Surface denaturation and amyloid fibril formation of insulin at model lipid-water interfaces

We consider the effects that different lipid surfaces have upon the denaturation and subsequent formation of amyloid fibrils of bovine insulin. The adsorption and unfolding kinetics of insulin being adsorbed onto the different lipid surfaces under denaturing conditions are studied using FTIR ATR spectroscopy and are compared to the bulk solution behavior of the protein. Atomic force microscopy studies are also performed to compare the fibrils growing on the different surfaces. This study shows that both the adsorption and unfolding kinetics of insulin can be described by a sum of exponential processes and that different surfaces behave differently, with respect both to one another and to the bulk protein solution. The proteins adsorbed onto the surfaces are observed to have faster unfolding kinetics than those in the bulk, and the fibril-like structures formed at the surfaces are shown to be different in a number of ways from those found in bulk solution. The beta-sheet content and growth kinetics of the adsorbed proteins also differ from those of the bulk system. An attempt is made to describe the observed behavior in terms of simple physical arguments involving adsorption, unfolding, and aggregation of the proteins.     

The effect of macromolecular crowding on protein aggregation and amyloid fibril formation

Macromolecular crowding is expected to have several significant effects on protein aggregation; the major effects will be those due to excluded volume and increased viscosity. In this report we summarize data demonstrating that macromolecular crowding may lead to a dramatic acceleration in the rate of protein aggregation and formation of amyloid fibrils, using the protein alpha-synuclein. The aggregation of alpha-synuclein has been implicated as a critical factor in development of Parkinson's disease. Various types of polymers, from neutral polyethylene glycols and polysaccharides (Ficolls, dextrans) to inert proteins, are shown to accelerate alpha-synuclein fibrillation. The stimulation of fibrillation increases with increasing length of polymer, as well as increasing polymer concentration. At lower polymer concentrations (typically up to approximately 100 mg/ml) the major effect is ascribed to excluded volume, whereas at higher polymer concentrations evidence of opposing viscosity effects become apparent. Pesticides and metals, which are linked to increased risk of Parkinson's disease by epidemiological studies, are shown to accelerate alpha-synuclein fibrillation under conditions of molecular crowding.     

Effect of surfaces on amyloid fibril formation

Using atomic force microscopy (AFM) we investigated the interaction of amyloid beta (Aβ) (1–42) peptide with chemically modified surfaces in order to better understand the mechanism of amyloid toxicity, which involves interaction of amyloid with cell membrane surfaces. We compared the structure and density of Aβ fibrils on positively and negatively charged as well as hydrophobic chemically-modified surfaces at physiologically relevant conditions. We report that due to the complex distribution of charge and hydrophobicity amyloid oligomers bind to all types of surfaces investigated (CH₃, COOH, and NH₂) although the charge and hydrophobicity of surfaces affected the structure and size of amyloid deposits as well as surface coverage. Hydrophobic surfaces promote formation of spherical amorphous clusters, while charged surfaces promote protofibril formation. We used the nonlinear Poisson-Boltzmann equation (PBE) approach to analyze the electrostatic interactions of amyloid monomers and oligomers with modified surfaces to complement our AFM data.     

Ultrasonication-dependent acceleration of amyloid fibril formation

Amyloid fibrils, similar to crystals, form through nucleation and growth. Because of the high free-energy barrier of nucleation, the spontaneous formation of amyloid fibrils occurs only after a long lag phase. Ultrasonication is useful for inducing amyloid nucleation and thus for forming fibrils, while the use of a microplate reader with thioflavin T fluorescence is suitable for detecting fibrils in many samples simultaneously. Combining the use of ultrasonication and microplate reader, we propose an efficient approach to studying the potential of proteins to form amyloid fibrils. With β₂-microglobulin, an amyloidogenic protein responsible for dialysis-related amyloidosis, fibrils formed within a few minutes at pH 2.5. Even under neutral pH conditions, fibrils formed after a lag time of 1.5 h. The results propose that fibril formation is a physical reaction that is largely limited by the high free-energy barrier, which can be effectively reduced by ultrasonication. This approach will be useful for developing a high-throughput assay of the amyloidogenicity of proteins.     

