Dissecting the role of disulfide bonds on the amyloid formation of insulin
Dissecting the role of disulfide bonds on the amyloid formation of insulin
复制标题
剖析二硫键对胰岛素淀粉样蛋白形成的作用
DOI：
10.1016/j.bbrc.2012.05.133
复制
发表时间：
2012-06-29
影响因子：
3.1
通讯作者：
Huang, Kun
中科院分区：
文献类型：
--
作者：
Li, Yang;Gong, Hao;Huang, Kun
二硫键在蛋白质的稳定性和折叠中起着关键作用。在这里，我们使用胰岛素作为模型系统，以研究其在胰岛素淀粉样蛋白形成过程中的作用。用三（2-羧乙基）膦（TCEP）还原猪胰岛素中三个二硫键中的两个，然后用碘乙酰胺将还原的二硫键烷基化。获得三种二硫键修饰的胰岛素类似物，INS-2（缺乏A6-A11）、INS-3（缺乏A7-B7）和INS-6（缺乏A6-A11和A7-B7）。远紫外圆二色性（CD）光谱结果表明，在中性条件下INS-2的二级结构最接近胰岛素，其次是INS-3和INS-6，而在酸性溶液中，所有类似物基本上是未折叠的。为了测试这些修饰如何影响胰岛素的淀粉样蛋白生成性，进行了硫磺素-T（ThT）荧光和透射电子显微镜（TEM）。我们的结果表明，所有类似物都比胰岛素更容易聚集，聚集率的顺序为INS-6> INS-3> INS-2。未修饰蛋白质的交联（PICUP）测定结果表明，不含A6-A11的类似物（INS-2和INS-6）具有比胰岛素和INS-3更高的寡聚化潜力，这伴随着更高的细胞毒性，如人红细胞的溶血测定所示。结果表明，A7-B7的断裂比A6-A11的断裂诱导更多的胰岛素结构的解折叠和更高的淀粉样变性，但A6-A11的断裂导致显著的细胞毒性增加和更高的形成高级毒性寡聚体的能力。(C)2012 Elsevier Inc. All rights reserved.
Disulfide bonds play a critical role in the stability and folding of proteins. Here, we used insulin as a model system, to investigate the role of its individual disulfide bond during the amyloid formation of insulin. Tris(2-carboxyethyl)phosphine (TCEP) was applied to reduce two of the three disulfide bonds in porcine insulin and the reduced disulfide bonds were then alkylated by iodoacetamide. Three disulfide bond-modified insulin analogs, INS-2 (lack of A6-A11), INS-3 (lack of A7-B7) and INS-6 (lack of both A6-A11 and A7-B7), were obtained. Far-UV circular dichroism (CD) spectroscopy results indicated that the secondary structure of INS-2 was the closest to insulin under neutral conditions, followed by INS-3 and INS-6, whereas in an acidic solution all analogs were essentially unfolded. To test how these modifications affect the amyloidogenecity of insulin, thioflavin-T (ThT) fluorescence and transmission electronic microscopy (TEM) were performed. Our results showed that all analogs were more prone to aggregation than insulin, with the order of aggregation rates being INS-6 > INS-3 > INS-2. Cross-linking of unmodified proteins (PICUP) assay results showed that analogs without A6-A11 (INS-2 and INS-6) have a higher potential for oligomerization than insulin and INS-3, which is accompanied with a higher cytotoxicity as the hemolytic assays of human erythrocytes suggested. The results indicated that breakage of A7-B7 induced more unfolding of the insulin structure and a higher amyloidogenicity than breakage of A6-A11, but breakage of A6-A11 caused a significant cytotoxicity increase and a higher potency to form high order toxic oligomers. (C) 2012 Elsevier Inc. All rights reserved.