Conformational changes during amyloid fibril formation of pancreatic thiol proteinase inhibitor: effect of copper and zinc

Pancreatic thiol proteinase inhibitor (PTPI), a variant of cystatin superfamily of cysteine protease inhibitors, has been isolated from pancreas of Capra hircus. In the present study, we examined the effects of acid denaturation and a co-solvent on PTPI with a focus on protein conformational changes and amyloid fibril formation. The results demonstrate that PTPI can form amyloid like fibrils. Acid denaturation as studied by CD and fluorescence spectroscopy showed that PTPI populates three partly unfolded species, a native like state at pH 3.0, a structured molten globule at pH 1.0 and partly unfolded species at pH 2.0, from each of which amyloid like fibrils grow as assessed by Thioflavin T (ThT) spectroscopy. Effect of trifluoroethanol (TFE) on acid induced states of PTPI was analyzed. TFE stabilized each of the three acid-induced intermediates at predenaturational concentrations (10%) and accelerated fibril formation. Morphology of the protein species at the beginning and end of reactions was observed using transmission electron microscopy. Solvent conditions were decisive for final fibril morphology. Biometals, Cu²⁺ and Zn²⁺ produced a concentration dependent decline in ThT fluorescence suggesting deaggregation of the fibrils. When added prior to amyloid fibril initiation 50 μM Cu²⁺ or 10 μM Zn²⁺ prevented any amyloid aggregation. Implications for therapeutics in view of Cu²⁺ and Zn²⁺ as essential micronutrients are suggested.     

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.     

Amyloid fibril formation by peptide LYS (11-36) in aqueous trifluoroethanol

Peptide LYS (11-36), derived from the beta-sheet region of T4 lysozyme, forms an amyloid fibril in aqueous trifluoroethanol (TFE) at elevated temperature. The peptide has a moderate alpha-helix content in 20 and 50% (v/v) TFE solution; large quantities of fibrils were formed after incubation at 55 degrees C for 2 weeks as monitored by a thioflavin T fluorescence assay. No fibrils were observed when the peptide initially existed predominantly as a random coil or as a complete alpha helix. Our results suggest that a moderate amount of alpha helix and random coil present in the peptide initially facilitates the fibril-formation process, but a high alpha-helix content inhibits fibril formation. Transmission electron microscopy revealed several types of fibril morphologies at different TFE concentrations. The fibrils were highly twisted and consisted of interleaved protofilaments in 50% TFE, while smooth and flat ribbonlike fibrils were found in 20% TFE. In 50% TFE, the fibril growth rate of LYS (11-36) was found to depend strongly on peptide concentration and seeding but was insensitive to solution pH and ionic strength.     

Amyloid fibril formation by a domain of rat cell adhesion molecule

Amyloid fibrils are protein aggregates implicated in several amyloidotic diseases. Cellular membranes with local decrease in pH and dielectric constant are associated with the amyloid formation. In this study, domain 1 of cell adhesion molecule CD2 (CD2-1) is used for studying amyloid fibril formation in a water/trifluoroethanol (TFE) mixture. CD2-1 is an all beta-sheet protein similar in topology to the amyloidogenic light chain variable domain, which deposits as amyloid in light chain amyloidosis at acidic pH. When incubated at pH 2.0 in the presence of 18% TFE, CD2-1 initiates the process to assemble into amyloid fibrils. It has been shown that TFE induces CD2-1 conformational change with a chemical transition (C(m)) of 18% (v/v). ANS (1-anilinonapthalene-8-sulfonic acid) binding was used to show that the hydrophobic surface becomes exposed under these solvent conditions. Our studies indicate that partial formation of a non-native conformation and the exposure of the hydrophobic interior could be the origins of oligomerization and fibril formation of CD2-1.     