High pressure induces scrapie-like prion protein misfolding and amyloid fibril formation

Our understanding of conformational conversion of proteins in diseases is essential for any diagnostic and therapeutic approach. Although not fully understood, misfolding of the prion protein (PrP) is implicated in the pathogenesis of prion diseases. Despite several efforts to produce the pathologically misfolded conformation in vitro from a recombinant PrP, no positive result has yet been obtained. Within the protein-only hypothesis, the reason for this hindrance may be that the experimental conditions used did not allow selection of the pathway adopted in vivo resulting in conversion into the infectious form. Here, using a pressure perturbation approach, we show that recombinant PrP is converted to a novel misfolded conformer, which is prone to aggregate and ultimately form amyloid fibrils. A short incubation at high pressure (600 MPa) of the truncated form of hamster prion protein (SHaPrP(90-231)) resulted in the formation of pre-amyloid structures. The mostly globular aggregates were characterized by ThT and ANS binding, and by a beta-sheet-rich secondary structure. After overnight incubation at 600 MPa, amyloid fibrils were formed. In contrast to pre-amyloid structures, they showed birefringency of polarized light after Congo red staining and a strongly decreased ANS binding capacity, but enhanced ThT binding. Both aggregate types were resistant to digestion by PK, and can be considered as potential scrapie-like forms or precursors. These results may be useful for the search for compounds preventing pathogenic PrP misfolding and aggregation.     

Pathways and intermediates of amyloid fibril formation

The lack of understanding of amyloid fibril formation at the molecular level is a major obstacle in devising strategies to interfere with the pathologies linked to peptide or protein aggregation. In particular, little is known on the role of intermediates and fibril elongation pathways as well as their dependence on the intrinsic tendency of a polypeptide chain to self-assembly by β-sheet formation (β-aggregation propensity). Here, coarse-grained simulations of an amphipathic polypeptide show that a decrease in the β-aggregation propensity results in a larger heterogeneity of elongation pathways, despite the essentially identical structure of the final fibril. Protofibrillar intermediates that are thinner, shorter and less structured than the final fibril accumulate along some of these pathways. Moreover, the templated formation of an additional protofilament on the lateral surface of a protofibril is sometimes observed as a collective transition. Conversely, for a polypeptide model with a high β-aggregation propensity, elongation proceeds without protofibrillar intermediates. Therefore, changes in intrinsic β-aggregation propensity modulate the relative accessibility of parallel routes of aggregation.     

Studying the effects of chaperones on amyloid fibril formation

The results of cell and animal model studies demonstrate that molecular chaperones play an important role in controlling the processes of protein misfolding and amyloid formation in vivo. In addition, chaperones are involved in the appearance, propagation and clearance of prion phenotypes in yeast. The effect of chaperones on amyloid formation has been studied in great detail in recent years in order to elucidate the underlying mechanisms. An important approach is the direct study of effects of chaperones on amyloid fibril formation in vitro. This review introduces the methods and techniques that are commonly used to control and monitor the time course of fibril formation, and to detect interactions between chaperones and fibril-forming proteins. The techniques we address include thioflavin T binding fluorescence and filter retardation assays, size-exclusion chromatography, dynamic light scattering, and biosensor assays. Our aim in this review is to provide guidance on how to embark on study of the effect of chaperones on amyloid fibril formation, and how to avoid common problems that may be encountered, using examples and experience from the authors' lab and from the wider literature.     

