Oligomeric forms of insulin amyloid aggregation disrupt outgrowth and complexity of neuron-like PC12 cells

Formation of protein amyloid fibrils consists of a series of intermediates including oligomeric aggregates, proto-fibrillar structures, and finally mature fibrils. Recent studies show higher toxicity for oligomeric and proto-fibrillar intermediates of protein relative to their mature fibrils. Here the kinetic of the insulin amyloid fibrillation was evaluated using a variety of techniques including ThT fluorescence, Congo red absorbance, circular dichroism, and atomic force microscopy (AFM). The solution surface tension changes were attributed to hydrophobic changes in insulin structure and were detected by Du Noüy Ring method. Determination of the surface tension of insulin oligomeric, proto-fibrillar and fibrillar forms indicated that the hydrophobicity of solution is enhanced by the formation of the oligomeric forms of insulin compared to other forms. In order to investigate the toxicity of the different forms of insulin we monitored morphological alterations of the differentiated neuron-like PC12 cells following incubation with native, oligomeric aggregates, proto-fibrillar, and fibrillar forms of insulin. The cell body area, average neurite length, neurite width, number of primary neurites, and percent of bipolar cells and node/primary neurite ratios were used to assess the growth and complexity of PC12 cells exposed to different forms of insulin. We observed that the oligomeric form of insulin impaired the growth and complexity of PC12 cells compared to other forms. Together our data suggest that the lower surface tension of oligomers and their perturbation affects the morphology of PC12 cells, mainly due to their enhanced hydrophobicity and detergent-like structures.     

Solvation-assisted pressure tuning of insulin fibrillation: from novel aggregation pathways to biotechnological applications

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.     

Aggregation-induced conformation changes dictate islet amyloid polypeptide (IAPP) membrane affinity

Islet amyloid polypeptide (IAPP) is a 37 residue intrinsically disordered protein whose aggregation is associated with Type II diabetes. Like most amyloids, it appears that the intermediate aggregates (“oligomers”) of IAPP are more toxic than the mature fibrils, and interaction with the cell membrane is likely to be an integral component of the toxicity. Here we probe the membrane affinity and the conformation of the peptide as a function of its aggregation state. We find that the affinity of the peptide for artificial lipid bilayers is more than 15 times higher in the small oligomeric state (hydrodynamic radius ~ 1.6 nm) compared to the monomeric state (hydrodynamic radius ~ 0.7 nm). Binding with RIN-m5F cell membranes also shows qualitatively similar behavior. The monomeric state, as determined by Forster Resonance Energy Transfer, has a much larger end to end distance than the oligomeric state, suggesting conformational change between the monomers and the oligomers. Raman and Infrared spectroscopic measurements show the presence of considerable alpha helical content in the oligomers, whereas the larger aggregates have largely beta sheet character. Therefore, the conformation of the small oligomers is distinct from both the smaller monomers and the larger oligomers, and this is associated with an enhanced membrane affinity. This provides a possible structural basis for the enhanced toxicity of amyloid oligomers. Such change is also reminiscent of amyloid beta, another aggregation prone amyloidogenic peptide, though the nature of the conformational change is quite different in the two cases. We infer that conformational change underlying oligomer formation is a key factor in determining the enhanced membrane affinity of disease causing oligomers, but the toxic “oligomer fold” may not be universal.     

Existence of Different Structural Intermediates on the Fibrillation Pathway of Human Serum Albumin

