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Amyloid fibril reduction through covalently modified lysine in HEWL and insulin

Rezaei, M ; Sharif University of Technology | 2022

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  1. Type of Document: Article
  2. DOI: 10.1016/j.abb.2022.109350
  3. Publisher: Academic Press Inc , 2022
  4. Abstract:
  5. Proteins possess a variety of nucleophiles, which can carry out different reactions in the functioning cells. Proteins endogenously and synthetically can be modified through their nucleophilic sites. The roles of these chemical modifications have not been completely revealed. These modifications can alter the protein folding process. Protein folding directly affects the function of proteins. If an error in protein folding occurs, it may cause protein malfunction leading to several neurodegenerative disorders such as Alzheimer's and Parkinson's. In this study, Hen Egg White Lysozyme (HEWL) and bovine insulin, as model proteins for studying the amyloid formation, were covalently attached with 5(6)-thiophenolfluorescein. The amyloid formation of the covalently labeled lysozyme and insulin were compared with the native proteins. Interestingly, the results indicated that the covalent attachment of fluorescein slowed down the amyloid formation of HEWL and insulin significantly. The amyloid formation was examined using Thioflavin T (ThT) fluorescence assay, circular dichroism, FTIR, and gel electrophoresis. Tandem mass spectrometry was employed to identify the sites of covalent modifications in HEWL. It turned out that two surface lysine residues (K97 and K 116) in HEWL were modified. Computational studies, including docking and molecular simulations, revealed that 5(6)-thiophenolfluorescein makes several non-covalent interactions with HEWL residues, including Lys 97, leading to the reduction of the β-sheet in the protein. Additionally, AFM analysis confirmed the amyloid fibril reduction of lysine-modified bovine insulin and HEWL. Altogether, our results expand mechanistic insights into preventing amyloid formation by providing an approach for reducing amyloid formation by modifying specific lysine residues in the proteins. © 2022 Elsevier Inc
  6. Keywords:
  7. Amyloid formation ; HEWL ; Insulin ; Protein covalent modification ; Protein misfolding ; Bovine insulin ; Bovine serum albumin ; Egg white ; Fluorescein derivative ; Lysine ; Lysozyme ; Thioflavine ; Agar gel electrophoresis ; Aqueous solution ; Atomic force microscopy ; Beta sheet ; Computer model ; Conformational transition ; Controlled study ; Covalent bond ; Female ; Fluorescence analysis ; Fourier transform infrared spectroscopy ; Hen ; Hydrogen bond ; Molecular docking ; Nonhuman ; Protein conformation ; Protein folding ; Protein modification ; protein secondary structure ; Reduction (chemistry) ; Synthesis ; Tandem mass spectrometry ; X ray diffraction ; Animal ; Bovine ; Chemistry ; Chicken ; Metabolism ; Amyloid ; Animals ; Cattle ; Chickens ; Circular Dichroism ; Insulin ; Muramidase
  8. Source: Archives of Biochemistry and Biophysics ; Volume 727 , 2022 ; 00039861 (ISSN)
  9. URL: https://pubmed.ncbi.nlm.nih.gov/35830943