Freezing of a fish antifreeze protein results in amyloid fibril formation

Amyloid is associated with a number of diseases including Alzheimer's, Huntington's, Parkinson's, and the spongiform encephalopathies. Amyloid fibrils have been formed in vitro from both disease and nondisease related proteins, but the latter requires extremes of pH, heat, or the presence of a chaotropic agent. We show, using fluorescence spectroscopy, electron microscopy, and solid-state NMR spectroscopy, that the alpha-helical type I antifreeze protein from the winter flounder forms amyloid fibrils at pH 4 and 7 upon freezing and thawing. Our results demonstrate that the freezing of some proteins may accelerate the formation of amyloid fibrils.     

Cellular mechanism of fibril formation from serum amyloid A1 protein

Serum amyloid A1 (SAA1) is an apolipoprotein that binds to the high‐density lipoprotein (HDL) fraction of the serum and constitutes the fibril precursor protein in systemic AA amyloidosis. We here show that HDL binding blocks fibril formation from soluble SAA1 protein, whereas internalization into mononuclear phagocytes leads to the formation of amyloid. SAA1 aggregation in the cell model disturbs the integrity of vesicular membranes and leads to lysosomal leakage and apoptotic death. The formed amyloid becomes deposited outside the cell where it can seed the fibrillation of extracellular SAA1. Our data imply that cells are transiently required in the amyloidogenic cascade and promote the initial nucleation of the deposits. This mechanism reconciles previous evidence for the extracellular location of deposits and amyloid precursor protein with observations the cells are crucial for the formation of amyloid.     

Aromatic interactions are not required for amyloid fibril formation by islet amyloid polypeptide but do influence the rate of fibril formation and fibril morphology

Amyloid formation has been implicated in a wide range of human diseases, and a diverse set of proteins is involved. There is considerable interest in elucidating the interactions which lead to amyloid formation and which contribute to amyloid fibril stability. Recent attention has been focused upon the potential role of aromatic-aromatic and aromatic-hydrophobic interactions in amyloid formation by short to midsized polypeptides. Here we examine whether aromatic residues are necessary for amyloid formation by islet amyloid polypeptide (IAPP). IAPP is responsible for the formation of islet amyloid in type II diabetes which is thought to play a role in the pathology of the disease. IAPP is 37 residues in length and contains three aromatic residues, Phe-15, Phe-23, and Tyr-37. Structural models of IAPP amyloid fibrils postulate that Tyr-37 is near one of the phenylalanine residues, and it is known that Tyr-37 interacts with one of the phenylalanines during fibrillization; however, it is not known if aromatic-aromatic or aromatic-hydrophobic interactions are absolutely required for amyloid formation. An F15L/F23L/Y37L triple mutant (IAPP-3XL) was prepared, and its ability to form amyloid was tested. CD, thioflavin binding assays, AFM, and TEM measurements all show that the triple leucine mutant readily forms amyloid fibrils. The substitutions do, however, decrease the rate of fibril formation and alter the tendency of fibrils to aggregate. Thus, while aromatic residues are not an absolute requirement for amyloid formation by IAPP, they do play a role in the fibril assembly process.     

Amyloid fibril formation in the context of full-length protein: effects of proline mutations on the amyloid fibril formation of beta2-microglobulin

Beta2-microglobulin (beta2-m), a typical immunoglobulin domain made of seven beta-strands, is a major component of amyloid fibrils formed in dialysis-related amyloidosis. To understand the mechanism of amyloid fibril formation in the context of full-length protein, we prepared various mutants in which proline (Pro) was introduced to each of the seven beta-strands of beta2-m. The mutations affected the amyloidogenic potential of beta2-m to various degrees. In particular, the L23P, H51P, and V82P mutations significantly retarded fibril extension at pH 2.5. Among these, only L23P is included in the known "minimal" peptide sequence, which can form amyloid fibrils when isolated as a short peptide. This indicates that the residues in regions other than the minimal sequence, such as H51P and V82P, determine the amyloidogenic potential in the full-length protein. To further clarify the mutational effects, we measured their stability against guanidine hydrochloride of the native state at pH 8.0 and the amyloid fibrils at pH 2.5. The amyloidogenicity of mutants showed a significant correlation with the stability of the amyloid fibrils, and little correlation was observed with that of the native state. It has been proposed that the stability of the native state and the unfolding rate to the amyloidogenic precursor as well as the conformational preference of the denatured state determine the amyloidogenicity of the proteins. The present results reveal that, in addition, stability of the amyloid fibrils is a key factor determining the amyloidogenic potential of the proteins.     

