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MODIFICATION OF ALPHA-CRYSTALLIN CHAPERONE FUNCTION

α-晶状体蛋白伴侣功能的修饰

基本信息

批准号：
6518548
负责人：
Edathara C Abraham
金额：
$ 24.81万
依托单位：
UNIV OF ARKANSAS FOR MED SCIS
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-06-01 至 2004-05-31
项目状态：
已结题

项目摘要

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.

本提案的目的是阐明 α-晶状体蛋白（αA 和 αB）的一些常见翻译后修饰对分子伴侣特性的影响，即 α-晶状体蛋白保护其他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，αA-和αB-晶状体蛋白的不同结构域将被靶向进行体外结构修饰，这反过来又与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-晶状体蛋白的分子伴侣样特性下降的体内修饰。这是有史以来第一项将体内发生的翻译后修饰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。该提案的具体目标基于我们最近的发现，即糖化、氧化和混合二硫键形成对α-晶状体蛋白伴侣功能具有抑制作用，并且衰老和糖尿病都会降低伴侣特性。以下具体目标是假设驱动的，每个目标旨在检验特定假设：1) 通过用 H2O2 氧化、用抗坏血酸和果糖糖化以及用 GSSG 形成混合二硫键对小牛或年轻人类 α-晶状体蛋白进行体外修饰，将用于检验这样的假设：此类翻译后修饰将影响分子伴侣功能（由分子伴侣的能力决定）。 α-晶状体蛋白可保护 β-和 γ-晶状体蛋白免受热变性和聚集。此外，我们将研究不止一种修饰的综合效果，希望显示两种治疗的协同效应。 2)对于1个月至90岁的人类晶状体的αL和αH分数，将显示随着年龄的增加，具有改变的伴侣功能的翻译后修饰的α-晶状体蛋白的水平是否会增加。 3) 由于糖尿病患者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化，我们将测试糖尿病人或大鼠晶状体中α-晶状体蛋白伴侣功能发生更大改变的可能性。 4) 我们将通过质谱分析鉴定将在体外引入或在体内发生的αA-和αB-晶状体蛋白中的蛋白质修饰，并与伴侣功能的变化（包括伴侣-靶蛋白结合）相关。这些分析将确定导致伴侣特性降低的修饰类型。 5）我们将研究衰老或糖尿病过程中由于体外修饰或体内修饰而导致伴侣功能丧失的机制。我们将测试翻译后修饰影响伴侣蛋白与靶蛋白结合从而导致伴侣功能降低的假设。