The role of conformational memory effect in propagation of structural variants of insulin amyloid fibrils

W sprzyjających warunkach fizykochemicznych molekuły natywnego białka mogą ulec rozwinięciu i asocjacji tworząc tzw. włókna (fibryle) amyloidowe – nierozpuszczalne, β-kartkowe agregaty. Struktury te nie tylko pozbawione są (z reguły) prawidłowej aktywności biologicznej swoich rozpuszczalnych prekursorów, ale ich pojawienie się w organizmie może być powiązane z tzw. chorobami konformacyjnymi m.in. chorobami Alzheimera, Parkinsona, czy Creutzfeldta-Jakoba (choroba prionowa). To sprawia, że badanie molekularnych mechanizmów amyloidogenezy oraz różnorodności struktur i właściwości biochemicznych agregatów amyloidowych jest nie tylko ciekawe, ale przede wszystkim ważne klinicznie. W niniejszej pracy odtworzono w kontrolowanych warunkach in vitro agregację modelowego, niepatogennego białka insuliny. Celem przeprowadzonych badań było podjęcie problematyki związanej z zależną od zasiewania proliferacją zarodków amyloidowych insuliny, a w szczególności: (1) wyjaśnienie kwestii termicznej stabilności zarodków amyloidu insuliny w odniesieniu do ich zdolności do indukowania pokoleń potomnych fibryli, (2) badanie wpływu punktowych podstawień w sekwencji aminokwasowej insuliny poza tzw. rejonem rdzenia amyloidowego na polimorfizm strukturalny powstających fibryli amyloidowych, (3) analiza stabilności fenotypu amyloidowego zarodka w trakcie propagacji w pokoleniach potomnych oraz badanie addytywności efektów pochodzących od konkurencyjnych fenotypów.Under permissive i.e. slightly destabilizing conditions, proteins tend to misfold and aggregate into highly ordered linear β-aggregates – the so-called amyloid fibrils. These structures are linked to several human degenerative disorders, including Alzheimer’s disease, Parkinson’s disease, Creutzfeldt-Jakob disease (“prion disease”). These features make protein amyloidogenesis not only an interesting but also clinically important research topic. In this work aggregation of model, nonpathogenic protein – insulin – was reproduced in vitro under controlled conditions. The central goal was to study seeding-dependent fibrillation patterns of this protein. Particular attention was paid to following issues: (1) thermal stability of amyloid fibrils in terms of capacity to seed daughter fibrils, (2) relation between amyloid polymorphism and variations in the amino acid sequence beyond the critical amyloidogenic regions, and (3) mechanisms of stable propagation of mother amyloid phenotype in daughter generation of fibrils

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

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

On the Function and Fate of Chloride Ions in Amyloidogenic Self-Assembly of Insulin in an Acidic Environment: Salt-Induced Condensation of Fibrils

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

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

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

On the Heat Stability of Amyloid-Based Biological Activity: Insights from Thermal Degradation of Insulin Fibrils

<div><p>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.</p></div

Infrared absorption (A) and second derivative (B) spectra of amyloid fibrils before and after exposure to high temperatures.

<p>Inset in panel A shows normalized spectra in the amide I/II spectral region. Second derivative FT-IR spectra in panel B are shown only for the amide I/II region with omission of featureless 750°C spectrum.</p

TEM (left) and SEM (right) images of amyloid fibrils before and after heating.

<p>Scale bars are 200 nm.</p

Temporal profiles of high temperature incubations of amyloid samples.

<p>Powder sample were heated to 100, 200, 250, 350°C with a linear ramp 10°C/min under helium atmosphere. After desired temperature was reached, isothermal conditions were held for 10 minutes, followed by gradual cooling of the samples down to room temperature.</p

Thermal stability of human insulin in the amyloid (A, C) and native (B, D) states.

<p>Panels A and B presents thermogravimetric curves (black lines) together with the corresponding heat flow profiles (red lines) obtained through heating of samples form 50 to 750°C with a linear ramp 10°C/min under helium atmosphere. Panels C and D show TGA-MS evolution profiles of gases released during thermal analysis at selected m/z channels and assigned to different molecules.</p

Aggregation kinetics of human insulin seeded at 37°C with fibrils exposed to high temperatures, probed by ThT fluorescence.

<p>Error bars correspond to standard deviations of fluorescence intensity calculated for six microplate wells with identical samples.</p