Search

In a simplified approach to the in vivo situation, where pathogenic fibrillar protein deposits are often found associated with cellular membranes, the aggregation kinetics of insulin in the presence of various model biomembranes were investigated using the Thioflavin T (ThT) fluorescence assay. The lipid dynamics near the gel-fluid transition, the chain length of saturated lipids and the presence of DOPE or DOPS in DOPC-vesicles modulate the aggregation kinetics of insulin in an indifferent, an aggregation-accelerating or an aggregation-inhibiting manner, subtly depending on the pH-value and the presence of salt. The rate of insulin aggregation in bulk solution dominates the overall aggregation process in most cases at low pH, where the lipid additives exert no effect on the aggregation kinetics. The occurrence of dynamic line defects near the gel-fluid transition temperature of DSPC facilitates a partial membrane insertion of the protein, which in turn shields exposed hydrophobic protein patches from intermolecular association and hence inhibit aggregation. An exclusively aggregation-accelerating effect was observed in the presence of 0.1M NaCl for all lipid additives investigated, which is likely due to an enhanced surface accumulation of the protein. Apart from weak dipole-dipole, dipole-monopole and hydrogen bonding interactions, the release of curvature elastic stress in mixed DOPC/DOPE-membranes and preferred interactions of insulin with carboxylic groups in DOPC/DOPS-membranes favour an increased surface accumulation. At neutral pH, a partial insertion of insulin into the lipid bilayer is favoured, which accounts for the aggregation-inhibiting effect of all lipid bilayer systems studied except those containing DOPS. Generally, the extent of inhibition increases with the lipid chain length and the extent of curvature stress in mixed unsaturated lipid membranes and also when the gel-fluid transition temperature of pure phospholipids is approached. The accelerating effect of DOPS on the aggregation of insulin under net electrostatic repulsion at pH 7.4 remains to be elucidated, yet, it might result from increased surface accumulation and/or faster/more extensive unfolding of the protein without a subsequent membrane insertion. These results demonstrate that a delicate interplay between different physical and chemical properties of lipid membranes has to be taken into account in a detailed discussion of membrane-associated protein aggregation phenomena.

Solvational perturbations were employed to selectively tune the aggregational preferences of insulin at 60 degrees C in vitro in purely aqueous acidic solution and in the presence of the model co-solvent ethanol (EtOH) (at 40%(w/w)). Dynamic light scattering (DLS), thioflavin T (ThT)-fluorescence, Fourier transform infrared (FTIR) and atomic force microscopy (AFM) techniques were employed to characterize these pathways biophysically with respect to the pre-aggregational assembly of the protein, the aggregation kinetics, and finally the aggregate secondary structure and morphology. Using cell viability assays, the results were subsequently correlated with the cytotoxicity of the insulin species that form in the two distinct aggregation pathways. In the cosolvent-free solution, predominantly dimeric insulin self-assembles via the well-known amyloidogenic pathway, yielding exclusively fibrillar aggregates, whereas in the solution containing EtOH, the aggregation of predominantly monomeric insulin proceeds via a pathway that leads to exclusively non-fibrillar, amorphous aggregates. Initially present native insulin assemblies as well as partially unfolded monomeric species and low molecular mass oligomeric aggregates could be ruled out as direct and major cytotoxic species. Apart from the slower overall aggregation kinetics under amorphous aggregate promoting conditions, which is due to the chaotropic nature of high EtOH concentrations, however, both pathways were unexpectedly found to evoke insulin aggregates that were cytotoxic to cultured rat insulinoma cells. The observed kinetics of the decrease of cell viabilities correlated well with the results of the DLS, ThT, FTIR and AFM studies, revealing that the formation of cytotoxic species correlated well with the formation of large-sized, beta-sheet-rich assemblies (>500 nm) of both fibrillar and amorphous nature. These results suggest that large-sized, beta-sheet-rich insulin assemblies of both fibrillar and amorphous nature are toxic to pancreatic beta-cells. In the light of the ongoing discussion about putative cytotoxic effects of prefibrillar and fibrillar amyloid aggregates, our results support the hypothesis that, in the case of insulin, factors other than the specific secondary or quarternary structural features of the various different aggregates may define their cytotoxic properties. Two such factors might be the aggregate size and the aggregate propensity to expose hydrophobic surfaces to a polar environment.