The fibrillation propensity of the multidomain protein human serum albumin (HSA) was analyzed under different solution conditions. The aggregation kinetics, protein conformational changes upon self-assembly, and structure of the different intermediates on the fibrillation pathway were determined by means of thioflavin T (ThT) fluorescence and Congo Red absorbance; far- and near-ultraviolet circular dichroism; tryptophan fluorescence; Fourier transform infrared spectroscopy; x-ray diffraction; and transmission electron, scanning electron, atomic force, and microscopies. HSA fibrillation extends over several days of incubation without the presence of a lag phase, except for HSA samples incubated at acidic pH and room temperature in the absence of electrolyte. The absence of a lag phase occurs if the initial aggregation is a downhill process that does not require a highly organized and unstable nucleus. The fibrillation process is accompanied by a progressive increase in the β-sheet (up to 26%) and unordered conformation at the expense of α-helical conformation, as revealed by ThT fluorescence and circular dichroism and Fourier transform infrared spectroscopies, but changes in the secondary structure contents depend on solution conditions. These changes also involve the presence of different structural intermediates in the aggregation pathway, such as oligomeric clusters (globules), bead-like structures, and ring-shaped aggregates. We suggest that fibril formation may take place through the role of association-competent oligomeric intermediates, resulting in a kinetic pathway via clustering of these oligomeric species to yield protofibrils and then fibrils. The resultant fibrils are elongated but curly, and differ in length depending on solution conditions. Under acidic conditions, circular fibrils are commonly observed if the fibrils are sufficiently flexible and long enough for the ends to find themselves regularly in close proximity to each other. These fibrils can be formed by an antiparallel arrangement of β-strands forming the β-sheet structure of the HSA fibrils as the most probable configuration. Very long incubation times lead to a more complex morphological variability of amyloid mature fibrils (i.e., long straight fibrils, flat-ribbon structures, laterally connected fibers, etc.). We also observed that mature straight fibrils can also grow by protein oligomers tending to align within the immediate vicinity of the fibers. This filament + monomers/oligomers scenario is an alternative pathway to the otherwise dominant filament + filament manner of the protein fibril's lateral growth. Conformational preferences for a certain pathway to become active may exist, and the influence of environmental conditions such as pH, temperature, and salt must be considered.     

Fibrillation of carrier protein albebetin and its biologically active constructs. Multiple oligomeric intermediates and pathways

We showed that the genetically engineered carrier-protein albebetin and its biologically active constructs with interferon-alpha(2) octapeptide LKEKKYSP or differentiation factor hexapeptide TGENHR are inherently highly amyloidogenic at physiological pH. The kinetics of fibrillation were monitored by thioflavine-T (ThT) binding and the morphological changes by atomic force microscopy. Fibrillation proceeds via multiple pathways and includes a hierarchy of amyloid structures ranging from oligomers to protofilaments and fibrils. Comparative height and volume microscopic measurements allowed us to identify two distinct types of oligomeric intermediates: pivotal oligomers ca. 1.2 nm in height comprised of 10-12 monomers and on-pathway amyloid-competent oligomers ca. 2 nm in height constituted of 26-30 molecules. The former assemble into chains and rings with "bead-on-string morphology", in which a "bead" corresponds to an individual oligomer. Once formed, the rings and chains remain in solution simultaneously with fibrils. The latter give rise to protofilaments and fibrils, and their formation is concomitant with an increasing level of ThT binding. The amyloid nature of filamentous structures was confirmed by a pronounced ThT and Congo red binding and beta-sheet-rich far-UV circular dichroism. We suggest that transformation of the pivotal oligomers into the amyloid-prone ones is a limiting stage in amyloid assembly. Peptides, either fused to albebetin or added into solution, and an increased ionic strength promote fibrillation of albebetin (net charge of -12) by counterbalancing critical electrostatic repulsions. This finding demonstrates that the fibrillation of newly designed polypeptide-based products can produce multimeric amyloid species with a potentially "new" functionality, raising questions about their safety.     

Biochemical and biophysical features of both oligomer/fibril and cell membrane in amyloid cytotoxicity

A great deal must still be learnt on the structural features of amyloid assemblies, particularly prefibrillar aggregates, and the relationship of the latter with amyloid cytotoxicity. Presently, it is recognized that the population of unstable, heterogeneous amyloid oligomers and protofibrils is mainly responsible for amyloid cytotoxicity. Conversely, mature fibrils are considered stable, harmless reservoirs of molecular species devoid of toxicity in the polymerized state. This view has been modified by recent reports showing that mature fibrils grown at different conditions can display different structural features and stabilities, possibly leading them to undergo disassembly with the leak of toxic oligomers. Fibril polymorphism is paralleled by oligomer polymorphism and both can be traced back to amyloid growth from differently destabilized monomers with distinct structural features at differing conditions. Recent research has started to unravel oligomer structural and biophysical features and the relationship between the latter and oligomer cytotoxicity. These data have led to the proposal that, together, both oligomer and membrane physical features determine the extent of oligomer-membrane interaction with the resulting disruption of membrane integrity and cell impairment. Such a view can help to explain the variable vulnerability of different cell types to the same amyloids and the lack of relationship between amyloid load and the severity of clinical symptoms. It also stresses the importance, for cell/tissue impairment, of the presence, in tissue, in addition to toxic oligomers, of fibrils conformers of reduced stability as a possible source of toxic oligomers, whose leakage can be favoured upon interaction with suitable surfaces or by other environmental conditions.     