Characterization of the oligomeric states of insulin in self-assembly and amyloid fibril formation by mass spectrometry

The self-assembly and aggregation of insulin molecules has been investigated by means of nanoflow electrospray mass spectrometry. Hexamers of insulin containing predominantly two, but up to four, Zn(2+) ions were observed in the gas phase when solutions at pH 4.0 were examined. At pH 3.3, in the absence of Zn(2+), dimers and tetramers are observed. Spectra obtained from solutions of insulin at millimolar concentrations at pH 2.0, conditions under which insulin is known to aggregate in solution, showed signals from a range of higher oligomers. Clusters containing up to 12 molecules could be detected in the gas phase. Hydrogen exchange measurements show that in solution these higher oligomers are in rapid equilibrium with monomeric insulin. At elevated temperatures, under conditions where insulin rapidly forms amyloid fibrils, the concentration of soluble higher oligomers was found to decrease with time yielding insoluble high molecular weight aggregates and then fibrils. The fibrils formed were examined by electron microscopy and the results show that the amorphous aggregates formed initially are converted to twisted, unbranched fibrils containing several protofilaments. Fourier transform infrared spectroscopy shows that both the soluble form of insulin and the initial aggregates are predominantly helical, but that formation of beta-sheet structure occurs simultaneously with the appearance of well-defined fibrils.     

Oxidation inhibits amyloid fibril formation of transthyretin

The role of amino acid side chain oxidation in the formation of amyloid assemblies has been investigated. Chemical oxidation of amino acid side chains has been used as a facile method of introducing mutations on protein structures. Oxidation promotes changes within tertiary contacts that enable identification of residues and interactions critical in stabilizing protein structures. Transthyretin (TTR) is a soluble human plasma protein. The wild-type (WT) and several of its variants are prone to fibril formation, which leads to amyloidosis associated with many clinical syndromes. The effects of amino acid side chain oxidations were investigated by comparing the kinetics of fibril formation of oxidized and unoxidized proteins. The WT and V30M TTR mutant (valine 30 substituted with methionine) were allowed to react over a time range of 10 min to 12 h with hydroxy radical and other reactive oxygen species. In these timescales, up to five oxygen atoms were incorporated into WT and V30M TTR proteins. Oxidized proteins retained their tetrameric structures, as determined by cross-linking experiments. Side chain modification of methionine residues at position 13 and 30 (the latter for V30M TTR only) were dominant oxidative products. Mono-oxidized and dioxidized methionine residues were identified by radical probe mass spectometry employing a footprinting type approach. Oxidation inhibited the initial rates and extent of fibril formation for both the WT and V30M TTR proteins. In the case of WT TTR, oxidation inhibited fibril growth by approximately 76%, and for the V30M TTR by nearly 90%. These inhibiting effects of oxidation on fibril growth suggest that domains neighboring the methionine residues are critical in stabilizing the tetrameric and folded monomer structures.     

