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Alzheimer's disease (AD) is one of the most common of the multifactorial diseases and is characterized by a range of abnormal molecular processes, such as the accumulation of extracellular plaques containing the amyloid-β (Aβ) peptides and dyshomeostasis of copper in the brain. In this study, we have investigated the effect of CuII on the aggregation of Aβ1-40 and Aβ4-40 , representing the two most prevalent families of Aβ peptides, that is, the full length and N-truncated peptides. Both families are similarly abundant in healthy and AD brains. For either of the studied peptides, substoichiometric CuII concentrations accelerated aggregation, whereas superstoichiometric CuII inhibited fibril formation, likely by stabilizing the oligomers. The addition of either Aβ4-40 or substoichiometric CuII affected the aggregation profile of Aβ1-40 , by yielding shorter and thicker fibrils; amorphous aggregates were formed in the presence of a molar excess of CuII . The similarity of these two effects can be attributed to the increase in the positive charge on the Aβ N terminus, caused both by CuII complexation and N truncation at position 4. Our findings provide a better understanding of the biological Aβ aggregation process as these two Aβ species and CuII coexist and interact under physiological conditions.

Serum amyloid A (SAA) is an acute-phase protein (APP) to which multiple immunological functions have been attributed. Regardless, the true biological role of SAA remains poorly understood. SAA is remarkably conserved in mammalian evolution, thereby suggesting an important biological function. Since its discovery in the 1970s, the majority of researchers have investigated SAA using recombinant forms made available through bacterial expression. Nevertheless, recent studies indicate that these recombinant forms of SAA are unreliable. Indeed, commercial SAA variants have been shown to be contaminated with bacterial products including lipopolysaccharides and lipoproteins. As such, biological activities and receptor usage (TLR2, TLR4) revealed through the use of commercial SAA variants may not reflect the inherent nature of this APP. Within this review, we discuss the biological effects of SAA that have been demonstrated through more solid experimental approaches. SAA takes part in the innate immune response via the recruitment of leucocytes and executes, through pathogen recognition, antimicrobial activity. Knockout animal models implicate SAA in a range of functions, such as regulation of T-cell-mediated responses and monopoiesis. Moreover, through its structural motifs, not only does SAA function as an extracellular matrix protein, but it also binds extracellular matrix proteins. Finally, we here also provide an overview of definite SAA receptor-mediated functions and highlight those that are yet to be validated. The role of FPR2 in SAA-mediated leucocyte recruitment has been confirmed; nevertheless, SAA has been linked to a range of other receptors including CD36, SR-BI/II, RAGE and P2RX7. ispartof: IMMUNOLOGY vol:163 issue:2 pages:115-127 ispartof: location:England status: published

The difficulty of synthesizing and purifying the amyloid β (Aβ) peptide, combined with its high aggregation propensity and low solubility under physiological conditions, leads to a wide variety of experimental results from kinetic assays to biological activity. Thus, it becomes challenging to reproduce outcomes, and this limits our ability to rely on reported results as the foundation for new research. This article examines variability of the Aβ peptide from different sources, comparing purity, and oligomer and fibril formation propensity side by side. The results highlight the importance of performing rigorous controls so that meaningful biophysical, biochemical, and neurobiological results can be obtained to improve our understanding on Aβ.

Elucidating local dynamics of protein aggregation is crucial for understanding the mechanistic details of protein amyloidogenesis. Herein, we studied the residue-specific dynamics and local environmental changes of Aβ40 along the course of aggregation by using para-cyanophenylalanine (PheCN ) as a fluorescent and vibrational probe. Our results show that the PheCN residues introduced at various positions all exhibited an immediate decay of fluorescence intensity, indicating a relatively synergistic process in early oligomer formation. The fast decreases in the fluorescence intensities of residues 19 and 20 in the central hydrophobic core region and residue 10 in the N-terminal region suggest that they play crucial roles in the formation of the oligomeric core. The PheCN 4 residue exhibits a remarkably slower decrease in fluorescence intensity, implicating its dynamic conformational characteristics in oligomer and fibril formation. Our results also suggest that the N-terminal residues in fibrils are surrounded by a relatively hydrophobic local environment, as opposed to being solvated.