Elongation in a beta-structure promotes amyloid-like fibril formation of human lysozyme

To understand the mechanism of amyloid fibril formation of a protein, we examined wild-type and three mutant human lysozymes containing both amyloidogenic and non-amyloidogenic proteins: I56T (amyloidogenic); EAEA, which has four additional residues (Glu-Ala-Glu-Ala-) at the N-terminus located on a beta-structure; and EAEA-I56T, which is an I56T mutant of EAEA. All formed amyloid-like fibrils through an in the increase contents of alpha-helix with increasing concentration of ethanol. The order of propensity for amyloid-like fibril formation in highly concentrated ethanol solution is EAEA-I56T > EAEA > I56T > wild-type. This order is almost the reverse of the order of conformational stability of these proteins, wild-type > EAEA > I56T > EAEA-I56T. The important views in this work are as follows. (i) Artificially modified proteins formed amyloid fibrils in vitro. This means that amyloid formation is a generic property of polypeptide chains. (ii) The amyloidogenic mutation Ile56 to Thr caused the destabilization and promoted fibril formation in the wild-type and EAEA human lysozymes, indicating that instability facilitates amyloid formation. (iii) The mutant protein EAEA human lysozyme had higher propensity for fibril formation than the amyloidogenic mutant protein, indicating that amyloid formation is controlled not only by stability but also by other factors. In this case, appending polypeptide chains to a beta-structure accelerated amyloid formation.     

Optimum amyloid fibril formation of a peptide fragment suggests the amyloidogenic preference of beta2-microglobulin under physiological conditions

Beta(2)-microglobulin (beta(2)m) is a major component of amyloid fibrils deposited in patients with dialysis-related amyloidosis. Although full-length beta(2)m readily forms amyloid fibrils in vitro by seed-dependent extension with a maximum at pH 2.5, fibril formation under physiological conditions as detected in patients has been difficult to reproduce. A 22-residue K3 peptide of beta(2)m, Ser(20)-Lys(41), obtained by digestion with Acromobacter protease I, forms amyloid fibrils without seeding. To obtain further insight into the mechanism of fibril formation, we studied the pH dependence of fibril formation of the K3 peptide and its morphology using a ThT fluorescence assay and electron microscopy, respectively. K3 peptide formed amyloid fibrils over a wide range of pH values with an optimum around pH 7 and contrasted with the pH profile of the seed-dependent extension reaction of full-length beta(2)m. This suggests that once the rigid native-fold of beta(2)m is unfolded and additional factors triggering the nucleation process are provided, full-length beta(2)m discloses an intrinsic potential to form amyloid fibrils at neutral pH. The fibril formation was strongly promoted by dimerization of K3 through Cys(25). The morphology of the fibrils varied depending on the fibril formation conditions and the presence or absence of a disulfide bond. Various fibrils had the potential to seed fibril formation of full-length beta(2)m accompanied with a characteristic lag phase, suggesting that the internal structures are similar.     

Theophylline-induced apoptosis is paralleled by protein kinase A-dependent tissue transglutaminase activation in cancer cells

beta(2)-Microglobulin (beta2M), the light chain of the type I major histocompatibility complex, is a major component of dialysis-related amyloid fibrils. beta2M in the native state has a typical immunoglobulin fold with a buried intrachain disulfide bond. The conformation and stability of recombinant beta2M in which the intrachain disulfide bond was reduced were studied by CD, tryptophan fluorescence, and one-dimensional NMR. The conformation of the reduced beta2M in the absence of denaturant at pH 8.5 was similar to that of the intact protein unless the thiol groups were modified. However, reduction of the disulfide bond decreased the stability as measured by denaturation in guanidine hydrochloride. Intact beta2M formed amyloid fibrils at pH 2.5 by extension reaction using sonicated amyloid fibrils as seeds. Under the same conditions, reduced beta2M did not form typical amyloid fibrils, although it inhibited fibril extension competitively, suggesting that the conformation defined by the disulfide bond is important for amyloid fibril formation of beta2M.     

