Kinetically controlled thermal response of beta2-microglobulin amyloid fibrils

Calorimetric measurements were carried out using a differential scanning calorimeter in the temperature range from 10 to 120 degrees C for characterizing the thermal response of beta2-microglobulin amyloid fibrils. The thermograms of amyloid fibril solution showed a remarkably large decrease in heat capacity that was essentially released upon the thermal unfolding of the fibrils, in which the magnitude of negative heat capacity change was not explicable in terms of the current accessible surface area model of protein structural thermodynamics. The heat capacity-temperature curve of amyloid fibrils prior to the fibril unfolding exhibited an unusual dependence on the fibril concentration and the heating rate. Particularly, the heat needed to induce the thermal response was found to be linearly dependent on the heating rate, indicating that its thermal response is under a kinetic control and precluding the interpretation in terms of equilibrium thermodynamics. Furthermore, amyloid fibrils of amyloid beta peptides also exhibited a heating rate-dependent exothermic process before the fibril unfolding, indicating that the kinetically controlled thermal response may be a common phenomenon to amyloid fibrils. We suggest that the heating rate-dependent negative change in heat capacity is coupled to the association of amyloid fibrils with characteristic hydration pattern.     

Core and heterogeneity of beta2-microglobulin amyloid fibrils as revealed by H/D exchange

Dialysis-related amyloidosis, which occurs in the patients receiving a long-term hemodialysis with high frequency, accompanies the deposition of amyloid fibrils composed of beta(2)-microglobulin (beta2-m). In vitro, beta2-m forms two kinds of fibrous structures at acidic pH. One is a rigid "mature fibril", and the other is a flexible thin filament often called an "immature fibril". In addition, a 22-residue peptide (K3 peptide) corresponding to Ser20 to Lys41 of intact beta2-m forms rigid amyloid-like fibrils similar to mature fibrils. We compared the core of these three fibrils at single-residue resolution using a recently developed hydrogen/deuterium (H/D) exchange method with the dissolution of fibrils by dimethylsulfoxide (DMSO). The exchange time-course of these fibrils showed large deviations from a single exponential curve showing that, because of the supramolecular structures, the same residue exists in different environments from molecule to molecule, even in a single fibril. The exchange profiles revealed that the core of the immature fibril is restricted to a narrow region compared to that of the mature fibril. In contrast, all residues were protected from exchange in the K3 fibril, indicating that a whole region of the peptide is engaged in the beta-sheet network. These results suggest the mechanism of amyloid fibril formation, in which the core beta-sheet formed by a minimal sequence propagates to form a rigid and extensive beta-sheet network.     

Thiol compounds inhibit the formation of amyloid fibrils by beta 2-microglobulin at neutral pH

Dialysis-related amyloidosis frequently develops in patients undergoing long-term hemodialysis, in which the major component of fibrils is β₂-microglobulin (β2-m). To prevent the disease, it is important to stop the formation of fibrils. β2-m has one disulfide bond, which stabilizes the native structure, and amyloid fibrils. Here, the effects of reductants (i.e., dithiothreitol and cysteine) on the formation of β2-m amyloid fibrils were examined at neutral pH. Fibrils were generated by three methods: seed-dependent, ultrasonication-induced, and salt-and-heat-induced fibrillation. Thioflavin T fluorescence, electron microscopy, and far-UV circular dichroism revealed that the addition of reductants significantly inhibits seed-dependent and ultrasonication-induced fibrillation. For salt-and-heat-induced fibrillation, where the solution of β2-m was strongly agitated, formation of amyloid fibrils was markedly reduced in the presence of reductants, although a small number of fibrils formed even after the reduction of the disulfide bond. The results suggest that reductants such as cysteine and dithiothreitol would be useful for preventing the formation of β2-m amyloid fibrils under physiological conditions.     

