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.

Classical and Nonclassical Nucleation Mechanisms of Insulin Crystals

“…Link and Heng (2022) (insulin concentration values below 1 mg mL –1 ) and Jensen et al (2014) (insulin concentration around 0.6 mg mL –1 ) obtained a hydrodynamic radius below 3 nm and a diffusion coefficient around 1 × 10 –10 m 2 s –1 . Yousefi et al (2016) (insulin concentration of 0.2 mg mL –1 ) reported values for the hydrodynamic radius around 1 nm.…”

Interaction of ZnO Nanostructures with Proteins: In Vitro Fibrillation/Antifibrillation Studies and in Silico Molecular Docking Simulations

“…The third phase is a static phase (plateau regime) . Acetylsalicylic acid (ASA) was also included in our studies as a positive control, since it induces the formation of insoluble fibril aggregates via changing the protein structure from helical to sheet form . The fluorescence emission spectra are percent normalized, with the 100% value corresponding to the solution incubated solely with protein monomers/oligomers.…”

“…ASA causes an increase in the fluorescence signal of both proteins with the respective intensities reaching 123% and 206%. Treatment with ASA boosts the fibril formation, since it destabilizes the oligomers in a way that leads to amyloid and insoluble plaque conformations . The more pronounced effect in the case of albumin may reflect the different structural characteristics of albumin .…”

“…7 Acetylsalicylic acid (ASA) was also included in our studies as a positive control, since it induces transition from α-helix to β-sheet and increased propensity for protein aggregation. 36 The fluorescence spectra are expressed as % protein fibrillation, with the fluorescence signal corresponding to 100% fibrillation status in case of agitation of the maximum concentration of neat protein.…”

“…Acetylation with ASA enhances the process of fibrillation, since this type of modification has significant structural destabilizing effect which finally makes the protein more vulnerable for pathogenic aggregation. 36 The more pronounced effect in case of albumin may reflect the different structural characteristics of albumin. 37 The same tendency was preserved in the presence of the nanoparticles for albumin; ZnO nanoflowers caused a slight increase in the intensity (106%), whereas ZnO@PEG NPs provoked a rather significant increase (177%).…”