pH-dependent amyloid and protofibril formation by the ABri peptide of familial British dementia

The ABri is a 34 residue peptide that is the major component of amyloid deposits in familial British dementia. In the amyloid deposits, the ABri peptide adopts aggregated beta-pleated sheet structures, similar to those formed by the Abeta peptide of Alzheimer's disease and other amyloid forming proteins. As a first step toward elucidating the molecular mechanisms of the beta-amyloidosis, we explored the ability of the environmental variables (pH and peptide concentration) to promote beta-sheet fibril structures for synthetic ABri peptides. The secondary structures and fibril morphology were characterized in parallel using circular dichroism, atomic force microscopy, negative stain electron microscopy, Congo red, and thioflavin-T fluorescence spectroscopic techniques. As seen with other amyloid proteins, the ABri fibrils had characteristic binding with Congo red and thioflavin-T, and the relative amounts of beta-sheet and amyloid fibril-like structures are influenced strongly by pH. In the acidic pH range 3.1-4.3, the ABri peptide adopts almost exclusively random structure and a predominantly monomeric aggregation state, on the basis of analytical ultracentrifugation measurements. At neutral pH, 7.1-7.3, the ABri peptide had limited solubility and produced spherical and amorphous aggregates with predominantly beta-sheet secondary structure, whereas at slightly acidic pH, 4.9, spherical aggregates, intermediate-sized protofibrils, and larger-sized mature amyloid fibrils were detected by atomic force microscopy. With aging at pH 4.9, the protofibrils underwent further association and eventually formed mature fibrils. The presence of small amounts of aggregated peptide material or seeds encourage fibril formation at neutral pH, suggesting that generation of such seeds in vivo could promote amyloid formation. At slightly basic pH, 9.0, scrambling of the Cys5-Cys22 disulfide bond occurred, which could lead to the formation of covalently linked aggregates. The presence of the protofibrils and the enhanced aggregation at slightly acidic pH is consistent with the behavior of other amyloid-forming proteins, which supports the premise that a common mechanism may be involved in protein misfolding and beta-amyloidosis.     

Helix-turn-helix peptides that form alpha-helical fibrils: turn sequences drive fibril structure

Models of apolipoprotein A-I (apo A-I), the main protein of high-density lipoprotein, predict that it contains 10 amphiphilic, alpha-helical segments connected by turns. We synthesized four peptides with two identical 18-residue, amphiphilic, alpha-helical segments (Anantharamaiah, G. M., et al. (1985) J. Biol. Chem. 260, 10248-10255) connected by putative turn sequences from apo A-I: (1) Ac-DWLKAFYDKVAEKLKEAFKVEPLRADWLKAFYDKVAEKLKEAF-NH2, (2) Ac-DWLKAFYDKVAEKLKEAFGLLPVLEDWLKAFYDKVAEKLKEAF-NH2, (3) Ac-DWLKAFYDKVAEKLKEAFKVQPYLDDWLKAFYDKVAEKLKEAF-NH2, and (4) Ac-DWLKAFYDKVAEKLKEAFNGGARLADWLKAFYDKVAEKLKEAF-NH2. Surprisingly, peptides 1-3 formed fibrils after incubation (37 degrees C, 10 mM sodium phosphate, pH 7.60), but in contrast to beta-sheet amyloid fibrils, these did not bind thioflavin T and they induced a blue shift in the spectrum of Congo red. CD (peptides 1-3) and FTIR (peptides 1 and 2) of the fibrils showed significant alpha-helical character. Synchrotron X-ray fiber diffraction on a magnetically aligned sample of 1 confirmed the alpha-helical character in the fibrils and indicated that the helical axes are oriented perpendicular to the fibril axis. In contrast, peptide 4, containing two Gly residues but no Pro in the turn, formed only a small amount of nonfibrillar precipitate after prolonged incubation. Peptide 4P (peptide 4 with a Pro in place of the central Ala) and peptide 5, containing a PEG block in lieu of the central turn, were similar to peptide 4 in not forming fibrils, possibly because the region linking the helices was unstructured. These studies indicate that varying turn sequences between longer amphiphilic alpha-helical segments can drive the structure of fibrils.     

