Crystallin-Derived Anti-Chaperones in the Lens

性状（由申请方提供）：认为晶体蛋白（主要透镜蛋白）结构的变化是老化导致透镜透明度丧失的原因。与年轻的非白内障晶状体相比，老年和白内障的人晶状体显示出增加的晶状体蛋白水解和聚集。然而，与年龄相关的蛋白质聚集的分子机制尚未完全了解。我们推测，老化透镜（>40岁）中的蛋白质聚集是由新型蛋白酶的作用引发的，这些蛋白酶识别晶状体蛋白中的特定序列，并负责切割晶状体蛋白和产生具有抗分子伴侣活性和毒性特性的晶状体蛋白片段。具有抗分子伴侣活性的晶状体蛋白片段在分子伴侣位点与1A-和1B-晶状体蛋白相互作用，导致已知在老化、不太透明的晶状体和白内障晶状体中发生的1-晶状体蛋白分子伴侣活性的丧失。此外，晶状体蛋白片段与完整的、修饰的或截短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-晶状体蛋白在透镜的核区域中的不溶解，导致透明度丧失和白内障形成。为了支持我们的假设，即堆叠衍生的抗分子伴侣肽在与年龄相关的透镜透明度丧失中具有关键作用，我们已经证明了体内产生的人透镜晶状体蛋白片段产生H2 O2，显示抗分子伴侣活性并诱导透镜蛋白聚集和沉淀。将在人类晶状体和年龄相关性白内障发生的豚鼠模型中进行研究。我们建议进行研究，以确定晶体蛋白片段的起源，并表征负责晶体蛋白的切割和抗伴侣肽的产生的蛋白水解机制。使用国家的最先进的质谱工具和分析技术，我们将扩大我们的理解的分子机制参与堆叠衍生肽介导的蛋白质聚集和白内障。1）研究年轻人、成年人、老年人和白内障患者晶状体以及正常豚鼠和高压氧（HBO）处理豚鼠晶状体中存在的低分子量晶状体蛋白片段。2)表征堆叠衍生的抗分子伴侣肽，并确定其在体外和离体诱导1-晶状体蛋白聚集的能力。3)研究序列特异性蛋白酶或非酶裂解是否负责产生堆叠衍生的抗分子伴侣肽，并分离和表征所涉及的新型蛋白酶。 公共卫生相关性：了解为什么透镜如此普遍地发展白内障可能导致预防这种常见原因的视力丧失的方法的发展。在老化过程中，由于某些未知的原因，透镜开始积累源自晶体蛋白（肽）的肽（分解产物），晶体蛋白是透镜中的主要蛋白质。随着时间的推移，这些肽在透镜中积累时变得有毒，导致透镜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.