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.     

Molecular determinants of amyloid deposition in Alzheimer's disease: conformational studies of synthetic beta-protein fragments

The amyloid beta-protein (1-42) is a major constituent of the abnormal extracellular amyloid plaque that characterizes the brains of victims of Alzheimer's disease. Two peptides, with sequences derived from the previously unexplored C-terminal region of the beta-protein, beta 26-33 (H2N-SNKGAIIG-CO2H) and beta 34-42 (H2N-LMVGGVVIA-CO2H), were synthesized and purified, and their solubility and conformational properties were analyzed. Peptide beta 26-33 was found to be freely soluble in water; however, peptide beta 34-42 was virtually insoluble in aqueous media, including 6 M guanidinium thiocyanate. The peptides formed assemblies having distinct fibrillar morphologies and different dimensions as observed by electron microscopy of negatively stained samples. X-ray diffraction revealed that the peptide conformation in the fibrils was cross-beta. A correlation between solubility and beta-structure formation was inferred from FTIR studies: beta 26-33, when dissolved in water, existed as a random coil, whereas the water-insoluble peptide beta 34-42 possessed antiparallel beta-sheet structure in the solid state. Solubilization of beta 34-42 in organic media resulted in the disappearance of beta-structure. These data suggest that the sequence 34-42, by virtue of its ability to form unusually stable beta-structure, is a major contributor to the insolubility of the beta-protein and may nucleate the formation of the fibrils that constitute amyloid plaque.     

Assembly and aggregation properties of synthetic Alzheimer's A4/beta amyloid peptide analogs.

The amyloid A4 or beta peptide is a major component of extracellular amyloid deposits that are a characteristic feature of Alzheimer's disease. We synthesized a series of peptide analogs of the A4/beta peptide which are progressively longer at their carboxyl termini, including 42- and 39-residue peptides which represent the major forms of the A4/beta peptide in senile plaque and the hereditary cerebral hemorrhage with amyloidosis form, respectively. All peptides tested, beta 1-28 through beta 1-42, formed amyloid-like fibrils and previously unreported thin sheet-like structures which stained with thioflavin T and Congo Red. The solubility of beta 1-42 and shorter peptides was pH and concentration dependent, with a broad insolubility profile in the pH range of 3.5-6.5 and at concentrations above 0.75 mg/ml. Only peptides of 42 residues or longer were significantly insoluble at pH 7.4. beta 1-47 and beta 1-52 peptides are highly insoluble in aqueous media but are soluble at 40 mg/ml in the alpha helix-promoting solvent, 1,1,1,3,3,3-hexafluoro-2-propanol. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the beta 1-42 peptide migrates as a series of higher molecular mass aggregates whereas shorter peptides migrate as monomers. Aggregation is also dependent on pH, peptide concentration, and time of incubation in aqueous medium. These results indicate that the length of the hydrophobic carboxyl terminus of the A4/beta peptide is important in determining the solubility and aggregation properties of the A4/beta peptide and that acid pH environment, high peptide concentration, and long incubation time would be predicted to be important factors in promoting amyloid deposition.     

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.     

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.     

Beta-helix core packing within the triple-stranded oligomerization domain of the P22 tailspike

A right-handed parallel beta-helix of 400 residues in 13 tightly packed coils is a major motif of the chains forming the trimeric P22 tailspike adhesin. The beta-helix domains of three identical subunits are side-by-side in the trimer and make predominantly hydrophilic inter-subunit contacts (Steinbacher S et al., 1994, Science 265:383-386). After the 13th coil the three individual beta-helices terminate and the chains wrap around each other to form three interdigitated beta-sheets organized into the walls of a triangular prism. The beta-strands then separate and form antiparallel beta-sheets, but still defining a triangular prism in which each side is a beta-sheet from a different subunit (Seckler R, 1998, J Struct Biol 122:216-222). The subunit interfaces are buried in the triangular core of the prism, which is densely packed with hydrophobic side chains from the three beta-sheets. Examination of this structure reveals that its packed core maintains the same pattern of interior packing found in the left-handed beta-helix, a single-chain structure. This packing is maintained in both the interdigitated parallel region of the prism and the following antiparallel sheet section. This oligomerization motif for the tailspike beta-helices presumably contributes to the very high thermal and detergent stability that is a property of the native tailspike adhesin.     

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.     

Investigation of a peptide responsible for amyloid fibril formation of beta 2-microglobulin by achromobacter protease I.