Partially folded intermediates as critical precursors of light chain amyloid fibrils and amorphous aggregates

Light chain, or AL, amyloidosis is a pathological condition arising from systemic extracellular deposition of monoclonal immunoglobulin light chain variable domains in the form of insoluble amyloid fibrils, especially in the kidneys. Substantial evidence suggests that amyloid fibril formation from native proteins occurs via a conformational change leading to a partially folded intermediate conformation, whose subsequent association is a key step in fibrillation. In the present investigation, we have examined the properties of a recombinant amyloidogenic light chain variable domain, SMA, to determine whether partially folded intermediates can be detected and correlated with aggregation. The results from spectroscopic and hydrodynamic measurements, including far- and near-UV circular dichroism, FTIR, NMR, and intrinsic tryptophan fluorescence and small-angle X-ray scattering, reveal the build-up of two partially folded intermediate conformational states as the pH is decreased (low pH destabilized the protein and accelerated the kinetics of aggregation). A relatively nativelike intermediate, I(N), was observed between pH 4 and 6, with little loss of secondary structure, but with significant tertiary structure changes and enhanced ANS binding, indicating exposed hydrophobic surfaces. At pH below 3, we observed a relatively unfolded, but compact, intermediate, I(U), which was characterized by decreased tertiary and secondary structure. The I(U) intermediate readily forms amyloid fibrils, whereas I(N) preferentially leads to amorphous aggregates. Except at pH 2, where negligible amorphous aggregate is formed, the amorphous aggregates formed significantly more rapidly than the fibrils. This is the first indication that different partially folded intermediates may be responsible for different aggregation pathways (amorphous and fibrillar). The data support the hypothesis that amyloid fibril formation involves the ordered self-assembly of partially folded species that are critical soluble precursors of fibrils.     

Conformational prerequisites for formation of amyloid fibrils from histones

We demonstrate that bovine core histones are natively unfolded proteins in solutions with low ionic strength due to their high net positive charge at pH 7.5. Using a variety of biophysical techniques we characterized their conformation as a function of pH and ionic strength, as well as correlating the conformation with aggregation and amyloid fibril formation. Tertiary structure was absent under all conditions except at pH 7.5 and high ionic strength. The addition of trifluoroethanol or high ionic strength induced significant alpha-helical secondary structure at pH 7.5. At low pH and high salt concentration, small-angle X-ray scattering and SEC HPLC indicate the histones are present as a hexadecamer of globular subunits. The secondary structure at low pH was independent of the ionic strength or presence of TFE, as judged by FTIR. The data indicate that histones are able to adopt five different relatively stable conformations; this conformational variability probably reflects, in part, their intrinsically disordered structure. Under most of the conditions studied the histones formed amyloid fibrils with typical morphology as seen by electron microscopy. In contrast to most aggregation/amyloidogenic systems, the kinetics of fibrillation showed an inverse dependence on histone concentration; we attribute this to partitioning to a faster pathway leading to non-fibrillar self-associated aggregates at higher protein concentrations. The rate of fibril formation was maximal at low pH, and decreased to zero by pH 10. The kinetics of fibrillation were very dependent on the ionic strength, increasing with increasing salt concentration, and showing marked dependence on the nature of the ions; interestingly Gdn.HCl increased the rate of fibrillation, although much less than NaCl. Different ions also differentially affected the rate of nucleation and the rate of fibril elongation.     

Role of the helical structure of the N-terminal region of Plasmodium falciparum merozoite surface protein 2 in fibril formation and membrane interaction

Merozoite surface protein 2 (MSP2), an abundant glycosylphosphatidylinositol-anchored protein on the surface of Plasmodium falciparum merozoites, is a promising malaria vaccine candidate. MSP2 is intrinsically disordered and forms amyloid-like fibrils in solution under physiological conditions. The 25 N-terminal residues (MSP2(1-25)) play an important role in both fibril formation and membrane binding of the full-length protein. In this study, the fibril formation and solution structure of MSP2(1-25) in the membrane mimetic solvents sodium dodecyl sulfate (SDS), dodecylphosphocholine (DPC), and trifluoroethanol (TFE) have been investigated by transmission electronic microscopy, turbidity, thioflavin T fluorescence, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy. Turbidity data showed that the aggregation of MSP2(1-25) was suppressed in the presence of membrane mimetic solvents. CD spectra indicated that helical structure in MSP2(1-25) was stabilized in SDS and DPC micelles and in high concentrations of TFE. The structure of MSP2(1-25) in 50% aqueous TFE, determined using NMR, showed that the peptide formed an amphipathic helix encompassing residues 10-24. Low concentrations of TFE favored partially folded helical conformations, as demonstrated by CD and NMR, and promoted MSP2(1-25) fibril formation. Our data suggest that partially folded helical conformations of the N-terminal region of MSP2 are on the pathway to amyloid fibril formation, while higher degrees of helical structure stabilized by high concentrations of TFE or membrane mimetics suppress self-association and thus inhibit fibril formation. The roles of the induced helical conformations in membrane interactions are also discussed.     

