Department of Chemistry, Pharmaqam, Université du Québec à Montréal, Montreal, Quebec H3C 3P8, Canada; Quebec Network for Research on Protein Function, Engineering, and Applications, PROTEO, Quebec G1V 0A6, Canada.
Department of Chemistry, Pharmaqam, Université du Québec à Montréal, Montreal, Quebec H3C 3P8, Canada; Quebec Network for Research on Protein Function, Engineering, and Applications, PROTEO, Quebec G1V 0A6, Canada.
J Biol Chem. 2019 May 24;294(21):8452-8463. doi: 10.1074/jbc.RA118.006454. Epub 2019 Apr 11.
The islet amyloid polypeptide (IAPP) is a 37-residue peptide hormone whose deposition as amyloid fibrils in the pancreatic islets is associated with type 2 diabetes. Previous studies have suggested that residue Asn-21 plays a critical role in the self-assembly of IAPP. Herein, we studied structure-self-assembly relationships focusing on position 21 to gain detailed insights into the molecular mechanisms of IAPP self-assembly and to probe the conformational nature of the toxic assemblies associated with β-cell death. Thioflavin T (ThT) fluorescence, CD spectroscopy, and transmission EM analysis revealed that the Asn-21 amide side chain is not required for IAPP nucleation and amyloid elongation, as N21A and N21G variants assembled into prototypical fibrils. In contrast, Asn-21 substitution with the conformationally constrained and turn-inducing residue Pro accelerated IAPP self-assembly. Successive substitutions with hydrophobic residues led to the formation of ThT-negative β-sheet-rich aggregates having high surface hydrophobicity. Cell-based assays revealed no direct correlation between the amyloidogenicity of these variants and their toxicity. In contrast, leakage of anionic lipid vesicles disclosed that membrane disruption is closely associated with cytotoxicity. We observed that the N21F variant self-assembles into worm-like aggregates, causing loss of lipid membrane structural integrity and inducing β-cell apoptosis. These results indicate that specific intra- and intermolecular interactions involving Asn-21 promote IAPP primary nucleation events by modulating the conformational conversion of the oligomeric intermediates into amyloid fibrils. Our study identifies position 21 as a hinge residue that modulates IAPP amyloidogenicity and cytotoxicity.
胰岛淀粉样多肽 (IAPP) 是一种 37 个残基的肽激素，其在胰岛中的淀粉样纤维沉积与 2 型糖尿病有关。先前的研究表明，残基 Asn-21 在 IAPP 的 自组装中起着关键作用。在此，我们研究了结构-自组装关系，重点关注位置 21，以深入了解 IAPP 自组装的分子机制，并探测与β细胞死亡相关的有毒组装的构象性质。硫黄素 T (ThT) 荧光、CD 光谱和透射电镜分析表明，Asn-21 酰胺侧链不是 IAPP 成核和淀粉样伸长所必需的，因为 N21A 和 N21G 变体组装成典型的纤维。相比之下，用构象受限和诱导转角的残基 Pro 取代 Asn-21 加速了 IAPP 的自组装。连续用疏水性残基取代导致形成 ThT 阴性的富含β-折叠的聚集物，具有高表面疏水性。基于细胞的测定显示，这些变体的 淀粉样形成性与其毒性之间没有直接相关性。相比之下，阴离子脂质囊泡的泄漏表明膜破坏与细胞毒性密切相关。我们观察到 N21F 变体自组装成蠕虫样聚集物，导致脂质膜结构完整性丧失，并诱导β细胞凋亡。这些结果表明，涉及 Asn-21 的特定的 分子内和分子间相互作用通过调节寡聚中间体向淀粉样纤维的构象转化来促进 IAPP 的初级成核事件。我们的研究确定位置 21 是一个铰链残基，它调节 IAPP 的淀粉样形成性和细胞毒性。
