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

项目摘要 白内障是由眼透镜晶体蛋白的进行性聚集引起的。老年性白内障的严重程度 与老化过程中积累的晶状体蛋白中的特定翻译后修饰有关。两个重要 与白内障相关的修饰类型是Trp残基氧化成更亲水的产物， Cys残基氧化产生二硫键。我们之前的研究揭示了一个关键的协同作用， the two.人γD晶状体蛋白的W 42 Q变体（Trp氧化模拟物）和W 42 R先天性白内障 变体，是不稳定的，但在还原条件下良好折叠和可溶，但形成非天然的 内部二硫键（Cys 32-Cys 41）将它们动力学地捕获在部分未折叠的构象中间体中， 在体外生理相关温度、pH和浓度下产生快速和稳健的聚集。 我们已经开发了一种快速的原子蒙特-卡罗建模方法， 潜在的，这是唯一适合于构象中间体的研究，包括在多个多肽 它们同时展开以揭示新的蛋白质-蛋白质相互作用。我们已经应用了这个 方法对γD晶体蛋白及其变体进行预测，不仅可以预测易于聚集的中间体的结构 而且，第一次，一个原子模型的聚合状态。通过实验，我们最近发现了一种 在人γD晶状体蛋白中具有新的氧化还原酶活性，并证明WT中的天然状态二硫化物可以 转移以在W 42 Q中产生非天然的促进聚集的二硫化物。我们发现了一个 令人惊讶的WT/突变体相互作用-结构域界面窃取-允许WT催化突变体的聚集 即使在存在大量外部二硫化物源的情况下。我们现在将（1）研究物理原理， 动力学，进化和疾病的影响，新的接口窃取相互作用的组合 我们正在开发的计算、生物化学和蛋白质水解/质谱分析方法;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 新改进的原子模型，以合理设计基于结构的肽抑制剂的聚集过程。 虽然我们的研究集中在W 42 Q/R变异，但其他白内障相关变异（V75 D，L5 S） 表现得很相似此外，我们确定天然和非天然二硫化物都是罪魁祸首。 在聚集过程中，老年人和白内障患者的组织蛋白质组学完全支持 在没有任何基因突变的情况下。因此，我们将测试假设，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。我们将 将聚集的详细机制和结构图片推广到其他γ-晶体蛋白和其他白内障- 相关变体测试人γC和γS晶体蛋白是否也具有氧化还原活性并能够介导 偷东西更一般的理解之间的协同作用的结构不稳定和氧化还原化学， 白内障将改进可在现有的白内障遗传小鼠模型上测试的聚集抑制剂的设计。

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.