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.     

Polymorphism of β2-microglobulin amyloid fibrils manifested by ultrasonication-enhanced fibril formation in trifluoroethanol

The polymorphic property of amyloid structures has been focused on as a molecular basis of the presence and propagation of different phenotypes of amyloid diseases, although little is known about the molecular mechanism for expressing diverse structures from only one protein sequence. Here, we have found that, in combination with an enhancing effect of ultrasonication on nucleation, β(2)-microglobulin, a protein responsible for dialysis-related amyloidosis, generates distinct fibril conformations in a concentration-dependent manner in the presence of 2,2,2-trifluoroethanol (TFE). Although the newly formed fibrils all exhibited a similar needle-like morphology with an extensive cross-β core, as suggested by Fourier transform infrared absorption spectra, they differed in thioflavin T intensity, extension kinetics, and tryptophan fluorescence spectra even in the same solvents, representing polymorphic structures. The hydrophobic residues seemed to be more exposed in the fibrils originating at higher concentrations of TFE, as indicated by the increased binding of 1-anilinonaphthalene-8-sulfonic acid, suggesting that the modulation of hydrophobic interactions is critical to the production of polymorphic amyloid structures. Interestingly, the fibrils formed at higher TFE concentrations showed significantly higher stability against guanidium hydrochloride, the perturbation of ionic strength, and, furthermore, pressurization. The cross-β structure inside the fibrils seems to have been more idealized, resulting in increased stability when nucleation occurred in the presence of the alcohol, indicating that a weaker contribution of hydrophobic interactions is intrinsically more amenable to the formation of a non-defective amyloid structure.     

A single mutation induces amyloid aggregation in the alpha-spectrin SH3 domain: analysis of the early stages of fibril formation

The Src-homology region 3 domain of chicken alpha-spectrin (Spc-SH3) is a small two-state folding protein, which has never been described to form amyloid fibrils under any condition investigated so far. We show here that the mutation of asparagine 47 to alanine at the distal loop, which destabilises similarly the native and folding transition states of the domain, induces the formation of amyloid fibrils under mild acid conditions. Amyloid aggregation of the mutant is enhanced by the increase in temperature, protein concentration and NaCl concentration. The early stages of amyloid formation have been monitored as a function of time and temperature using a variety of biophysical methods. Differential scanning calorimetry experiments under conditions of amyloid formation have allowed the identification of different thermal transitions corresponding to conformational and aggregation processes as well as to the high-temperature disaggregation and unfolding of the amyloid fibrils. Aggregation is preceded by a rapid conformational change in the monomeric domain involving about 40% of the global unfolding enthalpy, considerable change in secondary structure, large loss of tertiary structure and exposure of hydrophobic patches to the solvent. The conformational change is followed by formation of a majority of oligomeric species with apparent hydrodynamic radius between 2.5 nm and 10nm, depending on temperature, together with the appearance and progressive growth of protofibrillar aggregates. After these early aggregation stages, long and curved fibrils of up to several micrometers start to develop by elongation of the protofibrils. The calorimetric data indicate that the specific enthalpy of fibril disaggregation and unfolding is relatively low, suggesting a low density of interactions within the fibril structure as compared to the native protein and a main entropy contribution to the stability of the amyloid fibrils.     