To obtain insight into the mechanism of amyloid fibril formation from beta(2)-microglobulin (beta2-m), we prepared a series of peptide fragments using a lysine-specific protease from Achromobacter lyticus and examined their ability to form amyloid fibrils at pH 2.5. Among the nine peptides prepared by the digestion, the peptide Ser(20)-Lys(41) (K3) spontaneously formed amyloid fibrils, confirmed by thioflavin T binding and electron microscopy. The fibrils composed of K3 peptide induced fibril formation of intact beta2-m with a lag phase, distinct from the extension reaction without a lag phase observed for intact beta2-m seeds. Fibril formation of K3 peptide with intact beta2-m seeds also exhibited a lag phase. On the other hand, the extension reaction of K3 peptide with the K3 seeds occurred without a lag phase. At neutral pH, the fibrils composed of either intact beta2-m or K3 peptide spontaneously depolymerized. Intriguingly, the depolymerization of K3 fibrils was faster than that of intact beta2-m fibrils. These results indicated that, although K3 peptide can form fibrils by itself more readily than intact beta2-m, the K3 fibrils are less stable than the intact beta2-m fibrils, suggesting a close relation between the free energy barrier of amyloid fibril formation and its stability.     

Amyloid-forming peptides from beta2-microglobulin-Insights into the mechanism of fibril formation in vitro

Beta(2)-Microglobulin (beta(2)m) is one of over 20 proteins known to be involved in human amyloid disease. Peptides equivalent to each of the seven beta-strands of the native protein, together with an eighth peptide (corresponding to the most stable region in the amyloid precursor conformation formed at pH 3.6, that includes residues in the native strand E plus the eight succeeding residues (named peptide E')), were synthesised and their ability to form fibrils investigated. Surprisingly, only two sequences, both of which encompass the region that forms strand E in native beta(2)m, are capable of forming amyloid-like fibrils in vitro. These peptides correspond to residues 59-71 (peptide E) and 59-79 (peptide E') of intact beta(2)m. The peptides form fibrils under the acidic conditions shown previously to promote amyloid formation from the intact protein (pH <5 at low and high ionic strength), and also associate to form fibrils at neutral pH. Fibrils formed from these two peptides enhance fibrillogenesis of the intact protein. No correlation was found between secondary structure propensity, peptide length, pI or hydrophobicity and the ability of the peptides to associate into amyloid-like fibrils. However, the presence of a relatively high content of aromatic side-chains correlates with the ability of the peptides to form amyloid fibrils. On the basis of these results we propose that residues 59-71 may be important in the self-association of partially folded beta(2)m into amyloid fibrils and discuss the relevance of these results for the assembly mechanism of the intact protein in vitro.     

Apolipoprotein E inhibits the depolymerization of beta 2-microglobulin-related amyloid fibrils at a neutral pH.

beta 2-Microglobulin-related (A beta 2M) amyloidosis is a common and serious complication in patients on long-term hemodialysis, and beta 2-microglobulin (beta 2-m) is a major structural component of A beta 2M amyloid fibrils. Fluorescence spectroscopic analysis with thioflavin T and electron microscopic study revealed that A beta 2M amyloid fibrils readily depolymerize into monomeric beta 2-m at a neutral to basic pH. Circular dichroism analysis revealed that soon after the initiation of the depolymerization reaction at pH 7.5, the characteristic spectrum of beta 2-m in A beta 2M amyloid fibrils changes to resemble that of monomeric beta 2-m at pH 7.5. Apolipoprotein E (apoE), a representative amyloid-associated protein, formed a stable complex with A beta 2M amyloid fibrils and inhibited the depolymerization of A beta 2M amyloid fibrils dose-dependently in a range of 0--10 microM. These results showed that apoE could enhance the deposition of amyloid fibrils in vivo, possibly by binding directly to the surface of the fibrils and stabilizing the conformation of beta 2-m in the fibrils.     

Role of the single disulphide bond of beta(2)-microglobulin in amyloidosis in vitro

The aggregation of beta(2)-microglobulin (beta(2)m) into amyloid fibrils occurs in the condition known as dialysis-related amyloidosis (DRA). The protein has a beta-sandwich fold typical of the immunoglobulin family, which is stabilized by a highly conserved disulphide bond linking Cys25 and Cys80. Oxidized beta(2)m forms amyloid fibrils rapidly in vitro at acidic pH and high ionic strength. Here we investigate the role of the single disulphide bond of beta(2)m in amyloidosis in vitro. We show that reduction of the disulphide bond destabilizes the native protein such that non-native molecules are populated at neutral pH. These species are prone to oligomerization but do not form amyloid fibrils when incubated for up to 8 mo at pH 7.0 in 0.4 M NaCl. Over the pH range 4.0-1.5 in the presence of 0.4 M NaCl, however, amyloid fibrils of reduced beta(2)m are formed. These fibrils are approximately 10 nm wide, but are shorter and assemble more rapidly than those produced from the oxidized protein. These data show that population of non-native conformers of beta(2)m at neutral pH by reduction of its single disulphide bond is not sufficient for amyloid formation. Instead, association of one or more specific partially unfolded molecules formed at acid pH are necessary for the formation of beta(2)m amyloid in vitro. Further experiments will now be needed to determine the role of different oligomeric species of beta(2)m in the toxicity of the protein in vivo.     