Topological investigation of amyloid fibrils obtained from beta2-microglobulin

Amyloid fibrils of patients treated with regular hemodialysis essentially consists of beta2-microglobulin (beta2-m) and its truncated species DeltaN6beta2-m lacking six residues at the amino terminus. The truncated fragment has a more flexible three-dimensional structure and constitutes an excellent candidate for the analysis of a protein in the amyloidogenic conformation. The surface topology of synthetic fibrils obtained from intact beta2-m and truncated DeltaN6beta2-m was investigated by the limited proteolysis/mass spectrometry approach that appeared particularly suited to gain insights into the structure of beta2-m within the fibrillar polymer. The distribution of prefential proteolytic sites observed in both fibrils revealed that the central region of the protein, which had been easily cleaved in the full-length globular beta2-m, was fully protected in the fibrillar form. In addition, the amino- and carboxy-terminal regions of beta2-m became exposed to the solvent in the fibrils, whereas they were masked completely in the native protein. These data indicate that beta2-m molecules in the fibrils consist of an unaccessible core comprising residues 20-87 with the strands I and VIII being not constrained in the fibrillar polymer and exposed to the proteases. Moreover, proteolytic cleavages observed in vitro at Lys 6 and Lys 19 reproduce specific cleavages that have to occur in vivo to generate the truncated forms of beta2-m occurring in natural fibrils. On the basis of these data, a possible mechanism for fibril formation from native beta2-m is discussed and an explanation for the occurrence of truncated protein species in natural fibrils is given.     

Heat-induced conversion of beta(2)-microglobulin and hen egg-white lysozyme into amyloid fibrils

Thermodynamic parameters characterizing protein stability can be obtained for a fully reversible folding/unfolding system directly by differential scanning calorimetry (DSC). However, the reversible DSC profile can be altered by an irreversible step causing aggregation. Here, to obtain insight into amyloid fibrils, ordered and fibrillar aggregates responsible for various amyloidoses, we studied the effects on human beta(2)-microglobulin and hen egg-white lysozyme of a combination of agitation and heating. Aggregates formed by mildly agitating protein solutions in the native state in the presence of NaCl were heated in the cell of the DSC instrument. For beta(2)-microglobulin, with an increase in the concentration of NaCl at neutral pH, the thermogram began to show an exothermic transition accompanied by a large decrease in heat capacity, followed by a kinetically controlled thermal response. Similarly, the aggregated lysozyme at a high concentration of NaCl revealed a similar distinct transition in the DSC thermogram over a wide pH range. Electron microscopy demonstrated the conformational change into amyloid fibrils. Taken together, the combined use of agitation and heating is a powerful way to generate amyloid fibrils from two proteins, beta(2)-microglobulin and hen egg-white lysozyme, and to evaluate the effects of heat on fibrillation, in which the heat capacity is crucial to characterizing the transition.     

Structure of interacting segments in the growing amyloid fibril of beta2-microglobulin probed with IR spectroscopy

Inter-segmental interaction at the growing tip of the amyloid fibril of beta2-microglobulin (beta2m) was investigated using IR microscopy. Cross-seeded fibril formation was implemented, in which the amyloid fibril of the #21-31 fragment of beta2m (fA[#21-31]) was generated on the beta2m amyloid fibril (fA[beta2m]) as a seed. Differences between the IR spectra of the cross-seeded fibril and those of the seed were attributed to the contribution from the tip, whose structure is discussed. The results indicated that 6.5 +/- 1.0 out of 11 residues of the fA[#21-31] tip on fA[beta2m] are contained in a beta-sheet at pH 2.5, which was smaller than the corresponding value (7.5 +/- 1.1 residues) of the spontaneous fA[#21-31] at pH 2.5. The tip was suggested to have a planar structure, indicating the planarity of the interacting segment. The N-terminal region of fA[#21-31] in the fibril is more exposed to the solvent than that in the tip, and vice versa for the C-terminal region. This is consistent with the different protonation levels of these regions, and the direction of peptide in the fibrils is determined from these results.     

