Proteins of normal and cataractous lenses

描述（由申请人提供）：拟议工作的广泛目标是更好地了解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透镜混浊类型，也是最可能需要手术的类型。待检验的总体假设是分子氧（O2）以及大小<3.5 kDa的低分子量（LMW）晶状体蛋白片段均有助于形成高分子量（HMW）蛋白质聚集体，导致核光散射增加，最终导致核性白内障。主要关注点是使用PI完善的高压氧（HBO）/豚鼠体内模型治疗核性白内障。目的1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。这里的假设是，晶状体蛋白谷胱甘肽化（谷胱甘肽与蛋白质的结合），但可能不是半胱氨酸化（半胱氨酸的结合），防止HBO/豚鼠体内模型中的二硫化物交联和晶状体蛋白不溶。最先进的质谱（MS）方法，包括靶向蛋白质组学分析，电子转移解离和选择性反应监测，将用于鉴定谷胱甘肽化和半胱氨酸化的特定晶状体蛋白位点，以及蛋白质二硫化物形成。设计实验以鉴定O2应激透镜核中的哪些晶状体蛋白，特别是哪些晶状体蛋白半胱氨酸残基，最有可能结合谷胱甘肽和半胱氨酸，与其他晶状体蛋白交联，并留在溶液中或形成HMW聚集体和沉淀。还将使用分离和重组的αA-、α-、γ-和α-晶状体蛋白进行体外实验。目的2将检验以下假设，即透镜核中O2水平的升高导致作为正常老化的结果在核中形成的大小为6-12 kDa的晶状体蛋白片段进一步截短为<3.5 Da的LMW肽，其加速蛋白质聚集。将采用成像质谱的强大技术来视觉地跟踪豚鼠晶状体薄组织切片的各个区域中截短的晶状体蛋白片段的形成和消失，作为年龄和HBO治疗的函数。将采用对照品和HBO处理的豚鼠透镜提取物的LC/MS分析，以确定细胞核中的某些特定LMW肽是否由于O2诱导的应激而与水溶性HMW聚集体和水不溶性蛋白质结合。使用重组人截短α A-晶状体蛋白片段的体外实验将用于研究O2诱导透镜核中6-12 kDa片段消失的机制。影响：这些研究旨在阐明成熟型核性白内障以及玻璃体切除术诱导的核性白内障的机制，这是美国每年进行的150万例白内障手术的主要贡献者。了解衰老的人透镜核中蛋白质聚集的机制将有助于开发治疗剂来减缓该过程，并延迟人核性白内障的发作。

DESCRIPTION (provided by applicant): The broad objective of the proposed work is to better understand 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 to be tested is that both molecular oxygen (O2), as well as low molecular weight (LMW) crystallin fragments <3.5 kDa in size, contribute to the formation of high molecular weight (HMW) protein aggregates, causing increased nuclear light scatter and, eventually, nuclear cataract. A major focus is the use of the PI's well-established hyperbaric oxygen (HBO)/guinea pig in vivo model for nuclear cataract. Aim 1 will investigate the mechanism of O2-induced disulfide-crosslinking of lens crystallins, a modification strongly associated with human nuclear cataract. The hypothesis here is that crystallin glutathiolation (the binding of glutathione to a protein), but possibly not cysteinylation (the binding of cysteine, protects against disulfide-crosslinking and crystallin insolubilization in the HBO/guinea pig in vivo model. State-of-the-art mass spectrometry (MS) methods, including targeted proteomic analysis, electron-transfer dissociation and selective reaction monitoring, will be used to identif specific crystallin sites of glutathiolation and cysteinylation, and protein disulfide formation. Experiments are designed to identify which crystallins in the O2-stressed lens nucleus, and specifically which crystallin cysteine residues, are most likely to bind glutathione and cysteine, crosslink with other crystallins, and either stay in solution or form HMW aggregates and precipitate. In vitro experiments using isolated and recombinant αA-, ß-, γ- and ζ- crystallins wil also be employed. Aim 2 will test the hypothesis that an elevation in the level of O2 in the lens nucleus causes further truncation of crystallin fragments 6-12 kDa in size, formed in the nucleus as a result of normal aging, to LMW peptides <3.5 Da, which accelerate protein aggregation. The powerful technique of imaging mass spectrometry will be employed to visually follow the formation and disappearance of truncated crystallin fragments in various regions of thin tissue sections of guinea pig lenses, as a function of age and HBO- treatment. LC/MS analysis of control and HBO-treated guinea pig lens extracts will be employed to determine whether certain specific LMW peptides in the nucleus become associated with water-soluble HMW aggregates and water-insoluble protein as a result of O2-induced stress. In vitro experiments using recombinant human truncated αA-crystallin fragments will be used to investigate the mechanism of O2-induced disappearance of the 6-12 kDa fragments in the lens nucleus. Impact: The studies are designed to elucidate the mechanism of maturity-onset nuclear cataract, as well as vitrectomy-induced nuclear cataract, which are major contributors to the 1.5 million cataract surgeries performed in the United States each year. Understanding mechanisms involved in aggregation of proteins in the aging human lens nucleus will aid in developing therapeutic agents to slow the process, and delay the onset of human nuclear cataract.