Department of Chemistry and Biochemistry, Loyola Marymount University, 1 LMU Drive, Los Angeles, California 90045, USA.
Biochemistry. 2010 Sep 14;49(36):7783-9. doi: 10.1021/bi100337p.
The aggregation of the 37-residue protein, islet amyloid polypeptide (IAPP), as either insoluble amyloid or as small oligomers, appears to play a direct role in the death of pancreatic beta-islet cells in type II diabetes. While IAPP has been known to be the primary component of type II diabetes amyloid, the molecular interactions responsible for this aggregation have not been identified. To identify the aggregation-prone region(s), we constructed a library of randomly generated point mutants of IAPP. This mutant IAPP library was expressed in Escherichia coli as genetic fusions to the reporter protein enhanced green fluorescent protein (EGFP). Because IAPP aggregates rapidly, both independently and when fused to EGFP, the fusion protein does not yield a functional, fluorescent EGFP. However, mutations of IAPP that result in nonamyloidogenic sequences remain soluble and allow EGFP to fold and fluoresce. Using this screen, we identified 22 single mutations, 4 double mutations, and 2 triple mutations of IAPP that appear to be less amyloidogenic than wild-type human IAPP. A comparison of these sequences suggests residues 13 and 15-17 comprise an additional aggregation-prone region outside of the main amyloidogenic region of IAPP.
37 个氨基酸组成的蛋白，胰岛淀粉样多肽（IAPP），不论是不溶性淀粉样物还是小的寡聚物的聚集，似乎在 II 型糖尿病中直接导致胰岛β细胞的死亡。虽然 IAPP 已被确认为 II 型糖尿病淀粉样物的主要成分，但导致这种聚集的分子相互作用尚未确定。为了确定易于聚集的区域，我们构建了 IAPP 的随机点突变文库。该突变体 IAPP 文库在大肠杆菌中表达为与报告蛋白增强型绿色荧光蛋白（EGFP）的遗传融合。由于 IAPP 迅速聚集，无论是独立的还是与 EGFP 融合时，融合蛋白都不能产生功能性的荧光 EGFP。然而，导致非淀粉样序列的 IAPP 突变仍然是可溶的，并允许 EGFP 折叠和发光。使用该筛选，我们鉴定了 22 个 IAPP 的单突变、4 个双突变和 2 个三突变，它们似乎比野生型人 IAPP 具有较低的淀粉样形成性。对这些序列的比较表明，残基 13 和 15-17 构成了 IAPP 主要淀粉样形成区域之外的另一个易于聚集的区域。
