Early Events in the Fibrillation of Monomeric Insulin

Abstract

Insulin has a largely ␣-helical structure and exists as a mixture of hexameric, dimeric, and monomeric states in solution, depending on the conditions: the protein is monomeric in 20% acetic acid. Insulin forms amyloid-like fibrils under a variety of conditions, especially at low pH. In this study we investigated the fibrillation of monomeric human insulin by monitoring changes in CD, attenuated total reflectance-Fourier transform infrared spectroscopy, 8-anilinonaphthalenesulfonic acid fluorescence, thioflavin T fluorescence, dynamic light scattering, and H/D exchange during the initial stages of the fibrillation process to provide insight into early events involving the monomer. The results demonstrate the existence of structural changes occurring before the onset of fibril formation, which are detectable by multiple probes. The data indicate at least two major populations of oligomeric intermediates between the native monomer and fibrils. Both have significantly non-native conformations, and indicate that fibrillation occurs from a betarich structure significantly distinct from the native fold.A number of human diseases are caused by the pathogenic deposition of proteins in the form of amyloid-like fibrils (1-7). Several non-pathogenic proteins and peptides also undergo amyloid like fibril formation on destabilization of their native state (7-10). The fact that structurally and sequentially non-homologous proteins are able to self-assemble into fibrils possessing similar morphology (e.g. 10 -18 nm width, birefringence to polarized light, and cross-␤ structure) suggests a common molecular mechanism in the fibrillation pathways. A variety of hypotheses for the mechanism of fibril formation have been proposed.Insulin is a 51-residue hormone with a largely ␣-helical structure. It exists as a mixture of hexameric, dimeric, and monomeric states in solution, with the relative population of different oligomeric species being strongly dependent on the environmental conditions: the protein is predominantly monomeric in 20% acetic acid, dimeric in 20 mM HCl, and hexameric at pH 7.5 in the presence of zinc. Insulin forms amyloidlike fibrils under a variety of conditions (11-13), with various overall morphologies depending on the arrangement of constituent protofilaments (14, 15). Insulin fibrils pose a variety of problems in biomedical and biotechnological applications. Amyloid deposits of insulin have been observed in patients with diabetes after repeated injection and in normal aging, as well as after subcutaneous insulin infusion (16,17).In our previous work we have shown the important role of partially folded intermediates in insulin fibrillation in vitro (13, 18 -20). In fact, our studies showed that insulin, which is a hexamer at physiological pH, undergoes rapid fibrillation starting at relatively low concentrations of guanidine hydrochloride and urea. The predominant species characterized by various biophysical techniques under these conditions was shown to be a partially folded, expanded monomer, which is presen...

Supersaturation-limited Amyloid Fibrillation of Insulin Revealed by Ultrasonication

“…It should be noted that the alcohol concentrations in this article are (v/v) percentages. The concentration of insulin was determined spectrophotometrically using absorbance at 276 nm with an extinction coefficient of 1.0 mg ml Ϫ1 (37). The final concentrations of insulin used were 1.5 and 3.0 mg ml…”

Synchrotron-Infrared Microscopy Analysis of Amyloid Fibrils Irradiated by Mid-Infrared Free-Electron Laser

“…In contrast, IR absorption spectral measurements are comparatively simple, and the spectra are sensitive to the secondary structures of the peptides [18]. Moreover, the normal structure of a peptide can be easily distinguished from the amyloid fibril structure in IR spectra because a peak of the amide I band shifts to as smaller wavenumber as the content of β-sheet structure increases during fibrillation of the peptide [19] [20]; IR absorption measurements are therefore also useful for detecting amyloid fibrils, as reviewed in a recent paper [21]. IR microscopy analysis is often employed for studying the local structure of organic materials and biomaterials [22] [23], and its advantages lie in the fact that no labeling or pre-treatment of the sample is required, which means samples can be re-used.…”

Cytotoxicity of albebetin oligomers depends on cross‐β‐sheet formation

“…In order to produce amyloid structures albebetin Abbreviations: AFM, atomic force microscopy; CD, circular dichroism; SPIP, scanning probe image processor was incubated at a concentration of 1.2 mM in 20 mM HEPES buffer, 50 mM NaCl, 0.2% sodium azide, pH 7.4. Ab [25][26][27][28][29][30][31][32][33][34][35] and cell apoptotic agent D D-sphingosine were purchased from Sigma (USA). Ab 25-35 amyloid was produced upon 3 days of incubation at the same conditions as albebetin.…”

“…In control measurements we have shown that there was no difference in the viability of untreated cells and cells with added buffer used for amyloid incubation. Ab [25][26][27][28][29][30][31][32][33][34][35] amyloid structures and apoptotic agent D D-sphingosine were used as positive controls of induced cytotoxicity. Upon addition of Ab [25][26][27][28][29][30][31][32][33][34][35] amyloid the survival of granular neurons decreased by ca.…”