Role of Deamidation in Human Beta-Crystallin Structure

描述（由申请方提供）：脱酰胺是透镜中最常见的翻译后修饰，并随年龄增长而增加。所有主要晶体蛋白都是脱酰胺的，其中β-晶体蛋白脱酰胺最严重。我们的实验室最近已经确定了几个特定的晶体蛋白脱酰胺的体内位点，使我们能够研究这种修饰的功能后果。几个脱酰胺位点位于β亚基之间的高度保守区域，表明这些脱酰胺发生在关键结构区域。然而，脱酰胺对晶状体蛋白结构和稳定性的影响尚不清楚。晶体蛋白的稳定性是透镜透明度的基础。白内障的形成只有在我们开始阐明 改变晶状体蛋白稳定性的因素。 我们研究的长期目标是确定脱酰胺对人类透镜中β-晶体蛋白的影响。本授权申请的目的是表征β-晶体蛋白脱酰胺的几个特定体内位点。我们假设，在关键区域的脱酰胺破坏结构和稳定性促进不溶性，而在非关键区域的脱酰胺促进溶解度。我们的假设是来自我们以前的研究表明，在结构域之间的界面脱酰胺降低稳定性，而在其他网站的脱酰胺增加稳定性，或几乎没有影响。为了验证我们的假设，我们将使用定点诱变在预测结构显著的区域中引入体内脱酰胺。然后，我们将使用先进的生物物理技术，如多角度激光光散射和动态光散射来表征它们的结构，稳定性和聚集趋势。只有了解脱酰胺作用如何改变晶状体蛋白结构后，我们才能将特定的脱酰胺作用与白内障形成联系起来。然后，我们可以开始研究防止脱酰胺的方法或设计防止这些位点结构变化的分子伴侣。

DESCRIPTION (provided by applicant): Deamidation is the most prevalent post-translational modification in the lens, and increases with age. All of the major crystallins are deamidated, with the beta-crystallins the most heavily deamidated. Our laboratory has recently identified several specific in vivo sites of deamidation in crystallins allowing us to investigate the functional consequences of this modification. Several deamidation sites are in highly conserved regions between beta-subunits, suggesting that these deamidations occur in critical structural regions. However, the effects of deamidation on crystallin structure and stability are not known. The stability of crystallins is fundamental to the transparency of the lens. The formation of cataracts will only be understood when we begin to elucidate factors that alter crystallin stability. The long-term goal of our research is to determine the effects of deamidation on beta-crystallins in the human lens. The objective of this grant application is to characterize several specific in vivo sites of deamidation of beta-crystallins. We hypothesize that deamidation in critical regions disrupt structure and stability promoting insolubilization, while deamidations in non-critical regions foster solubility. Our hypothesis is derived from our previous studies showing that a deamidation at the interface between domains decreased stability, while deamidations at other sites increased stability, or had little effect. To test our hypothesis, we will use site directed mutagenesis to introduce in vivo deamidations in regions predicted to be structurally significantly. We will then characterize their structure, stability, and tendency to aggregate using advanced biophysical techniques, such as multi-angle laser light scattering, and dynamic light scattering. Only after we understand how deamidation alters crystallin structure can we correlate specific deamidations to cataract formation. We can then begin to investigate methods to prevent deamidation or design chaperones that prevent structural changes at these sites.