26 research outputs found
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
The Division of Amyloid Fibrils: Systematic Comparison of Fibril Fragmentation Stability by Linking Theory with Experiments.
The division of amyloid protein fibrils is required for the propagation of the amyloid state and is an important contributor to their stability, pathogenicity, and normal function. Here, we combine kinetic nanoscale imaging experiments with analysis of a mathematical model to resolve and compare the division stability of amyloid fibrils. Our theoretical results show that the division of any type of filament results in self-similar length distributions distinct to each fibril type and the conditions applied. By applying these theoretical results to profile the dynamical stability toward breakage for four different amyloid types, we reveal particular differences in the division properties of disease-related amyloid formed from α-synuclein when compared with non-disease associated model amyloid, the former showing lowered intrinsic stability toward breakage and increased likelihood of shedding smaller particles. Our results enable the comparison of protein filaments' intrinsic dynamic stabilities, which are key to unraveling their toxic and infectious potentials
Master and Slave Relationship Between Two Types of Self-Propagating Insulin Amyloid Fibrils
Cross-seeding
of fibrils of bovine insulin (BI) and Lys<sup>B31</sup>-Arg<sup>B32</sup> 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<sup>–</sup> 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<sup>B31</sup>-Arg<sup>B32</sup> 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<sup>B31</sup>-Arg<sup>B32</sup> 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
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
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
TEM (left) and SEM (right) images of amyloid fibrils before and after heating.
<p>Scale bars are 200 nm.</p