Department of Chemistry, Quebec Network for Research on Protein Function, Engineering and Applications, PROTEO, University of Québec in Montreal , C.P. 8888, Succursale Centre-Ville, Montreal, Québec H3C 3P8, Canada.
Bioconjug Chem. 2018 Feb 21;29(2):517-527. doi: 10.1021/acs.bioconjchem.7b00827. Epub 2018 Feb 12.
Amyloid fibril formation and tissue deposition are associated with many diseases. Studies have shown that prefibrillar intermediates, such as oligomers, are the most toxic proteospecies of the amyloidogenic cascade. Thus, understanding the mechanisms of formation and the conformational ensemble of prefibrillar species is critical. Due to their transient and heterogeneous nature, detection and characterization of prefibrillar species remain challenging. The fluorogenic probe fluorescein arsenical hairpin (FlAsH), which recognizes a tetracysteine motif, has been recently used to detect the oligomerization of amyloidogenic peptides encompassing a Cys-Cys tag. In this study, we extended the FlAsH detection method to gain novel kinetic and conformational insights into the self-assembly of islet amyloid polypeptide (IAPP), a 37-residue peptide hormone whose deposition is associated with type II diabetes. By positional scanning of the Cys-Cys motif, the stability of the noncontiguous tetracysteine FlAsH-binding sites formed during self-assembly was evaluated and revealed rapid monomer self-recognition through the convergence of C-terminal domains. On the other hand, the N-terminal domains come close to each other only upon the formation of the cross-β-sheet amyloid structure. We demonstrated that this method is well-suited to detect thioflavin T-negative fibrils and to screen inhibitors of amyloid formation. This study highlights that with positional scanning of the split-tetracysteine motif (Cys-Cys), the FlAsH detection method offers unique time-dependent conformational insights on the proteospecies assembled throughout the amyloidogenic pathway.
淀粉样纤维的形成和组织沉积与许多疾病有关。研究表明，原纤维中间态，如寡聚物，是淀粉样蛋白级联中最具毒性的蛋白物种。因此，了解形成机制和原纤维物种的构象整体对于理解疾病的发生至关重要。由于其瞬态和异质性，检测和鉴定原纤维物种仍然具有挑战性。荧光探针荧光素砷发夹（FlAsH）可以识别四半胱氨酸基序，最近被用于检测包含半胱氨酸-半胱氨酸标签的淀粉样肽的寡聚化。在这项研究中，我们扩展了 FlAsH 检测方法，以获得关于胰岛淀粉样多肽（IAPP）自组装的新的动力学和构象见解，IAPP 是一种 37 个残基的激素肽，其沉积与 II 型糖尿病有关。通过对 Cys-Cys 基序的位置扫描，评估了在自组装过程中形成的非连续四半胱氨酸 FlAsH 结合位点的稳定性，并通过 C 末端结构域的收敛揭示了单体的快速自我识别。另一方面，只有在形成交叉β-折叠淀粉样结构时，N 末端结构域才会彼此靠近。我们证明，这种方法非常适合检测硫黄素 T 阴性纤维，并筛选淀粉样形成抑制剂。这项研究强调，通过对半胱氨酸-半胱氨酸分裂四半胱氨酸基序的位置扫描，FlAsH 检测方法为整个淀粉样蛋白途径中组装的蛋白物种提供了独特的时间依赖性构象见解。
