Racemization of the amyloidal beta Asp1 residue blocks the acceleration of fibril formation caused by racemization of the Asp23 residue

Amyloid β proteins extracted from the amyloid cores of neuritic plaques are considerably racemized at their Asp residues. To assess the impact of d-Asp on amyloid β_1-42 conformation and on initiation of amyloid fibril formation, we used wild-type amyloid β_1-42 and analogs in which d-Asp was substituted for l-Asp at residues 1, 7, 23, and all combinations of these residues. Amyloid fibril formation was enhanced by d-Asp²³; modulation of Asp chirality at N-terminal position 1 blocked this enhancement and modulation at position 7 augmented it. Knowledge of such chirality modifications may help to develop potent inhibitors of amyloid fibril formation.     

Mutations enhance the aggregation propensity of the Alzheimer's A beta peptide

Aggregation of the amyloid β (Aβ) peptide plays a key role in the molecular etiology of Alzheimer’s disease. Despite the importance of this process, the relationship between the sequence of Aβ and the propensity of the peptide to aggregate has not been fully elucidated. The sequence determinants of aggregation can be revealed by probing the ability of amino acid substitutions (mutations) to increase or decrease aggregation. Numerous mutations that decrease aggregation have been isolated by laboratory-based studies. In contrast, very few mutations that increase aggregation have been reported, and most of these were isolated from rare individuals with early-onset familial Alzheimer’s disease. To augment the limited data set of clinically derived mutations, we developed an artificial genetic screen to isolate novel mutations that increase aggregation propensity. The screen relies on the expression of Aβ-green fluorescent protein fusion in Escherichia coli. In this fusion, the ability of the green fluorescent protein reporter to fold and fluoresce is inversely correlated with the aggregation propensity of the Aβ sequence. Implementation of this screen enabled the isolation of 20 mutant versions of Aβ with amino acid substitutions at 17 positions in the 42-residue sequence of Aβ. Biophysical studies of synthetic peptides corresponding to sequences isolated by the screen confirm the increased aggregation propensity and amyloidogenic behavior of the mutants. The mutations were isolated using an unbiased screen that makes no assumptions about the sequence determinants of aggregation. Nonetheless, all 16 of the most aggregating mutants contain substitutions that reduce charge and/or increase hydrophobicity. These findings provide compelling evidence supporting the hypothesis that sequence hydrophobicity is a major determinant of Aβ aggregation.     

Ultrasonication-dependent acceleration of amyloid fibril formation

Amyloid fibrils, similar to crystals, form through nucleation and growth. Because of the high free-energy barrier of nucleation, the spontaneous formation of amyloid fibrils occurs only after a long lag phase. Ultrasonication is useful for inducing amyloid nucleation and thus for forming fibrils, while the use of a microplate reader with thioflavin T fluorescence is suitable for detecting fibrils in many samples simultaneously. Combining the use of ultrasonication and microplate reader, we propose an efficient approach to studying the potential of proteins to form amyloid fibrils. With β₂-microglobulin, an amyloidogenic protein responsible for dialysis-related amyloidosis, fibrils formed within a few minutes at pH 2.5. Even under neutral pH conditions, fibrils formed after a lag time of 1.5 h. The results propose that fibril formation is a physical reaction that is largely limited by the high free-energy barrier, which can be effectively reduced by ultrasonication. This approach will be useful for developing a high-throughput assay of the amyloidogenicity of proteins.     

Effect of amino acid variations in the central region of human serum amyloid A on the amyloidogenic properties

Human serum amyloid A (SAA) is a precursor protein of the amyloid fibrils that are responsible for AA amyloidosis. Of the four human SAA genotypes, SAA1 is most commonly associated with AA amyloidosis. Furthermore, SAA1 has three major isoforms (SAA1.1, 1.3, and 1.5) that differ by single amino acid variations at two sites in their 104-amino acid sequences. In the present study, we examined the effect of amino acid variations in human SAA1 isoforms on the amyloidogenic properties. All SAA1 isoforms adopted α-helix structures at 4 °C, but were unstructured at 37 °C. Heparin-induced amyloid fibril formation of SAA1 was observed at 37 °C, as evidenced by the increased thioflavin T (ThT) fluorescence and β-sheet structure formation. Despite a comparable increase in ThT fluorescence, SAA1 molecules retained their α-helix structures at 4 °C. At both temperatures, no essential differences in ThT fluorescence and secondary structures were observed among the SAA1 isoforms. However, the fibril morphologies appeared to differ; SAA1.1 formed long and curly fibrils, whereas SAA1.3 formed thin and straight fibrils. The peptides corresponding to the central regions of the SAA1 isoforms containing amino acid variations showed distinct amyloidogenicities, reflecting their direct effects on amyloid fibril formation. These findings may provide novel insights into the influence of amino acid variations in human SAA on the pathogenesis of AA amyloidosis.     