Acceleration of the depolymerization of amyloid β fibrils by ultrasonication

Amyloid fibrils, rigid and filamentous aggregates associated with various diseases, are often difficult to depolymerize into monomers. Ultrasonication is a strong agitation that accelerates nucleation above the critical concentration of amyloid fibrillation. We examined the effects of ultrasonication on the fibrils of amyloid β(1–40) as well as on monomers. Ultrasonic pulses accelerated spontaneous fibrillation when the peptide concentration was above 1 μM. On the other hand, ultrasonic pulses accelerated the depolymerization of fibrils into monomers at 1 μM. These results indicate that, although amyloid fibrillation is a reversible process determined by thermodynamic stability, kinetically trapped supersaturation and physical difficulty of dissolving rigid fibrils prevent the smooth phase transitions. We propose that, in addition to accelerating the nucleation of fibrillation and fragmentation of fibrils above the critical concentration, ultrasonication is useful for dissolving fibrils below the critical concentration.     

Instantaneous amyloid fibril formation of alpha-synuclein from the oligomeric granular structures in the presence of hexane

Amyloid fibrils found in various neurodegenerative disorders are also recognized as high-performance protein nanomaterials with a formidable rigidity. Elucidation of an underlying molecular mechanism of the amyloid fibril formation is crucial not only to develop controlling strategy toward the diseases, but also to apply the protein fibrils for future nanobiotechnology. alpha-Synuclein is an amyloidogenic protein responsible for the radiating filament formation within Lewy bodies of Parkinson's disease. The amyloid fibril formation of alpha-synuclein has been shown to be induced from the oligomeric granular species of the protein acting as a growing unit by experiencing structural rearrangement within the preformed oligomeric structures in the presence of an organic solvent of hexane. This granule-based concerted amyloid fibril formation model would parallel the prevalent notion of nucleation-dependent fibrillation mechanism in the area of amyloidosis.     

Tyrosine Autofluorescence as a Measure of Bovine Insulin Fibrillation

The traditional approach to investigating the partial unfolding and fibrillation of insulin, and proteins at large, has involved use of the dyes 1-anilinonaphthalene-8-sulphonic acid (ANS) and Thioflavin T (ThT), respectively. We compare the kinetic profiles of ThT, ANS, light scattering, and intrinsic Tyr fluorescence during insulin fibrillation. The data reveal that the sequence of structural changes (dimers → monomers → partially unfolded monomers → oligomeric aggregates → fibrils) accompanying insulin fibrillation can be detected directly using intrinsic Tyr fluorescence. The results indicate that at least two distinguishable structural intermediates precede fibril development. There is no evidence of tyrosinate or dityrosine during insulin aggregation. Obtaining such critical information from the protein itself is complementary to existing aggregation probes and affords the advantage of directly examining structural changes that occur at the molecular level, providing concrete details of the early events preceding fibrillation.     

Instantaneous Amyloid Fibril Formation of α-Synuclein from the Oligomeric Granular Structures in the Presence of Hexane

Amyloid fibrils found in various neurodegenerative disorders are also recognized as high-performance protein nanomaterials with a formidable rigidity. Elucidation of an underlying molecular mechanism of the amyloid fibril formation is crucial not only to develop controlling strategy toward the diseases, but also to apply the protein fibrils for future nanobiotechnology. α-Synuclein is an amyloidogenic protein responsible for the radiating filament formation within Lewy bodies of Parkinson's disease. The amyloid fibril formation of α-synuclein has been shown to be induced from the oligomeric granular species of the protein acting as a growing unit by experiencing structural rearrangement within the preformed oligomeric structures in the presence of an organic solvent of hexane. This granule-based concerted amyloid fibril formation model would parallel the prevalent notion of nucleation-dependent fibrillation mechanism in the area of amyloidosis.     