Aggregation and conformational studies on a pentapeptide derivative

Most of the disease causing proteins such as beta amyloid, amylin, and huntingtin protein, which are natively disordered, readily form fibrils consisting of beta-sheet polymers. Though all amyloid fibrils are made up of beta-sheet polymers, not all peptides with predominant beta-sheet content in the native state develop into amyloid fibrils. We hypothesize that stable amyloid like fibril formation may require mixture of different conformational states in the peptide. We have tested this hypothesis on amyloid forming peptide namely HCl(Ile)(5)NH(CH(2)CH(2)O)(3)CH(3) (I). We show peptide I, has propensity to form self-assembled structures of beta-sheets in aqueous solutions. When incubated over a period of time in aqueous buffer, I self assembled into beta sheet like structures with diameters ranging from 30 to 60 A that bind with amyloidophilic dyes like Congo red and Thioflavin T. Interestingly peptide I developed into unstable fibrils after prolonged aging at higher concentration in contrast with the general mature fibril-forming propensity of various amyloid petides known to date.     

Amyloid-like features of polyglutamine aggregates and their assembly kinetics

The repeat length-dependent tendency of the polyglutamine sequences of certain proteins to form aggregates may underlie the cytotoxicity of these sequences in expanded CAG repeat diseases such as Huntington's disease. We report here a number of features of various polyglutamine (polyGln) aggregates and their assembly pathways that bear a resemblance to generally recognized defining features of amyloid fibrils. PolyGln aggregation kinetics displays concentration and length dependence and a lag phase that can be abbreviated by seeding. PolyGln aggregates exhibit classical beta-sheet-rich circular dichroism spectra consistent with an amyloid-like substructure. The fundamental structural unit of all the in vitro aggregates described here is a filament about 3 nm in width, resembling the protofibrillar intermediates in amyloid fibril assembly. We observed these filamentous structures either as isolated threads, as components of ribbonlike sheets, or, rarely, in amyloid-like twisted fibrils. All of the polyGln aggregates described here bind thioflavin T and shift its fluorescence spectrum. Although all polyGln aggregates tested bind the dye Congo red, only aggregates of a relatively long polyGln peptide exhibit Congo red birefringence, and this birefringence is only observed in a small portion of these aggregates. Remarkably, a monoclonal antibody with high selectivity for a generic amyloid fibril conformational epitope is capable of binding polyGln aggregates. Thus, polyGln aggregates exhibit most of the characteristic features of amyloid, but the twisted fibril structure with Congo red birefringence is not the predominant form in the polyGln repeat length range studied here. We also find that polyGln peptides exhibit an unusual freezing-dependent aggregation that appears to be caused by the freeze concentration of peptide and/or buffer components. This is of both fundamental and practical significance. PolyGln aggregation is revealed to be a highly specific process consistent with a significant degree of order in the molecular structure of the product. This ordered structure, or the assembly process leading to it, may be responsible for the cell-specific neuronal degeneration observed in Huntington's and other expanded CAG repeat diseases.     

The HET-s prion protein of the filamentous fungus Podospora anserina aggregates in vitro into amyloid-like fibrils.

The HET-s protein of Podospora anserina is a fungal prion. This protein behaves as an infectious cytoplasmic element that is transmitted horizontally from one strain to another. Under the prion form, the HET-s protein forms aggregates in vivo. The specificity of this prion model compared with the yeast prions resides in the fact that under the prion form HET-s causes a growth inhibition and cell death reaction when co-expressed with the HET-S protein from which it differs by 13 residues. Herein we describe the purification and initial characterization of recombinant HET-s protein expressed in Escherichia coli. The HET-s protein self-associates over time into high molecular weight aggregates. These aggregates greatly accelerate precipitation of the soluble form. HET-s aggregates appear as amyloid-like fibrils using electron microscopy. They bind Congo Red and show birefringence under polarized light. In the aggregated form, a HET-s fragment of approximately 7 kDa is resistant to proteinase K digestion. CD and FTIR analyses indicate that upon transition to the aggregated state, the HET-s protein undergoes a structural rearrangement characterized by an increase in antiparallel beta-sheet structure content. These results suggest that the [Het-s] prion element propagates in vivo as an infectious amyloid.     

