title: Aspirin-mediated acetylation induces structural alteration and aggregation of bovine pancreatic insulin.
abstract: The simple aggregation of insulin under various chemical and physical stresses is still an important challenge for both pharmaceutical production and clinical formulation. In the storage form, this protein is subjected to various chemical modifications which alter its physicochemical and aggregation properties. Aspirin (acetylsalicylic acid) which is the most widely used medicine worldwide has been indicated to acetylate a large number of proteins both in vitro and in vivo. In this study, as insulin treated with aspirin at 37°C, a significant level of acetylation was observed by flourescamine and o-phthalaldehyde assay. Also, different spectroscopic techniques, gel electrophoresis, and microscopic assessment were applied to compare the structural variation and aggregation/fibrillation propensity among acetylated and non-acetylated insulin samples. The results of spectroscopic assessments elucidate that acetylation induces insulin unfolding which is accompanied with the exposure of protein hydrophobic patches, a transition from alpha-helix to beta-sheet and increased propensity of the protein for aggregation. The kinetic studies propose that acetylation increases aggregation rate of insulin under both thermal and chemical stresses. Also, gel electrophoresis and dynamic light scattering experiments suggest that acetylation induces insulin oligomerization. Additionally, the results of Thioflavin T fluorescence study, Congo red absorption assessment, and microscopic analysis suggest that acetylation with aspirin enhances the process of insulin fibrillation. Overall, the increased susceptibility of acetylated insulin for aggregation may reflect the fact that this type of modification has significant structural destabilizing effect which finally makes the protein more vulnerable for pathogenic aggregation/fibrillation.
authors.affiliation: a Protein Chemistry Laboratory (PCL), Department of Biology , College of Sciences, Shiraz University , Shiraz , Iran.|b Institute of Biotechnology , Shiraz University , Shiraz , Iran.
authors.forename: Reza
authors.initials: R
authors.lastname: Yousefi
authors.affiliation: a Protein Chemistry Laboratory (PCL), Department of Biology , College of Sciences, Shiraz University , Shiraz , Iran.
authors.forename: Behnaz
authors.initials: B
authors.lastname: Taheri
authors.affiliation: a Protein Chemistry Laboratory (PCL), Department of Biology , College of Sciences, Shiraz University , Shiraz , Iran.|f Diagnostic Laboratory Sciences and Technology Research Center , School of Paramedical Sciences, Shiraz University of Medical Sciences , Shiraz , Iran.
authors.forename: Parnian
authors.initials: P
authors.lastname: Alavi
authors.affiliation: a Protein Chemistry Laboratory (PCL), Department of Biology , College of Sciences, Shiraz University , Shiraz , Iran.
authors.forename: Mohammad Bagher
authors.initials: MB
authors.lastname: Shahsavani
authors.affiliation: c Department of Chemistry , College of Sciences, Shiraz University , Shiraz , Iran.
authors.forename: Zahra
authors.initials: Z
authors.lastname: Asadi
authors.affiliation: a Protein Chemistry Laboratory (PCL), Department of Biology , College of Sciences, Shiraz University , Shiraz , Iran.
authors.forename: Maryam
authors.initials: M
authors.lastname: Ghahramani
authors.affiliation: b Institute of Biotechnology , Shiraz University , Shiraz , Iran.
authors.forename: Ali
authors.initials: A
authors.lastname: Niazi
authors.affiliation: d Department of Chemistry, College of Sciences , Shiraz University of Technology , Shiraz , Iran.
authors.forename: Mohammad Mehdi
authors.initials: MM
authors.lastname: Alavianmehr
authors.affiliation: e Institute of Biochemistry and Biophysics (IBB) , The University of Tehran , Tehran , Iran.
authors.forename: Ali Akbar
authors.initials: AA
authors.lastname: Moosavi-Movahedi
mesh_terms: D000107:Acetylation / Q000187:drug effects; D000595:Amino Acid Sequence; D000818:Animals; D001241:Aspirin / Q000494:pharmacology*; D002417:Cattle; D002942:Circular Dichroism; D000067493:Dynamic Light Scattering; D004591:Electrophoresis, Polyacrylamide Gel; D057446:Hydrodynamics; D007328:Insulin / Q000737:chemistry* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010179:Pancreas / Q000378:metabolism*; D066329:Protein Aggregates / Q000187:drug effects*; D017433:Protein Structure, Secondary
chemical_list: D007328:Insulin; D066329:Protein Aggregates; D001241:Aspirin
keywords: acetylation; aggregation; aspirin; gel electrophoresis; insulin; unfolding