Racemates often have lower solubility than enantiopure compounds, and mixing of enantiomers can enhance aggregation propensity of peptides. Amyloid β (Aβ) 42 is an aggregation-prone peptide, believed to play a key role in Alzheimer's Disease. Soluble Aβ42 aggregation intermediates (oligomers) have emerged as particularly neurotoxic. We hypothesized that addition of mirror image (D-) Aβ42 should reduce the concentration of toxic oligomers formed by natural (L-) Aβ42. We synthesized L- and D-Aβ42 and found their equimolar mixing to lead to accelerated fibril formation. Confocal microscopy with fluorescently labeled analogs of the enantiomers showed their co-localization in racemic fibrils. Reflecting enhanced fibril formation propensity, racemic Aβ42 was less prone to form soluble oligomers. This resulted in protection of cells from toxicity of L-Aβ42 at concentrations ranging up to 50 μM. In summary, mixing of Aβ42 enantiomers induces accelerated formation of non-toxic fibrils.

Glycosaminoglycans are known to be associated with extracellular amyloid deposits of various amyloidogenic proteins. In this study, we found that the glycosaminoglycan heparin greatly accelerates the elongation step in fibril formation by the N-terminal 1-83 fragment of human apolipoprotein A-I (apoA-I), especially in the amyloidogenic W50R variant. Using fragment peptides, we demonstrate that heparin significantly promotes β-transition and fibril formation of the highly amyloidogenic region spanning residues 44-65 and co-localizes with fibrils formed by the W50R variant. These results suggest the possible role of glycosaminoglycans in fibril formation by amyloidogenic apoA-I variants. This article is protected by copyright. All rights reserved.

Multidrug resistance of bacteria is a major challenge due to the wide-spread use of antibiotics. While a range of strategies have been developed in recent years, suppression of bacterial activity and virulence via their network of extracellular amyloid has rarely been explored, especially with nanomaterials. Here, silver nanoparticles and nanoclusters (AgNPs and AgNCs) capped with cationic branched polyethylenimine polymer are synthesized, and their antimicrobial potentials are determined at concentrations safe to mammalian cells. Compared with the ultrasmall AgNCs, AgNPs entail stronger binding to suppress the fibrillization of FapC, a major protein constituent of the extracellular amyloid matrix of Pseudomonas aeruginosa. Both types of nanoparticles exhibit concentration-dependent antibiofilm and antimicrobial properties against P. aeruginosa. At concentrations of 1 × 10-6 m or below, both the bactericidal activity of AgNCs and the antibiofilm capacity of AgNPs are associated with their structure-mediated bio-nano interactions but not ion release. For AgNPs, specifically, their antibiofilm potency correlates with their capacity of FapC fibrillization inhibition, but not with their bactericidal activity. This study demonstrates the antimicrobial potential of safe nanotechnology through the novel route of amyloidosis inhibition.

Amyloid β (Aβ), a 39-42 amino acid peptide derived from amyloid precursor protein, is deposited as fibrils in Alzheimer's disease brains, and is considered to play a major role in the pathogenesis of the disease. We have investigated the effects of a water-soluble Zn-phthalocyanine, ZnPc(COONa)_8, a macrocyclic compound with near-infrared optical properties, on Aβ fibril formation in vitro. A thioflavin T fluorescence assay showed that ZnPc(COONa)_8 significantly inhibited Aβ fibril formation, increasing the lag time and dose-dependently decreasing the plateau level of fibril formation. Moreover, it destabilized pre-formed Aβ fibrils, resulting in an increase in low-molecular-weight species. After fibril formation in the presence of ZnPc(COONa)_8, immunoprecipitation of Aβ_ using Aβ-specific antibody followed by near-infrared scanning demonstrated binding of ZnPc(COONa)_8 to Aβ_. A study using the hydrophobic fluorescent probe 8-anilino-1-naphthalenesulfonic acid showed that ZnPc(COONa)_8 decreased the hydrophobicity during Aβ_ fibril formation. CD spectroscopy showed an increase in the α helix structure and a decrease in the β sheet structure of Aβ_ in fibril-forming buffer containing ZnPc(COONa)_8. SDS/PAGE and a dot-blot immunoassay showed that ZnPc(COONa)_8 delayed the disappearance of low-molecular-weight species and the appearance of higher-molecular-weight oligomeric species of Aβ_. A cell viability assay showed that ZnPc(COONa)_8 was not toxic to a neuronal cell line (A1), but instead protected A1 cells against Aβ_-induced toxicity. Overall, our results indicate that ZnPc(COONa)_8 binds to Aβ and decreases the hydrophobicity, and this change is unfavorable for Aβ oligomerization and fibril formation.

