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.     

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.     

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.     

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.     

Low concentrations of sodium dodecyl sulfate induce the extension of beta 2-microglobulin-related amyloid fibrils at a neutral pH

In beta(2)-microglobulin-related (Abeta2M) amyloidosis, partial unfolding of beta(2)-microglobulin (beta2-m) is believed to be prerequisite to its assembly into Abeta2M amyloid fibrils in vivo. Although low pH or 2,2,2-trifluoroethanol at a low concentration has been reported to induce partial unfolding of beta2-m and subsequent amyloid fibril formation in vitro, factors that induce them under near physiological conditions have not been determined. Using fluorescence spectroscopy with thioflavin T, circular dichroism spectroscopy, and electron microscopy, we here show that at low concentrations, sodium dodecyl sulfate (SDS) converts natively folded beta2-m monomers into partially folded, alpha-helix-containing conformers. Surprisingly, this results in the extension of Abeta2M amyloid fibrils at neutral pH, which could be explained basically by a first-order kinetic model. At low concentrations, SDS also stabilized the fibrils at neutral pH. These SDS effects were concentration-dependent and maximal at approximately 0.5 mM, around the critical micelle concentration of SDS (0.67 mM). As the concentration of SDS was increased above 1 mM, the alpha-helix content of beta2-m rose to approximately 10%, while the beta-sheet content decreased to approximately 20%, a change paralleled by a complete cessation of fibril extension and the destabilization of the fibrils. Detergents of other classes had no significant effect on the extension of fibrils. These findings are consistent with the hypothesis that in vivo, specific factors (e.g., phospholipids) that affect the conformation and stability of beta2-m and amyloid fibrils will have significant effects on the kinetics of Abeta2M fibril formation.     

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.     

Lysozyme amyloidogenesis is accelerated by specific nicking and fragmentation but decelerated by intact protein binding and conversion

We have revisited the well-studied heat and acidic amyloid fibril formation pathway (pH 1.6, 65 degrees C) of hen egg-white lysozyme (HEWL) to map the barriers of the misfolding and amyloidogenesis pathways. A comprehensive kinetic mechanism is presented where all steps involving protein hydrolysis, fragmentation, assembly and conversion into amyloid fibrils are accounted for. Amyloid fibril formation of lysozyme has multiple kinetic barriers. First, HEWL unfolds within minutes, followed by irreversible steps of partial acid hydrolysis affording a large amount of nicked HEWL, the 49-101 amyloidogenic fragment and a variety of other species over 5-40 h. Fragmentation forming the 49-101 fragment is a requirement for efficient amyloid fibril formation, indicating that it forms the rate-determining nucleus. Nicked full-length HEWL is recruited efficiently into amyloid fibrils in the fibril growth phase or using mature fibrils as seeds, which abolished the lag phase completely. Mature amyloid fibrils of HEWL are composed mainly of nicked HEWL in the early equilibrium phase but go through a "fibril shaving" process, affording fibrils composed of the 49-101 fragment and 53-101 fragment during more extensive maturation (incubation for longer than ten days). Seeding of the amyloid fibril formation process using sonicated mature amyloid fibrils accelerates the fibril formation process efficiently; however, addition of intact full-length lysozyme at the end of the lag phase slows the rate of amyloidogenesis. The intact full-length protein, in contrast to nicked lysozyme, slows fibril formation due to its slow conversion into the amyloid fold, probably due to inclusion of the non-amyloidogenic 1-48/102-129 portion of HEWL in the fibrils, which can function as a "molecular bumper" stalling further growth.     

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.     

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.     

Direct observation of amyloid fibril growth monitored by thioflavin T fluorescence

Real-time monitoring of fibril growth is essential to clarify the mechanism of amyloid fibril formation. Thioflavin T (ThT) is a reagent known to become strongly fluorescent upon binding to amyloid fibrils. Here, we show that, by monitoring ThT fluorescence with total internal reflection fluorescence microscopy (TIRFM), amyloid fibrils of beta2-microgobulin (beta2-m) can be visualized without requiring covalent fluorescence labeling. One of the advantages of TIRFM would be that we selectively monitor fibrils lying along the slide glass, so that we can obtain the exact length of fibrils. This method was used to follow the kinetics of seed-dependent beta2-m fibril extension. The extension was unidirectional with various rates, suggesting the heterogeneity of the amyloid structures. Since ThT binding is common to all amyloid fibrils, the present method will have general applicability for the analysis of amyloid fibrils. We confirmed this with the octapeptide corresponding to the C terminus derived from human medin and the Alzheimer's amyloid beta-peptide.     