Water-soluble beta-sheet models which self-assemble into fibrillar structures.

Self-assembly of beta-sheet domains resulting in the formation of pathogenic, fibrillar protein aggregates (amyloids) is a characteristic feature of various medical disorders. These include neurodegenerative diseases, such as Alzheimer's, Huntington's, and Creutzfeldt-Jacob's. A significant problem in studying such aggregation processes is the poor solubility of these beta-sheet complexes. The present work describes water-soluble de novo beta-sheet peptides which self-assemble into fibrillar structures. The model peptides enable studies of the relationship between beta-sheet stability and association behavior. The peptides [DPKGDPKG-(VT)n-GKGDPKPD-NH2, n = 3-8] are composed of a central beta-sheet-forming domain (VT-sequence), and N- and C-terminal nonstructured octapeptide sequences which promote water solubility. Conformational analyses by circular dichroism and Fourier transform infrared spectroscopy indicate the influence of peptide length, D-amino acid substitution, and concentration on the ability of the peptides to form stable beta-sheet structures. The association behavior investigated by analytical ultracentrifugation and dynamic light scattering was found to correlate strongly with the stability of a beta-sheet conformation. Model peptides with n >/= 6 form stable, water-soluble beta-sheet complexes with molecular masses of more than 2000 kDa, which are organized in fibrillar structures. The fibrils examined by Congo Red staining and electron microscopy show some similarities with naturally occurring amyloid fibrils.     

Structural requirements for antimicrobial versus chemoattractant activities for dermaseptin S9

Dermaseptin S9 (Drs S9), GLRSKIWLWVLLMIWQESNKFKKM, isolated from frog skin, does not resemble any of the cationic and amphipathic antimicrobial peptides identified to date, having a highly hydrophobic core sequence flanked at either side by cationic termini. Previous studies [Lequin O, Ladram A, Chabbert A, Bruston F, Convert O, Vanhoye D, Chassaing G, Nicolas P & Amiche M (2006) Biochemistry45, 468-480] demonstrated that this peptide adopted a non-amphipathic alpha-helical conformation in trifluoroethanol/water mixtures, but was highly aggregated in aqueous solutions and in the presence of sodium dodecyl sulfate micelles. Circular dichroism, FTIR and attenuated total reflectance FTIR spectroscopies, combined with a surface plasmon resonance study, show that Drs S9 forms stable and ordered beta-sheet aggregates in aqueous buffers or when bound to anionic or zwitterionic phospholipid vesicles. These structures slowly assembled into amyloid-like fibrils in aqueous environments via spherical intermediates, as revealed by electron microscopy and Congo red staining. Drs S9 induced the directional migration of neutrophils, T lymphocytes and monocytes. Interestingly, the antimicrobial and chemotactic activities of Drs S9 are modulated by its amyloid-like properties. Whereas spherical oligomers of Drs S9 exhibit antimicrobial activity, the soluble, weakly self-associated forms of Drs S9 act on human leukocytes to promote chemotaxis and/or immunological response activation in the same range of concentration as amyloidogenic peptides Abeta(1-42), the most fibrillogenic isoform of amyloid beta peptides, and the prion peptide PrP(106-126).     

