Proteins of normal and cataractous lenses

描述（由申请方提供）：拟定工作的目的是评价氧化应激在人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透镜混浊类型，也是最可能需要手术的类型。该提案的总体假设是，分子氧（O2）和UVA光都有助于核性白内障的形成。目的1将研究玻璃体液液化（老年人眼中常见的事件）、玻璃体液中O2水平升高与核性白内障之间的可能联系。该目的将采用实验动物中玻璃体液的酶辅助液化，以及使用高灵敏度光纤装置测量体内玻璃体和透镜O2水平。技术将包括裂隙灯生物显微镜检查、体外晶状体激光扫描、各种生化分析、SDS-PAGE、HPLC和电子显微镜检查，以确定玻璃体液化是否会对透镜产生有害影响，从而导致核性白内障。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探讨氧诱导透镜蛋白二硫键交联的机制，这是一种与人类核性白内障密切相关的修饰。Pi的假设是透镜晶状体蛋白的蛋白S-谷胱甘肽化（谷胱甘肽与蛋白的结合）在HBO/豚鼠体内模型中保护晶状体蛋白免受二硫化物交联和晶状体蛋白不溶。质谱和2-D凝胶电泳将用于鉴定谷胱甘肽化的特定位点，以及已变成水不溶性的特定晶状体蛋白。所有豚鼠透镜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。目的3将检验以下假设：UVA光与O2和毒性UVA发色团（例如可随年龄在人透镜核中积累的发色团）组合将产生H2 O2，并在体外诱导透镜晶状体蛋白聚集。该目的将使用含有内源性UVA发色团-β-晶状体蛋白和结合的NADPH的豚鼠透镜上清液。将使用其他UVA发色团（包括游离NADPH、游离犬尿氨酸和合成犬尿氨酸肽）进行选定实验，以模拟人透镜中存在的UVA发色团。该目标还将确定GSH以及重组α-晶状体蛋白是否可以防止UVA诱导的透镜晶状体蛋白聚集。相关性：这些研究旨在阐明成熟型核性白内障的形成机制，这是美国每年进行的150万例白内障手术的主要原因。研究结果为保护老化的人透镜免受氧和UVA的损伤，预防核性白内障的形成提供了有价值的信息。

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.