Cytotoxicity of albebetin oligomers depends on cross-beta-sheet formation

Prefibrillar cytotoxicity was suggested as a common amyloid characteristic. We showed two types of albebetin prefibrillar oligomers are formed during incubation at pH 7.3. Initial round-shaped oligomers consist of 10-15 molecules determined by atomic force microscopy, do not bind thioflavin-T and do not affect viability of granular neurons and SH-SY5Y cells. They are converted into ca. 30-40-mers possessing cross-beta-sheet and reducing viability of neuronal cells. Neither monomers nor fibrils possess cytotoxicity. We suggest that oligomeric size is important for stabilising cross-beta-sheet core critical for cytotoxicity. As albebetin was used as a carrier-protein for drug delivery, examination of amyloidogenicity is required prior polypeptide biomedical applications.     

Vortex-Induced Formation of Insulin Amyloid Superstructures Probed by Time-Lapse Atomic Force Microscopy and Circular Dichroism Spectroscopy

Misfolding and aggregation of proteins are multipathway processes that result in polymorphism of amyloid fibrils. While agitation is one of the most common means of inducing structural variants of fibrils (the so-called ‘amyloid strains’), there is as yet no mechanistic explanation for this effect. In this study, time-lapse atomic force microscopy has been employed to probe insulin fibrillation upon intensive agitation. At 60 °C, the initial stages of aggregation in agitated samples are similar to those in quiescent solutions; however, in vortexed samples, an abrupt and highly cooperative collapse of early filaments occurs, yielding twisted and laterally aligned aggregates with defined chiroptical properties. In the absence of any detectable birefringence and linear dichroism, the observed strong Cotton effect is attributed to twisted chiral amyloid superstructures. Early fibrils formed in agitated samples, but transferred to quiescent conditions before the collapse event, do not form the superstructures. On the other hand, mature insulin fibrils grown in quiescent samples and later subjected to rapid vortexing transform into clumps of finely broken fibers lacking the superstructural chirality and chiroptical properties of the continuously agitated samples. A generalized mechanism of inducing structural variants of amyloid fibrils by hydrodynamic forces favoring secondary nucleation events over elongation of fibrils is put forward. We propose that competition between low-aspect-ratio and high-aspect-ratio amyloidogenic pathways driven by fluid dynamics may play an important role in promoting distinct amyloid strains.     

TiO2 nanoparticles promote beta-amyloid fibrillation in vitro

Alzheimer’s disease (AD) is the most common neurodegenerative disease in the world. The pathogenesis of AD is associated with β-amyloid (Aβ) fibrillation. Nanoparticles have large surface area and can access the brain. But no investigation has been made to study the relationship between nanoparticles and AD. In our study, we observed Aβ fibril formation in the presence of six kinds of nanoparticles and found that TiO₂ nanoparticles can promote Aβ fibrillation by shortening nucleation process, which is the key rate-determining step of fibrillation. Hereby the interaction between Aβ and nanoparticles may contribute to AD etiology.     

Elongation of amyloid fibrils through lateral binding of monomers revealed by total internal reflection fluorescence microscopy

Amyloid fibrils are fibrillar aggregates of denatured proteins associated with a large number of amyloidoses. The formation of amyloid fibrils has been considered to occur by nucleation and elongation. Real-time imaging of the elongation as well as linear morphology of amyloid fibrils suggests that all elongation events occur at the growing ends of fibrils. On the other hand, we suggested that monomers also bind to the lateral sides of preformed fibrils during the seed-dependent elongation, diffuse to the growing ends, and finally make further conformation changes to the mature amyloid fibrils. To examine lateral binding during the elongation of fibrils, we used islet amyloid polypeptide (IAPP), which has been associated with type II diabetes, and prepared IAPP modified with the fluorescence dye, Alexa532. By monitoring the elongation process with amyloid specific thioflavin T and Alexa532 fluorescence, we obtained overlapping images of the two fluorescence probes, which indicated lateral binding. These results are similar to the surface diffusion-dependent growth of crystals, further supporting the similarities between amyloid fibrillation and the crystallization of substances.     

