pH-Driven Polymorphism of Insulin Amyloid-Like Fibrils.

Prions are infective proteins, which can self-assemble into different strain conformations, leading to different disease phenotypes. An increasing number of studies suggest that prion-like self-propagation may be a common feature of amyloid-like structures. Thus it is important to unravel every possible factor leading to the formation of different amyloid strains. Here we report on the formation of two types of insulin amyloid-like fibrils with distinct infrared spectroscopic features grown under slightly different pH conditions. Similar to prion strains, both insulin fibril types are able to self-propagate their conformational template under conditions, favoring spontaneous formation of different type fibrils. The low-pH-induced insulin amyloid strain is structurally very similar to previously reported strains formed either in the presence of 20% ethanol, or by modification of the amino acid sequence of insulin. A deeper analysis of literature data in the context of our current findings suggests a shift of the monomer-dimer equilibrium of insulin as a possible factor controlling the formation of different strains.

Flavone derivatives as inhibitors of insulin amyloid-like fibril formation.

Several natural and synthetic flavone derivatives have been reported to inhibit formation of amyloid fibrils or to remodel existing fibrils. These studies suggest that the numbers and positions of hydroxyl groups on the flavone rings determine their effectiveness as amyloid inhibitors. In many studies the primary method for determining the effectiveness of inhibition is measuring Thioflavin T (ThT) fluorescence. This method demonstrably results in a number of false positives for inhibition. We studied the effects of 265 commercially available flavone derivatives on insulin fibril formation. We enhanced the effectiveness of ThT fluorescence measurements by fitting kinetic curves to obtain halftime of aggregation (t50). Maximal values of ThT fluorescence varied two fold or more in one third of all cases, but this did not correlate with changes in t50. Changes in t50 values were more accurate measures of inhibition of amyloid formation. We showed that without a change in an assay, but just by observing complete kinetic curves it is possible to eliminate numbers of false positive and sometimes even false negative results. Examining the data from all 265 flavones we confirmed previous observations that identified the importance of hydroxyl groups for inhibition. Our evidence suggests the importance of hydroxyl groups at locations 5, 6, 7, and 4', and the absence of a hydroxyl group at location 3, for inhibiting amyloid formation. However, the main conclusion is that the positions are not additive. The structures and their effects must be thought of in the context of the whole molecule.

Amyloid-like fibril elongation follows michaelis-menten kinetics.

A number of proteins can aggregate into amyloid-like fibrils. It was noted that fibril elongation has similarities to an enzymatic reaction, where monomers or oligomers would play a role of substrate and nuclei/fibrils would play a role of enzyme. The question is how similar these processes really are. We obtained experimental data on insulin amyloid-like fibril elongation at the conditions where other processes which may impact kinetics of fibril formation are minor and fitted it using Michaelis-Menten equation. The correlation of the fit is very good and repeatable. It speaks in favour of enzyme-like model of fibril elongation. In addition, obtained [Formula: see text] and [Formula: see text] values at different conditions may help in better understanding influence of environmental factors on the process of fibril elongation.