Title
Site-specific covalent modifications of human insulin by catechol estrogens: Reactivity and induced structural and functional changes
Author
Ming-Chun Ku, Chieh-Ming Fang, Juei-Tang Cheng, Huei-Chen Liang, Tzu-Fan Wang, Chih-Hsing Wu, Chiao-Chen Chen, Jung-Hsiang Tai, Shu-Hui Chen
Year
2016
Journal
Scientific Reports
Abstract
Proteins, covalently modified by catechol estrogens (CEs), were identified recently from the blood serum of diabetic patients and referred to as estrogenized proteins. Estrogenization of circulating insulin may occur and affect its molecular functioning. Here, the chemical reactivity of CEs towards specific amino acid residues of proteins and the structural and functional changes induced by the estrogenization of insulin were studied using cyclic voltammetry, liquid chromatography-mass spectrometry, circular dichroism spectroscopy, molecular modeling, and bioassays. Our results indicate that CEs, namely, 2- and 4-hydroxyl estrogens, were thermodynamically and kinetically more reactive than the catechol moiety. Upon co-incubation, intact insulin formed a substantial number of adducts with one or multiple CEs via covalent conjugation at its Cys 7 in the A or B chain, as well as at His10 or Lys29 in the B chain. Such conjugation was coupled with the cleavage of inter-chain disulfide linkages. Estrogenization on these sites may block the receptor-binding pockets of insulin. Insulin signaling and glucose uptake levels were lower in MCF-7 cells treated with modified insulin than in cells treated with native insulin. Taken together, our findings demonstrate that insulin molecules are susceptible to active estrogenization, and that such modification may alter the action of insulin.
Full Article
Instrument
J-815
Keywords
Circular dichroism, Secondary structure, Biochemistry