Seeding-dependent propagation and maturation of beta2-microglobulin amyloid fibrils under high pressure

High hydrostatic pressure reversibly transforms the amyloid fibrils of beta2-microglobulin (beta2-m) into a more tightly packed, reorganized structure, which has provided insight into the polymorphic properties of amyloid fibrils. Here, to further investigate the molecular mechanism that controls fibril structure, seed-dependent fibril growth from an acid-unfolded monomeric form under high pressure was studied. At all pressures up to 400 MPa, the fibril growth could be approximated by a single-exponential kinetics, although pressure above 300 MPa decreased the growth rate significantly. The fibrils formed at high pressure were similar to the reorganized fibrils formed initially at ambient pressure and then pressurized, suggesting that the reorganized fibrils were formed directly at high pressure. A systematic investigation of the extension rate under various pressures indicated that the activation free energies for the original and reorganized fibrils are significantly different, suggesting that different amino acid contacts are involved in these two types of fibrils. On the other hand, for the seed-dependent extension reactions of both types of fibrils, the activation volume was much smaller than the change in reaction volume, implying that only small numbers of side-chain interactions are achieved in the transition state. Importantly, we observed a marked acceleration of fibril growth, i.e., maturation, on repeated self-seeding above 300 MPa, revealing the coexistence of another type of fibril with a similar structure but with an increased growth-rate under high pressure.     

Structures of a peptide fragment of beta2-microglobulin studied by replica-exchange molecular dynamics simulations - towards the understanding of the mechanism of amyloid formation

We investigate in detail the structural properties of the monomeric peptide fragment that corresponds to residues 21-31 of beta(2)-microglobulin. As a first step towards the understanding of the mechanism of the amyloid formation, we have performed a replica-exchange molecular dynamics simulation of this peptide with explicit water molecules. We analyze various structural properties as functions of temperature. Although the corresponding part of the native protein is a fully extended beta-strand, our results show that beta-hairpin structures are formed with high frequency around 310 K. We conjecture that this beta-hairpin formation is closely related to the amyloid fibrillogenesis.     

Conformation of amyloid fibrils of beta2-microglobulin probed by tryptophan mutagenesis

Beta2-microglobulin (beta2-m), a protein responsible for dialysis-related amyloidosis, adopts an immunoglobulin domain fold in its native state. Although beta2-m has Trp residues at positions 60 and 95, both are located near the surface of the domain. Hence, beta2-m does not have a conserved Trp common to other immunoglobulin domains, which is buried in close proximity to the disulfide bond. To study the structure of amyloid fibrils in relation to their native fold, we prepared a series of Trp mutants. Trp60 and Trp95 were both replaced with Phe, and a single Trp was introduced at various positions. Among various mutants, W39-beta2-m, in which a Trp was introduced at the position corresponding to the conserved Trp, exhibited a remarkable quenching of fluorescence in the native state, as observed for other immunoglobulin domains. An x-ray structural analysis revealed that W39-beta2-m assumes the native fold with Trp39 located in the vicinity of the disulfide bond. Comparison of the fluorescence spectra of various mutants for the native and fibrillar forms indicated that, while the Trp residues introduced in the middle of the beta2-m sequence tend to be buried in the fibrils, those located in the C-terminal region are more exposed. In addition, the fluorescence spectra of fibrils prepared at pH 2.5 and 7.0 revealed a large difference in the fluorescence intensity for W60-beta2-m, implying a major structural difference between them.     

Inhibition of beta2-microglobulin amyloid fibril formation by alpha2-macroglobulin

The relationship between various amyloidoses and chaperones is gathering attention. In patients with dialysis-related amyloidosis, α(2)-macroglobulin (α2M), an extracellular chaperone, forms a complex with β(2)-microglobulin (β2-m), a major component of amyloid fibrils, but the molecular mechanisms and biological implications of the complex formation remain unclear. Here, we found that α2M substoichiometrically inhibited the β2-m fibril formation at a neutral pH in the presence of SDS, a model for anionic lipids. Binding analysis showed that the binding affinity between α2M and β2-m in the presence of SDS was higher than that in the absence of SDS. Importantly, SDS dissociated tetrameric α2M into dimers with increased surface hydrophobicity. Western blot analysis revealed that both tetrameric and dimeric α2M interacted with SDS-denatured β2-m. At a physiologically relevant acidic pH and in the presence of heparin, α2M was also dissociated into dimers, and both tetrameric and dimeric α2M interacted with β2-m, resulting in the inhibition of fibril growth reaction. These results suggest that under conditions where native β2-m is denatured, tetrameric α2M is also converted to dimeric form with exposed hydrophobic surfaces to favor the hydrophobic interaction with denatured β2-m, thus dimeric α2M as well as tetrameric α2M may play an important role in controlling β2-m amyloid fibril formation.     