We have synthesized a 17-mer peptide (ER17p) that is issued from the hinge region of the estrogen receptor and which activates the proliferation of breast carcinoma cells in steroid-deprived conditions. In the present paper, we show that at a concentration of similar to 50M, it rapidly forms amyloid-like fibrils with the assistance of electrostatic interactions and that at higher concentrations, it spontaneously forms a hydrogel. By using biophysical, spectral and rheological techniques, we have explored the structural, biophysical and mechanical characteristics of ER17p with respect to fibril formation and gelation.

Pretangles are cytoplasmic tau immunoreactivity in neurons without apparent formation of fibrillary structures. In Alzheimer disease, such tau deposition is considered to represent a premature state prior to fibril formation (AD-pretangles), later to form neurofibrillary tangles and finally ghost tangles. This morphological evolution from pretangles to ghost tangles is in parallel with their profile shift from four repeat (4R) tau-positive pretangles to three repeat (3R) tau-positive ghost tangles with both positive neurofibrillary tangles in between. This complementary shift of tau profile from 4R to 3R suggests that these tau epitopes are represented interchangeably along tangle evolution. Similar tau immunoreactivity without fibril formation is also observed in corticobasal degeneration (CBD-pretangles). CBD-pretangles and AD-pretangles share: (i) selective 4R tau immunoreactivity without involvement of 3R tau; and (ii) argyrophilia with Gallyas silver impregnation. However, CBD-pretangles neither evolve into ghost tangles nor exhibit 3R tau immunoreactivity even at the advanced stage. Because electron microscopic studies on these pretangles are quite limited, it remains to be clarified whether such differences in later evolution are related to their primary ultrastructures, potentially distinct between AD and CBD. As double staining for 3R and 4R tau clarified complementary shift from 4R to 3R tau along evolution from pretangles to ghost tangles, double immunoelectron microscopy, if possible, may clarify similar profile shifts in relation to each tau fibril at the ultrastructural dimension. This will provide a unique viewpoint on how molecular (epitope) representations are related to pathogenesis of fibrillary components.

BACKGROUND Viticultural residues from commercial viticultural activities represent a potentially important source of bioactive stilbenes such as resveratrol. The main aim of the present study was therefore to isolate, identify and perform biological assays against amyloid-β peptide aggregation of original stilbenes from Vitis vinifera shoots. RESULTS A new resveratrol oligomer, (Z)-cis-miyabenol C (3), was isolated from Vitis vinifera grapevine shoots together with two newly reported oligostilbenes from Vitis vinifera shoots, vitisinol C (1) and (E)-cis-miyabenol C (2), and six known compounds: piceatannol, resveratrol, (E)-e-viniferin (trans-e-viniferin), ω-viniferin, vitisinol C and (E)-miyabenol C. The structures of these resveratrol derivatives were established on the basis of detailed spectroscopic analysis including nuclear magnetic resonance experiments. All the newly reported compounds were tested for their anti-aggregative activity against amyloid-β fibril formation. Vitisinol C was found to exert a significant activity against amyloid-β aggregation. CONCLUSION Vitis vinifera grapevine shoots are potentially interesting as a source of new bioactive stilbenes, such as vitisinol C. © 2013 Society of Chemical Industry

Recent mutagenesis studies using the hydrophobic segment of Aβ suggest that aromatic π-stacking interactions may not be critical for fibril formation. We have tested this conjecture by probing the effect of Leu, Ile, and Ala mutation of the aromatic Phe residues at positions 19 and 20, on the double-layer hexametric chains of Aβ fragment Aβ16–22 using explicit solvent all-atom molecular dynamics. As these simulations rely on the accuracy of the utilized force fields, we first evaluated the dynamic and stability dependence on various force fields of small amyloid aggregates. These initial investigations led us to choose AMBER99SB-ILDN as force field in multiple long molecular dynamics simulations of 100 ns that probe the stability of the wild-type and mutants oligomers. Single-point and double-point mutants confirm that size and hydrophobicity are key for the aggregation and stability of the hydrophobic core region (Aβ16–22). This suggests as a venue for designing Aβ aggregation inhibitors the substitution of residues (especially, Phe 19 and 20) in the hydrophobic region (Aβ16–22) with natural and non-natural amino acids of similar size and hydrophobicity.