Ultrasonication-induced amyloid fibril formation of beta2-microglobulin

To obtain insight into the mechanism of fibril formation, we examined the effects of ultrasonication, a strong agitator, on beta2-microglobulin (beta2-m), a protein responsible for dialysis-related amyloidosis. Upon sonication of an acid-unfolded beta2-m solution at pH 2.5, thioflavin T fluorescence increased markedly after a lag time of 1-2 h with a simultaneous increase of light scattering. Atomic force microscopy images showed the formation of a large number of short fibrils 3 nm in diameter. When the sonication-induced fibrils were used as seeds in the next seeding experiment at pH 2.5, a rapid and intense formation of long fibrils 3 nm in diameter was observed demonstrating seed-dependent fibril growth. We then examined the effects of sonication on the native beta2-m at neutral pH, conditions under which amyloid deposits occur in patients. In the presence of 0.5 mm sodium dodecyl sulfate, a model compound of potential trigger and stabilizer of amyloid fibrils in patients, a marked increase of thioflavin T fluorescence was observed after 1 day of sonication at pH 7.0. The products of sonication caused the accelerated fibril formation at pH 7.0. Atomic force microscopy images showed that the fibrils formed at pH 7.0 have a diameter of more than 7 nm, thicker than those prepared at pH 2.5. These results indicate that ultrasonication is one form of agitation triggering the formation of amyloid fibrils of beta2-m, producing fibrils adapted to the respective pH.     

Lysophosphatidylcholine modulates fibril formation of amyloid beta peptide

Phospholipids are known to influence fibril formation of amyloid beta (Aβ) peptide. Here, we show that lysophosphatidylcholine (LPC), a polar phospholipid, enhances Aβ(1-42) fibril formation, by decreasing the lag time and the critical peptide concentration required for fibril formation, and increasing the fibril elongation rate. Conversely, LPC did not have an enhancing effect on Aβ(1-40) fibril formation, and appeared to be inhibitory. Tyrosine fluorescence spectroscopy showed that LPC altered the fluorescence spectra of Aβ(1-40) and Aβ(1-42) in opposite ways. Further, 8-anilino-1-naphthalene sulfonic acid fluorescence spectroscopy showed that LPC significantly increased the hydrophobicity of Aβ(1-42), but not of Aβ(1-40). Tris-tricine gradient SDS/PAGE revealed that LPC increased the formation of higher-molecular-weight species of Aβ(1-42), including trimers and tetramers. LPC had no such effect on Aβ(1-40), and thus may specifically influence the oligomerization and nucleation processes of Aβ(1-42) in a manner dependent on its native structure. Dot-blot assays confirmed that LPC induced Aβ(1-42) oligomer formation at an early time point. Thus our results indicate that LPC specifically enhances the formation of Aβ(1-42) fibrils, the main component of senile plaques in Alzheimer's disease patients, and may be involved in Alzheimer's disease pathology.     

Thermodynamic modulation of light chain amyloid fibril formation

To obtain further insight into the pathogenesis of amyloidosis and develop therapeutic strategies to inhibit fibril formation we investigated: 1) the relationship between intrinsic physical properties (thermodynamic stability and hydrogen-deuterium (H-D) exchange rates) and the propensity of human immunoglobulin light chains to form amyloid fibrils in vitro; and 2) the effects of extrinsically modulating these properties on fibril formation. An amyloid-associated protein readily formed amyloid fibrils in vitro and had a lower free energy of unfolding than a homologous nonpathological protein, which did not form fibrils in vitro. H-D exchange was much faster for the pathological protein, suggesting it had a greater fraction of partially folded molecules. The thermodynamic stabilizer sucrose completely inhibited fibril formation by the pathological protein and shifted the values for its physical parameters to those measured for the nonpathological protein in buffer alone. Conversely, urea sufficiently destabilized the nonpathological protein such that its measured physical properties were equivalent to those of the pathological protein in buffer, and it formed fibrils. Thus, fibril formation by light chains is predominantly controlled by thermodynamic stability; and a rational strategy to inhibit amyloidosis is to design high affinity ligands that specifically increase the stability of the native protein.     