The role of disulfide bond in the amyloidogenic state of beta(2)-microglobulin studied by heteronuclear NMR

beta(2)-Microglobulin (beta2-m) is a major component of dialysis-related amyloid fibrils. Although recombinant beta2-m forms needle-like fibrils by in vitro extension reaction at pH 2.5, reduced beta2-m, in which the intrachain disulfide bond is reduced, cannot form typical fibrils. Instead, thinner and flexible filaments are formed, as shown by atomic force microscopy images. To clarify the role of the disulfide bond in amyloid fibril formation, we characterized the conformations of the oxidized (intact) and reduced forms of beta2-m in the acid-denatured state at pH 2.5, as well as the native state at pH 6.5, by heteronuclear NMR. [(1)H]-(15)N NOE at the regions between the two cysteine residues (Cys25-Cys80) revealed a marked difference in the pico- and nanosecond time scale dynamics between that the acid-denatured oxidized and reduced states, with the former showing reduced mobility. Intriguingly, the secondary chemical shifts, DeltaCalpha, DeltaCO, and DeltaHalpha, and (3)J(HNHalpha) coupling constants indicated that both the oxidized and reduced beta2-m at pH 2.5 have marginal alpha-helical propensity at regions close to the C-terminal cysteine, although it is a beta-sheet protein in the native state. The results suggest that the reduced mobility of the denatured state is an important factor for the amylodogenic potential of beta2-m, and that the marginal helical propensity at the C-terminal regions might play a role in modifying this potential.     

Heparin strongly enhances the formation of beta2-microglobulin amyloid fibrils in the presence of type I collagen

The tissue specificity of fibrillar deposition in dialysis-related amyloidosis is most likely associated with the peculiar interaction of beta2-microglobulin (beta2-m) with collagen fibers. However, other co-factors such as glycosaminoglycans might facilitate amyloid formation. In this study we have investigated the role of heparin in the process of collagen-driven amyloidogenesis. In fact, heparin is a well known positive effector of fibrillogenesis, and the elucidation of its potential effect in this type of amyloidosis is particularly relevant because heparin is regularly given to patients subject to hemodialysis to prevent blood clotting. We have monitored by atomic force microscopy the formation of beta2-m amyloid fibrils in the presence of collagen fibers, and we have discovered that heparin strongly accelerates amyloid deposition. The mechanism of this effect is still largely unexplained. Using dynamic light scattering, we have found that heparin promotes beta2-m aggregation in solution at pH 6.4. Morphology and structure of fibrils obtained in the presence of collagen and heparin are highly similar to those of natural fibrils. The fibril surface topology, investigated by limited proteolysis, suggests that the general assembly of amyloid fibrils grown under these conditions and in vitro at low pH is similar. The exposure of these fibrils to trypsin generates a cleavage at the C-terminal of lysine 6 and creates the 7-99 truncated form of beta2-m (DeltaN6beta2-m) that is a ubiquitous constituent of the natural beta2-m fibrils. The formation of this beta2-m species, which has a strong propensity to aggregate, might play an important role in the acceleration of local amyloid deposition.     

Dynamic behavior of small heat shock protein inhibition on amyloid fibrillization of a small peptide (SSTSAA) from RNase A

Small heat shock proteins, a class of molecular chaperones, are reported to inhibit amyloid fibril formation in vitro, while the mechanism of inhibition remains unknown. In the present study, we investigated the mechanism by which Mj HSP16.5 inhibits amyloid fibril formation of a small peptide (SSTSAA) from RNase A. A model peptide (dansyl-SSTSAA-W) was designed by introducing a pair of fluorescence resonance energy transfer (FRET) probes into the peptide, allowing for the monitoring of fibril formation by this experimental model. Mj HSP16.5 completely inhibited fibril formation of the model peptide at a molar ratio of 1:120. The dynamic process of fibril formation, revealed by FRET, circular dichroism, and electron microscopy, showed a lag phase of about 2 h followed by a fast growth period. The effect of Mj HSP16.5 on amyloid fibril formation was investigated by adding it into the incubation solution during different growth phases. Adding Mj HSP16.5 to the incubating peptide before or during the lag phase completely inhibited fibril formation. However, introducing Mj HSP16.5 after the lag phase only slowed down the fibril formation process by adhering to the already formed fibrils. These findings provide insight into the inhibitory roles of small heat shock proteins on amyloid fibril formation at the molecular level.     