A de novo designed helix-turn-helix peptide forms nontoxic amyloid fibrils

We report here that a monomeric de novo designed alpha-helix-turn-alpha-helix peptide, alpha t alpha, when incubated at 37 degrees C in an aqueous buffer at neutral pH, forms nonbranching, protease resistant fibrils that are 6-10 nm in diameter. These fibrils are rich in beta-sheet and bind the amyloidophilic dye Congo red. alpha t alpha fibrils thus display the morphologic, structural, and tinctorial properties of authentic amyloid fibrils. Surprisingly, unlike fibrils formed by peptides such as the amyloid beta-protein or the islet amyloid polypeptide, alpha t alpha fibrils were not toxic to cultured rat primary cortical neurons or PC12 cells. These results suggest that the potential to form fibrils under physiologic conditions is not limited to those proteins associated with amyloidoses and that fibril formation alone is not predictive of cytotoxic activity.     

Poly-(L-alanine) expansions form core beta-sheets that nucleate amyloid assembly

Expansion to a total of 11-17 sequential alanine residues from the normal number of 10 in the polyadenine-binding protein nuclear-1 (PABPN1) results in formation of intranuclear, fibrillar inclusions in skeletal muscle and hypothalamic neurons in adult-onset, dominantly inherited oculopharyngeal muscular dystrophy (OPMD). To understand the role that homopolymeric length may play in the protein misfolding that leads to the inclusions, we analyzed the self-assembly of synthetic poly-(L-alanine) peptides having 3-20 residues. We found that the conformational transition and structure of polyalanine (polyAla) assemblies in solution are not only length-dependent but also are determined by concentration, temperature, and incubation time. No beta-sheet complex was detected for those peptides characterized by n < 8, where n is number of alanine residues. A second group of peptides with 7 < n < 15 showed varying levels of complex formation, while for those peptides having n > 15, the interconversion process from the monomeric to the beta-sheet complex was complete under any of the tested experimental conditions. Unlike the typical tinctorial properties of amyloid fibrils, polyalanine fibrils did not show fluorescence with thioflavin T or apple-green birefringence with Congo red; however, like amyloid, X-ray diffraction showed that the peptide chains in these fibrils were oriented normal to the fibril axis (i.e., in the cross-beta arrangement). Neighboring beta-sheets are quarter-staggered in the hydrogen-bonding direction such that the alanine side-chains were closely packed in the intersheet space. Strong van der Waals contacts between side-chains in this arrangement likely account for the high stability of the macromolecular fibrillar complex in solution over a wide range of temperature (5-85 degrees C), and pH (2-10.5), and its resistance to denaturant (< 8 M urea) and to proteases (protease K, trypsin). We postulate that a similar stabilization of an expanded polyalanine stretch could form a core beta-sheet structure that mediates the intermolecular association of mutant proteins into fibrillar inclusions in human pathologies.     

Techniques to study amyloid fibril formation in vitro

Amyloid fibrils are ordered aggregates of peptides or proteins that are fibrillar in structure and contribute to the complications of many diseases (e.g., type 2 diabetes mellitus, Alzheimer's disease, and primary systemic amyloidosis). These fibrils can also be prepared in vitro and there are three criteria that define a protein aggregate as an amyloid fibril: green birefringence upon staining with Congo Red, fibrillar morphology, and beta-sheet secondary structure. The purpose of this review is to describe the techniques used to study amyloid fibril formation in vitro, address common errors in the collection and interpretation of data, and open a discussion for a critical review of the criteria currently used to classify a protein aggregate as an amyloid fibril.     

Fibrils from Designed Non-Amyloid-Related Synthetic Peptides Induce AA-Amyloidosis during Inflammation in an Animal Model

Background

Mouse AA-amyloidosis is a transmissible disease by a prion-like mechanism where amyloid fibrils act by seeding. Synthetic peptides with no amyloid relationship can assemble into amyloid-like fibrils and these may have seeding capacity for amyloid proteins.

Principal Findings

Several synthetic peptides, designed for nanotechnology, have been examined for their ability to produce fibrils with Congo red affinity and concomitant green birefringence, affinity for thioflavin S and to accelerate AA-amyloidosis in mice. It is shown that some amphiphilic fibril-forming peptides not only produced Congo red birefringence and showed affinity for thioflavin S, but they also shortened the lag phase for systemic AA-amyloidosis in mice when they were given intravenously at the time of inflammatory induction with silver nitride. Peptides, not forming amyloid-like fibrils, did not have such properties.