Several natural polyphenols with potent inhibitory effects on amyloid fibril formation have been reported. Herein, we studied modulation of transthyretin (TTR) fibrillogenesis by selected polyphenols. We demonstrate that both curcumin and nordihydroguaiaretic acid (NDGA) bind to TTR and stabilize the TTR tetramer. However, while NDGA slightly reduced TTR aggregation, curcumin strongly suppressed TTR amyloid fibril formation by generating small “off-pathway” oligomers and EGCG maintained most of the protein in a non-aggregated soluble form. This indicates alternative mechanisms of action supported by the occurrence of different non-toxic intermediates. Moreover, EGCG and curcumin efficiently disaggregated pre-formed TTR amyloid fibrils. Our studies, together with the safe toxicological profile of these phytochemicals may guide a novel pharmacotherapy for TTR-related amyloidosis targeting different steps in fibrillogenesis.Structured summary of protein interactionsTTR binds to TTR by comigration in gel electrophoresis (View interaction)TTR binds to TTR by dynamic light scattering (View interaction)TTR binds to TTR by electron microscopy (View interaction)

The difficulty in identifying the toxic agents in all amyloid-related diseases is likely due to the complicated kinetics and thermodynamics of the nucleation process and subsequent fibril formation. The slow progression of these diseases suggests that the formation, incorporation, and/or action of toxic agents are possibly rate limiting. Candidate toxic agents include precursors (some at very low concentrations), also called oligomers and protofibrils, and the fibrils. Here, we investigate the kinetic and thermodynamic behavior of human insulin oligomers (imaged by cryo-EM) under fibril-forming conditions (pH 1.6 and 65 degrees C) by probing the reaction pathway to insulin fibril formation using two different types of experiments-cooling and seeding-and confirm the validity of the nucleation model and its effect on fibril growth. The results from both the cooling and seeding studies confirm the existence of a time-changing oligomer reaction process prior to fibril formation that likely involves a rate-limiting nucleation process followed by structural rearrangements of intermediates (into beta-sheet rich entities) to form oligomers that then form fibrils. The latter structural rearrangement step occurs even in the absence of nuclei (i.e., with added heterologous seeds). Nuclei are formed at the fibrillation conditions (pH 1.6 and 65 degrees C) but are also continuously formed during cooling at pH 1.6 and 25 degrees C. Within the time-scale of the experiments, only after increasing the temperature to 65 degrees C are the trapped insulin nuclei and resultant structures able to induce the structural rearrangement step and overcome the energy barrier to form fibrils. This delay in fibrillation and accumulation of nuclei at low temperature (25 degrees C) result in a decrease in the mean length of the fibers when placed at 65 degrees C. Fits of an empirical model to the data provide quantitative measures of the delay in the lag-time during the nucleation process and subsequent reduction in fibril growth rate resulting from the cooling. Also, the seeding experiments, within the time-scale of the measurements, demonstrate that fibers can initiate fast fibrillation with dissolved insulin (fresh or taken during the lag-period) but not with other fibers. Qualitatively this is explained with a conjectual free-energy space plot.

Only a minute fraction of all possible protein sequences can exist in the genomes of all life forms. To explore whether physicochemical constraints or a lack of need causes the paucity of different protein folds, we set out to construct protein libraries without any restriction of topology. We generated different libraries (all alpha-helix, all beta-strand, and alpha-helix plus beta-strand) with an average length of 100 amino acid residues, composed of designed secondary structure modules (alpha-helix, beta-strand, and beta-turn) in various proportions, based primarily on the patterning of polar and nonpolar residues. We wished to explore that part of sequence space that is rich in secondary structure. The analysis of randomly chosen clones from each of the libraries showed that, despite the low sequence homology to known protein sequences, a substantial proportion of the library members containing alpha-helix modules were indeed helical, possess a defined oligomerization state, and showed cooperative chemical unfolding behavior. On the other hand, proteins composed of mainly beta-strand modules tended to form amyloid-like fibrils and were among the least soluble proteins ever reported. We found that a large fraction of members in non-beta-strand-containing protein libraries that are distant from natural proteins in sequence space possess unexpectedly favorable properties. These results reinforce the efficacy of applying binary patterning to design proteins with native-like properties despite lack of restriction in topology. Because of the intrinsic tendency of beta-strand modules to aggregate, their presence requires precise topologic arrangement to prevent fibril formation.