Interaction-based evaluation of the propensity for amyloid formation with cross-beta structure

In order to reveal the requirements for amino acid sequences prone to form amyloid fibrils, a novel prediction method based on the original structural model of amyloids was developed. As a working hypothesis, two fundamental conditions were introduced into the design of the present system for the evaluation of the propensity for amyloidogenicity. The first of these two conditions was to ensure that the hydrophobic and hydrogen-bonding interactions between residues on neighboring antiparallel beta-strands were formed along a fibril axis. The other condition was that the hydrophobic interacting residues appeared on both faces of the protofibril, which gave line-matching interactions. Most peptides with sequences exhibiting high scores, as evaluated by this method, were found to easily form amyloids with the aid of a turn-inducing structure designed as a connection of two beta-strands. On the other hand, peptides with low-scoring native sequences and those modified by an internal residue-residue exchange (the latter yielding a null score) did not lead to amyloid formation. These data demonstrated the validity of this method for the prediction of amyloid structures. Moreover, the present study provided support for the proposed model of the essential structure associated with the above working hypothesis. The predicted high-scoring regions were in good agreement with the putative amyloid core regions reported thus far.     

A systematic investigation into the effect of protein destabilisation on beta 2-microglobulin amyloid formation

Beta-2-microglobulin (beta(2)m) has been shown to form amyloid fibrils with distinct morphologies under acidic conditions in vitro. Short, curved fibrils (<600 nm in length), form rapidly without a lag phase, with a maximum rate at pH 3.5. By contrast, fibrils with a long (approximately 1 microm), straight morphology are produced by incubation of the protein at pH< or =3.0. Both fibril types display Congo red birefringence, bind Thioflavin-T and have X-ray fibre diffraction patterns consistent with a cross-beta structure. In order to investigate the role of different partially folded states in generating fibrils of each type, and to probe the effect of protein stability on amyloid formation, we have undertaken a detailed mutagenesis study of beta(2)m. Thirteen variants containing point mutations in different regions of the native protein were created and their structure, stability and fibril forming propensities were investigated as a function of pH. By altering the stability of the native protein in this manner, we show that whilst destabilisation of the native state is important in the generation of amyloid fibrils, population of specific denatured states is a pre-requisite for amyloid formation from this protein. Moreover, we demonstrate that the formation of fibrils with different morphologies in vitro correlates with the relative population of different precursor states.     

Increase in the conformational flexibility of beta 2-microglobulin upon copper binding: a possible role for copper in dialysis-related amyloidosis

A key pathological event in dialysis-related amyloidosis is the fibril formation of beta(2)-microglobulin (beta 2-m). Because beta 2-m does not form fibrils in vitro, except under acidic conditions, predisposing factors that may drive fibril formation at physiological pH have been the focus of much attention. One factor that may be implicated is Cu(2+) binding, which destabilizes the native state of beta 2-m and thus stabilizes the amyloid precursor. To address the Cu(2+)-induced destabilization of beta 2-m at the atomic level, we studied changes in the conformational dynamics of beta 2-m upon Cu(2+) binding. Titration of beta 2-m with Cu(2+) monitored by heteronuclear NMR showed that three out of four histidines (His13, His31, and His51) are involved in the binding at pH 7.0. (1)H-(15)N heteronuclear NOE suggested increased backbone dynamics for the residues Val49 to Ser55, implying that the Cu(2+) binding at His51 increased the local dynamics of beta-strand D. Hydrogen/deuterium exchange of amide protons showed increased flexibility of the core residues upon Cu(2+) binding. Taken together, it is likely that Cu(2+) binding increases the pico- to nanosecond fluctuation of the beta-strand D on which His51 exists, which is propagated to the core of the molecule, thus promoting the global and slow fluctuations. This may contribute to the overall destabilization of the molecule, increasing the equilibrium population of the amyloidogenic intermediate.     

