Proteins of normal and cataractous lenses

描述（由申请人提供）：拟议工作的目的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中最常见的晶状体混浊类型，也是最可能需要手术的类型。该建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将研究玻璃体房体液化（在衰老的人眼中常见的事件）、玻璃体房体中氧水平的增加和核性白内障之间的可能联系。该目标将在实验动物中使用酶辅助液化玻璃体，并使用高灵敏度光纤装置测量体内玻璃体和晶状体02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、SDS-PAGE、HPLC和电子显微镜，以确定玻璃体液化是否会对晶状体产生有害影响，导致核性白内障。两种核性白内障的体内实验动物模型，高压氧（HBO）和UVA光，也将被采用。目的2将研究臭氧诱导晶状体晶体蛋白二硫交联的机制，这是一种与人类核性白内障密切相关的修饰。Pi的假设是，在HBO/豚鼠体内模型中，晶状体晶体蛋白的s -谷胱甘肽化（谷胱甘肽与蛋白质的结合）可以防止二硫化物交联和晶体蛋白不溶化。质谱法和二维凝胶电泳将用于鉴定谷胱甘肽化的特定位点，以及已成为不溶于水的特定结晶蛋白。所有豚鼠晶体蛋白的序列现在都可以在NEIBank在线数据库中获得，这一事实将有助于实现这一目标。Aim 3将测试UVA光与O2和有毒的UVA发色团（如随着年龄的增长可以在人类晶状体核中积累的发色团）结合会产生H2O2的假设，并诱导晶状体结晶蛋白在体外聚集。该目的将使用含有内源性UVA发色团^-结晶蛋白的豚鼠晶状体上清，并结合NADPH。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肽，以模拟人类晶状体中存在的UVA发色团。目的还将确定谷胱甘肽和重组a-结晶蛋白是否可以防止uva诱导的晶状体结晶蛋白聚集。相关性：本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，核性白内障占很大比例。该结果将为保护老化的人体晶状体免受氧气和uva引起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.