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.

Cross-seeding of fibrils of bovine insulin (BI) and Lys(B31)-Arg(B32) human insulin analog (KR) induces self-propagating amyloid variants with infrared features inherited from mother seeds. Here we report that when native insulin (BI or KR) is simultaneously seeded with mixture of equal amounts of both templates (i.e., of separately grown fibrils of BI and KR), the phenotype of resulting daughter fibrils is as in the case of the purely homologous seeding: heterologous cotemplates accelerate the fibrillation but do not determine infrared traits of the daughter amyloid. This implies that fibrillation-promoting and structure-imprinting properties of heterologous seeds become uncoupled in the presence of homologous seeds. We argue that explanation of such behavior requires that insulin molecules partly transformed through interactions with heterologous fibrils are subsequently recruited by homologous seeds. The selection bias toward homologous daughter amyloid is exceptional: more than 200-fold excess of heterologous seed is required to imprint its structural phenotype upon mixed seeding. Our study captures a snapshot of elusive docking interactions in statu nascendi of elongation of amyloid fibril and suggests that different types of seeds may collaborate in sequential processing of soluble protein into fibrils.

Typically, elongation of an amyloid fibril entails passing conformational details of the mother seed to daughter generations of fibrils with high fidelity. There are, however, several factors that can potentially prevent such transgenerational structural imprinting from perpetuating, for example heterogeneity of mother seeds or so-called conformational switching. Here, we examine phenotypic persistence of bovine insulin amyloid ([BI]) upon multiple rounds of self-seeding under quiescent conditions. According to infrared spectroscopy, with the following passages of homologous seeding, daughter fibrils gradually depart from the mother seed’s spectral characteristics. We note that this transgenerational structural drift in [BI] amyloid leads toward fibrils with infrared, chiroptical, and morphological traits similar to those of the superstructural variant of fibrils which normally forms upon strong agitation of insulin solutions. However, in contrast to agitation-induced insulin amyloid, the superstructural assemblies of daughter fibrils isolated through self-seeding are sonication-resistant. Our results suggest that formation of single amyloid fibrils is not a dead-end of the amyloidogenic self-assembly. Instead, the process appears to continue toward the self-assembly of higher-order structures although on longer time-scales. From this perspective, the fast agitation-induced aggregation of insulin appears to be a shortcut to amyloid superstructures whose formation under quiescent conditions is slow.

Formation of amyloid fibrils is often associated with intriguing far-from-equilibrium phenomena such as conformational memory effects or flow-driven self-assembly. Insulin is a model amyloidogenic polypeptide forming distinct structural variants of fibrils, which self-propagate through seeding. According to infrared absorption, fibrils from bovine insulin ([BI]) and Lys(B31)-Arg(B32) human insulin analogue ([KR]) cross-seed each other and imprint distinct structural features in daughter fibrils. In the absence of preformed [KR] amyloid seeds, bovine insulin agitated at 60 °C converts into chiral amyloid superstructures exhibiting negative extrinsic Cotton effect in bound thioflavin T. However, when agitated bovine insulin is simultaneously cross-seeded with [KR] amyloid, daughter fibrils reveal a positive extrinsic Cotton effect. Our study indicates that dramatic changes in global properties of amyloid superstructures may emerge from subtle conformational-level variations in single fibrils (e.g., alignment and twist of β-strands) that are encoded by memory effects.

Formation of amyloid fibrils in vivo has been linked to disorders such as Alzheimer’s disease and prion-associated transmissible spongiform encephalopathies. One of the characteristic features of amyloid fibrils is the high thermodynamic stability relative both to native and disordered states which is also thought to underlie the perplexingly remarkable heat resistance of prion infectivity. Here, we are comparing high-temperature degradation of native and fibrillar forms of human insulin. Decomposition of insulin amyloid has been studied under helium atmosphere and in the temperature range from ambient conditions to 750°C using thermogravimetry and differential scanning calorimetry coupled to mass spectrometry. While converting native insulin into amyloid does upshift onset of thermal decomposition by ca. 75°C, fibrils remain vulnerable to covalent degradation at temperatures below 300°C, as reflected by mass spectra of gases released upon heating of amyloid samples, as well as morphology and infrared spectra of fibrils subjected to incubation at 250°C. Mass spectra profiles of released gases indicate that degradation of fibrils is much more cooperative than degradation of native insulin. The data show no evidence of water of crystallization trapped within insulin fibrils. We have also compared untreated and heated amyloid samples in terms of capacity to seed daughter fibrils. Kinetic traces of seed-induced insulin fibrillation have shown that the seeding potency of amyloid samples decreases significantly already after exposure to 200°C, even though corresponding electron micrographs indicated persisting fibrillar morphology. Our results suggest that amyloid-based biological activity may not survive extremely high temperature treatments, at least in the absence of other stabilizing factors.

Formation of amyloid fibrils is often facilitated in the presence of specific charge-compensating ions. Dissolved sodium chloride is known to accelerate insulin fibrillation at low pH that has been attributed to the shielding of electrostatic repulsion between positively charged insulin molecules by chloride ions. However, the subsequent fate of Cl(-) anions; that is, possible entrapment within elongating fibrils or escape into the bulk solvent, remains unclear. Here, we show that, while the presence of NaCl at the onset of insulin aggregation induces structural variants of amyloid with distinct fingerprint infrared features, a delayed addition of salt to fibrils that have been already formed in its absence and under quiescent conditions triggers a "condensation effect": amyloid superstructures with strong chiroptical properties are formed. Chloride ions appear to stabilize these superstructures in a manner similar to stabilization of DNA condensates by polyvalent cations. The concentration of residual chloride ions trapped within bovine insulin fibrils grown in 0.1 M NaCl, at pD 1.9, and rinsed extensively with water afterward is less than 1 anion per 16 insulin monomers (as estimated using ion chromatography) implying absence of defined solvent-sequestered nesting sites for chloride counterions. Our results have been discussed in the context of mechanisms of insulin aggregation.

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