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 白内障是由眼晶状体蛋白逐渐聚集引起的。 与衰老过程中积累的晶状体蛋白的特定翻译后修饰有关。 与白内障相关的修饰类别包括色氨酸残基氧化成更亲水的产物和 我们之前的研究揭示了半胱氨酸残基氧化生成二硫键之间的重要协同作用。 人类 γD 晶状体蛋白（色氨酸氧化模拟物）的 W42Q 变体和 W42R 先天性白内障。 变体，不稳定，但在还原条件下折叠良好且可溶，但形成非天然 内部二硫键（Cys32-Cys41）在动力学上将它们捕获在部分展开的构象中间体中， 在体外生理相关温度、pH 和浓度下产生快速且稳定的聚集。 我们开发了一种快速、原子的蒙特卡洛建模方法，具有基于知识的统计 潜力，特别适合构象中间体的研究，包括多种多肽 链同时展开以揭示新的蛋白质-蛋白质相互作用，我们已经应用了这一点。 γD 晶状体蛋白及其变体的方法不仅可以预测易于聚集的中间体的结构 而且，我们最近在实验上首次发现了聚合状态的原子模型。 人 γD 晶状体蛋白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 转移以在 W42Q 中生成非天然的、促进聚集的二硫键。 令人惊讶的 WT/突变体相互作用——域界面窃取——使得 WT 能够催化突变体的聚集 即使存在丰富的外部二硫化物源，我们现在将 (1) 研究物理原理， 新型界面窃取相互作用的动力学、进化和疾病影响 我们正在开发计算、生物化学和蛋白水解/质谱方法；以及 (2) 区分聚合前体和聚合状态的原子模型，并 (3) 应用这些模型 新完善的原子模型可以合理设计基于结构的肽抑制剂的聚集过程。 尽管我们的研究重点是 W42Q/R 变异，但其他与白内障相关的变异（V75D、L5S） 此外，我们确定的罪魁祸首是天然和非天然二硫化物。 聚集过程完全得到老年人和白内障人类组织蛋白质组学的支持 因此，我们将测试许多突变或后遗症的假设。 翻译修饰集中在几个决定聚集的构象中间体上。 将聚集的详细机械和结构图推广到其他 γ-晶状体蛋白和其他白内障- 相关变体测试人类 γC 和 γS 晶状体蛋白是否也具有氧化还原活性并能够界面 对结构不稳定和氧化还原化学之间的协同作用有更全面的了解。 白内障将改进可在现有白内障遗传小鼠模型上进行测试的聚集抑制剂的设计。