Conclusions

These observations should caution researchers and those who work with synthetic peptides and their derivatives to be aware of the potential health concerns.     

An engineered PrPsc-like molecule from the chimera of mammalian prion protein and yeast Ure2p prion-inducing domain

Production of the pathogenic prion isoform PrP^sc-like molecules is thought to be useful forunderstanding the mysterious mechanism of conformational conversion process of prion diseases andproving the “protein-only“ hypothesis. In this report, an engineered PrP^sc-like conformation was producedfrom a chimera of mammalian bovine prion protein (bPrP) and yeast Ure2p prion-inducing domain (UPrD).Compared with the normal form of bPrP, the engineered recombinant protein, termed bPrP-UPrD,spontaneously aggregated into ordered fibrils under physiological condition, displaying amyloid-likecharacteristics, such as fibrillar morphology, birefringence upon binding to Congo red and increasedfluorescence intensity with Thioflavine T. Limited resistance to protease K digestion and CD spectroscopyexperiments suggested that the structure of bPrP-UPrD had been changed, and adopted a new, high contentB-sheet conformation during the fibrils formation. Moreover, bPrP-UPrD amyloid fibrils could recruit moresoluble forms into the aggregates. Therefore, the engineered molecules could mimic significant behaviors of PrP^se and will be helpful for further understanding the mechanism of conformational conversion process.     

Structural transitions involved in a novel amyloid-like beta-sheet assemblage of tripeptide derivatives

Self-assembly of two tripeptide derivatives containing three nonpolar isoleucine moieties and polar oxyethylene groups are studied in methanol. Peptide A [CH3(OCH2CH2)3OCH2CO(Ile)3OCH3] and peptide B [CH3(OCH2CH2)3OCH2CO(Ile)3NH (CH2CH2O)3CH3] take a mixture of unordered and helical conformation at low concentration (8.5 x 10(-4) M). However, at high concentration (2 x 10(-3) M), both the peptide showed significant increase in the helical conformation. An interesting conformational transition of peptides A and B at various methanol contents was observed in the solvated films of these compounds by spectroscopic methods like the far-uv circular dichroism and Fourier transform infrared (FT-IR) techniques. Peptide B, which contains more polar oxyethylene groups than A, showed a highly cooperative conformational transition when the methanol content was decreased. This transition was characterized by a large increase of beta-sheet, retaining a alpha-helical contribution. Peptide A showed a conformational transition resulting in a beta-sheet in the aggregated state. From the CD spectra, the ratio in the ellipticity indicates that peptide B forms twisted antiparallel beta-sheet conformation, whereas peptide A takes a parallel beta-sheet conformation. The results obtained in this work indicates the role of polar derivatization on the conformational preference of peptides having similar sequence.     

Is Congo red an amyloid-specific dye?

Congo red (CR) binding, monitored by characteristic yellow-green birefringence under crossed polarization has been used as a diagnostic test for the presence of amyloid in tissue sections for several decades. This assay is also widely used for the characterization of in vitro amyloid fibrils. In order to probe the structural specificity of Congo red binding to amyloid fibrils we have used an induced circular dichroism (CD) assay. Amyloid fibrils from insulin and the variable domain of Ig light chain demonstrate induced CD spectra upon binding to Congo red. Surprisingly, the native conformations of insulin and Ig light chain also induced Congo red circular dichroism, but with different spectral shapes than those from fibrils. In fact, a wide variety of native proteins exhibited induced CR circular dichroism indicating that CR bound to representative proteins from different classes of secondary structure such as alpha (citrate synthase), alpha + beta (lysozyme), beta (concavalin A), and parallel beta-helical proteins (pectate lyase). Partially folded intermediates of apomyoglobin induced different Congo red CD bands than the corresponding native conformation, however, no induced CD bands were observed with unfolded protein. Congo red was also found to induce oligomerization of native proteins, as demonstrated by covalent cross-linking and small angle x-ray scattering. Our data suggest that Congo red is sandwiched between two protein molecules causing protein oligomerization. The fact that Congo red binds to native, partially folded conformations and amyloid fibrils of several proteins shows that it must be used with caution as a diagnostic test for the presence of amyloid fibrils in vitro.     