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.     

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.     

Amyloid fibril formation by pentapeptide and tetrapeptide fragments of human calcitonin

The process of amyloid fibril formation by the human calcitonin hormone is associated with medullary thyroid carcinoma. Based on the effect of pH on the fibrillization of human calcitonin, the analysis of conformationally constrained analogues of the hormone, and our suggestion regarding the role of aromatic residues in the process of amyloid fibril formation, we studied the ability of a short aromatic charged peptide fragment of calcitonin (NH(2)-DFNKF-COOH) to form amyloid fibrils. Here, using structural and biophysical analysis, we clearly demonstrate the ability of this short peptide to form well ordered amyloid fibrils. A shorter truncated tetrapeptide, NH(2)-DFNK-COOH, also formed fibrils albeit less ordered than those formed by the pentapeptide. We could not detect amyloid fibril formation by the NH(2)-FNKF-COOH tetrapeptide, the NH(2)-DFN-COOH tripeptide, or the NH(2)-DANKA-COOH phenylalanine to the alanine analogue of the pentapeptide. The formation of amyloid fibrils by rather hydrophilic peptides is quite striking, because it was speculated that hydrophobic interactions might play a key role in amyloid formation. This is the first reported case of fibril formation by a peptide as short as a tetrapeptide and one of very few cases of amyloid formation by pentapeptides. Because the aromatic nature seems to be the only common property of the various very short amyloid-forming peptides, it further supports our hypothesis on the role of aromatic interactions in the process of amyloid fibril formation.     

Alzheimer beta-amyloid homodimers facilitate A beta fibrillization and the generation of conformational antibodies

We reported previously that stabilized beta-amyloid peptide dimers were derived from mutant amyloid precursor protein with a single cysteine in the ectodomain juxtamembrane position. In vivo studies revealed that two forms of SDS-stable A beta homodimers exist, species ending at A beta 40 and A beta 42. The phenomenon of the transformation of the initially deposited 42-residue beta-amyloid peptide into the amyloid fibrils of Alzheimer's disease plaques remains to be explained in physical terms, i.e. energetically and structurally. We therefore performed spectroscopic analyses revealing that engineered dimeric peptides ending at residue 42 displayed a much more pronounced beta-structural transition than corresponding monomers. Specifically, the single chemically induced dimerization of A beta peptides significantly increased the beta-sheet content by a factor of 2. The C-terminal residues Ile-41 and Ala-42 of dimeric forms further increased the beta-sheet content by roughly one-third. In contrast to A beta 42, the beta-sheet content of the alpha- and gamma-secretase-generated p3 fragments did not necessarily correlate with the tendency to form fibrils, although p3/17-42 had a pronounced thread forming character with fibril lengths of up to 2.5 microM. Electron microscopic images show that forms of p3/17-42 generated smaller granular particles than forms ending at residue 40. We discuss these findings in terms of A beta 1-42 dimers representing paranuclei, which self-aggregate into ribbon-like ordered fibrils by elongation. Based on A beta 42 dimer-specific titers of a polyclonal antiserum we propose that the A beta homodimer represents a nidus for plaque formation and a well defined novel therapeutic target.     

Beta(2)-microglobulin and its deamidated variant, N17D form amyloid fibrils with a range of morphologies in vitro.

Amyloid fibrils formed by incubation of recombinant wild-type human beta(2)-microglobulin (beta(2)M) ab initio in vitro at low pH and high ionic strength are short and highly curved. By contrast, fibrils extracted from patients suffering from haemodialysis-related amyloidosis and those formed by seeding growth of the wild-type protein in vitro with fibrils ex vivo are longer and straighter than those previously produced ab initio in vitro. Here we explore the effect of growth conditions on morphology of beta(2)M fibrils formed ab initio in vitro from the wild-type protein, as well as a variant form of beta(2)M in which Asn17 is deamidated to Asp (N17D). We show that deamidation results in significant destabilisation of beta(2)M at neutral pH. Despite this, acidification is still necessary to form amyloid from the mutant protein in vitro. Interestingly, at low pH and low ionic strength long, straight fibrils of recombinant beta(2)M are formed in vitro. The fibrils comprise three distinct morphological types when examined using electron microscopy (EM) and atomic force microscopy (AFM) that vary in periodicity and the number of constituent protofibrils. Using kinetic experiments we suggest that the immature fibrils observed previously do not represent intermediates in the assembly of fully mature amyloid, at least under the conditions studied here.     

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.     