Seeding-dependent maturation of beta2-microglobulin amyloid fibrils at neutral pH

Beta2-microglobulin (beta2-m) is a major component of amyloid fibrils deposited in patients with dialysis-related amyloidosis. Recent studies have focused on the mechanism by which amyloid fibrils are formed under physiological conditions, which had been difficult to reproduce quantitatively. Yamamoto et al. (Yamamoto, S., Hasegawa, K., Yamaguchi, I., Tsutsumi, S., Kardos, J., Goto, Y., Gejyo, F. & Naiki, H. (2004) Biochemistry 43, 11075-11082) showed that a combination of seed fibrils prepared under acidic conditions and a low concentration of sodium dodecyl sulfate below its critical micelle concentration enabled extensive fibril formation at pH 7.0. Here, we found that repeated self-seeding at pH 7.0 with fibrils formed at the same pH causes a marked acceleration of growth, indicating the maturation of fibrils. The observed maturation can be simulated by assuming the existence of two types of fibrils with different growth rates. Importantly, some mutations of beta2-m or the addition of a low concentration of urea, both destabilizing the native conformation, were not enough to extend the fibrils at pH 7.0, and a low concentration of sodium dodecyl sulfate (i.e. 0.5 mM) was essential. Thus, even though the first stage fibrils in patients are unstable and require stabilizing factors to remain at neutral pH, they can adapt to a neutral pH with repeated self-seeding, implying a mechanism of development of amyloid deposition after a long latent period in patients.     

Conformation of beta 2-microglobulin amyloid fibrils analyzed by reduction of the disulfide bond

Beta2-microglobulin (beta2-m), a major component of dialysis-related amyloid fibrils, has an intrachain disulfide bond buried inside the native structure. We examined the conformation of beta2-m amyloid fibrils by analyzing the reactivity of the disulfide bond to a reducing reagent, dithiothreitol. Although the disulfide bond in the native structure was highly protected from reduction, the disulfide bonds in the amyloid fibrils prepared at pH 2.5 were progressively reduced at pH 8.5 by 50 mm dithiothreitol. Because beta2-m amyloid fibrils prepared under acidic conditions have been known to depolymerize at a neutral pH, we examined the relation between depolymerization and reduction of the disulfide bond. The results indicate that the disulfide bonds in the amyloid fibrils were protected from reduction, and the reduction occurred during depolymerization. On the other hand, the disulfide bonds of immature filaments, the thin and flexible filaments prepared under conditions of high salt at pH 2.5, were reduced at pH 8.5 more readily than those of amyloid fibrils, suggesting that the disulfide bonds are exposed to the solvent. Taken together, the disulfide bond once exposed to the solvent upon acid denaturation may be progressively buried in the interior of the amyloid fibrils during its formation.     

Growth of beta(2)-microglobulin-related amyloid fibrils by non-esterified fatty acids at a neutral pH

Abeta2M (beta(2)-microglobulin-related) amyloidosis is a frequent and serious complication in patients on long-term dialysis. Partial unfolding of beta2-m (beta(2)-microglobulin) may be essential to its assembly into Abeta2M amyloid fibrils in vivo. Although SDS around the critical micelle concentration induces partial unfolding of beta2-m to an alpha-helix-containing aggregation-prone amyloidogenic conformer and subsequent amyloid fibril formation in vitro, the biological molecules with similar activity under near-physiological conditions are still unknown. The effect of various NEFAs (non-esterified fatty acids), which are representative anionic amphipathic compounds in the circulation, on the growth of Abeta2M amyloid fibrils at a neutral pH was examined using fluorescence spectroscopy with thioflavin T, CD spectroscopy, and electron microscopy. Physiologically relevant concentrations of laurate, myristate, oleate, linoleate, and mixtures of palmitate, stearate, oleate and linoleate, induced the growth of fibrils at a neutral pH by partially unfolding the compact structure of beta2-m to an aggregation-prone amyloidogenic conformer. In the presence of human serum albumin, these NEFAs also induced the growth of fibrils when their concentrations exceeded the binding capacity of albumin, indicating that the unbound NEFAs rather than albumin-bound NEFAs induce the fibril growth reaction in vitro. These results suggest the involvement of NEFAs in the development of Abeta2M amyloidosis, and in the pathogenesis of Abeta2M amyloidosis.     

