Proteins of normal and cataractous lenses

描述(申请人提供)：拟议工作的目标是评估氧化应激在人类核性白内障发展中的作用，核性白内障是老年人最常见的晶状体混浊类型，也是最有可能需要手术的类型。该提议的总体假设是，分子氧(O2)和UVA光都可以促进核性白内障的形成。目的1将研究玻璃体体液液化(在老化的人眼中发生的一种常见事件)、玻璃体体液中氧气水平的增加与核性白内障之间的可能联系。这一目标将采用酶辅助液化实验动物的玻璃体体液，并使用高灵敏度的光纤设备测量体内玻璃体和晶状体02的水平。技术将包括裂隙灯生物显微镜、体外晶状体激光扫描、各种生化分析、十二烷基硫酸钠-PAGE、高效液相和电子显微镜，以确定玻璃体液化是否会对晶状体造成有害影响，导致核性白内障。核性白内障的两个体内实验动物模型，高压氧(HBO)和UVA光，也将被使用。目的2研究O2诱导晶状体蛋白二硫键交联的机制，这种修饰与人类核性白内障密切相关。Pi的假设是，在高压氧/豚鼠体内模型中，晶状体蛋白的蛋白质S-谷胱甘肽与蛋白质的结合(谷胱甘肽与蛋白质的结合)可以保护晶状体蛋白免受二硫键交联和晶状体蛋白不溶解的影响。质谱学和双向凝胶电泳法将被用来识别谷胱甘肽的特定位置，以及已经变得不能溶于水的特定晶体蛋白。所有豚鼠晶状体蛋白的序列现在都可以在NEIBank在线数据库中找到，这将有助于实现这一目标。目的3将验证UVA光与O2和有毒的UVA发色团(如随年龄积累在人晶状体细胞核中的发色团)在体外会产生过氧化氢并诱导晶状体晶体蛋白聚集的假设。这一目标将使用含有内源性UVA发色团的晶体蛋白与NADPH结合的豚鼠晶状体上清液。选定的实验将与其他UVA发色团进行，包括游离NADPH、游离犬尿氨酸和合成犬尿氨酸多肽，以模拟存在于人类晶状体中的UVA发色团。目的还将确定GSH以及重组的a-晶状体蛋白是否可以防止UVA诱导的晶状体晶状体蛋白聚集。相关性：这些研究旨在阐明成熟性核性白内障的形成机制，在美国每年进行的150万例白内障手术中，核性白内障是主要原因。这一结果将为保护老化的人类晶状体免受氧和UVA的损伤，以及防止核性白内障的形成提供有价值的信息。

DESCRIPTION (provided by applicant): The objective of the proposed work is to evaluate the role of oxidative stress in the development of human nuclear cataract, the most common type of lens opacity in older adults, and the type most likely to require surgery. The overall hypothesis of the proposal is that both molecular oxygen (O2) and UVA light can contribute to the formation of nuclear cataract. Aim 1 will investigate possible links between liquefaction of the vitreous humor (a common event occurring in the aging human eye), an increase in the level of O2 in the vitreous humor and nuclear cataract. This aim will employ enzyme-assisted liquefaction of vitreous humor in experimental animals, and the measurement of vitreal and lens 02 levels in vivo using a highly sensitive fiber optic device. Techniques will include slit-lamp biomicroscopy, laser scanning of lenses in vitro, a variety of biochemical analyses, SDS-PAGE, HPLC and electron microscopy to determine whether vitreous liquefaction can induce detrimental effects on the lens, leading to nuclear cataract. Two in vivo experimental animal models for nuclear cataract, hyperbaric O2 (HBO) and UVA light, will also be employed. Aim 2 will investigate the mechanism of O2-induced disulfide-crosslinking of lens crystallins, a modification strongly associated with human nuclear cataract. The Pi's hypothesis is that protein S-glutathiolation (the binding of glutathione to a protein) of lens crystallins protects against disulfide-crosslinking and crystallin insolubilization in an HBO/guinea pig in vivo model. Mass spectrometry and 2-D gel electrophoresis will be used to identify specific sites of glutathiolation, as well as specific crystallins that have become water- insoluble. The aim will be aided by the fact that sequences of all guinea pig lens crystallins are now available in the NEIBank on-line database. Aim 3 will test the hypothesis that UVA light in combination with O2 and a toxic UVA chromophore (such as that which can accumulate in the human lens nucleus with age) will generate H2O2, and induce aggregation of lens crystallins in vitro. This aim will employ guinea pig lens supernatants containing the endogenous UVA chromophore ^-crystallin with bound NADPH. Selected experiments will be conducted with other UVA chromophores including free NADPH, free kynurenine and a synthetic kynurenine peptide to mimic UVA chromophores present in the human lens. The aim will also determine whether GSH, as well as recombinant a-crystallin, can protect against UVA-induced aggregation of lens crystallins. Relevance: The studies are designed to elucidate the mechanism of formation of maturity-onset nuclear cataract, which is the cause for a major proportion of the 1.5 million cataract surgeries conducted in the United States each year. The results will provide valuable information on protecting the aging human lens against oxygen- and UVA-induced damage, and on guarding against formation of nuclear cataract.