Mechanism of A beta(1-40) fibril-induced fluorescence of (trans,trans)-1-bromo-2,5-bis(4-hydroxystyryl)benzene (K114)

K114, (trans,trans)-1-bromo-2,5-bis(4-hydroxystyryl)benzene, is a fluorescent Congo Red analogue that binds tightly to amyloid fibrils, but not the monomeric proteins, with a concomitant enhancement in fluorescence. The mechanism for the low aqueous fluorescence and the subsequent enhancement by A beta(1-40) fibrils was investigated by fluorescence spectroscopy and binding analysis. K114's unusually low buffer fluorescence is due to self-quenching in sedimentable aggregates or micelles which upon interacting with amyloid fibrils undergo an enhancement in fluorescence intensity and shifts in the excitation and emission spectra. These spectral changes are suggestive of a stabilization of the phenolate anion, perhaps by hydrogen bonding, rather than an increase in the microenvironment dielectric constant or dye immobilization. 1,4-Bis(4-aminophenylethenyl)-2-methoxybenzene, which lacks the phenol moiety, and X-34, which contains a stabilized phenol (pK approximately 13.4), do not display the phenolate anion fluorescence in the presence of fibrils. The apparent affinity of K114 for fibril binding is 20-30 nM with a stoichiometry of 2.2 mol of K114/mol of A beta(1-40) monomer. Competition studies indicate that K114 and Congo Red share a site, but K114 does not bind to sites on A beta(1-40) fibrils for neutral benzothiazole (BTA-1), cationic thioflavin T, or the hydrophobic (S)-naproxen and (R)-ibuprofen molecules. Comparison of benzothiazole binding stoichiometry which has been suggested to reflect disease-relevant amyloid structures to that of Congo Red analogues which reflect total fibril content may be useful in defining biologically pertinent conformational forms of amyloid.     

pH-dependent structural transitions of Alzheimer amyloid peptides.

To understand the molecular interactions leading to the assembly of beta/44 protein into the hallmark fibrils of Alzheimer's disease (AD), we have examined the ability of synthetic peptides that correspond to the beta/A4 extracellular sequence to form fibrils over the range of pH 3-10. Peptides included the sequences 1-28, 19-28, 17-28, 15-28, 13-28, 11-28, and 9-28 of beta/A4. The model fibrils were compared with isolated amyloid with respect to morphology, conformation, tinctorial properties, and stability under denaturing conditions. Electron microscopy, Fourier-transform infrared (FT-IR) spectroscopy, and x-ray diffraction revealed that the ionization states of the amino acid sidechains appeared to be a crucial feature in fibril formation. This was reflected by the ability of several peptides to undergo fibril assembly and disassembly as a function of pH. Comparisons between different beta/A4 sequences demonstrated that the fibrillar structure representative of AD amyloid was dependent upon electrostatic interactions, likely involving His-13 and Asp-23, and hydrophobic interactions between uncharged sidechains contained within residues 17-21. The results also indicated an exclusively beta-sheet conformation for the synthetic (and possibly AD fibrils) in contrast to certain other (e.g., systemic) amyloids.     

A systematic study of the effect of physiological factors on beta2-microglobulin amyloid formation at neutral pH

Beta(2)-microglobulin (beta(2)m) forms amyloid fibrils that deposit in the musculo-skeletal system in patients undergoing long-term hemodialysis. How beta(2)m self-assembles in vivo is not understood, since the monomeric wild-type protein is incapable of forming fibrils in isolation in vitro at neutral pH, while elongation of fibril-seeds made from recombinant protein has only been achieved at low pH or at neutral pH in the presence of detergents or cosolvents. Here we describe a systematic study of the effect of 11 physiologically relevant factors on beta(2)m fibrillogenesis at pH 7.0 without denaturants. By comparing the results obtained for the wild-type protein with those of two variants (DeltaN6 and V37A), the role of protein stability in fibrillogenesis is explored. We show that DeltaN6 forms low yields of amyloid-like fibrils at pH 7.0 in the absence of seeds, suggesting that this species could initiate fibrillogenesis in vivo. By contrast, high yields of amyloid-like fibrils are observed for all proteins when assembly is seeded with fibril-seeds formed from recombinant protein at pH 2.5 stabilized by the addition of heparin, serum amyloid P component (SAP), apolipoprotein E (apoE), uremic serum, or synovial fluid. The results suggest that the conditions within the synovium facilitate fibrillogenesis of beta(2)m and show that different physiological factors may act synergistically to promote fibril formation. By comparing the behavior of wild-type beta(2)m with that of DeltaN6 and V37A, we show that the physiologically relevant factors enhance fibrillogenesis by stabilizing fibril-seeds, thereby allowing fibril extension by rare assembly competent species formed by local unfolding of native monomers.