FT-Raman spectroscopy as diagnostic tool of Congo red binding to amyloids

Chorion is the major component of silkmoth eggshell. More than 95% of its dry mass consists of the A and B families of low molecular weight structural proteins, which have remarkable mechanical and chemical properties protecting the oocyte and developing embryo from environmental hazards. We present data from FT-Raman spectroscopy of silkmoth chorion and amyloid-like fibrils formed from peptide analogues of chorion proteins, both unstained and stained by Congo red. The results show that FT-Raman spectroscopy is not a straightforward diagnostic tool for the specific interactions of Congo red with amyloids: a dilute aqueous solution of the Congo red dye at pH 5.5 and a thin solid film of the dye cast from this solution exhibit the same "diagnostic" Raman shifts relative to the neat Congo red dry powder as do amyloid fibrils formed from peptide analogues of chorion proteins stained by Congo red. An important consequence of this finding is that these shifts of the Raman active modes of Congo red are probably due to the formation of supramolecular dye aggregates in the presence of water. Therefore, this is not an appropriate diagnostic test for Congo red binding to amyloids.     

Amyloid-like properties of a synthetic peptide corresponding to the carboxy terminus of beta-amyloid protein precursor.

A synthetic peptide whose sequence corresponds to the 20 carboxy-terminal amino acids of beta-amyloid protein precursor (APP) was found to form fibrils in vitro. These fibrils showed birefringence in polarized light when stained with Congo red, fluoresced when bound with thioflavin S, were resistant to proteases, and had a cross-beta conformation. By contrast, peptides with other sequences from the intracellular domain of APP and a peptide corresponding to this entire domain did not exhibit the full range of beta-amyloid properties. These results suggest that a fragment from the C-terminus of the beta-amyloid protein precursor could bind to intraneuronal paired helical filaments and account for some of its amyloid-like properties.     

Identification of an amyloidogenic region on keratoepithelin via synthetic peptides

Mutations of keratoepithelin (KE) gene in human chromosome 5q31 have been linked with corneal epithelial or stromal dystrophies characterized by the abnormal deposits of amyloid fibrils and/or non-amyloid aggregations in corneal tissue. We report herein that synthetic peptide containing amino acid (a.a.) residues of 515-532 of native KE protein can readily form beta-sheet-containing amyloid fibrils in vitro. Amyloid fibrils formed in various conditions from short synthetic peptides (containing a.a. 515-532 and 515-525, respectively) were characterized by thioflavin T (ThT) fluorescence assay, Congo red staining, electron microscopy (EM) and circular dichroism (CD). Triple-N-methylation of the synthetic peptides prevented the beta-sheet polymerization and related amyloid fibril formation. Comparison study with ThT fluorescence further demonstrated that synthetic peptides containing corneal dystrophy-related mutations within this region formed amyloid fibrils to various extents. Our results suggest that each individual dystrophy-related mutation by itself does not necessarily potentiate amyloid fibril formation of KE. Roles of these intrinsically amyloidogenic foci in abnormal KE aggregations and amyloid deposits of stromal corneal dystrophies await further investigation.     

