MODIFICATION OF ALPHA-CRYSTALLIN CHAPERONE FUNCTION

本提案的目的是阐明一些常见的 α-晶状体蛋白（α A和α B）的翻译后修饰 分子伴侣特性，即，α-晶状体蛋白 保护其他蛋白质免于变性和聚集。 通过选择 生物学相关修饰α A-和 α B-晶体蛋白将被靶向用于体外结构修饰 这又将与伴侣蛋白功能的变化相关。 体外修饰的研究将有助于表征在体外的修饰。 体内修饰导致蛋白伴侣样性质的下降， 人α-晶状体蛋白。 这是有史以来第一个 在体内发生的翻译后修饰， 在体外造成的伤害将与伴侣活性相关， 伴侣蛋白-靶蛋白结合。 这项建议的具体目标是基于我们最近的调查结果 糖化、氧化和混合二硫键形成具有抑制作用， 对α-晶状体蛋白伴侣功能的影响， 糖尿病降低了伴侣蛋白的特性。 具体目标如下： 假设驱动，每个设计用于测试特定的假设：1）在 通过氧化对小牛或年轻人α-晶状体蛋白的体外修饰 用H2 O2，用抗坏血酸和果糖糖化， 将使用GSSG形成二硫化物来检验以下假设： 这种翻译后修饰将影响分子伴侣的功能 如通过α-晶状体蛋白保护β-和 γ-晶状体蛋白的热变性和聚集。 此外，本发明还提供了一种方法， 我们将研究一种以上改性的综合效果 希望通过两种处理来显示协同效应。2)与alphaL 从1个月到90岁的人类晶状体中的α H分数， 显示随着年龄的增长， 具有改变的伴侣蛋白功能的修饰的α-晶状体蛋白积累。第三章 由于糖尿病镜片暴露于更高水平的氧化， 糖化比年龄匹配的非糖尿病镜片，我们将测试 α-晶状体蛋白伴侣蛋白功能改变的可能性， 糖尿病患者或大鼠的晶状体中更多。4)我们将鉴定出 将引入的α A-和α B-晶体蛋白中的修饰 体外或体内发生，并与 分子伴侣功能的变化，包括分子伴侣-靶蛋白结合。 这些分析将确定产生 减少伴侣的属性。5)我们将研究其机制 由于体外修饰或体内修饰导致的伴侣蛋白功能丧失 在衰老或糖尿病期间的变化。 我们将检验这个假设， 翻译后修饰影响伴侣蛋白靶蛋白 结合导致伴侣蛋白功能降低。

The objective of this proposal is to elucidate the effect of some common posttranslational modifications of alpha-crystallin (alphaA and alphaB) on the molecular chaperone property, i.e., the ability of alpha-crystallin to protect other proteins from denaturation and aggregation. By choosing biologically relevant modifications different domains of alphaA- and alphaB-crystallins will be targeted for in vitro structural modifications which in turn will be correlated with changes in chaperone function. Studying in vitro modification will facilitate the characterization of in vivo modifications that cause a decline in chaperone like property of human alpha-crystallin. This is the first study ever where posttranslational modifications that occur in vivo and that can be inflicted in vitro will be correlated with chaperone activity and chaperone-target protein binding. The specific aims of this proposal are based on our most recent findings that glycation, oxidation and mixed disulfide formation have inhibitory effects on alpha-crystallin chaperone function and that both aging and diabetes decrease the chaperone property. The following specific aims are hypothesis driven, each designed to test a specific hypothesis: 1) In vitro modifications of calf or young human alpha-crystallin by oxidation with H2O2, by glycation with ascorbic acid and fructose, and by mixed disulfide formation with GSSG will be used to test the hypothesis that such posttranslational modifications will influence the chaperone function as determined by the ability of alpha-crystallin to protect beta- and gamma-crystallin from thermal denaturation and aggregation. In addition, we will study the combined effect of more than one type of modification hoping to show a synergistic effect by two treatments. 2) With both alphaL and alphaH fractions from 1 month to 90 years old human lenses it will be shown whether with increasing age increasing levels of posttranslationally modified alpha-crystallin with altered chaperone function accumulates. 3) Since diabetic lenses are exposed to higher levels of oxidation and glycation than age-matched non-diabetic lenses we will test the possibility that the alpha-crystallin chaperone function is altered even more in diabetic human or rat lenses. 4) We will identify the protein modifications in alphaA- and alphaB-crystallins that will be introduced in vitro or that occur in vivo by mass spectral analysis and correlate with changes in chaperone function including chaperone-target protein binding. These analyses will identify the types of modifications that produce a decrease in the chaperone property. 5) We will investigate the mechanism of the loss of chaperone function due to in vitro modifications or in vivo modifications during aging or diabetes. We will test the hypothesis that posttranslational modifications affect the chaperone-target protein binding leading to reduced chaperone function.