From the Departments of Biochemistry Case Western Reserve University, Cleveland, Ohio 44106, USA.
J Biol Chem. 2010 Apr 2;285(14):10806-21. doi: 10.1074/jbc.M109.067850. Epub 2010 Jan 27.
Insulin fibrillation provides a model for a broad class of amyloidogenic diseases. Conformational distortion of the native monomer leads to aggregation-coupled misfolding. Whereas beta-cells are protected from proteotoxicity by hexamer assembly, fibrillation limits the storage and use of insulin at elevated temperatures. Here, we have investigated conformational distortions of an engineered insulin monomer in relation to the structure of an insulin fibril. Anomalous (13)C NMR chemical shifts and rapid (15)N-detected (1)H-(2)H amide-proton exchange were observed in one of the three classical alpha-helices (residues A1-A8) of the hormone, suggesting a conformational equilibrium between locally folded and unfolded A-chain segments. Whereas hexamer assembly resolves these anomalies in accordance with its protective role, solid-state (13)C NMR studies suggest that the A-chain segment participates in a fibril-specific beta-sheet. Accordingly, we investigated whether helicogenic substitutions in the A1-A8 segment might delay fibrillation. Simultaneous substitution of three beta-branched residues (Ile(A2) --> Leu, Val(A3) --> Leu, and Thr(A8) --> His) yielded an analog with reduced thermodynamic stability but marked resistance to fibrillation. Whereas amide-proton exchange in the A1-A8 segment remained rapid, (13)Calpha chemical shifts exhibited a more helical pattern. This analog is essentially without activity, however, as Ile(A2) and Val(A3) define conserved receptor contacts. To obtain active analogs, substitutions were restricted to A8. These analogs exhibit high receptor-binding affinity; representative potency in a rodent model of diabetes mellitus was similar to wild-type insulin. Although (13)Calpha chemical shifts remain anomalous, significant protection from fibrillation is retained. Together, our studies define an "Achilles' heel" in a globular protein whose repair may enhance the stability of pharmaceutical formulations and broaden their therapeutic deployment in the developing world.
胰岛素纤维提供了广泛的淀粉样变性疾病模型。天然单体的构象扭曲导致聚集相关的错误折叠。虽然β细胞通过六聚体组装免受蛋白毒性的影响，但纤维状会限制胰岛素在高温下的储存和使用。在这里，我们研究了工程胰岛素单体的构象扭曲与胰岛素纤维的结构之间的关系。激素的三个经典α螺旋（残基 A1-A8）之一中观察到异常的（13）C NMR 化学位移和快速（15）N 检测到的（1）H-（2）H 酰胺质子交换，表明局部折叠和未折叠 A 链段之间存在构象平衡。虽然六聚体组装根据其保护作用解决了这些异常，但固态（13）C NMR 研究表明 A 链段参与了纤维特异性的β-折叠。因此，我们研究了 A1-A8 片段中的螺旋取代是否会延迟纤维状。同时取代三个β-分支残基（Ile（A2）→Leu、Val（A3）→Leu 和 Thr（A8）→His）得到的类似物热力学稳定性降低，但对纤维状的抵抗力显著增强。尽管 A1-A8 片段中的酰胺质子交换仍然迅速，但（13）Calpha 化学位移显示出更具螺旋性的模式。然而，由于 Ile（A2）和 Val（A3）定义了保守的受体接触，这种类似物基本上没有活性。为了获得活性类似物，取代仅限于 A8。这些类似物表现出高受体结合亲和力；在糖尿病啮齿动物模型中的代表性效力与野生型胰岛素相似。尽管（13）Calpha 化学位移仍然异常，但保留了对纤维状的显著保护。总之，我们的研究定义了一种“阿喀琉斯之踵”在球状蛋白中，其修复可能会提高药物制剂的稳定性，并扩大其在发展中国家的治疗应用。