Seeding-dependent propagation and maturation of amyloid fibril conformation

Recent studies of amyloid fibrils have focused on the presence of multiple amyloid forms even with one protein and their propagation by seeding, leading to conformational memory. To establish the structural basis of these critical features of amyloid fibrils, we used the amyloidogenic fragment Ser20-Lys41 (K3) of beta2-microglobulin, a protein responsible for dialysis-related amyloidosis. In 20% (v/v) 2,2,2-trifluoroethanol and 10 mM HCl (pH approximately 2), K3 peptide formed two types of amyloid-like fibrils, f218 and f210, differing in the amount of beta-sheet as measured by circular dichroism spectroscopy and Fourier transform infrared spectroscopy. Atomic force microscopy showed that the fibril with a larger amount of beta-sheet (f210) is thinner and longer. Both fibrils were reproduced by seeding, showing the template-dependent propagation of a fibril's conformation. However, upon repeated self-seeding, f218 fibrils were gradually transformed into f210 fibrils, revealing the conformational maturation. The observed maturation can be explained fully by a competitive propagation of two fibrils. The maturation of amyloid fibrils might play a role during the development of amyloidosis.     

Aβ40(L17A/F19A) mutant diminishes the aggregation and neurotoxicity of Aβ40

Aggregated β-amyloid peptides (Aβ) are neurotoxic and responsible for neuronal death both in vitro and in vivo. From the structural point of view, Aβ self-aggregation involves a conformational change in the peptide. Here, we investigated the relationship between conformational changes and amino acid residues of Aβ₄₀. Urea unfolding in combination with NMR spectroscopy was applied to probe the stabilization of Aβ₄₀ conformation. L17 and F19 residues were found more sensitive to environmental changes than the other residues. Replacement of these two residues with alanine could stabilize the conformation of Aβ₄₀. Further analysis indicated that the Aβ₄₀(L17A/F19A) mutant could diminish the aggregation and reduce the neurotoxicity. These results suggest that L17 and F19 are the critical residues responsible for conformational changes which may trigger neurotoxic cascade of Aβ₄₀.     

Anti-oligomeric Abeta single-chain variable domain antibody blocks Abeta-induced toxicity against human neuroblastoma cells

The Amyloid-β (Aβ) peptide is a major component of the amyloid plaques associated with Alzheimer's disease (AD). Recent studies suggest that the most toxic forms of Aβ are small, soluble oligomeric aggregates. Here, we report the isolation and characterization of a single-chain variable domain (scFv) antibody isolated against oligomeric Aβ using a protocol developed in our laboratory that combines phage display technology and atomic force microscopy (AFM). Starting with a randomized, single framework phage display library, after three rounds of selection against oligomeric Aβ, we identified an scFv that bound oligomeric Aβ specifically, but not monomeric or fibrillar forms. The anti-oligomeric scFv inhibits Aβ aggregation and toxicity, and reduces the toxicity of preformed oligomeric Aβ towards human neuroblastoma cells. When used to probe samples of human brain tissue, the scFv reacted with AD tissue but not a healthy control or Parkinson's disease brain samples. The anti-oligomeric Aβ scFv therefore has potential therapeutic and diagnostic applications in specifically targeting or identifying the toxic morphologies of Aβ in AD brains.     

Methods for structural characterization of prefibrillar intermediates and amyloid fibrils

Protein fibrillation is first and foremost a structural phenomenon. Adequate structural investigation of the central conformational individuals of the fibrillation process is however exceedingly difficult. This is due to the nature of the process, which may be described as a dynamically evolving equilibrium between a large number of structural species. These are furthermore of highly diverging sizes and present in very uneven amounts and timeframes. Different structural methods have different strengths and limitations. These, and in particular recent advances within solution analysis of the undisturbed equilibrium using small angle X-ray scattering, are reviewed here.     

Small organic probes as amyloid specific ligands - Past and recent molecular scaffolds

Molecular probes for selective staining and imaging of protein aggregates, such as amyloid, are important to advance our understanding of the molecular mechanisms underlying protein misfolding diseases and also for obtaining an early and accurate clinical diagnosis of these disorders. Since normal immunohistochemical reagents, such as antibodies have shown limitation for identifying protein aggregates both in vitro and in vivo, small organic probes have been utilized as amyloid specific markers. In this review, past and recent molecular scaffolds that have been utilized for the development of small organic amyloid imaging agents are discussed.     

