生的抗伴侣多肽在与年龄相关的晶状体透明度丧失中起关键作用，我们证明了在体内产生的人晶体蛋白片段产生过氧化氢，显示出抗伴侣活性，并诱导晶状体蛋白聚集和沉淀。研究将在人类晶状体和年龄相关性白内障形成的豚鼠模型中进行。我们建议开展研究，以确定晶体蛋白片段的来源，并表征导致晶体蛋白裂解和抗伴侣蛋白多肽产生的蛋白分解机制。使用最先进的质谱学工具和分析技术，我们将扩大我们对晶体蛋白衍生肽介导的蛋白质聚集和白内障发生的分子机制的理解。所提出的研究将实现以下特定目标：1)研究低分子量晶体蛋白片段存在于年轻、成人、老年和白内障人晶状体以及正常和高压氧(HBO)处理的豚鼠晶状体中。2)鉴定晶体蛋白衍生的抗伴侣蛋白多肽，并测定它们在体外和体外诱导1-晶体蛋白聚集的能力。3)研究序列特异性蛋白水解酶或非酶裂解是否导致晶体蛋白衍生的抗伴侣蛋白多肽的产生，并分离和鉴定涉及的新的蛋白水解酶。 与公共健康相关：了解为什么晶状体如此普遍地发展为白内障，可能会导致开发预防这种常见的视力丧失原因的方法。在老化过程中，由于某些未知的原因，晶状体中的主要蛋白质--晶体蛋白(多肽)开始积累多肽(分解产物)。随着时间的推移，这些多肽在晶状体中积累时会变得有毒，导致晶状体透明度丧失和白内障。

DESCRIPTION (provided by applicant): Changes in the structure of crystallins, the major lens protein, are thought to account for the loss of lens transparency that occurs with aging. Aged and cataractous human lenses show increased crystallin proteolysis and aggregation as compared to young, non-cataractous lenses. However, the molecular mechanisms for age-related protein aggregation are not fully understood. We hypothesize that protein aggregation in the aging lens (>40 years old) is initiated by the actions of novel proteases, which recognize a specific sequence in crystallins and are responsible for cleavage of crystallin and generation of crystallin fragments with anti-chaperone activity and toxic properties. The crystallin fragments with anti-chaperone activity interact with 1A- and 1B-crystallins at chaperone sites, contributing to the loss of the 1-crystallin chaperone activity known to occur in aged, less transparent lenses and cataractous lenses. Additionally the interactions between crystallin fragments and intact, modified or truncated crystallins lead to age-related protein aggregation and insolubilization of 1-crystallin in the nuclear region of the lens, resulting in loss of transparency and cataract formation. In support of our hypothesis that crystallin-derived anti-chaperone peptides have a key role in the age-related loss of lens transparency, we have demonstrated that in vivo-generated human lens crystallin fragments generate H2O2, display anti-chaperone activity and induce lens protein aggregation and precipitation. Studies will be performed in both human lenses and in the guinea pig model of age-related cataractogenesis. We propose to undertake studies to identify the origin of crystallin fragments and to characterize the proteolytic mechanisms responsible for the cleavage of crystallins and the generation of anti-chaperone peptides. Using state-of-the-art mass spectrometric tools and analytical techniques, we will expand our understanding of the molecular mechanisms involved in crystallin-derived-peptide mediated protein aggregation and cataractogenesis. The proposed studies will accomplish the following Specific Aims: 1) Investigate the low-molecular weight crystallin fragments present in young, adult, aged and cataractous human lenses and in lenses of normal and hyperbaric oxygen (HBO)-treated guinea pigs. 2) Characterize the crystallin-derived anti-chaperone peptides and determine their ability to induce 1- crystallin aggregation in vitro and ex vivo. 3) Investigate whether sequence-specific proteases or non-enzymatic cleavages are responsible for the generation of crystallin-derived anti-chaperone peptides and isolate and characterize the novel protease involved. PUBLIC HEALTH RELEVANCE: Understanding why the lens so commonly develops a cataract may lead to the development of methods for preventing this common cause of vision loss. During aging, for some unknown reason, the lens begins to accumulate peptides (breakdown products) derived from crystallin (peptides), the major protein in the lens. Over time, these peptides become toxic in the lens as they accumulate, leading to loss of lens transparency and cataract.