Solvation-assisted pressure tuning has been employed to unravel unknown structural and kinetic aspects of the insulin aggregation and fibrillation process. Our approach, using fluorescence, Fourier transform infrared and atomic force microscopy techniques in combination with pressure and solvent perturbation, reveals new insights into the pre-aggregated regime as well as mechanistic details about two concurrent aggregation pathways and the differential stability of insulin aggregates. Pressure uniformly fosters the dissociation of native insulin oligomers, whereas the aggregation pathways at elevated temperatures are affected by pressure differently and in a cosolvent-dependent manner. Moderate pressures accelerate the amyloid pathway in the presence of EtOH (leading to essentially monomeric aggregating species) via relatively dehydrated transition states with negative activation volumes for nucleation and elongation. Alternatively, a novel, fast equilibrium pathway to distinct beta-sheet-rich oligomers with thioflavin T-binding capability is accessible to partially unfolded insulin monomers at pressures below approximately 200 bar in the absence of EtOH. These oligomers, probably off the normal fibrillation pathway, are stabilized mainly by electrostatic and hydrophobic interactions, lacking the precise packing of mature insulin fibrils, which renders them susceptible to quantitative pressure-induced dissociation. Due to a highly negative activation volume for dissociation (-70(+/-16)ml/mol), pressure dissociation is fast and technologically feasible at ambient temperatures and moderate pressures. Becoming kinetically very labile above 35 degrees C, the pressurized oligomers can re-enter the slower, ultimately irreversible, fibrillation pathway at higher temperatures. At pressures above approximately 1000 bar, the partial unfolding of insulin monomers, accompanied by a volumetric expansion, dominates the aggregation kinetics, which manifests in a progressive inhibition of the fibrillation. Unlike their precursors, the pressure-insensitivity of mature insulin fibrils demonstrates that an extensive hydrogen bonding network and optimized side-chain packing are crucial for their stability.

Solvational perturbations, accomplished by the addition of the three model cosolvents glycerol, ethanol and trifluoroethanol, exert pronounced and diversified effects on the unfolding, non-native assembly and fibril formation of the amyloidogenic protein insulin. Fluorescence, CD and UV-spectroscopic methods as well as atomic force microscopy imaging have been employed to reveal distinct structural and kinetic features upon the aggregation of insulin under different solvational perturbations, which ultimately manifest in morphological variations of mature aggregates and fibrils. In particular, fluorescence anisotropy studies proved to be very valuable in characterizing the corresponding aggregation nuclei. Glycerol stabilizes, through enhanced hydration, native oligomerization and retards fibrillar aggregation at all concentrations studied (up to 40% (w/w)). In contrast, both monoalcohols facilitate the formation of aggregation-prone intermediates by destabilization of the native assembly. The reversal from a kosmotropic to a merely chaotropic solvational behaviour can explain the accelerating effect on ordered fibrillation of low concentrations and the inhibitory nature of high concentrations of ethanol and trifluoroethanol, ultimately leading to amorphous aggregate structures. Mechanistically, dimer dissociation under stabilizing and nucleation under destabilizing conditions have been identified to be the rate-limiting steps that account for the non-monotonic concentration effects of the monoalcohols on the aggregation kinetics. A rationale as to how solvational constraints can tune the stability of the species on the native self-assembly and non-native aggregation pathway, and the energetic barriers that need to be overcome for the required structural interconversions has been put forward. We may propose that the concept of perturbed solvation is generally applicable to phenomena that are related to pathogenic amyloidogenesis of proteins and, in general, solvational effects, besides other aspects of the cellular environment, may play a significant role in a reshaping of the folding/aggregation funnel of proteins.

A model cosolvent, ethanol, has profound and diversified effects on the amyloidogenic self-assembly of insulin, yielding spectroscopically and morphologically distinguishable forms of beta-aggregates. The alcohol reduces hydrodynamic radii of insulin molecules, decreases enthalpic costs associated with aggregation-prone intermediate states, and accelerates the aggregation itself. Increasing the concentration of the cosolvent promotes curved, amorphous, and finally donut-shaped forms. According to FT-IR data, inter-beta-strand hydrogen bonding is stronger in fibrils formed in the presence of ethanol. Mechanisms underlying the polymorphism of insulin aggregates were investigated by spectroscopic (CD, FT-IR, and fluorescence anisotropy) and calorimetric (DSC and PPC) methods. The nonmonotonic character of the influence of ethanol on insulin aggregation suggests that both preferential exclusion (predominant at the low concentrations) and direct alcohol-protein interactions are involved. The perturbed hydration of aggregation nuclei appears to be a decisive factor in selection of a dominant mode of beta-strand alignment. It may override unfavorable structural consequences of an alternative strand-to-strand stacking, such as strained hydrogen bonding. A hypothetical mechanism of inducing different amyloid "strains" has been put forward. The cooperative character of fibril assembly creates enormous energy barriers for any interstrain transition, which renders the energy landscape comblike-shaped.

Amyloids, initially associated with certain degenerative diseases, and recently with the prions and prion-based inheritance in yeasts, are linearly-ordered beta-sheet-rich protein aggregates, presently thought to represent a rather common generic trait of proteins as polymers. Regardless of genetic origins and properties of precursor protein molecules, amyloids share many physicochemical properties, including the linear fibrillar morphology. Here, we show that under high hydrostatic pressure insulin forms amyloids of a unique circular morphology. Despite a degree of size-distribution, the smallest forms of the approximate radius of 340-420 nm are most abundant among the ring-shaped structures. The circular amyloid is accompanied by bent 20-100 nm long fibrils. The pressure-enhancement of a ring-like supramolecular fold suggests an anisotropic distribution of void volumes in regular amyloid fibres. While the ability of high pressure to evoke such drastic perturbations on an amyloidogenic pathway may help tune conformation of amyloid templates (e.g. inducing the PrP(Sc)-type infectivity in amyloids grown in vitro from recombinant PrP), the very finding raises new questions concerning possible consequences for high-pressure food processing.