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

γ-D 晶状体蛋白聚集中构象中间体的结构和相互作用及其预防白内障的靶向作用

• 批准号: 10608130
• 负责人: EUGENE I SHAKHNOVICH
• 金额: $ 41.62万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10608130
• 关键词: Acceleration; Address; Affect; Age; Age of Onset; Aging; American; Biochemical; Biochemistry; Biological; Biological Assay; Blindness; C-terminal; Catalysis; Cataract; Chemicals; Chemistry; Collaborations; Computing Methodologies; Coupled; Crystalline Lens; Crystallins; DNA Sequence Alteration; Data; Development; Disease; Disulfides; Evolution; Eye Surgeon; FDA approved; Future; Genetic; Homo; Human; In Vitro; Kinetics; Knowledge; Lead; Link; Maps; Mass Spectrum Analysis; Measures; Medical; Methods; Modeling; Modification; Molecular Conformation; Monte Carlo Method; Mutation; N-terminal; Operative Surgical Procedures; Oxidation-Reduction; Oxidoreductase; Pathway interactions; Patients; Peptides; Pharmaceutical Preparations; Physical Chemistry; Physiological; Population; Post-Translational Protein Processing; Prevention; Prions; Process; Property; Protein Engineering; Proteins; Proteolysis; Proteomics; Research; Severities; Site; Source; Structural Models; Structure; Temperature; Testing; Theft; Therapeutic; Thermodynamics; Time; Tissues; United States National Institutes of Health; Variant; Visual impairment; Work; aged; analytical ultracentrifugation; congenital cataract; cost; crosslink; deamidation; design; disulfide bond; gamma-Crystallins; hydrophilicity; improved; in silico; in vitro testing; in vivo; inhibitor; kinetic model; knowledge base; lens; light scattering; mathematical model; migration; mouse model; mutant; non-Native; novel; oxidation; peptidomimetics; polypeptide; prevent; prion-like; protein aggregation; protein protein interaction; protein structure; rational design; residence; simulation; solid state nuclear magnetic resonance; synergism

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.

项目摘要 白内障是由眼镜晶体蛋白的进行性聚集而产生的。年龄率的白内障的严重程度 我们与在衰老过程中积累的结晶蛋白中的特定翻译后修饰有关。两个很重要 白内障相关的修饰类别是将TRP残基氧化与更多亲水产物和 Cys残基的氧化以产生二硫键。我们先前的研究表明 两个。人γD晶体的W42Q变体（一种TRP氧化模拟物）和W42R先天性 - 卡拉克 变体是不稳定的，但在还原条件下折叠良好且坚固，但形成了非本地 内部二硫键键（Cys32-Cys41）会在部分展开的构象中间体中捕获它们 在体外在物理相关的温度，pH和浓度下产生快速，稳健的聚集。 我们开发了一种基于知识的统计的快速，原子化的蒙特卡洛建模方法 潜力，这是独特的，适合于构象中间体的研究，包括多种多肽 链时，它们只是展开了新的蛋白质 - 蛋白质相互作用。我们已经应用了 γD结晶蛋白及其变体的方法不仅可以预测聚集的中间体的结构 而且，这也是第一次是汇总状态的原子模型。在实验上，我们最近发现了一个 在人γD晶体中的新型氧化还原酶活性，并证明WT中的天然状态二硫化物可以是 转移以生成W42Q中的非本地，聚合促进的二硫化物。我们发现了更多 令人惊讶的WT/突变体相互作用 - 域接口窃取 - 允许WT催化突变体的聚集 即使存在绝对的外部二硫化物来源。现在我们将（1）调查物​​理原理， 动力学以及新界面的进化和疾病的影响，通过合并的 我们现在正在发展的计算，生化和蛋白水解/质谱法。 （2） 区分聚合前体和聚合状态的原子模型，以及（3）应用这些 新精炼的原子模型以合理设计的基于结构的肽抑制剂的聚集过程。 尽管我们的研究集中在W42Q/R变体上，但其他与白内障相关的变体（V75D，L5S） 表现似乎很相似。此外，我们确定为罪魁祸首的天然和非本地二硫化物 在聚集过程中，已完全由衰老和白内障人类的组织蛋白质组学支持 在没有任何遗传突变的情况下镜片。因此，我们将检验以下假设：许多突变或之后的许多突变 翻译修饰会收敛于几个决定聚集的构象中间体。我们将 将聚集的详细机械和结构图概括为其他γ-晶体和其他白内障 相关的变体测试人γC和γs结晶蛋白是否也具有氧化还原活性并且能够界面 偷窃。对结构不稳定和氧化还原化学之间的协同作用的更一般的了解 白内障将改善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。