Biophysics Program, The University of Michigan, Ann Arbor, MI 48109, USA; Department of Chemistry, Biomedical Engineering, and Macromolecular Science and Engineering, The University of Michigan, Ann Arbor, MI 48109, USA.
College of Veterinary Medicine, University of Florida, Gainesville, FL 32611, USA.
J Mol Biol. 2022 Jan 30;434(2):167385. doi: 10.1016/j.jmb.2021.167385. Epub 2021 Dec 6.
Human amylin forms structurally heterogeneous amyloids that have been linked to type-2 diabetes. Thus, understanding the molecular interactions governing amylin aggregation can provide mechanistic insights in its pathogenic formation. Here, we demonstrate that fibril formation of amylin is altered by synthetic amphipathic copolymer derivatives of the styrene-maleic-acid (SMAQA and SMAEA). High-speed AFM is used to follow the real-time aggregation of amylin by observing the rapid formation of de novo globular oligomers and arrestment of fibrillation by the positively-charged SMAQA. We also observed an accelerated fibril formation in the presence of the negatively-charged SMAEA. These findings were further validated by fluorescence, SOFAST-HMQC, DOSY and STD NMR experiments. Conformational analysis by CD and FT-IR revealed that the SMA copolymers modulate the conformation of amylin aggregates. While the species formed with SMAQA are α-helical, the ones formed with SMAEA are rich in β-sheet structure. The interacting interfaces between SMAEA or SMAQA and amylin are mapped by NMR and microseconds all-atom MD simulation. SMAEA displayed π-π interaction with Phe23, electrostatic π-cation interaction with His18 and hydrophobic packing with Ala13 and Val17; whereas SMAQA showed a selective interaction with amylin's C terminus (residues 31-37) that belongs to one of the two β-sheet regions (residues 14-19 and 31-36) involved in amylin fibrillation. Toxicity analysis showed both SMA copolymers to be non-toxic in vitro and the amylin species formed with the copolymers showed minimal deformity to zebrafish embryos. Together, this study demonstrates that chemical tools, such as copolymers, can be used to modulate amylin aggregation, alter the conformation of species.
人胰岛淀粉样多肽形成结构异构的淀粉样纤维，与 2 型糖尿病有关。因此，了解控制胰岛淀粉样多肽聚集的分子相互作用可以为其致病形成提供机制见解。在这里，我们证明了苯乙烯-马来酸（SMAQA 和 SMAEA）的合成两亲共聚物衍生物改变了胰岛淀粉样多肽的纤维形成。高速 AFM 用于通过观察新形成的球状寡聚物的快速形成以及正电荷 SMAQA 对纤维形成的抑制作用来实时跟踪胰岛淀粉样多肽的聚集。我们还观察到在带负电荷的 SMAEA 存在下，纤维形成加速。这些发现通过荧光、SOFAST-HMQC、DOSY 和 STD NMR 实验进一步得到验证。通过 CD 和 FT-IR 的构象分析表明，SMA 共聚物调节胰岛淀粉样多肽聚集物的构象。虽然与 SMAQA 形成的物质是 α-螺旋，与 SMAEA 形成的物质富含β-折叠结构。通过 NMR 和微秒全原子 MD 模拟映射 SMAEA 或 SMAQA 与胰岛淀粉样多肽之间的相互作用界面。SMAEA 与 Phe23 显示π-π 相互作用，与 His18 显示静电π-阳离子相互作用，与 Ala13 和 Val17 显示疏水性堆积；而 SMAQA 显示与胰岛淀粉样多肽的 C 末端（残基 31-37）的选择性相互作用，该末端属于两个β-折叠区（残基 14-19 和 31-36）之一，参与胰岛淀粉样多肽的纤维形成。毒性分析表明，两种 SMA 共聚物在体外均无毒性，与共聚物形成的胰岛淀粉样多肽对斑马鱼胚胎的畸形程度最小。总之，这项研究表明，化学工具，如共聚物，可以用于调节胰岛淀粉样多肽的聚集，改变物质的构象。