Early mechanisms of amyloid fibril nucleation in model and disease-related proteins

Protein amyloid aggregation is a hallmark in neuropathologies and other diseases of tremendous impact such as Alzheimer’s or Parkinson’s diseases. During the last decade, it has become increasingly evident that neuronal death is mainly induced by proteinaceous oligomers rather than the mature amyloid fibrils. Therefore, the earliest molecular events occurring during the amyloid aggregation cascade represent a growing interest of study.Important breakthroughs have been achieved using experimental data from different proteins, used as models, as well as systems related to diseases. Here, we summarize the structural properties of amyloid oligomeric and fibrillar aggregates and review the recent advances on how biophysical techniques can be combined with quantitative kinetic analysis and theoretical models to study the detailed mechanism of oligomer formation and nucleation of fibrils.These insights into the mechanism of early oligomerization and amyloid nucleation are of relevant interest in drug discovery and in the design of preventive strategies against neurodegenerative diseases.     

Fibrillation of human insulin A and B chains

Human insulin, which consists of disulfide cross-linked A and B polypeptide chains, readily forms amyloid fibrils under slightly destabilizing conditions. We examined whether the isolated A and B chain peptides of human insulin would form fibrils at neutral and acidic pH. Although insulin exhibits a pH-dependent lag phase in fibrillation, the A chain formed fibrils without a lag at both pHs. In contrast, the B chain exhibited complex concentration-dependent fibrillation behavior at acidic pH. At higher concentrations, e.g., >0.2 mg/mL, the B chains preferentially and rapidly formed stable protofilaments rather than mature fibrils upon incubation at 37 degrees C. Surprisingly, these protofilaments did not convert into mature fibrils. At lower B chain concentrations, however, mature fibrils were formed. The explanation for the concentration dependence of B chain fibrillation is as follows. The B chains exist as soluble oligomers at acidic pH, have a beta-sheet rich conformation as determined by CD, and bind ANS strongly, and these oligomers rapidly form dead-end protofilaments. However, under conditions in which the B chain monomer is present, such as low B chain concentration (<0.2 mg/mL) or in the presence of low concentrations of GuHCl, which dissociates the soluble oligomers, mature fibrils were formed. Thus, both A and B chain peptides can form amyloid fibrils, and both are likely to be involved in the interactions leading to the fibrillation of intact insulin.     

Structural polymorphism and possible pathways of amyloid fibril formation on the example of insulin protein

In this review we analyze the main works on amyloid formation of insulin. There are many environmental factors affecting the formation of insulin amyloid fibrils (and other amyloidogenic proteins) such as: protein concentration, pH, ionic strength of solution, medium composition (anions, cations), presence of denaturants (urea, guanidine chloride) or stabilizers (saccharose), temperature regime, agitation. Since polymorphism is potentially crucial for human diseases and may underlie the natural variability of some amyloid diseases, in this review we focus attention on polymorphism that is an important biophysical difference between native protein folding suggesting correspondence between the amino acid sequence and unique folding state, and formation of amyloid fibrils, when the same amino acid sequence can form amyloid fibrils of different morphology. At present, according to the literature data, we can choose three ways of polymerization of insulin molecules depending on the nucleus size. The first suggests that fibrillogenesis can occur through assembly of insulin monomers. The second suggests that precursors of fibrils are dimers, and the third assumes that precursors of fibrils are oligomers. Additional experimental works and new methods of investigation and assessment of results are needed to clarify the general picture of insulin amyloid formation.     

Prefibrillar transthyretin oligomers and cold stored native tetrameric transthyretin are cytotoxic in cell culture

Recent studies suggest that soluble, oligomeric species, which are intermediates in the fibril formation process in amyloid disease, might be the key species in amyloid pathogenesis. Soluble oligomers of human wild type transthyretin (TTR) were produced to elucidate oligomer properties. Employing ThT fluorescence, time-resolved fluorescence anisotropy of pyrene-labeled TTR, chemical cross-linking, and electron microscopy we demonstrated that early formed soluble oligomers (within minutes) from A-state TTR comprised on the average 20-30 TTR monomers. When administered to neuroblastoma cells these early oligomers proved highly cytotoxic and induced apoptosis after 48 h of incubation. More mature fibrils (>24 h of fibrillation) were non-toxic. Surprisingly, we also found that native tetrameric TTR, when purified and stored under cold conditions (4 °C) was highly cytotoxic. The effect could be partially restored by increasing the temperature of the protein. The cytotoxic effects of native tetrameric TTR likely stems from a hitherto unexplored low temperature induced rearrangement of the tetramer conformation that possibly is related to the conformation of misfolded TTR in amyloigogenic oligomers.     