Amyloid fibril formation of the mouse V(L) domain at acidic pH

The recombinant V(L) domain that represents the variable part of the light chain (type kappa) of mouse monoclonal antibody F11 directed against human spleen ferritin was found to form amyloid fibrils at acidic pH as evidenced by electron microscopy, thioflavin T binding, and apple-green birefringence after Congo red staining. This is the first demonstration of amyloid fibril formation of the mouse V(L) domain. To understand the mechanism of acidic pH-induced amyloid fibril formation, conformational changes of the V(L) domain were studied by one-dimensional NMR, differential scanning calorimetry, analytical ultracentrifugation, hydrophobic dye binding, far-UV circular dichroism, and tryptophan fluorescence. The results indicated accumulation of two intermediate states during acid unfolding, which might be responsible for amyloid fibril formation. The more structured intermediate that exhibited maximal accumulation at pH 3 retained the nativelike secondary structure and a hydrophobic core, but exposed hydrophobic surfaces that bind 8-anilino-1-naphthalenesulfonate. Below pH 2, a more disordered intermediate with dequenched tryptophan fluorescence but still retaining the beta-sheet structure accumulated. The optimal pH of amyloid fibril formation (i.e., pH 4) was close to the optimal pH of the accumulation of the nativelike intermediate, suggesting that the amyloid fibrils might be formed through this intermediate.     

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.     

Association of thin filaments into thick filaments revealing the structural hierarchy of amyloid fibrils

Beta2-Microglobulin (beta2-m) is a major structural component of dialysis-related amyloid fibrils. Kozhukh et al. [J. Biol. Chem. 277 (2002) 1310] prepared a series of peptide fragments of beta2-m by the protease digestion and examined their ability to form amyloid fibrils in citrate buffer at pH 2.5. Among various peptides, a 22-residue K3 peptide corresponding to Ser20-Lys41 spontaneously formed amyloid fibrils in aqueous solution. This peptide also formed amyloid protofibrils in 20% (v/v) 2,2,2-trifluoroethanol (TFE). To investigate the influence of solvent conditions on fibril formation, we studied their structures by atomic force microscopy. In aqueous solution, fibrils had a diameter of 4 or 8 nm and tended to cluster each other. On the other hand, protofibrils in 20% (v/v) TFE had a diameter of 2 nm with no tendency of clustering. Intriguingly, when the K3 protofibrils were transferred from 20% (v/v) TFE to aqueous solution, some of them associated to form thicker fibrils with a diameter of 4-15 nm and a left-handed helical twist. TFE is a hydrophobic solvent, so that hydrophobic interactions between molecules may be weakened. The results suggest that the fibrils in aqueous conditions are formed by the cooperative association of protofibrils at the growing ends of the fibrils, in which hydrophobic interactions play a major role.     

Amyloid fibril formation in vitro from halophilic metal binding protein: Its high solubility and reversibility minimized formation of amorphous protein aggregations

Halophilic proteins are characterized by high net negative charges and relatively small fraction of hydrophobic amino acids, rendering them aggregation resistant. These properties are also shared by histidine‐rich metal binding protein (HP) from moderate halophile, Chromohalobacter salexigens, used in this study. Here, we examined how halophilic proteins form amyloid fibrils in vitro. His‐tagged HP, incubated at pH 2.0 and 58°C, readily formed amyloid fibrils, as observed by thioflavin fluorescence, CD spectra, and transmission or atomic force microscopies. Under these low‐pH harsh conditions, however, His‐HP was promptly hydrolyzed to smaller peptides most likely responsible for rapid formation of amyloid fibril. Three major acid‐hydrolyzed peptides were isolated from fibrils and turned out to readily form fibrils. The synthetic peptides predicted to form fibrils in these peptide sequences by Waltz software also formed fibrils. Amyloid fibril was also readily formed from full‐length His‐HP when incubated with 10–20% 2,2,2‐trifluoroethanol at pH 7.8 and 25°C without peptide bond cleavage.     

Solution conformation and amyloid-like fibril formation of a polar peptide derived from a beta-hairpin in the OspA single-layer beta-sheet