Human transthyretin (hTTR) is one of several proteins known to cause amyloid disease. Conformational changes in its native structure result in aggregation of the protein, leading to insoluble amylo ...

This mini-review focuses on the processes and consequences of protein folding and misfolding. The latter process often leads to protein aggregation and precipitation with the aggregates adopting either highly ordered (amyloid fibril) or disordered (amorphous) forms. In particular, the amyloid fibril is discussed because this form has gained considerable notoriety due to its close links to a variety of debilitating diseases including Alzheimer's, Parkinson's, Huntington's, and Creutzfeldt-Jakob diseases, and type-II diabetes. In each of these diseases a different protein forms fibrils, yet the fibrils formed have a very similar structure. The mechanism by which fibrils form, fibril structure, and the cytotoxicity associated with fibril formation are discussed. The generic nature of amyloid fibril structure suggests that a common target may be accessible to treat amyloid fibril-associated diseases. As such, the ability of some molecules, for example, the small heat-shock family of molecular chaperone proteins, to inhibit fibril formation is of interest due to their therapeutic potential.

Intranuclear fibrils due to poly-alanine expansions in the N-terminal domain of the poly(A) binding protein PABPN1 correlate with the disease oculopharyngeal muscular dystrophy (OPMD). For monitoring fibril formation by fluorescence and real-time NMR spectroscopy, tryptophans were introduced either into the middle or C-terminal of the poly-alanine segment. The kinetics of fibril formation which were monitored by fluorescence spectroscopy were matched by real-time NMR kinetics. Our results show that fibril formation is concomitant with the burial of the tryptophans in the fibrillar core. Since no soluble pre-fibrillar intermediate(s) was detected, fibril formation of this domain may be regarded as a two state conversion from an unfolded soluble into folded insoluble species.

Amyloid deposition is a common feature of Alzheimer's disease and type 2 diabetes related to beta-amyloid peptides (betaA) and human amylin (hA), respectively. Both betaA and hA form aggregates and fibrils and kill cultured cells. To investigate whether betaA and hA display peptide-specific toxicity on cultured islet beta-cells, we examined the effects of (1-40)betaA and (25-35)betaA peptides on hA-mediated cell death and [(125)I-Tyr(37)]hA precipitation. Synthetic hA aggregated in solution and evoked both conformation- and sequence-dependent cell death. While neither (1-40)betaA nor (25-35)betaA was toxic to islet beta-cells, they suppressed hA-evoked cell death in a concentration-dependent and saturable manner. Only (1-40)betaA, but not (25-35)betaA, showed trophic effects on cultured islet beta-cells and inhibited the precipitation of [(125)I]hA caused by hA. These results suggest that (25-35)betaA does not interfere with hA-mediated fibril formation. Suppression of hA-evoked death of cultured pancreatic islet beta-cells by the betaA peptides is likely to occur through a competing interaction at these cells.

The molecular chaperone, alpha-crystallin, has the ability to prevent the fibrillar aggregation of proteins implicated in human diseases, for example, amyloid beta peptide and alpha-synuclein. In this study, we examine, in detail, two aspects of alpha-crystallin's fibril-suppressing ability: (a) its temperature dependence, and (b) the nature of the aggregating species with which it interacts. First, the efficiency of alpha-crystallin to suppress fibril formation in kappa-casein and alpha-synuclein increases with temperature, despite their rate of fibrillation also increasing in the absence of alpha-crystallin. This is consistent with an increased chaperone ability of alpha-crystallin at higher temperatures to protect target proteins from amorphous aggregation [GB Reddy, KP Das, JM PetrashWK Surewicz (2000) J Biol Chem275, 4565-4570]. Second, dual polarization interferometry was used to monitor real-time alpha-synuclein aggregation in the presence and absence of alphaB-crystallin. In contrast to more common methods for monitoring the time-dependent formation of amyloid fibrils (e.g. the binding of dyes like thioflavin T), dual polarization interferometry data did not reveal any initial lag phase, generally attributed to the formation of prefibrillar aggregates. It was shown that alphaB-crystallin interrupted alpha-synuclein aggregation at its earliest stages, most likely by binding to partially folded monomers and thereby preventing their aggregation into fibrillar structures.