The contrasting effect of macromolecular crowding on amyloid fibril formation

Background

Amyloid fibrils associated with neurodegenerative diseases can be considered biologically relevant failures of cellular quality control mechanisms. It is known that in vivo human Tau protein, human prion protein, and human copper, zinc superoxide dismutase (SOD1) have the tendency to form fibril deposits in a variety of tissues and they are associated with different neurodegenerative diseases, while rabbit prion protein and hen egg white lysozyme do not readily form fibrils and are unlikely to cause neurodegenerative diseases. In this study, we have investigated the contrasting effect of macromolecular crowding on fibril formation of different proteins.

Methodology/Principal Findings

As revealed by assays based on thioflavin T binding and turbidity, human Tau fragments, when phosphorylated by glycogen synthase kinase-3β, do not form filaments in the absence of a crowding agent but do form fibrils in the presence of a crowding agent, and the presence of a strong crowding agent dramatically promotes amyloid fibril formation of human prion protein and its two pathogenic mutants E196K and D178N. Such an enhancing effect of macromolecular crowding on fibril formation is also observed for a pathological human SOD1 mutant A4V. On the other hand, rabbit prion protein and hen lysozyme do not form amyloid fibrils when a crowding agent at 300 g/l is used but do form fibrils in the absence of a crowding agent. Furthermore, aggregation of these two proteins is remarkably inhibited by Ficoll 70 and dextran 70 at 200 g/l.

Conclusions/Significance

We suggest that proteins associated with neurodegenerative diseases are more likely to form amyloid fibrils under crowded conditions than in dilute solutions. By contrast, some of the proteins that are not neurodegenerative disease-associated are unlikely to misfold in crowded physiological environments. A possible explanation for the contrasting effect of macromolecular crowding on these two sets of proteins (amyloidogenic proteins and non-amyloidogenic proteins) has been proposed.     

Influence of the carboxy terminus of serum amyloid A on protein oligomerization, misfolding, and fibril formation

The fibrillar deposition of serum amyloid A (SAA) has been linked to the disease amyloid A (AA) amyloidosis. We have used the SAA isoform, SAA2.2, from the CE/J mouse strain, as a model system to explore the inherent structural and biophysical properties of SAA. Despite its nonpathogenic nature in vivo, SAA2.2 spontaneously forms fibrils in vitro, suggesting that SAA proteins are inherently amyloidogenic. However, whereas the importance of the amino terminus of SAA for fibril formation has been well documented, the influence of the proline-rich and presumably disordered carboxy terminus remains poorly understood. To clarify the inherent role of the carboxy terminus in the oligomerization and fibrillation of SAA, we truncated the proline-rich final 13 residues of SAA2.2. We found that unlike full-length SAA2.2, the carboxy-terminal truncated SAA2.2 (SAA2.2ΔC) did not oligomerize to a hexamer or octamer, but formed a high molecular weight soluble aggregate. Moreover, SAA2.2ΔC also exhibited a pronounced decrease in the rate of fibril formation. Intriguingly, when equimolar amounts of denatured SAA2.2 and SAA2.2ΔC were mixed and allowed to refold together, the mixture formed an octamer and exhibited rapid fibrillation kinetics, similar to those for full-length SAA2.2. These results suggest that the carboxy terminus of SAA, which is highly conserved among SAA sequences in all vertebrates, might play important structural roles, including modulating the folding, oligomerization, misfolding, and fibrillation of SAA.     

The association of amyloid P-component (AP) with the amyloid fibril: an updated method for amyloid fibril protein isolation

An amyloid fibril isolation procedure is proposed which uses citrate as well as saline washes to dissociate the calcium dependent linkage of amyloid P-component (AP) from the amyloid fibril. In two amyloid rich tissues, the amount of AP was quantitated in each saline and citrate wash and totalled 13.8% and 20.8% of the amyloid fibrils isolated. The amount of AP removed from these and 22 additional amyloid rich tissues was greater than had previously been recognized. AP protein was present in tissue only when amyloid fibrils were present. It could not be found in normal non-amyloidotic tissue, nor could it be found in tissue sediment after the fibrils were removed.