Crystallin-Derived Anti-Chaperones in the Lens

晶状体中的晶状体蛋白衍生的反伴侣分子

• 批准号: 7992774
• 负责人: KRISHNA K SHARMA
• 金额: $ 37万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7992774
• 关键词: Abbreviations; Accounting; Age; Aging; Alanine; Alcohol dehydrogenase; Alkanesulfonates; Arts; Binding Sites; Biochemical; Blindness; Cataract; Cavia; Cell Nucleus; Cells; Cleaved cell; Complex; Crystallins; Disease; Epithelial Cells; Generations; Goals; Human; Hydrogen Peroxide; Hyperbaric Oxygen; In Vitro; Lasers; Lead; Lens Opacities; Mass Spectrum Analysis; Measures; Mediating; Methods; Mind; Modeling; Molecular; Molecular Chaperones; Molecular Weight; Nuclear; Peptide Hydrolases; Peptides; Physical condensation; Post-Translational Protein Processing; Precipitation; Predisposition; Property; Proteins; Proteolysis; Reaction; Relative (related person); Role; Sampling; Senile Cataract; Site; Spatial Distribution; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structure; Surface Plasmon Resonance; Systems Analysis; Techniques; Testing; Time; Toxic effect; Water; age related; aged; analytical tool; in vivo; inhibitor/antagonist; lens; lens protein; lens transparency; method development; novel; prevent; protein aggregate; protein aggregation; protein structure; public health relevance; tris(2-carboxyethyl)phosphine; young adult

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.

描述（由申请人提供）：晶状体蛋白（主要的晶状体蛋白）结构的变化被认为是随着老化而发生的晶状体透明度丧失的原因。与年轻的非白内障晶状体相比，老年和白内障的人类晶状体表现出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（> 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并生成具有抗伴侣活性和毒性的晶状体蛋白片段。特性。具有抗伴侣活性的晶状体蛋白片段与伴侣位点的 1A- 和 1B- 晶状体蛋白相互作用，导致 1- 晶状体蛋白伴侣活性的丧失，这种活性已知发生在老化的、不太透明的晶状体和白内障晶状体中。此外，晶状体蛋白片段与完整、修饰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以及1-晶状体蛋白在晶状体核区域的不溶解，导致透明度丧失和白内障形成。为了支持我们的假设，即晶状体蛋白衍生的抗伴侣肽在与年龄相关的晶状体透明度丧失中起着关键作用，我们已经证明体内产生的人晶状体蛋白片段产生H2O2，表现出抗伴侣活性并诱导晶状体蛋白质聚集和沉淀。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研究。我们建议进行研究，以确定晶状体蛋白片段的起源，并表征负责晶状体蛋白裂解和抗伴侣肽生成的蛋白水解机制。使用最先进的质谱工具和分析技术，我们将扩大对晶状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。拟议的研究将实现以下具体目标：1）研究年轻、成人、老年和白内障人类晶状体以及正常和高压氧（HBO）处理的豚鼠晶状体中存在的低分子量晶状体蛋白片段。 2) 表征晶状体蛋白衍生的抗伴侣肽并确定它们在体外和离体诱导 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晶状体蛋白衍生的抗伴侣肽，并分离和表征所涉及的新型蛋白酶。 公共健康相关性：了解晶状体为何如此常见地产生白内障可能有助于开发预防这种视力丧失常见原因的方法。在老化过程中，由于某种未知的原因，晶状体开始积累源自晶状体蛋白（肽）的肽（分解产物），晶状体蛋白是晶状体中的主要蛋白质。随着时间的推移，这些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。