alpha-Synuclein (alpha-Syn) has been identified as a component of intracellular fibrillar deposits in Parkinson's disease. Though the real pathogenesis is still unknown, many investigations have revealed that conformational alteration and fibril formation of alpha-Syn protein have an important role in causing the disease. In this work, we introduced the g-factor spectra of solid-state circular dichroism to estimate the secondary structure contents of alpha-Syn fragments in amyloids. Fourier-transform infrared (FTIR) was also applied to confirm the structural formation. The results suggest that the central hydrophobic region is critical for beta-sheet formation and the conformational alteration is the foundation of protein abnormal aggregation. The research provides a practical approach to estimate the secondary structure contents of protein amyloids and further insight into the relevance of structural transformation and amyloidogenesis. (c) 2006 Wiley Periodicals, Inc.

The identification of the driving factor for fibril formation is paramount to understand the molecular basis of amyloidogenic disease. Recently, an atomic-detail structure of a fibrillogenic aggregate was reported and revealed a tight packing of beta-sheets. However, there is not a single pair-wise interaction of significance between the beta-sheets, no hydrogen bond and no hydrophobic interaction. Instead, there is extensive burial of polar groups at the interface. These observations lead to the question: What factor drives the association of beta-sheets? This issue is addressed by combining all-atom molecular dynamics with an implicit-solvent analysis. The driving force for the association arises from the mechanical equivalent of the dehydration propensity of pre-formed intra-sheet hydrogen bonds and dipole-dipole interactions.

β-Amyloid peptide (Aβ) is the primary protein component of senile plaques in Alzheimer's disease and is believed to be responsible for the neurodegeneration associated with the disease. Aβ is toxic only when aggregated, however, the size and structure of the aggregated species associated with toxicity is unknown. In the present study, we developed a diffusion-based method to simultaneously separate and detect the biological activity of toxic Aβ oligomers and used the method to examine the relationship between size of aggregated protein and toxicity to SH-SY5Y cells. From these measurements, the effective diffusivity and hydrodynamic radius of the toxic oligomeric species of Aβ could be determined. A sensitivity analysis was performed to examine the effects of model assumptions used in data analysis on the effective diffusivity calculated. The method provides a new estimate of the size of small toxic Aβ species associated with fibril formation. This work contributes to our understanding of the relationship between Aβ structure and toxicity and with further refinements may aid in our ability to design agents which alter the Aβ aggregation/dissociation processes associated with neurotoxicity. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 50–59, 2002.

In spiders soluble proteins are converted to form insoluble silk fibres, stronger than steel. The final fibre product has long been the subject of study; however, little is known about the conversion process in the silk-producing gland of the spider. Here we describe a study of the conversion of the soluble form of the major spider-silk protein, spidroin, directly extracted from the silk gland, to a β-sheet enriched state using circular dichroism (CD) spectroscopy. Combined with electron microscopy (EM) data showing fibril formation in the β-sheet rich region of the gland and amino-acid sequence analyses linking spidroin and amyloids, these results lead us to suggest that the refolding conversion is amyloid like. We also propose that spider silk could be a valuable model system for testing hypotheses concerning β-sheet formation in other fibrilogenic systems, including amyloids.

Many neurodegenerative diseases, like Parkinson's, Alzheimer's, or Huntington's disease, occur as a result of amyloid protein fibril formation and cell death induced by this process. Cyclic peptides (CPs) and their derivatives form a new class of powerful inhibitors that prevent amyloid fibrillation and decrease the cytotoxicity of aggregates. The strategies for designing CPs are described, with respect to their amino acid sequence and/or conformational similarity to amyloid fibrils. The implications of CPs for the study and possible treatment of amyloid-related diseases are discussed.

