MODIFICATION OF ALPHA-CRYSTALLIN CHAPERONE FUNCTION

本研究的目的是阐明α -结晶蛋白（α a和α b）的一些常见翻译后修饰对分子伴侣特性的影响，即α -结晶蛋白保护其他蛋白质不变性和聚集的能力。通过选择生物学上相关的修饰，α -和α -晶体蛋白的不同结构域将成为体外结构修饰的目标，而体外结构修饰又与伴侣蛋白功能的变化相关。研究体外修饰将有助于表征体内修饰导致人α -晶体蛋白伴侣样性质下降的特性。这是有史以来第一个在体内和体外发生的翻译后修饰与伴侣活性和伴侣-靶蛋白结合相关的研究。这项提议的具体目的是基于我们最近的发现，糖基化，氧化和混合二硫的形成对α -结晶蛋白伴侣的功能有抑制作用，衰老和糖尿病都会降低伴侣的性质。以下具体目标是假设驱动的，每个目标都是为了检验一个特定的假设：1)通过H2O2氧化，抗坏血酸和果糖糖基化，以及与GSSG混合二硫形成对小牛或年轻人α -晶体蛋白的体外修饰，将用于验证这样的假设，即这些翻译后修饰会影响伴侣蛋白的功能，这是由α -晶体蛋白保护β -和γ -晶体蛋白免受热变性和聚集的能力决定的。此外，我们将研究一种以上修饰的联合作用，希望通过两种处理显示出协同效应。2)从1个月到90岁的人晶状体的α和α h分数将显示随着年龄的增长，翻译后修饰的α -晶体蛋白水平是否增加，伴蛋白功能改变。3)由于糖尿病晶状体比年龄匹配的非糖尿病晶状体暴露于更高水平的氧化和糖基化，我们将测试α -晶体蛋白伴侣功能在糖尿病人类或大鼠晶状体中发生更大改变的可能性。4)我们将通过质谱分析确定α -和α -晶体蛋白中的蛋白修饰，这些蛋白修饰将在体外引入或在体内发生，并与伴侣蛋白功能的变化相关，包括伴侣蛋白-靶蛋白结合。这些分析将确定产生伴侣性降低的修饰类型。5)我们将探讨在衰老或糖尿病过程中，伴侣蛋白在体外修饰或体内修饰导致其功能丧失的机制。我们将检验翻译后修饰影响伴侣-靶蛋白结合导致伴侣功能降低的假设。

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.