Growth-incompetent monomers of human calcitonin lead to a noncanonical direct relationship between peptide concentration and aggregation lag time
Growth-incompetent monomers of human calcitonin lead to a noncanonical direct relationship between peptide concentration and aggregation lag time
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DOI：
10.1074/jbc.m117.791236
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发表时间：
2017-09-08
影响因子：
4.8
通讯作者：
Ramamoorthy, Ayyalusamy
中科院分区：
文献类型：
--
作者：
Kamgar-Parsi, Kian;Hong, Liu;Ramamoorthy, Ayyalusamy
肽激素降钙素在骨骼保护中的作用导致其用作骨质疏松症的治疗性。然而，降钙素聚集到淀粉样蛋白原纤维中限制了其治疗功效，因此需要修改降钙素的聚集动力学。在这里，我们报告人类降钙素（HCT）浓度与聚集滞后时间之间的直接关系。这种动力学趋势与对淀粉样蛋白聚集的常规理解相反，并在一系列聚集条件下持续存在，如硫非类T动力学测定，CD光谱和传播EM所证实的那样。动态光散射，1H NMR实验和种子硫非类 - T分析结果表明，初始肽物种的差异对这一趋势的差异远远超过一级核形成速率的变化。在动力学建模的基础上，我们提出了一种机制，在将HCT单体的结构转化掺入原纤维之前。我们的动力学机制概括了肽浓度和滞后时间之间的实验观察到的关系，并代表了淀粉样蛋白聚集的新机制。有趣的是，低pH和鲑鱼降钙素（SCT）的HCT表现出浓度和滞后时间之间的规范反比关系。 HCT和SCT与分子动力学模拟和CD的比较研究表明，与中性pH HCT相比，SCT和低pH HCT HCT单体的α-螺旋结构增加，这表明α-螺旋单体代表了一种生长型物种，而非结构性的随机盘旋型线粒代表生长型物种。我们的发现，最初的单体浓度与HCT聚合中的滞后时间呈正相关，可以帮助您为改善CT的治疗应用的未来努力提供信息。
The role of the peptide hormone calcitonin in skeletal protection has led to its use as a therapeutic for osteoporosis. However, calcitonin aggregation into amyloid fibrils limits its therapeutic efficacy, necessitating a modification of calcitonin's aggregation kinetics. Here, we report a direct relationship between human calcitonin (hCT) concentration and aggregation lag time. This kinetic trend was contrary to the conventional understanding of amyloid aggregation and persisted over a range of aggregation conditions, as confirmed by thioflavin-T kinetics assays, CD spectroscopy, and transmission EM. Dynamic light scattering, 1H NMR experiments, and seeded thioflavin-T assay results indicated that differences in initial peptide species contribute to this trend more than variations in the primary nucleus formation rate. On the basis of kinetics modeling results, we propose a mechanism whereby a structural conversion of hCT monomers is needed before incorporation into the fibril. Our kinetic mechanism recapitulates the experimentally observed relationship between peptide concentration and lag time and represents a novel mechanism in amyloid aggregation. Interestingly, hCT at low pH and salmon calcitonin (sCT) exhibited the canonical inverse relationship between concentration and lag time. Comparative studies of hCT and sCT with molecular dynamics simulations and CD indicated an increased alpha-helical structure in sCT and low-pH hCT monomers compared with neutral-pH hCT, suggesting that alpha-helical monomers represent a growthcompetent species, whereas unstructured random coil monomers represent a growth-incompetent species. Our finding that initial monomer concentration is positively correlated with lag time in hCT aggregation could help inform future efforts for improving therapeutic applications of CT.