Role of crystallin racemization and isomerization in cataract

项目摘要 晶状体相关性白内障与晶状体蛋白的广泛脱酰胺、外消旋化和异构化有关， 透镜的主要折射蛋白质。我们的实验室和其他机构的广泛研究发现， 脱酰胺作用显著降低了晶体蛋白的稳定性，而结构扰动最小。然而，在这方面， 使用诱变产生的这些脱酰胺模拟物在体外不容易聚集。我们假设 这是因为它们缺乏外消旋化和异构化， 脱酰胺可能比单独脱酰胺对晶体蛋白结构更具破坏性。与脱酰胺不同， 这些修饰不能通过遗传手段引入晶体蛋白。这阻碍了研究， 衡量这些额外的修改在年龄相关性白内障的重要性。因此， 这些实验的目的是产生含有生理学相关的外消旋和 由老化相关的脱酰胺过程产生的异构化反丁烯二酸酯，并确定它们对 蛋白质结构具体目标是：1）使用合成重肽标准品和高分辨率质量 用分光光度法测定γ-S-γ-S中不同消旋化和异构化异构体的相对比例。 从老年白内障人晶状体的不溶性蛋白质中提取晶状体蛋白， 2）使用半合成的方法将这些外消旋和异构化的残基引入γ S-晶状体蛋白中， 以前从未在透镜领域中使用的工艺，以及3）检查这些修改的结果 利用蛋白质稳定性和光散射测量对γ S-晶状体蛋白结构的影响，氢/氘 交换质谱和核磁共振光谱。这些实验将为 第一次，严格测试年龄相关的消旋化和异构化对晶状体蛋白的潜在影响 结构这些结果可以通过开发药物来减缓白内障的发展， 特异性地防止老化晶状体中外消旋和异构化晶状体蛋白的聚集和光散射。

Project Summary Age-related cataract is associated with extensive deamidation, racemization, and isomerization of crystallins, the major refractive proteins of the lens. Extensive studies by our laboratories and others have found deamidation significantly decreases the stability of crystallins with minimal structural perturbations. However, these deamidation mimics created using mutagenesis did not readily aggregate in vitro. We hypothesize that this is because they were lacking racemization and isomerization, two modifications accompanying deamidation that may be more disruptive to crystallin structure than deamidation alone. Unlike deamidation, these modifications cannot be introduced into crystallins by genetic means. This has prevented studies to gauge the importance of these additional modifications in age-related cataract. Therefore, the purpose of these experiments is to create γS-crystallins containing specific sites of physiologically relevant racemized and isomerized aspartates that result from the age-related deamidation process and determine their effect on protein structure. The specific aims are to: 1) use synthetic heavy peptide standards and high-resolution mass spectrometry to determine the relative proportion of different racemized and isomerized aspartates in γS- crystallin from the insoluble protein of aged cataractous human lenses so that relevant species can be selected, 2) introduce these racemized and isomerized residues into γS-crystallin using semi-synthetic processes that have never before been used in the lens field, and 3) examine the result of these modifications on γS-crystallin structure using protein stability and light scattering measurements, hydrogen/deuterium exchange mass spectrometry, and nuclear magnetic resonance spectroscopy. These experiments will, for the first time, critically test the potential impact of age-related racemization and isomerization on crystallin structure. These results could play an important role in slowing cataract development by developing drugs that specifically prevent the aggregation and light scatter of racemized and isomerized crystallins in aged lenses.