Stepwise Organization of the β-Structure Identifies Key Regions Essential for the Propagation and Cytotoxicity of Insulin Amyloid Fibrils

Amyloid fibrils are supramolecular assemblies, the deposition of which is associated with many serious diseases including Alzheimer, prion, and Huntington diseases. Several smaller aggregates such as oligomers and protofibrils have been proposed to play a role in early stages of the fibrillation process; however, little is known about how these species contribute to the formation of mature amyloid fibrils with a rigid cross-β structure. Here, we identified a new pathway for the formation of insulin amyloid fibrils at a high concentration of salt in which mature fibrils were formed in a stepwise manner via a prefibrillar intermediate: minute prefibrillar species initially accumulated, followed by the subsequent formation of thicker amyloid fibrils. Fourier transform infrared spectra suggested the sequential formation of two types of β-sheets with different strength hydrogen bonds, one of which was developed concomitantly with the mutual assembly of the prefibrillar intermediate to form mature fibrils. Interestingly, fibril propagation and cellular toxicity appeared only after the later step of structural organization, and a comparison of β-sheet regions between the prefibrillar intermediate and mature fibrils using proteolysis led to the proposal of specific regions essential for manifestation of these properties.     

Oligomeric assembly of native-like precursors precedes amyloid formation by beta-2 microglobulin

The deposition of beta-2-microglobulin (beta2m) as amyloid fibers results in debilitating complications for renal failure patients who are treated by hemodialysis. In vitro, wild-type beta2m can be converted to amyloid under physiological conditions by exposure to biomedically relevant concentrations of Cu(2+). In this work, we have made comparative measurements of the structural and oligomeric changes in beta2m at time points preceding fibrillogenesis. Our results show Cu(2+) mediates the formation of a monomeric, activated state followed by the formation of a discrete dimeric intermediate. The dimeric intermediates then assemble into tetra- and hexameric forms which display little additional oligomerization on the time scales of their own formation (<1 h). Amyloid fiber formation progresses from these intermediate states but on much longer time scales (>1 week). Although Cu(2+) is necessary for the generation and stabilization of these intermediates, it is not required for the stability of mature amyloid fibers. This suggests that Cu(2+) acts as an initiating factor of amyloidosis by inducing oligomer formation. (1)H NMR and near-UV circular dichroism are used to establish that oligomeric intermediates are native-like in structure. The native-like structure and discrete oligomeric size of beta2m amyloid intermediates suggest that this protein forms fibrils by structural domain swapping.     

Partially folded intermediates in insulin fibrillation

Native zinc-bound insulin exists as a hexamer at neutral pH. Under destabilizing conditions, the hexamer dissociates, and is very prone to forming fibrils. Insulin fibrils exhibit the typical properties of amyloid fibrils, and pose a problem in the purification, storage, and delivery of therapeutic insulin solutions. We have carried out a systematic investigation of the effect of guanidine hydrochloride (Gdn.HCl)-induced structural perturbations on the mechanism of fibrillation of insulin. At pH 7.4, the addition of as little as 0.25 M Gdn.HCl leads to dissociation of insulin hexamers into dimers. Moderate concentrations of Gdn.HCl lead to formation of a novel partially unfolded dimer state, which dissociates into a partially unfolded monomer state. High concentrations of Gdn.HCl resulted in unfolded monomers with some residual structure. The addition of even very low concentrations of Gdn.HCl resulted in substantially accelerated fibrillation, although the yield of fibrils decreased at high concentrations. Accelerated fibrillation correlated with the population of the expanded (partially folded) monomer, which existed up to >6 M Gdn.HCl, accounting for the formation of substantial amounts of fibrils under such conditions. In the presence of 20% acetic acid, where insulin exists as the monomer, fibrillation was also accelerated by Gdn.HCl. The enhanced fibrillation of the monomer was due to the increased ionic strength at low denaturant concentrations, and due to the presence of the partially unfolded, expanded conformation at Gdn.HCl concentrations above 1 M. The data suggest that under physiological conditions, the fibrillation of insulin involves both changes in the association state (with rate-limiting hexamer dissociation) and conformational changes, leading to formation of the amyloidogenic expanded monomer intermediate.