MODIFICATION OF ALPHA-CRYSTALLIN CHAPERONE FUNCTION

这项建议的目的是阐明一些常见的 α-晶体蛋白(αA和αB)的翻译后修饰 分子伴侣的性质，即α-晶状体蛋白对 保护其他蛋白质不会变性和聚集。通过选择 与生物相关的不同结构域的αA-和 α-晶状体蛋白将成为体外结构修饰的靶点 这反过来又会与伴侣功能的变化相关。 研究In的体外修饰将有助于In的表征 导致伴侣样属性下降的体内修饰 人类α-晶状体蛋白。这是有史以来第一项研究 体内发生的翻译后修饰，这可能是 在体外造成的伤害将与伴侣活性和 伴侣-靶蛋白结合。 这项建议的具体目的是基于我们最新的发现 糖基化、氧化和混合二硫化物的形成具有抑制作用 对α-晶状体蛋白伴侣功能的影响及衰老和 糖尿病会降低伴侣的属性。以下是具体目标 假设驱动，每个都旨在测试特定的假设：1)在 小牛或青年人α-晶状体蛋白的体外氧化修饰 用过氧化氢，用抗坏血酸和果糖糖基化，通过混合 将使用GSSG形成二硫化物来检验以下假设 这种翻译后修饰会影响伴侣功能 由α-晶体蛋白保护β-和β-晶体蛋白的能力决定 热变性和凝聚产生的γ-晶状体蛋白。此外, 我们将研究不止一种类型的修改的综合效果 希望通过两种治疗方法显示出协同效应。2)两个字母均为L 和字母H分数从1个月到90岁的人类晶状体 显示了随着年龄的增长，翻译后水平是否增加 伴随功能改变的修饰的α-晶状体蛋白积累。3) 由于糖尿病晶状体暴露在更高水平的氧化和 糖基化比年龄匹配的非糖尿病晶状体，我们将测试 α-晶体蛋白伴侣功能甚至发生改变的可能性 糖尿病人或大鼠的晶状体中更多。4)我们将鉴定蛋白质 将在#年引入的αA和αB晶体蛋白的修饰 通过质谱分析在体外或体内发生的，并与 伴侣功能的变化，包括伴侣与靶蛋白的结合。 这些分析将确定产生 减少监护人属性。5)我们将调查这一机制 由于体外或体内修饰而导致的伴侣功能的丧失 在衰老或糖尿病期间的修改。我们将检验这一假设 翻译后修饰会影响伴侣靶蛋白 结合导致伴侣功能减弱。

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.