Cross-Seeding of Fibrils from Two Types of Insulin Induces New Amyloid Strains

Abstract

The irreversibility and autocatalytic character of amyloidogenesis and the polymorphism of amyloid fibrils underlie the phenomenon of self-propagating strains, wherein the mother seed, rather than the seeding environment, determines the properties of daughter fibrils. Here we study the formation of amyloid fibrils from bovine insulin and the recombinant Lys(B31)-Arg(B32) human insulin analog. The two polypeptides are similar enough to cross-seed but, upon spontaneous aggregation, form amyloid fibrils with distinct spectral features in the infrared amide I' band region. When bovine insulin is cross-seeded with the analog amyloid (and vice versa), the shape, absorption maximum, and even fine fingerprint features of the amide I' band are passed from the mother to daughter fibrils with a high degree of fidelity. Although the differences in primary structure between bovine insulin and the Lys(B31)-Arg(B32) analog of human insulin lie outside of the polypeptide's critical amyloidogenic regions, they affect the secondary structure of fibrils, possibly the formation of intermolecular salt bridges, and the susceptibility to dissection and denaturation with dimethyl sulfoxide (DMSO). All these phenotypic features of mother fibrils are imprinted in daughter amyloid upon cross-seeding. Analysis of noncooperative DMSO-induced denaturation of daughter fibrils suggests that the self-propagating polymorphism underlying the emergence of new amyloid strains is encoded on the level of secondary structure. Our findings have been discussed in the context of polymorphism of fibrils, amyloid strains, and possible implications for mechanisms of amyloidogenesis.

Master and Slave Relationship Between Two Types of Self-Propagating Insulin Amyloid Fibrils

“…27 When these fibrils are used as templates for crossseeding, the shape, absorption maximum and fine fingerprint traits in the amide I′ band region are passed from mother seeds to daughter fibrils with high fidelity. 25 Here, we report a counterintuitive outcome of seeding experiments in which insulin fibrillation is triggered by a mixture of preformed [BI] and [KR] fibrils. Instead of anticipated competition in recruiting soluble protein collaboration between the two structural variants has been observed: the heterologous seeds accelerate proliferation of the homologous phenotypes.…”

Highly Amyloidogenic Two-chain Peptide Fragments Are Released upon Partial Digestion of Insulin with Pepsin

“…1, freshly prepared samples containing 1% w/v BI in 0.1 M NaCl, 25 M ThT, pH 1.9, and specified concentrations of pepsin (added directly before measurements in small portions of acidified 4 mg/ml enzyme solution in 0.1 M NaCl) were swiftly mixed and transferred to a 96-well black plate. Aggregation of insulin samples at 37°C was monitored by measuring intensity of ThT fluorescence using Fluoroskan Ascent FL fluorometer (from Thermo) equipped with a pair of ex 440/ em 485-nm optical filters and a 96-well Microfluor 1 U-bottom plate (Thermo) and covered with dedicated transparent adhesive foil (40). Before each measurement, the plate was shaken at 300 rpm and 3 mm amplitude for 10 s. To assess reproducibility of aggregation kinetics, six microplate wells were filled with identical 150-l portions of each sample for parallel measurements.…”

“…Baseline correction was performed with GRAMS software (Thermo). All further experimental details were the same as specified earlier (40).…”

“…Additional spectra were collected after gradual re-acidification of the sample with diluted HCl first to pH 7.0 and then 1.9. All far UV-CD measurements were carried out at 40°C by accumulation of five independent spectra on Jasco J-815 S spectropolarimeter (Jasco, Japan) as specified in our previous work (40).…”

“…This is in contrast to re-association and sonication, which have virtually no impact on the band's features. Infrared absorption in the amide IЈ band region has often been shown to capture even tiny structural variations of fibrils, including insulin amyloid polymorphs (36,40 (Fig. 8F).…”