Identification of multiple amyloidogenic sequences in laminin-1

Amyloid fibril formation is associated with several pathologies, including Alzheimer's disease, Parkinson's disease, type II diabetes, and prion diseases. Recently, a relationship between basement membrane components and amyloid deposits has been reported. The basement membrane protein, laminin, may be involved in amyloid-related diseases, since laminin is present in amyloid plaques in Alzheimer's disease and binds to amyloid precursor protein. Recently, we showed that peptide A208 (AASIKVAVSADR), the IKVAV-containing peptide, formed amyloid-like fibrils. We previously identified 60 cell adhesive sequences in laminin-1 using a total of 673 12-mer synthetic peptides. Here, we screened for additional amyloidogenic sequences among 60 cell adhesive peptides derived from laminin-1. We first examined amyloid-like fibril formation by the 60 active peptides with Congo red, a histological dye binding to many amyloid-like proteins. Thirteen peptides were stained with Congo red. Four of the 13 peptides promoted cell attachment and neurite outgrowth like the IKVAV-containing peptide. The four peptides also showed amyloid-like fibril formation in both X-ray diffraction and electron microscopic analyses. The amyloidogenic peptides contain consensus amino acid components, including both basic and acidic amino acids and Ser and Ile residues. These results indicate that at least five laminin-derived peptides can form amyloid-like fibrils. We conclude that the laminin-derived amyloidogenic peptides have the potential to form amyloid-like fibrils in vivo, possibly when laminin-1 is degraded.     

Toxicity of non-abeta component of Alzheimer's disease amyloid, and N-terminal fragments thereof, correlates to formation of beta-sheet structure and fibrils.

The non-Abeta component of Alzheimer's disease amyloid (NAC) and its precursor alpha-synuclein have been linked to amyloidogenesis in Alzheimer's disease (AD), Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Previously we have shown that NAC forms beta-sheet structures and fibrils [El-Agnaf, O.M.A., Bodles, A.M., Guthrie, D.J.S., Harriott, P. & Irvine, G.B. (1998) Eur. J. Biochem. 258, 157-163]. As a measure of their neurotoxic potential we have examined the ability of fresh and aged NAC and fragments thereof to inhibit the reduction of the redox dye 3-(4, 5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide by rat pheochromocytoma PC12 cells. Micromolar concentrations of NAC and fragments thereof display varying degrees of toxicity. On immediate dissolution and after an incubation period for 3 days at 37 degrees C the full-length peptide and fragments NAC(3-18) and NAC(1-18) scrambled sequence [NAC(1-18 s)] were toxic, whereas fragments NAC(1-13) and NAC(6-14) were not. CD indicates that NAC(3-18) and NAC(1-18 s) exhibit beta-sheet secondary structure in aqueous solution, whereas NAC(1-13) and NAC(6-14) do not. NAC(3-18) aggregates, as indicated by concentration of peptide remaining in solution after 3 days measured by an HPLC assay, and forms fibrils, as determined by electron microscopy. However, although some fibrils were detected for NAC(1-18 s) it does not come out of solution to a significant degree. Fragments NAC(1-13) and NAC(6-14) form few fibrils and remain in solution. These findings indicate that the ability of the central region of NAC to form beta-sheet secondary structures is important for determining the toxicity of the peptide. This contrasts with what has been reported previously for most Abeta peptides as their toxicity appears to require the peptide to have formed fibrillary aggregates as well as displaying beta-sheet. These results suggest that an intermediate, which exhibits beta-sheet structure, may be responsible for the toxic properties of NAC and provides further evidence for the role of NAC in the pathogenesis of AD, PD and DLB.     

Formation of insulin amyloid fibrils followed by FTIR simultaneously with CD and electron microscopy

Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), and electron microscopy (EM) have been used simultaneously to follow the temperature-induced formation of amyloid fibrils by bovine insulin at acidic pH. The FTIR and CD data confirm that, before heating, insulin molecules in solution at pH 2.3 have a predominantly native-like alpha-helical structure. On heating to 70 degrees C, partial unfolding occurs and results initially in aggregates that are shown by CD and FT-IR spectra to retain a predominantly helical structure. Following this step, changes in the CD and FTIR spectra occur that are indicative of the extensive conversion of the molecular conformation from alpha-helical to beta-sheet structure. At later stages, EM shows the development of fibrils with well-defined repetitive morphologies including structures with a periodic helical twist of approximately 450 A. The results indicate that formation of fibrils by insulin requires substantial unfolding of the native protein, and that the most highly ordered structures result from a slow evolution of the morphology of the initially formed fibrillar species.