Fibrillation of Human Calcitonin and its Analogs: Effects of Phosphorylation and Disulfide Reduction.
Fibrillation of Human Calcitonin and its Analogs: Effects of Phosphorylation and Disulfide Reduction.
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DOI：
10.1016/j.bpj.2020.11.009
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发表时间：
2020-11
影响因子：
3.4
通讯作者：
Harshil K Renawala;K. B. Chandrababu;E. Topp
中科院分区：
文献类型：
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作者：
Harshil K Renawala;K. B. Chandrababu;E. Topp
一些治疗性多肽在溶液中自组装，形成有序的、不溶的、富含β片状的淀粉样蛋白纤维。这种物理不稳定性可能导致效力降低，导致免疫原性副作用，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。在这篇论文中，我们使用了酰胺氢-重离子交换-质谱仪(HDX-MS)结合蛋白质分解分析来鉴定导致人降钙素(HCT)纤颤的早期相互作用，HCT是一种在钙代谢中起重要作用的多肽激素。硫代黄素T和浊度测量表明，HCT纤颤动力学呈S形，有滞后期、增长期和平台期。纤颤Hct(pH 7.4；25℃)的HDX-MS显示，N端(1-11)和中心(12-19)片段在迟滞期早期参与相互作用，而C-末端片段(20-32和26-32)在此期间参与有限。残基水平信息被用来开发磷酸化的Hct类似物，显示依赖于磷酸化位点的修饰的纤颤。中心区域的磷酸化导致磷酸-Thr-13HCT类似物完全抑制纤颤，而N-末端和C-末端的磷酸化抑制但不能阻止纤颤。脉冲HDX-MS表明，Cys1-Cys7二硫键的减少导致更快的纤颤，并涉及不同的Hct残基。综上所述，结果表明微小的结构变化对HCT纤颤有显著影响，了解这些影响有助于合理开发抗纤颤的HCT类似物。
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 and 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, whereas 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.