Amino-Carbonyl Reactions in the Aging Human Lens

描述（由申请人提供）：我们研究的重点是调查抗坏血酸的氨基羰基反应在老化透镜的核硬化中起作用的假设。另一方面，最近的证据也表明，低谷胱甘肽（GSH）水平的氧化应激在年龄相关性核性白内障（ARNC）的形成中是重要的。为了检验这些假设，我们在过去五年中取得了许多重要突破，包括1）产生了在透镜中表达人源化水平的抗坏血酸的hSVCT 2小鼠，2）证明在12个月内透镜变黄并积累了存在于人透镜中的所有抗坏血酸化产物，3）发现口服和局部L-精氨酸都是体内晶状体蛋白损伤的有效抑制剂，4）LEGSKO的成功开发（透镜GSH合成条件性敲除）小鼠，其GSH水平模拟人透镜核中的低水平，和5）鉴定精氨酸残基为老化透镜中的主要损伤形式。在接下来的五年里，我们建议进行完整的循环并测试ARNC需要羰基和氧化应激才能形成的两击假设，具体来说，目标1将测试以下假设：1）对人类和小鼠γ-晶状体蛋白的精氨酸残基的羰基攻击导致蛋白质电荷改变，在相同位点脱鸟苷化成鸟苷酸，蛋白质解折叠，SH基团暴露，聚集，并且与1-晶体蛋白的相互作用增加; 2）聚集发生在易于浑浊化的位点;以及3）这些变化在体外和在hSVCT 2小鼠中用L-精氨酸可预防。目标2将定义低晶状体GSH水平在ARNC中的作用。将使用蛋白质组学方法在体外和新型LEGSKO小鼠中确定晶状体蛋白靶点、晶状体蛋白氧化的性质和位点及其在蛋白质聚集中的作用。将测试模拟谷氨酸盐和药理学抗氧化剂在体内预防混浊的能力。在这些实验中，将从小鼠透镜中的研究获得的结果与影响来自老的正常晶状体和ARN白内障晶状体的晶状体蛋白的变化进行比较。目的3是使hSVCT 2与LEGSKO小鼠杂交以产生具有组合的羰基和氧化剂应激的小鼠，我们假设这将促进蛋白质聚集和交联，如在旧的人透镜中一样。与对照菌株相比，将确定蛋白质聚集和晶状体混浊的动力学，并将测试羰基和氧化剂应激的药理学清除剂。我们希望，这种方法将成为开发用于临床预防人类ARNC的低成本药物的基础。为了实现这些目标，我们将使用强大的技术，结合联合收割机激光捕获显微切割，蛋白质组学和动态光散射（DLS）。 公共卫生相关性：由于缺乏合适的动物模型，对年龄相关性核性白内障的发病机制和预防的研究一直受到阻碍。我们假设，对透镜蛋白的两种主要形式的损伤源于破坏性的维生素C氧化产物和低抗氧化防御。我们现在建立了两个老化透镜的人源化小鼠模型，即一个具有人源化水平的维生素C氧化产物，另一个具有低抗氧化防御。利用这些模型，我们将研究损伤如何发生并导致白内障，并开发药物来预防它。

DESCRIPTION (provided by applicant): The focus of our research is to investigate the hypothesis that amino-carbonyl reactions from ascorbic acid play a role in nuclear sclerosis of the aging lens. On the other hand, recent evidence also suggests oxidant stress from low glutathione (GSH) levels is important in the formation of age-related nuclear cataract (ARNC). Toward testing these hypotheses, we made a number of important breakthroughs in the past five years which include 1) the creation of the hSVCT2 mouse which expresses humanized levels of ascorbic acid in the lens, 2) the demonstration that within 12 mos the lenses turn yellow and accumulate all ascorbylation products present in the human lens, 3) the discovery that both oral and topical L-arginine is a potent inhibitor of crystallin damage in vivo, 4) the successful development of the LEGSKO (lens GSH synthesis conditional knockout) mouse with GSH levels mimicking the low levels in the human lens nucleus, and 5) the identification of arginine residues as the major form of damage in the aging lens. In the coming five years we propose to go full circle and test the two-hit hypothesis that ARNC requires both carbonyl and oxidant stress to form, Specifically, Aim 1 will test the hypothesis that 1) carbonyl attack onto arginine residues of human and mouse gamma-crystallins results in altered protein charge, deguanylation into ornithine at identical sites, protein unfolding, exposure of SH groups, aggregation, and increased interaction with 1-crystallins 2) aggregation occurs at sites prone to opacification, and 3) these changes are preventable with L-arginine in vitro and in the hSVCT2 mouse. Aim 2 will define the role of low lenticular GSH levels in ARNC. Crystallin targets, nature and site of crystallin oxidation and their role in protein aggregation will be determined in vitro and in the novel LEGSKO mouse using a proteomics approach. Glutathione mimetics and pharmacological antioxidants will be tested for their ability to prevent opacification in vivo. Throughout these experiments, the results obtained from investigation in the mouse lens will be compared with changes affecting the crystallins from old normal and ARN cataractous lenses. Aim3 will be to hybridize the hSVCT2 with the LEGSKO mouse to create a mouse with combined carbonyl and oxidant stress, which we hypothesize will facilitate protein aggregation and crosslinking as in the old human lens. Kinetics of protein aggregation and lenticular opacification will be determined compared to control strains, and pharmacological scavengers of carbonyl and oxidant stress will be tested. This approach, we hope, will form the basis for the development of low cost agents for the clinical prevention of human ARNC. To achieve these goals we will use powerful techniques that combine laser capture microdissection, proteomics and dynamic light scattering (DLS). PUBLIC HEALTH RELEVANCE: Investigation into the mechanisms and prevention of age-related nuclear cataract has been hampered by the lack of appropriate animals models. The two major forms of damage to the lens proteins, we hypothesize, stem from damaging vitamin C oxidation products and low antioxidant defense. We now created two humanized mouse models of the aging lens, i.e. one that has humanized levels of vitamin C oxidation products, and one that has low antioxidant defense. Using these models we will study how the damage occurs and leads to cataract, and develop drugs to prevent it.