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.     

Stereospecific amyloid-like fibril formation by a peptide fragment of beta2-microglobulin

Understanding the role of the L/D-stereospecificity of amino acids is important in obtaining further insight into the mechanism of the formation of amyloid fibrils. Beta(2)-microglobulin is a major component of amyloid fibrils deposited in patients with dialysis-related amyloidosis. A 22-residue peptide of beta(2)-microglobulin, Ser20-Lys41 (L-K3 peptide), obtained by digestion with Acromobacter protease I, formed amyloid-like fibrils in 50% (v/v) 2,2,2-trifluoroethanol and 10 mM HCl at 25 degrees C, as confirmed by thioflavin T fluorescence, circular dichroism spectra, and atomic force microscopy images. A synthetic K3 peptide composed of D-amino acids (D-K3 peptide) formed similar fibrils but with opposite chirality as indicated by circular dichroism spectra. A mixture of L-K3 and D-K3 peptides also formed fibrils, although the L- and D-amino acid composition of each fibril is unknown. To examine the possible cross-reactivity between L- and D-enantiomers, we carried out seeding experiments in which preformed seeds were extended by monomers. The results revealed that only the homologous extensions proceed smoothly, i.e., the growth of L-seeds by L-monomers or D-seeds by D-monomers. The results suggest that, while the fibrils derived from L- and D-peptides form in a similar manner but with opposite stereochemistry, a cross-reaction between them is prevented because the geometry of the mixed sheet cannot satisfy dominant factors for beta-sheet stabilization.     

Establishment of a first-order kinetic model of light chain-associated amyloid fibril extension in vitro

Light chain-associated (AL) amyloidosis is a common and fatal systemic amyloidosis. AL amyloid fibrils (fAL) are composed of intact or fragmental monoclonal light chains (AL proteins). To elucidate the molecular mechanisms of fAL formation from AL proteins, we purified fAL and AL proteins from the amyloid-deposited organs of five AL amyloidosis patients. By electron microscopy and fluorometric thioflavin T method, we observed optimal fibril extension at pH 2.0-3.5 for the fibrils obtained from four patients, while at pH 7.5-8.0 for those obtained from one patient. Fragmental AL proteins were more efficient in the extension reaction than intact AL proteins. The fibrils obtained from all five patients showed clear fibril extension electron microscopically at pH 7.5. The extension of the fibrils obtained from all five patients could be explained by a first-order kinetic model, i.e., fibril extension proceeds via the consecutive association of AL proteins onto the ends of existing fibrils. Fibril extension was accelerated by dermatan sulfate proteoglycan, and inhibited by apolipoprotein E, alpha1-microglobulin, fibronectin, and an antioxidant nordihydroguaiaretic acid. These findings contribute to our understanding of the molecular mechanism underlying the pathogenesis of AL amyloidosis, and will be useful for developing a therapeutic strategy against the disease.     

Nuclear magnetic resonance characterization of the refolding intermediate of beta2-microglobulin trapped by non-native prolyl peptide bond

beta(2)-Microglobulin (beta2-m), a light chain of the major histocompatibility complex type I, is also found as a major component of amyloid fibrils formed in dialysis-related amyloidosis. Denaturation of beta2-m is considered to initiate the formation of fibrils. To clarify the mechanism of fibril formation, it is important to characterize the intermediate conformational states at the atomic level. Here, we investigated the refolding of beta2-m from the acid-unfolded state by heteronuclear magnetic resonance and circular dichroism spectroscopies. At low temperature, beta2-m refolded slowly, accumulating a rate-limiting intermediate with non-native chemical shift dispersions for several residues, but with compactness and secondary structures similar to those of the native protein. beta2-m has a cis proline residue at Pro32, located on the turn connecting the betaB and betaC strands. The slow refolding phase disappeared upon mutation of Pro32 to Val, indicating that Pro32 is responsible for the accumulation of the intermediate. The distribution of the perturbed residues in the intermediate suggests that the non-native prolyl peptide bond of Pro32 affects large areas of the molecule. A cis proline residue is common to various immunoglobulin domains involved in amyloidosis, implying that a non-native prolyl peptide bond that might occur under physiological conditions is related to the amyloidogenicity of these immunoglobulin domains.