Metastable Crystallins: Structure and Stabilization

描述(由申请人提供)：许多不同的疾病，包括白内障、某些类型的青光眼、阿尔茨海默病、糖尿病和癌症，现在已知具有聚集、错误折叠或修饰的蛋白质沉积的共同特征。这组疾病的病理特征是特定细胞、组织或器官中的蛋白质聚集和沉积。病理上的相似性表明，控制蛋白质相互作用的共同原则是蛋白质错误折叠退行性疾病的基础。白内障是蛋白质错误折叠和聚集性疾病的典型例子。白内障是世界上导致失明的主要原因之一，它是由于年龄相关的晶状体蛋白质的修饰和聚集而发展起来的。α-晶体蛋白占成人晶状体蛋白的近40%，但其结构与功能尚不完全清楚。类似于伴侣的活动 α-晶体蛋白被认为在维持晶状体透明度方面起着核心作用。在老化过程中，晶状体晶体蛋白被截断，这些修饰与晶状体晶体蛋白聚集体有关，这些聚集体负责光散射。众所周知，α-晶体蛋白的加入阻止了聚集蛋白的聚集和光散射的进一步增加。我们已经证明了α-晶体蛋白亚基中的特定序列抑制了变性蛋白的聚集。然而，α-晶体蛋白衍生的多肽(迷你伴侣)的全部潜力尚未实现。为了增强 根据我们对非天然相互作用在白内障形成中作用的理解，我们提出了以下具体目标：目的1.使用新的氢化交联剂、生物苷化试剂和多肽阵列，鉴定导致人类白内障形成的A-晶体蛋白突变体(αAR49C、αAF71V、αAG98R和αAR116H)中导致蛋白质聚集的αA-晶体蛋白亚单位异常相互作用所涉及的序列。目的2.研究αA-晶状体蛋白衍生的小分子伴侣阻止亚稳态αA-晶状体蛋白(突变体和截短晶状体蛋白)聚集和恢复伴侣活性的机制和效果。具体目标将使用新的交联剂和质谱学方法来实现。我们还将使用定点突变和细胞培养表达和分析系统来确认交叉连接数据。这些创新的研究将让我们对蛋白质错误折叠疾病的潜在干预有新的见解，不仅是眼睛，还有身体的其他部位。

DESCRIPTION (provided by applicant): Many diverse diseases, including cataract, some types of glaucoma, Alzheimer disease, diabetes and cancer to name only a few, are now known to share the common feature of aggregated, misfolded or modified protein deposits. The pathological hallmark in this group of diseases is protein aggregation and deposition in specific cells, tissues or organs. The pathological similarities indicate that common principles that govern protein interactions underlie protein misfolding degenerative diseases. Cataract is a classic example of a protein misfolding and aggregation disease. Cataract, a major cause of blindness in the world, develops as a result of age-related modifications and aggregation of the lens proteins. α-crystallin accounts for nearly 40% of the adult lens proteins but its structure-function is yet to be fully understood. The chaperone-like activity of α-crystallin is believed to play a central role in maintaining lens transparency. During aging, lens crystallins undergo truncation and these modifications correlate with lens crystallin aggregations responsible for light scattering. It is well established that the addition of α-crystallin to aggregating proteins stops a further increase in aggregation and light scattering. We have demonstrated that specific sequences in α-crystallin subunits suppress aggregation of denaturing proteins. However, the full potential of α-crystallin-derived peptides (mini-chaperones) is yet to be realized. To enhance our understanding of the role of non-native interactions in cataract formation, we propose the following specific aims: Aim 1. Identify the sequences involved in abnormal interactions of αA-crystallin subunits that lead to protein aggregation in human cataract-causing A-crystallin mutants (αAR49C, αAF71V, αAG98R and αAR116H) using novel deuterated cross linkers, biotynylated reagents and peptide arrays. Aim 2. Determine the mechanism and efficacy of αA-crystallin-derived mini-chaperone in preventing the aggregation of metastable αA- crystallins (mutants and truncated crystallins) and restoring chaperone activity. The specific aims will be accomplished using novel cross-linkers and mass spectrometric methods. We will also use site-directed mutagenesis and cell culture expression and analysis systems to confirm the cross linking data. These innovative studies will give us new insights into potential interventions for protein misfolding diseases, not only of the eye but also of other parts of the body.