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

Abstract

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) weremore » 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.« less

Authors:
;  [1];  [2]; ; ;  [1];  [2]; ;  [1];  [2];  [1];  [3]
  1. Tongji School of Pharmacy, Huazhong University of Science and Technology, Wuhan 430030 (China)
  2. College of Life Sciences, Wuhan University, Wuhan 430072 (China)
  3. (China)
Publication Date:
OSTI Identifier:
22207920
Resource Type:
Journal Article
Journal Name:
Biochemical and Biophysical Research Communications
Additional Journal Information:
Journal Volume: 423; Journal Issue: 2; Other Information: Copyright (c) 2012 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 0006-291X
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; CROSS-LINKING; DICHROISM; DISULFIDES; ERYTHROCYTES; FLUORESCENCE; HEMOLYSIS; HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY; INSULIN; PHOSPHINES; SPECTROSCOPY; TOXICITY; TRANSMISSION ELECTRON MICROSCOPY

Citation Formats

Li, Yang, Gong, Hao, Sun, Yue, Yan, Juan, Cheng, Biao, Zhang, Xin, Huang, Jing, Yu, Mengying, Guo, Yu, Zheng, Ling, E-mail: lzheng217@hotmail.com, Huang, Kun, E-mail: kunhuang2008@hotmail.com, and Centre for Biomedicine Research, Wuhan Institutes of Biotechnology, Wuhan 430070. Dissecting the role of disulfide bonds on the amyloid formation of insulin. United States: N. p., 2012. Web. doi:10.1016/J.BBRC.2012.05.133.
Li, Yang, Gong, Hao, Sun, Yue, Yan, Juan, Cheng, Biao, Zhang, Xin, Huang, Jing, Yu, Mengying, Guo, Yu, Zheng, Ling, E-mail: lzheng217@hotmail.com, Huang, Kun, E-mail: kunhuang2008@hotmail.com, & Centre for Biomedicine Research, Wuhan Institutes of Biotechnology, Wuhan 430070. Dissecting the role of disulfide bonds on the amyloid formation of insulin. United States. doi:10.1016/J.BBRC.2012.05.133.
Li, Yang, Gong, Hao, Sun, Yue, Yan, Juan, Cheng, Biao, Zhang, Xin, Huang, Jing, Yu, Mengying, Guo, Yu, Zheng, Ling, E-mail: lzheng217@hotmail.com, Huang, Kun, E-mail: kunhuang2008@hotmail.com, and Centre for Biomedicine Research, Wuhan Institutes of Biotechnology, Wuhan 430070. Fri . "Dissecting the role of disulfide bonds on the amyloid formation of insulin". United States. doi:10.1016/J.BBRC.2012.05.133.
@article{osti_22207920,
title = {Dissecting the role of disulfide bonds on the amyloid formation of insulin},
author = {Li, Yang and Gong, Hao and Sun, Yue and Yan, Juan and Cheng, Biao and Zhang, Xin and Huang, Jing and Yu, Mengying and Guo, Yu and Zheng, Ling, E-mail: lzheng217@hotmail.com and Huang, Kun, E-mail: kunhuang2008@hotmail.com and Centre for Biomedicine Research, Wuhan Institutes of Biotechnology, Wuhan 430070},
abstractNote = {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.},
doi = {10.1016/J.BBRC.2012.05.133},
journal = {Biochemical and Biophysical Research Communications},
issn = {0006-291X},
number = 2,
volume = 423,
place = {United States},
year = {2012},
month = {6}
}