Dissecting the role of disulfide bonds on the amyloid formation of insulin.
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.
Tongji School of Pharmacy, Huazhong University of Science and Technology, Wuhan 430030, PR China.
Yang
Y
Li
Hao
H
Gong
Yue
Y
Sun
Juan
J
Yan
Biao
B
Cheng
Xin
X
Zhang
Jing
J
Huang
Mengying
M
Yu
Yu
Y
Guo
Ling
L
Zheng
Kun
K
Huang
D000478:Alkylation; D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry*; D000818:Animals; D052160:Benzothiazoles; D002478:Cells, Cultured; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D004912:Erythrocytes / Q000187:drug effects / Q000473:pathology; D006461:Hemolysis; D006801:Humans; D007328:Insulin / Q000737:chemistry* / Q000494:pharmacology; D008969:Molecular Sequence Data; D013552:Swine; D013844:Thiazoles / Q000737:chemistry; D014466:Ultraviolet Rays
D000682:Amyloid; D052160:Benzothiazoles; D004220:Disulfides; D007328:Insulin; D013844:Thiazoles; C009462:thioflavin T