Role of deamidation in human beta-crystallin structure

描述(申请人提供)：我们研究的总体目标是确定脱酰胺在晶状体中的作用。在上一个资金周期中，我们令人信服地证明了脱酰胺导致了可溶性聚集体，破坏了-晶状体蛋白的稳定性，改变了-晶状体蛋白亚基之间的相互作用，并增加了伴侣-晶状体蛋白所需的-晶状体蛋白。这些结果提示了脱酰胺化诱导晶状体蛋白不溶解的机制，并有力地支持了脱酰胺化直接导致白内障的形成。根据我们的发现，我们假设去酰胺化降低了晶体蛋白的稳定性，导致聚集，最终触发白内障的形成。Aim 1的实验将识别晶状体中潜在的相关脱氨。下一步，我们将使用体内模型来确定先前发现的引起聚集或降低稳定性的脱氨基是否直接导致白内障的形成。目标2中的实验将通过识别β-晶体蛋白内部和与β-伴侣蛋白的变化相互作用来确定脱酰胺化触发聚集的机制。结晶蛋白中存在大量的脱酰亚胺位点。拟议的实验具有创新性，因为它们将区分功能相关的部位和有害的部位，使用最先进的方法直接测试体内脱酰胺的作用。未来的研究将筛选阻止晶体蛋白聚集的药物。与公共健康相关：白内障是世界范围内导致失明的主要原因，也是美国政府医疗保健系统的最大支出之一。我们的研究表明，晶状体中与年龄相关的主要修饰，脱酰胺，改变了晶状体晶体蛋白的结构和功能，并可能在体内诱导与白内障相关的聚集。能够阻止脱酰胺诱导的晶状体晶体蛋白聚集可能会预防白内障，并有助于预防其他聚集性疾病。

DESCRIPTION (provided by applicant): The overall goal of our research is to determine the role of deamidation in the lens. During the last funding cycle, we convincingly demonstrated that deamidation led to soluble aggregates, destabilized ?-crystallins, altered interactions between ? -subunits, and increased the ?-crystallin needed to chaperone ?-crystallins. These results suggest a mechanism for deamidation-induced insolubilization of crystallins and strongly support that deamidation contributes directly to cataract formation. Based on our findings, we hypothesize that deamidation decreases crystallin stability leading to aggregation that eventually triggers cataract formation. Experiments in Aim 1 will identify potentially relevant deamidations in the lens. Next, we will determine if deamidations previously identified to cause aggregation or decrease stability directly lead to cataract formation by using an in vivo model. Experiments in Aim 2 will determine the mechanism by which deamidation triggers aggregation, by identifying altered interactions within ? -crystallins and with the ?-chaperone. Numerous deamidation sites exist in the ? -crystallins. The proposed experiments are innovative in that they will distinguish between functionally relevant sites and those that are detrimental, using state-of-the art approaches to directly test the role of deamidation in vivo. Future studies will screen for agents that prevent crystallin aggregation. PUBLIC HEALTH RELEVANCE: Cataracts are the leading cause of blindness worldwide and one of the largest expenses to the U.S. government's health care system. Our research suggests that the major age-related modification in the lens, deamidation, alters structure and function of the lens crystallins and may induce aggregation associated with cataracts in vivo. Being able to prevent deamidation-induced aggregation of lens crystallins may prevent cataracts and help to prevent other aggregation diseases.