Characterisation of amyloid fibril formation by small heat-shock chaperone proteins human alphaA-, alphaB- and R120G alphaB-crystallins

AlphaB-Crystallin is a ubiquitous small heat-shock protein (sHsp) renowned for its chaperone ability to prevent target protein aggregation. It is stress-inducible and its up-regulation is associated with a number of disorders, including those linked to the deposition of misfolded proteins, such as Alzheimer's and Parkinson's diseases. We have characterised the formation of amyloid fibrils by human alphaB-crystallin in detail, and also that of alphaA-crystallin and the disease-related mutant R120G alphaB-crystallin. We find that the last 12 amino acid residues of the C-terminal region of alphaB-crystallin are predicted from their physico-chemical properties to have a very low propensity to aggregate. (1)H NMR spectroscopy reveals that this hydrophilic C-terminal region is flexible both in its solution state and in amyloid fibrils, where it protrudes from the fibrillar core. We demonstrate, in addition, that the equilibrium between different protofilament assemblies can be manipulated and controlled in vitro to select for particular alphaB-crystallin amyloid morphologies. Overall, this study suggests that there could be a fine balance in vivo between the native functional sHsp state and the formation of amyloid fibrils.     

Design, synthesis, and biophysical properties of a helical Abeta1-42 analog: Inhibition of fibrillogenesis and cytotoxicity

The aggregation of amyloid β-peptide (Aβ) into β-sheet-rich aggregates is a crucial step in the etiology of Alzheimer’s disease. Helical forms of Aβ have been suggested to be intermediates in the aggregation process of the peptide in aqueous phase, micelles and membranes. A stable helical Aβ analog would be useful to investigate the role of helical intermediates in fibrillization by Aβ. Here we designed a helical analog by simply cross-linking the Cys residues of A30C, G37C-Aβ1-42 with 1,6-bismaleimidohexane. The analog assumed a weak α-helical conformation in model membranes mimicking lipid raft microdomains of neuronal membranes under conditions in which the wild-type Aβ1-42 formed a β-sheet, indicating the cross-linking locally induced a helical conformation. Furthermore, addition of equimolar helical Aβ analog significantly reduced the amyloid formation and cytotoxicity by Aβ1-42. Thus, our helical Aβ1-42 is not only a model peptide to investigate the role of helical intermediates in fibrillization by Aβ, but also an inhibitor of Aβ-induced cytotoxicity.     

Identification and characterization of a novel fibril forming peptide in fungal starch binding domain

Scanty information is available regarding the chemical basis for structural alterations of the carbohydrate-binding modules (CBMs). The N-terminal starch binding domain (SBD) of Rhizopus oryzae glucoamylase (GA) forms fibrils under thermal stress, presenting an unusual conformational change from immunoglobulin-like to β-sheet-rich structure. Site-directed mutagenesis revealed that the C-terminal Lys of SBD played a crucial role in the fibril formation. The synthetic peptide (DNNNSANYQVSTSK) representing the C-terminal 14 amino acid residues of SBD was further demonstrated to act as a fibril-forming segment, in which terminal charges and an internal NNNxxNYQ motif were key fibril-forming determinants. The formation of fibril structure in a fungal SBD, caused by its chemical and biophysical requirements, was demonstrated for the first time.     

Interaction of dipeptydil peptidase IV with amyloid peptides

The aggregates of amyloid beta peptides (Aβs) are regarded as one of the main pathological hallmarks of Alzheimer’s disease (AD). An imbalance between the rates of synthesis and clearance of Aβs is considered to be a possible cause for the onset of AD. Dipeptidyl peptidases II and IV (DPPII and DPPIV) are serine proteases removing N-terminal dipeptides from polypeptides and proteins with proline or alanine on the penultimate position. Alanine is an N-terminal penultimate residue in Аβs, and we presumed that DPPII and DPPIV could cleave them. The results of present in vitro research demonstrate for the first time the ability of DPPIV to truncate the commercial Aβ40 and Aβ42 peptides, to hinder the fibril formation by them and to participate in the disaggregation of preformed fibrils of these peptides. The increase of absorbance at 334 nm due to complex formation between primary amines with o-phtalaldehyde was used to show cleaving of Aβ40 and Aβ42. The time-dependent increase of the quantity of primary amines during incubation of peptides in the presence of DPPIV suggested their truncation by DPPIV, but not by DPPII. The parameters of the enzymatic breakdown by DPPIV were determined for Aβ40 (K_m = 37.5 μM, k_cat/K_m = 1.7 × 10³ M⁻¹sec⁻¹) and Aβ42 (K_m = 138.4 μM, k_cat/K_m = 1.90 × 10² M⁻¹sec⁻¹). The aggregation-disaggregation of peptides was controlled by visualization on transmission electron microscope and by Thioflavin-T fluorescence on spectrofluorimeter and fluorescent microscope. DPPIV hindered the peptide aggregation/fibrillation during 3-4 days incubation in 20 mM phosphate buffer, pH 7.4, 37 °C by 50–80%. Ovalbumin, BSA and DPPII did not show this effect. In the presence of DPPIV, the preformed fibrils were disaggregated by 30–40%. Conclusion: for the first time it was shown that the Aβ40 and Aβ42 are substrates of DPPIV. DPPIV prohibits the fibrillation of peptides and promotes disaggregation of their preformed aggregates.     

