Proteins of normal and cataractous lenses

正常和白内障晶状体的蛋白质

• 批准号: 7847891
• 负责人: Frank Joseph Giblin
• 金额: $ 2.89万
• 依托单位: OAKLAND UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2011-08-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7847891
• 关键词: Age; Aging; Animals; Binding; Biochemical; Cataract; Cataract Extraction; Cavia; Cell Nucleus; Collaborations; Crystallins; Databases; Development; Devices; Dialysis procedure; Disulfides; Elderly; Electron Microscopy; Electrophoresis; Enzymes; Event; Experimental Animal Model; Eye; Felis catus; Fiber Optics; Glutathione; Goals; High Pressure Liquid Chromatography; Human; Human Development; Hydrogen Peroxide; Immunoprecipitation; In Vitro; Kynurenine; Laboratories; Lasers; Lens Opacities; Link; Mass Spectrum Analysis; Measurement; Measures; Methods; Modification; Molecular; NADP; Nuclear; Operative Surgical Procedures; Oxidative Stress; Oxygen; Partial Pressure; Peptides; Permeability; Procedures; Process; Proteins; Rattus; Recombinants; Research Design; Retina; Role; Scanning; Site; Spermophilus; Techniques; Testing; Time; Two-Dimensional Gel Electrophoresis; UVA induced; Ultraviolet A radiation; United States; Vitrectomy; Vitreous humor; Water; Work; antioxidant therapy; chromophore; crosslink; in vivo; in vivo Model; irradiation; lens; lens protein; light scattering; prevent; research study

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.

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，也是最有可能需要手术的类型。该提案的总体假设是分子氧 (O2) 和 UVA 光均可导致核性白内障的形成。目标 1 将调查玻璃体液液化（老化人眼中发生的常见事件）、玻璃体液中 O2 水平增加与核性白内障之间可能存在的联系。该目标将在实验动物中采用酶辅助液化玻璃体液，并使用高灵敏度光纤装置测量体内玻璃体和晶状体 O2 水平。技术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，从而导致核性白内障。还将采用两种核性白内障体内实验动物模型：高压氧 (HBO) 和 UVA 光。目标 2 将研究 O2 诱导晶状体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，在 HBO/豚鼠体内模型中，晶状体蛋白的蛋白质 S-谷胱甘肽化（谷胱甘肽与蛋白质的结合）可防止二硫键交联和晶状体蛋白不溶。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，以及已变得不溶于水的特定晶状体蛋白。所有豚鼠晶状体蛋白的序列现已在 NEIBank 在线数据库中提供，这一事实将有助于实现这一目标。目标 3 将测试以下假设：UVA 光与 O2 和有毒 UVA 发色团（例如随着年龄的增长而在人类晶状体核中积累的发色团）结合会产生 H2O2，并在体外诱导晶状体蛋白聚集。该目标将使用含有内源性UVA发色团β-晶状体蛋白和结合NADPH的豚鼠晶状体上清液。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）进行选定的实验，以模拟人类晶状体中存在的 UVA 发色团。该目的还将确定 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，这是美国每年进行的 150 万例白内障手术中大部分的原因。研究结果将为保护老化的人类晶状体免受氧气和 UVA 引起的损伤以及预防核性白内障的形成提供有价值的信息。