Dissecting the role of disulfide bonds on the amyloid formation of insulin

Journal Article · Fri Jun 29 04:00:00 EDT 2012 · Biochemical and Biophysical Research Communications

DOI:https://doi.org/10.1016/J.BBRC.2012.05.133· OSTI ID:22207920

Li, Yang; Gong, Hao ^[1]; Sun, Yue ^[2]; Yan, Juan; Cheng, Biao; Zhang, Xin ^[1]; Huang, Jing ^[2]; Yu, Mengying; Guo, Yu ^[1]; Zheng, Ling ^[2]; Huang, Kun ^[1]

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1. Tongji School of Pharmacy, Huazhong University of Science and Technology, Wuhan 430030 (China)
2. College of Life Sciences, Wuhan University, Wuhan 430072 (China)

Highlights: Black-Right-Pointing-Pointer We dissect how individual disulfide bond affects the amyloidogenicity of insulin. Black-Right-Pointing-Pointer A controlled reduction system for insulin is established in this study. Black-Right-Pointing-Pointer Disulfide breakage is associated with unfolding and increased amyloidogenicity. Black-Right-Pointing-Pointer Breakage of A6-A11 is associated with significantly increased cytotoxicity. Black-Right-Pointing-Pointer Analogs without A6-A11 have a higher potency to form high order toxic oligomers. -- Abstract: 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 amyloidogenicity 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.

• OSTI ID: 22207920
• Journal Information: Biochemical and Biophysical Research Communications, Journal Name: Biochemical and Biophysical Research Communications Journal Issue: 2 Vol. 423; ISSN 0006-291X; ISSN BBRCA9
• Country of Publication: United States
• Language: English

Related Subjects

60 APPLIED LIFE SCIENCES
CROSS-LINKING
DICHROISM
DISULFIDES
ERYTHROCYTES
FLUORESCENCE
HEMOLYSIS
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY
INSULIN
PHOSPHINES
SPECTROSCOPY
TOXICITY
TRANSMISSION ELECTRON MICROSCOPY