MEK inhibitors suppress β-amyloid production by altering the level of a β-C-terminal fragment of amyloid precursor protein in neuronal cells

β-Amyloid peptide (Aβ) is generated via sequential proteolysis of amyloid precursor protein (APP) by β- and γ-secretases. Cell-based screening experiments disclosed that the MEK (MAP kinase kinase) inhibitors, U0126 and PD184352, suppress Aβ secretion from human neuronal SH-SY5Y cells expressing Swedish mutant APP. These inhibitors did not affect the cellular levels of APP but significantly reduced those of the APP β-C-terminal fragment (β-CTF). Additionally, β-CTF levels were markedly reduced by these inhibitors in cells expressing the fragment in a γ-secretase-independent and proteasome-dependent manner. Our results suggest that MEK inhibitors reduce Aβ generation via secretase-independent alteration of β-CTF levels.     

Amyloid β (1-42) Folding Multiplicity and Single-Molecule Binding Behavior Studied with STM

The fine folding and assembling characteristics of amyloid β (Aβ) peptides are important to pharmaceutical studies of drug molecules and to the pathological analysis of neurodegenerative disorders such as Alzheimer's disease at the molecular level. Here we present observations of the multiple folding characteristics of amyloid peptide Aβ₄₂ lamellae using scanning tunneling microscopy. Molecularly resolved core regions of Aβ₄₂ hairpins and unfolded peptide assembly structures are identified. The parallel assembling characteristics of Aβ₄₂ hairpins can be confirmed in the study. In addition, single-molecule binding characteristics of Congo red and thioflavin T have been shown to bind at the groove regions of peptide assemblies. This study demonstrates a complementary venue for studying molecular heterogeneity of peptide assemblies, as well as the binding characteristics of molecular modulators.     

Cholesterol Regulates Assembly of Human Islet Amyloid Polypeptide on Model Membranes

Amylin, a 37-aa pancreatic hormone, is the major constituent of islet amyloid, a hallmark of type II diabetes mellitus. Recent studies have revealed a pivotal role of anionic phospholipids in membrane-catalyzed amylin fibrillogenesis and aggregation. However, cholesterol, an integral component of eukaryotic cell membranes, also could have a role. In this study, we have examined the effect of cholesterol on amylin polymerization both on planar membranes and in solution. Using time-lapse atomic force microscopy, we have studied the dynamics and macromolecular organization of amylin on anionic and neutral planar membranes that lack or include cholesterol. On cholesterol-depleted planar membranes, amylin formed highly symmetrical tetrameric and pentameric pore-like supramolecular structures composed of 25- to 35-nm intermediate-sized globular structures or oligomers. Conversely, on membranes incorporating cholesterol, amylin formed highly compact ∼ 200- to 500-nm protein clusters that constituted seeds or nuclei for continuing amylin binding and aggregation. However, cholesterol inhibited amylin nucleation with a 7-fold decrease in the number of amylin particles. Consequently, cholesterol-containing membranes accumulated significantly less amyloid with some membrane areas completely free of amyloid particles. The inhibitory effect of cholesterol on amylin aggregation in solution was also demonstrated as a 16-fold decrease in the aggregation rate. Consistent with this, circular dichroism spectroscopy revealed a stable, soluble random-coil conformation for amylin in the presence of cholesterol that could explain the inhibitory effect of cholesterol on amylin polymerization in solution and on membranes. The modulatory effect of cholesterol was largely independent of membrane charge or phospholipids, suggesting a novel cholesterol-regulated amylin polymerization process.