A 23-residue peptide termed BH(9-10) was designed based on a beta-hairpin segment of the single-layer beta-sheet region of Borrelia OspA protein. The peptide contains a large number of charged amino acid residues, and it does not follow the amphipathic pattern that is commonly found in natural beta-sheets. In aqueous solution, the peptide was highly soluble and flexible, with a propensity to form a non-native beta-turn. Trifluoroethanol (TFE) stabilized a native-like beta-turn in BH(9-10). TFE also decreased the level of solubility of the peptide, resulting in peptide precipitation. The precipitation process accompanied a conformational conversion to a beta-sheet structure, as judged with circular dichroism spectroscopy. The precipitate was found to be fibrils similar to those associated with human amyloid diseases. The fibrillization kinetics depended on peptide and TFE concentrations, and had a nucleation step followed by an assembly step. The fibrillization was reversible, and the dissociation reaction involved two phases. TFE appears to induce the fibrils by stabilizing a beta-sheet conformation of the peptide that optimally satisfies hydrogen bonding and electrostatic complementarity. This TFE-induced fibrillization is quite unusual, because most amyloidogenic peptides form fibrils in aqueous solution and TFE disrupts these fibrils. Nevertheless, the BH(9-10) fibrils have similar structure to other fibrils, supporting the emerging idea that polypeptides possess an intrinsic ability to form amyloid-like fibrils. The high level of solubility of BH(9-10), the ability to precisely control fibril formation and dissociation, and the high-resolution structure of the same sequence in the beta-hairpin conformation in the OspA protein provide a tractable experimental system for studying the fibril formation mechanism.     

Template-directed self-assembly and growth of insulin amyloid fibrils

The formation of amyloid aggregates in tissue is a pathological feature of many neurodegenerative diseases and type II diabetes. Amyloid deposition, the process of amyloid growth by the association of individual soluble amyloid molecules with a pre-existing amyloid template (i.e., plaque), is known to be critical for amyloid formation in vivo. The requirement for a natural amyloid template, however, has made amyloid deposition study difficult and cumbersome. In the present work, we developed a novel, synthetic amyloid template by attaching amyloid seeds covalently onto an N-hydroxysuccinimide-activated surface, where insulin was chosen as a model amyloidogenic protein. According to ex situ atomic force microscopy observations, insulin monomers in solution were deposited onto the synthetic amyloid template to form fibrils, like hair growth. The fibril formation on the template occurred without lag time, and its rate was highly accelerated than in the solution. The fibrils were long, over 2 mum, and much thinner than those in the solution, which was caused by limited nucleation sites on the template surface and lack of lateral twisting between fibrils. According to our investigations using thioflavin T-induced fluorescence, birefringent Congo red binding, and circular dichroism, fibrils grown on the template were identified to be amyloids that formed through a conformational rearrangement of insulin monomers upon interaction with the template. The amyloid deposition rate followed saturation kinetics with respect to insulin concentration in the solution. The characteristics of amyloid deposition on the synthetic template were in agreement with previous studies performed with human amyloid plaques. It is demonstrated that the synthetic amyloid template can be used for the screening of inhibitors on amyloid deposition in vitro.     

Tetramer dissociation and monomer partial unfolding precedes protofibril formation in amyloidogenic transthyretin variants

Amyloid fibril formation and deposition is a common feature of a wide range of fatal diseases including spongiform encephalopathies, Alzheimer's disease, and familial amyloidotic polyneuropathies (FAP), among many others. In certain forms of FAP, the amyloid fibrils are mostly constituted by variants of transthyretin (TTR), a homotetrameric plasma protein. Recently, we showed that transthyretin in solution may undergo dissociation to a non-native monomer, even under close to physiological conditions of temperature, pH, ionic strength, and protein concentration. We also showed that this non-native monomer is a compact structure, does not behave as a molten globule, and may lead to the formation of partially unfolded monomeric species and high molecular mass soluble aggregates (Quintas, A., Saraiva, M. J. M., and Brito, R. M. M. (1999) J. Biol. Chem. 274, 32943-32949). Here, based on aging experiments of tetrameric TTR and chemically induced protein unfolding experiments of the non-native monomeric forms, we show that tetramer dissociation and partial unfolding of the monomer precedes amyloid fibril formation. We also show that TTR variants with the least thermodynamically stable non-native monomer produce the largest amount of partially unfolded monomeric species and soluble aggregates under conditions that are close to physiological. Additionally, the soluble aggregates formed by the amyloidogenic TTR variants showed morphological and thioflavin-T fluorescence properties characteristic of amyloid. These results allowed us to conclude that amyloid fibril formation by some TTR variants might be triggered by tetramer dissociation to a compact non-native monomer with low conformational stability, which originates partially unfolded monomeric species with a high tendency for ordered aggregation into amyloid fibrils. Thus, partial unfolding and conformational fluctuations of molecular species with marginal thermodynamic stability may play a crucial role on amyloid formation in vivo.