Dissolution of beta2-microglobulin amyloid fibrils by dimethylsulfoxide

Increasing numbers of proteins have been found to aggregate into insoluble fibers, collectively referred to as amyloid fibrils. To address the conformational stability of amyloid fibrils, we studied the effects of dimethylsulfoxide (DMSO), 2,2,2-trifluoroethanol (TFE), and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) on beta(2)-microglobulin amyloid fibrils by circular dichroism, thioflavin T fluorescence, light scattering, and electron microscopy. When measured by circular dichroism and thioflavin T fluorescence, HFIP, and TFE dissolved the fibrils, producing predominantly helical conformations. However, these alcohols did not dissolve the amyloid fibrils completely as monitored by light scattering and electron microscopy. On the other hand, DMSO completely dissolved the amyloid fibrils although a high concentration [i.e., 80% (v/v)] was required. These results are consistent with the important role of hydrogen bonds in stabilizing amyloid fibrils.     

Lysine 58-cleaved beta2-microglobulin is not detectable by 2D electrophoresis in ex vivo amyloid fibrils of two patients affected by dialysis-related amyloidosis

The lysine 58 cleaved and truncated variant of beta(2)-microglobulin (DeltaK58-beta2m) is conformationally unstable and present in the circulation of a large percentage of patients on chronic hemodialysis, suggesting that it could play a role in the beta2-microglobulin (beta2m) amyloid fibrillogenesis associated with dialysis-related amyloidosis (DRA). However, it has yet to be detected in the amyloid deposits of such patients. Here, we extracted amyloid fibrils, without denaturation or additional purification, from different amyloidotic tissues of two unrelated individuals suffering from DRA, and characterized them by high-sensitivity bidimensional gel electrophoresis (2D-PAGE), immunoblotting, MALDI time-of-flight mass spectrometry, and protein sequencing. To confirm whether or not this species could be identified by our proteomic approaches, we mapped its location in 2D-PAGE, in mixtures of pure DeltaK58-beta2m, and extracts of amyloid fibrils from patients, to a discrete region of the gel distinct from other isoforms of beta2m. Using this approach, the two known principal isoforms found in beta2m amyloid were identified, namely, the full-length protein and the truncated species lacking six N-terminal amino acid residues (DeltaN6-beta2m). In contrast, we found no evidence for the presence of DeltaK58-beta2m.     

Mechanism by which the amyloid-like fibrils of a beta 2-microglobulin fragment are induced by fluorine-substituted alcohols

Although the formation of an alpha-helix or partial unfolding of proteins has been suggested to be important for amyloid fibrils to form in alcohols, the exact mechanism involved remains elusive. To obtain further insight into the development of amyloid fibrils, we used a 22-residue peptide, K3, corresponding to Ser20 to Lys41 of intact beta2-microglobulin. Although K3 formed an alpha-helix at high concentrations of 2,2,2-trifluoroethanol (TFE) and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) in 10 mM HCl (pH approximately 2), the helical content was not high, indicating a low preference to do so. The partly alpha-helical conformation was converted with time into a highly ordered beta-sheet with a fibrillar morphology as revealed by atomic force microscopy. Importantly, the TFE and HFIP-induced fibrillation exhibited a concentration dependence with a maximum at approximately 20 and approximately 10% (v/v), respectively, slightly below the concentrations at which these alcohols form dynamic clusters. Focusing on the similarity of the effects of alcohol on proteins with those of sodium dodecyl sulfate (SDS), we examined the effects of SDS on K3. SDS also induced fibrils to form with a maximum at approximately 4 mM, slightly below the critical micelle concentration. These results indicate that, with an increase in the concentration of hydrophobic cosolvent (TFE, HFIP, or SDS), a delicate balance of decreasing hydrophobic interactions and increasing polar interactions (i.e. H-bonds) in and between peptides leads to the formation of ordered fibrils with a bell-shaped concentration dependence.     

Strong acids induce amyloid fibril formation of β2-microglobulin via an anion-binding mechanism

Amyloid fibrils, crystal-like fibrillar aggregates of proteins associated with various amyloidoses, have the potential to propagate via a prion-like mechanism. Among known methodologies to dissolve preformed amyloid fibrils, acid treatment has been used with the expectation that the acids will degrade amyloid fibrils similar to acid inactivation of protein functions. Contrary to our expectation, treatment with strong acids, such as HCl or H₂SO₄, of β₂-microglobulin (β2m) or insulin actually promoted amyloid fibril formation, proportionally to the concentration of acid used. A similar promotion was observed at pH 2.0 upon the addition of salts, such as NaCl or Na₂SO₄. Although trichloroacetic acid, another strong acid, promoted amyloid fibril formation of β2m, formic acid, a weak acid, did not, suggesting the dominant role of anions in promoting fibril formation of this protein. Comparison of the effects of acids and salts confirmed the critical role of anions, indicating that strong acids likely induce amyloid fibril formation via an anion-binding mechanism. The results suggest that although the addition of strong acids decreases pH, it is not useful for degrading amyloid fibrils, but rather induces or stabilizes amyloid fibrils via an anion-binding mechanism.     

