Graduate School of Engineering, Muroran Institute of Technology, Muroran 050-8585, Japan.
Int J Mol Sci. 2024 Oct 17;25(20):11132. doi: 10.3390/ijms252011132.
Protein aggregation is associated with various diseases caused by protein misfolding. Among them, amylin deposition is a prominent feature of type 2 diabetes. At present, the mechanism of amylin aggregation remains unclear, and this has hindered the treatment of type 2 diabetes. In this study, we analyzed the aggregation process of amylin using the quantum dot (QD) imaging method. QD fluorescence imaging revealed that in the presence of 100 μM amylin, aggregates appeared after 12 h of incubation, while a large number of aggregates formed after 24 h of incubation, with a standard deviation (SD) value of 5.435. In contrast, 50 μM amylin did not induce the formation of aggregates after 12 h of incubation, although a large number of aggregates were observed after 24 h of incubation, with an SD value of 2.883. Confocal laser microscopy observations revealed that these aggregates were deposited in three dimensions. Transmission electron microscopy revealed that amylin existed as misfolded fibrils in vitro and that QDs were uniformly bound to the amylin fibrils. In addition, using a microliter-scale high-throughput screening (MSHTS) system, we found that rosmarinic acid, a polyphenol, inhibited amylin aggregation at a half-maximal effective concentration of 852.8 μM. These results demonstrate that the MSHTS system is a powerful tool for evaluating the inhibitory activity of amylin aggregation. Our findings will contribute to the understanding of the pathogenesis of amylin-related diseases and the discovery of compounds that may be useful in the treatment and prevention of these diseases.
蛋白质聚集与多种由蛋白质错误折叠引起的疾病有关。其中，淀粉样蛋白沉积是 2 型糖尿病的一个显著特征。目前，淀粉样蛋白聚集的机制尚不清楚，这阻碍了 2 型糖尿病的治疗。在这项研究中，我们使用量子点（QD）成像方法分析了淀粉样蛋白的聚集过程。QD 荧光成像显示，在存在 100 μM 淀粉样蛋白的情况下，孵育 12 小时后出现聚集物，而孵育 24 小时后形成大量聚集物，标准偏差（SD）值为 5.435。相比之下，孵育 12 小时后，50 μM 淀粉样蛋白不会诱导聚集物的形成，尽管孵育 24 小时后观察到大量聚集物，但 SD 值为 2.883。共聚焦激光显微镜观察显示，这些聚集物是三维沉积的。透射电子显微镜显示，淀粉样蛋白在体外以错误折叠的纤维形式存在，并且 QD 均匀地结合在淀粉样蛋白纤维上。此外，使用微升规模高通量筛选（MSHTS）系统，我们发现迷迭香酸，一种多酚，在半最大有效浓度为 852.8 μM 时抑制淀粉样蛋白聚集。这些结果表明，MSHTS 系统是评估淀粉样蛋白聚集抑制活性的有力工具。我们的研究结果将有助于理解与淀粉样蛋白相关疾病的发病机制，并发现可能对这些疾病的治疗和预防有用的化合物。
