Deamidation in Human Beta-Crystallin Structure

描述(申请人提供)：脱酰胺化是晶状体中最普遍的翻译后修饰，并随着年龄的增长而增加。所有主要的晶状体蛋白都是去胺化的，其中β-晶状体蛋白去胺化最严重。我们的实验室最近在晶体蛋白中发现了几个特定的体内脱酰胺位点，这使得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-亚基之间高度保守的区域，这表明这些脱酰胺作用发生在关键的结构区域。然而，脱酰胺对晶体蛋白结构和稳定性的影响尚不清楚。晶状体蛋白的稳定性对晶状体的透明度至关重要。只有当我们开始阐明白内障的形成时，才能了解白内障的形成。 影响晶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晶状体中β-晶状体蛋白的影响。这项赠款申请的目的是表征几个特定的体内β-晶体蛋白的脱酰胺化位点。我们假设，临界区的脱酰胺作用破坏了结构和促进不溶性的稳定性，而非临界区的脱酰胺作用促进了溶解性。我们的假设源于我们之前的研究，该研究表明，结构域之间的界面上的脱酰胺化降低了稳定性，而其他位置的脱酰胺化提高了稳定性，或者影响很小。为了验证我们的假设，我们将使用定点突变在预测具有显著结构意义的区域中引入体内脱氨基。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。只有在我们了解了脱酰胺如何改变晶体蛋白结构之后，我们才能将特定的脱酰胺作用与白内障的形成联系起来。然后，我们可以开始研究防止脱酰胺的方法，或者设计防止这些部位结构变化的伴侣。

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.