Role of crystallin racemization and isomerization in cataract

项目摘要 年龄相关性白内障与晶体蛋白的广泛脱酰胺、外消旋和异构化有关， 晶状体的主要屈光蛋白。我们的实验室和其他机构进行的广泛研究发现 脱酰胺化显著降低了晶体蛋白的稳定性，并对其结构造成极小的扰动。然而， 这些通过诱变产生的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和异构化，伴随着两个修饰 可能比单独的脱酰胺作用对晶体蛋白结构更具破坏性的脱酰胺作用。与去酰胺化不同， 这些修饰不能通过遗传手段引入晶体蛋白中。这阻止了研究 评估这些额外修饰在老年性白内障中的重要性。因此，其目的是 这些实验是为了创建γS-含有特定位置的生理相关的消旋和消旋的晶体蛋白 与年龄相关的脱酰亚胺过程中产生的异构化天冬氨酸及其对 蛋白质结构。具体目标是：1)使用合成重肽标准品和高分辨质量 分光光度法测定γS中不同外消旋和异构化天冬氨酸的相对比例 从老年性白内障人晶状体中提取晶体蛋白，以便相关物种 选择，2)将这些外消旋和异构化残基引入γS晶体中，采用半合成 以前从未在镜头领域中使用过的工艺，以及3)检查这些修改的结果 用蛋白质稳定性和光散射方法研究γS晶体结构，氢/氢 交换质谱学和核磁共振光谱学。这些实验将，对于 第一次，严格测试与年龄相关的消旋和异构化对结晶蛋白的潜在影响 结构。这些结果可以通过开发药物在减缓白内障发展方面发挥重要作用 特别防止老化晶状体中消旋化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.