Crystallin-Derived Anti-Chaperones in the Lens

描述（由申请人提供）：晶体蛋白（晶状体的主要蛋白质）的结构变化被认为是晶状体随着年龄增长而失去透明度的原因。与年轻、非白内障的人晶状体相比，老年和白内障的人晶状体显示晶状体蛋白水解和聚集增加。然而，与年龄相关的蛋白质聚集的分子机制尚不完全清楚。我们假设老化晶状体（bbb40岁）中的蛋白质聚集是由新蛋白酶的作用引发的，该蛋白酶识别晶体蛋白中的特定序列，并负责晶体蛋白的切割和产生具有抗伴侣活性和毒性的晶体蛋白片段。具有抗伴侣蛋白活性的晶体蛋白片段与伴侣蛋白位点的1A-和1b -晶体蛋白相互作用，导致1-晶体蛋白伴侣蛋白活性的丧失，已知发生在老化、不透明的晶状体和白内障晶状体中。此外，晶体蛋白片段与完整的、修饰的或截短的晶体蛋白之间的相互作用导致年龄相关的蛋白质聚集和晶状体核区1-晶体蛋白的不溶化，导致透明度丧失和白内障的形成。为了支持我们的假设，即晶体衍生的抗伴侣肽在与年龄相关的晶状体透明度丧失中起关键作用，我们已经证明，在体内产生的人晶状体晶体蛋白片段产生H2O2，显示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与年龄相关的白内障发生的豚鼠模型中进行。我们建议开展研究，以确定晶体蛋白片段的起源，并表征晶体蛋白的裂解和抗伴侣肽的产生的蛋白水解机制。利用最先进的质谱工具和分析技术，我们将扩大我们对晶体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。拟开展的研究将实现以下具体目标：1)研究存在于年轻、成人、老年和白内障人晶状体以及正常和高压氧（HBO）处理豚鼠晶状体中的低分子量晶体蛋白片段。2)对晶体蛋白衍生的抗伴侣肽进行表征，并在体外和离体实验中确定其诱导1-晶体蛋白聚集的能力。3)研究序列特异性蛋白酶或非酶促裂解是否与晶体衍生的抗伴侣肽的产生有关，并分离和表征所涉及的新型蛋白酶。

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.