Integrative Biosciences, Oregon Health & Science University, Portland, Oregon, USA.
Research School of Chemistry, College of Science, The Australian National University, Acton, Australia.
Protein Sci. 2020 Sep;29(9):1945-1963. doi: 10.1002/pro.3915.
Age-related lens cataract is the major cause of blindness worldwide. The mechanisms whereby crystallins, the predominant lens proteins, assemble into large aggregates that scatter light within the lens, and cause cataract, are poorly understood. Due to the lack of protein turnover in the lens, crystallins are long-lived. A major crystallin, γS, is heavily modified by deamidation, in particular at surface-exposed N14, N76, and N143 to introduce negative charges. In this present study, deamidated γS was mimicked by mutation with aspartate at these sites and the effect on biophysical properties of γS was assessed via dynamic light scattering, chemical and thermal denaturation, hydrogen-deuterium exchange, and susceptibility to disulfide cross-linking. Compared with wild type γS, a small population of each deamidated mutant aggregated rapidly into large, light-scattering species that contributed significantly to the total scattering. Under partially denaturing conditions in guanidine hydrochloride or elevated temperature, deamidation led to more rapid unfolding and aggregation and increased susceptibility to oxidation. The triple mutant was further destabilized, suggesting that the effects of deamidation were cumulative. Molecular dynamics simulations predicted that deamidation augments the conformational dynamics of γS. We suggest that these perturbations disrupt the native disulfide arrangement of γS and promote the formation of disulfide-linked aggregates. The lens-specific chaperone αA-crystallin was poor at preventing the aggregation of the triple mutant. It is concluded that surface deamidations cause minimal structural disruption individually, but cumulatively they progressively destabilize γS-crystallin leading to unfolding and aggregation, as occurs in aged and cataractous lenses.
年龄相关性白内障是全球范围内导致失明的主要原因。尽管晶状体中的主要蛋白质——晶状蛋白聚合成大的聚集体，导致光线在晶状体中散射，从而引发白内障，但人们对其形成机制仍知之甚少。由于晶状体中缺乏蛋白质周转，晶状蛋白的寿命很长。一种主要的晶状蛋白γS 受到脱酰胺作用的强烈修饰，特别是在表面暴露的 N14、N76 和 N143 处引入负电荷。在本研究中，通过在这些位点突变天冬氨酸模拟脱酰胺化的 γS，并通过动态光散射、化学和热变性、氢氘交换以及对二硫键交联的敏感性来评估其对 γS 生物物理性质的影响。与野生型 γS 相比，每个脱酰胺突变体的一小部分都会迅速聚集形成大的、散射光的物质，这对总散射有很大的贡献。在盐酸胍或升高温度的部分变性条件下，脱酰胺作用导致更快的展开和聚集，并增加了氧化的敏感性。三重突变体进一步失稳，表明脱酰胺作用的影响是累积的。分子动力学模拟预测脱酰胺作用增强了 γS 的构象动力学。我们认为这些干扰破坏了 γS 的天然二硫键排列，并促进了二硫键连接的聚集体的形成。晶状体特异性伴侣蛋白αA-晶体蛋白在防止三重突变体聚集方面效果不佳。结论是，表面脱酰胺作用单独引起的结构破坏很小，但累积起来会逐渐使 γS 晶体蛋白失稳，导致展开和聚集，就像在衰老和白内障晶状体中发生的那样。
