Some therapeutic peptides self-assemble in solution to form ordered, insoluble, β-sheet rich amyloid fibrils. This physical instability can result in reduced potency, cause immunogenic side effects, and limit options for formulation. Understanding the mechanisms of fibrillation is key to developing rational mitigation strategies. Here, amide hydrogen/deuterium exchange with mass spectrometric analysis (HDX-MS) coupled with proteolytic digestion was used to identify the early stage interactions leading to fibrillation of human calcitonin (hCT), a peptide hormone important in calcium metabolism. hCT fibrillation kinetics was sigmoidal, with lag, growth and plateau phases as shown by thioflavin T and turbidity measurements. HDX-MS of fibrillating hCT (pH 7.4, 25 °C) suggested early involvement of the N-terminal (1-11) and central (12-19) fragments in interactions during the lag phase, whereas C-terminal fragments (20-32, 26-32) showed limited involvement during this period. The residue-level information was used to develop phosphorylated hCT analogs that showed modified fibrillation that depended on phosphorylation site. Phosphorylation in the central region resulted in complete inhibition of fibrillation for the phospho-Thr-13 hCT analog, while phosphorylation in the N-terminal and C-terminal regions inhibited but did not prevent fibrillation. Reduction of the Cys1-Cys7 disulfide bond resulted in faster fibrillation with involvement of different hCT residues as indicated by pulsed HDX-MS. Together, the results demonstrate that small structural changes have significant effects on hCT fibrillation, and that understanding these effects can inform the rational development of fibrillation-resistant hCT analogs.

中文翻译：

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人降钙素及其类似物的纤颤：磷酸化和二硫键还原的影响

一些治疗性肽在溶液中自组装形成有序的、不溶的、富含β-折叠的淀粉样蛋白原纤维。这种物理不稳定性会导致效力降低，引起免疫原性副作用，并限制制剂的选择。了解纤颤的机制是制定合理缓解策略的关键。在这里，酰胺氢/氘交换与质谱分析 (HDX-MS) 结合蛋白水解消化用于识别导致人降钙素 (hCT) 纤颤的早期相互作用，这是一种在钙代谢中很重要的肽激素。hCT 纤颤动力学是 S 型的，具有滞后期、生长期和平台期，如硫代黄素 T 和浊度测量所示。纤维化 hCT 的 HDX-MS（pH 7.4，25 °C) 表明 N 末端 (1-11) 和中心 (12-19) 片段在滞后期相互作用中的早期参与，而 C 末端片段 (20-32, 26-32) 在滞后期显示有限参与这一时期。残留水平信息用于开发磷酸化 hCT 类似物，该类似物显示出依赖于磷酸化位点的修饰纤颤。中心区域的磷酸化导致磷酸化 Thr-13 hCT 类似物的纤颤完全抑制，而 N 末端和 C 末端区域的磷酸化抑制但不阻止纤颤。如脉冲 HDX-MS 所示，Cys1-Cys7 二硫键的还原导致更快的原纤维化，其中涉及不同的 hCT 残基。总之，结果表明微小的结构变化对 hCT 纤颤有显着影响，