Amyloidogenic synthetic peptides of beta2-microglobulin--a role of the disulfide bond

To search for the essential regions responsible for the beta2-microglobulin (beta2-m) amyloid fibril formation, we synthesized six peptides corresponding to six of the seven beta-sheets in the native structure of beta2-m, and examined their amyloidogenicity. Among the peptides examined, peptide (21-31) (strand B) and the mixture of peptide (21-31) and (78-86) (strand F) showed fibril formation at both pH 2.5 and 7.5. Peptide (21-31) is the N-terminal half of the previously reported proteolytic fragment of beta2-m, Ser21-Lys41 (K3), suggesting that this region may be the essential core. Interestingly, the dimer formation of peptide (21-31) by the disulfide bond substantially facilitated the fibril formation, indicating that the disulfide bond is important for the structural stability of the fibrils.     

Novel method for quantitative determination of amyloid fibrils of alpha-synuclein and amyloid beta/A4 protein by using resveratrol

Amyloidosis producing insoluble fibrillar protein aggregates is the common pathological feature of various neurodegenerative disorders such as Parkinson's and Alzheimer's diseases in which alpha-synuclein and amyloid beta/A4 protein (Abeta) participate to form Lewy bodies and senile plaques, respectively. To develop a novel analytical tool for amyloidosis, resveratrol, the major phenolic constituent of red wine and isolatable from grapevines, was employed to monitor the amyloids of alpha-synuclein and Abeta. Specific interaction to the amyloids enhanced the intrinsic fluorescence of resveratrol at 395 nm with an advent of new shoulder peak at 440 nm following an excitation at 320 nm. An increase in the resveratrol binding fluorescence was proportional to the quantity of amyloids. Typical sigmoidal kinetics of the amyloidosis of alpha-synuclein assessed with the thioflavin-T binding fluorescence or the beta-sheet content was fully reproduced by the resveratrol binding fluorescence. The resveratrol binding to the amyloids became saturated as the dye concentration increased, whereas the enhanced thioflavin-T binding fluorescence was quenched by the unbound thioflavin-T at the high dye concentration. Because resveratrol does not require any adjustment of the amyloid/dye ratio to obtain optimal amyloid binding fluorescence, and it exerts a higher quantum yield than does thioflavin-T, resveratrol is suggested to be a specific and more reliable fluorescent probe to determine the amyloids quantitatively.     

Characterization of two distinct beta2-microglobulin unfolding intermediates that may lead to amyloid fibrils of different morphology

The self-assembly of beta(2)-microglobulin into fibrils leads to dialysis-related amyloidosis. pH-mediated partial unfolding is required for the formation of the amyloidogenic intermediate that then self-assembles into amyloid fibrils. Two partially folded intermediates of beta(2)-microglobulin have been identified experimentally and linked to the formation of fibrils of distinct morphology, yet it remains difficult to characterize these partially unfolded states at high resolution using experimental approaches. Consequently, we have performed molecular dynamics simulations at neutral and low pH to determine the structures of these partially unfolded amyloidogenic intermediates. In the low-pH simulations, we observed the formation of alpha-sheet structure, which was first proposed by Pauling and Corey. Multiple simulations were performed, and two distinct intermediate state ensembles were identified that may account for the different fibril morphologies. The predominant early unfolding intermediate was nativelike in structure, in agreement with previous NMR studies. The late unfolding intermediate was significantly disordered, but it maintained an extended elongated structure, with hydrophobic clusters and residual alpha-extended chain strands in specific regions of the sequence that map to amyloidogenic peptides. We propose that the formation of alpha-sheet facilitates self-assembly into partially unfolded prefibrillar amyloidogenic intermediates.