Structure and Interactions of Conformational Intermediates in gamma-D Crystallin Aggregation, and Their Targeting for Cataract Prevention

项目摘要 白内障是眼晶状体晶体蛋白逐渐聚集的结果。老年性白内障的严重程度有 与老化过程中积累的晶体蛋白中特定的翻译后修饰有关。两个重要的因素 与白内障相关的修饰类别是色氨酸残基氧化成更亲水的产品和 半胱氨酸残基的氧化生成二硫键。我们之前的研究揭示了一种关键的协同效应 两个人。人类γD晶状体蛋白的W42Q变异与W42R先天性白内障 变种，不稳定，但折叠良好，在还原条件下可溶，但形成非天然的 内部二硫键(Cys32-Cys41)以动力学方式将它们捕获在部分未折叠的构象中间体中， 在体外，在生理相关的温度、pH和浓度下产生快速和强劲的聚集。 我们开发了一种快速、原子化的蒙特卡罗建模方法，采用基于知识的统计方法 潜力，特别适合于构象中间体的研究，包括在多个多肽中 当它们同时展开以揭示新的蛋白质-蛋白质相互作用时。我们已经应用了这一点 一种γD晶体蛋白及其变异体的方法，不仅预测易于聚集的中间体的结构 而且第一次是聚集状态的原子模型。在实验上，我们最近发现了一种 人γD晶体蛋白中新的氧化还原酶活性和证明WT中的自然态二硫化物可以 在W42Q中转移以生成非天然的、促进聚集的二硫化物。我们发现了更多 令人惊讶的WT/突变体相互作用-区域界面窃取-允许WT催化突变体的聚集 即使在大量的外部二硫化物来源存在的情况下。我们现在将(1)研究物理原理， 通过组合的新界面窃取相互作用的动力学、进化和疾病含义 我们正在开发的计算、生化和蛋白质分解/质谱学方法；以及(2) 区分聚集前体和聚集状态的原子模型，以及(3)应用这些模型 最新改进的原子模型，以合理设计基于结构的多肽抑制剂的聚集过程。 虽然我们的研究主要集中在W42Q/R变异上，但其他与白内障相关的变异(V75D，L5S) 似乎表现得非常相似。此外，我们确认的本地和非本地二硫化物都是罪魁祸首 在聚集过程中完全由老年人和白内障患者的组织蛋白质组学支持 镜片中没有任何基因突变。因此，我们将检验这一假设，即许多突变或后 翻译修饰集中在少数决定聚集的构象中间体上。我们会 将聚集的详细机制和结构图推广到其他γ-晶体蛋白和其他白内障- 检测人γC和γS晶体蛋白是否也具有氧化还原活性和界面能力的相关变体 偷窃。对结构失稳和氧化还原化学之间的协同作用有更一般的理解 白内障将改进聚集抑制剂的设计，可在现有的遗传白内障小鼠模型上进行测试。

PROJECT ABSTRACT Cataracts result from progressive aggregation of eye lens crystallin proteins. Severity of age-onset cataract has been linked to specific post-translational modifications in crystallins that accumulate during aging. Two important classes of cataract-associated modifications are oxidation of Trp residues to more hydrophilic products and oxidation of Cys residues to generate disulfide bonds. Our prior research has revealed a crucial synergy between the two. The W42Q variant of human γD crystallin (a Trp oxidation mimic) and the W42R congenital-cataract variant, are destabilized but well folded and soluble under reducing conditions, yet formation of a non-native internal disulfide bond (Cys32-Cys41) kinetically traps them in a partially unfolded conformational intermediate, generating rapid and robust aggregation at physiologically relevant temperature, pH, and concentration in vitro. We have developed a rapid, atomistic Monte-Carlo modeling method, with a knowledge-based statistical potential, that is uniquely suited for the study of conformational intermediates, including in multiple polypeptide chains as they simultaneously unfold to reveal new protein-protein interactions. We have already applied this method to γD crystallin and its variants to predict not only the structure of the aggregation-prone intermediate but also, for the first time, an atomistic model of the aggregated state. Experimentally, we recently discovered a novel oxidoreductase activity in human γD crystallin and demonstrated that native-state disulfides in WT can be transferred to generate the non-native, aggregation-promoting disulfide in W42Q. We found an even more surprising WT/mutant interaction – domain interface stealing – that allows WT to catalyze mutants’ aggregation even in the presence of an abundant external disulfide source. We will now (1) investigate the physical principles, kinetics, and evolutionary and disease implications of the novel interface stealing interaction by a combined computational, biochemical, and proteolysis/mass spectrometry approach we are now developing; and (2) distinguish among atomistic models for the aggregation precursor and the aggregated state and (3) apply these newly refined atomistic models to rationally design structure-based peptide inhibitors of the aggregation process. Although our studies have focused on the W42Q/R variants, other cataract-associated variants (V75D, L5S) appear to behave quite similarly. Moreover, both the native and the non-native disulfide we identified as culprits in aggregation processes have been entirely supported by tissue proteomics of aged and cataractous human lenses in the absence of any genetic mutation. We will therefore test the hypothesis that many mutations or post- translational modifications converge on few conformational intermediates that determine aggregation. We will generalize the detailed mechanistic and structural picture of aggregation to other γ-crystallins and other cataract- associated variants testing whether human γC and γS crystallins are also redox-active and capable of interface stealing. A more general understanding of the synergy between structural destabilization and redox chemistry in cataract will improve design of aggregation inhibitors testable on existing genetic mouse models of cataract.