MODIFICATION OF ALPHA-CRYSTALLIN CHAPERONE FUNCTION

本提案的目的是阐明α-晶状体蛋白（α A和α B）的一些常见翻译后修饰对分子伴侣性质的影响，即，α-晶状体蛋白保护其它蛋白质免于变性和聚集的能力。通过选择生物学相关的修饰，α A-和α B-晶体蛋白的不同结构域将被靶向用于体外结构修饰，这反过来将与分子伴侣功能的变化相关。研究体外修饰将有助于表征导致人α-晶状体蛋白分子伴侣样性质下降的体内修饰。这是有史以来第一个研究，在体内发生的翻译后修饰，并可以在体外造成将与伴侣蛋白活性和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发现，即糖化，氧化和混合二硫化物形成对α-晶状体蛋白伴侣功能有抑制作用，衰老和糖尿病都会降低伴侣的特性。以下具体目标是假设驱动的，每个目标都旨在测试特定的假设：1）通过用H2 O2氧化，通过用抗坏血酸和果糖糖化，以及通过与GSSG形成混合二硫键，将用于检验这样的翻译后修饰将影响分子伴侣功能的假设，如通过α-晶状体蛋白以保护β-和γ-晶状体蛋白免于热变性和聚集。此外，我们将研究一种以上改性的组合效果，希望通过两种处理显示协同效应。2)对于1个月至90岁的人晶状体的α L和α H级分，将显示是否随着年龄的增加，具有改变的伴侣蛋白功能的后修饰的α-晶状体蛋白的水平增加。3)由于糖尿病镜片比年龄匹配的非糖尿病镜片暴露于更高水平的氧化和糖化，我们将测试α-晶状体蛋白伴侣蛋白功能在糖尿病人类或大鼠镜片中改变更多的可能性。4)我们将通过质谱分析确定体外引入或体内发生的α A和α B晶体蛋白中的蛋白质修饰，并与分子伴侣功能（包括分子伴侣-靶蛋白结合）的变化相关。这些分析将确定类型的修改，产生伴侣的财产减少。5)我们将研究由于衰老或糖尿病期间的体外修饰或体内修饰而导致的伴侣蛋白功能丧失的机制。我们将测试的假设，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.