Amyloid proteins and peptides comprise a diverse group of molecules that vary both in size and amino-acid sequence, yet assemble into amyloid fibrils that have a common core structure. Kinetic studies of amyloid fibrillogenesis have revealed that certain amyloid proteins form oligomeric intermediates prior to fibril formation. We have investigated fibril formation with a peptide corresponding to residues 195–213 of the human prion protein. Through a combination of kinetic and equilibrium studies, we have found that the fibrillogenesis of this peptide proceeds as an all-or-none reaction where oligomeric intermediates are not stably populated. This variation in whether oligomeric intermediates are stably populated during fibril formation indicates that amyloid proteins assemble into a common fibrillar structure; however, they do so through different pathways.

We have proposed that amyloid fibrils contain subunits (protofibrils) that are formed from beta-strands wound into continuous 2-3 nm-diameter beta-helices. Subsequent lateral aggregation of the beta-helices to form the widely observed 5-12 nm-diameter fibrils could be promoted by hydrophobic residues on the exterior of the postulated beta-helix. A number of short peptide fragments of the amyloid-beta (A beta) proteins, such as A beta34-42 [LMVGGVVIA], the nine-residue, carboxyl-terminal portion of A beta1-42, can also form amyloid fibrils. In the present study, it was found that a beta-helix formed from A beta34-42 accounts for features suggested by published rotational resonance solid-state NMR data, including an anomalous conformation about the Gly-37-Gly-38 region and exaggerated pleating. An analogue of A beta34-42 was synthesized in which the hydrophobic groups on the exterior of the postulated beta-helix were replaced with glutamates, giving LEVGGVEIE. The analogue was completely soluble at pH 7, but at pH 2.5 it produced 2-2.5 nm-diameter fibrils which did not associate into larger-diameter bundles. The results of this study support the proposal that amyloid fibrils are formed from beta-helical subunits.

The crystal structure of despentapeptide insulin, a monomeric insulin, has been refined at 1.3 A spacing and subsequently used to predict and model the organization in the insulin fibril. The model makes use of the contacts in the densely packed despentapeptide insulin crystal, and takes into account other experimental evidence, including binding studies with Congo red. The dimensions of this model fibril correspond well with those measured experimentally, and the monomer–monomer contacts within the fibril are in accordance with the known physical chemistry of insulin fibrils. Using this model, it may be possible to predict mutations in insulin that might alleviate problems associated with fibril formation during insulin therapy. © 1997 Wiley-Liss Inc.

4H-1,4-oxazines were designed as transthyretin (TTR) amyloid fibril inhibitors based on an analysis of the interactions between known small molecule inhibitors and TTR by molecular docking. A series of 2,4,6-triaryl-4H-1,4-oxazines was synthesized by the cyclization of N,N-bis(phenacyl)anilines with POCl3 in pyridine. Inhibition of TTR amyloid fibril was evaluated by a fibril formation assay. The results indicate that 4H-1,4-oxazines significantly inhibit TTR amyloid fibril at a concentration of 7.2 μM.

Put your coat on: It is well recognized that the surfaces of nanomaterials in biological media are covered by various biomolecules (e.g., proteins). A) The protein corona creates a shell over different nanomaterials, regardless of their physicochemical properties (e.g., composition and shape), resulting in reduced levels of amyloid beta fibril formation. B) Pristine nanomaterials might have acceleratory effects on the fibrillation of amyloid beta.

Alzheimer's disease (AD) is the most common chronic, progressive neurodegenerative disease, which affects an increasing number of elderly patients. So far, there is no cure for the disease. Early diagnosis to predict dementia outcome is of great importance. Biomarkers, as physiological indicators of AD, hold great promise for clinical diagnosis and drug discovery. Sensitive and selective methods that are amenable to the detection of biomarkers are much preferred. Surface plasmon resonance (SPR) is a sensitive optical technique capable of measuring analyte concentrations as well as kinetics of biomolecular interactions. It exhibits a variety of fascinating properties, such as high sensitivity, label-free and real-time analysis, and low sample consumption. In this review, we address the recent development on sensitive detection of a series of biomarkers for AD using SPR. Furthermore, amyloid-β (Aβ) fibril formation and interaction monitored by SPR have been documented. The challenges and perspectives inherent in the detection of the biomarkers are also highlighted.

X-PLOR modelling of collagen dimers containing Gly-Glu-Arg in each chain has been carried out. The interaction between molecules when two Gly-Glu-Arg are present on each chain is found to be substantially less than two times that obtained with one per chain, implying that relative tilting of two collagen molecules does not offset the disadvantages of misalignment of the interacting moieties. This implies that if multiple (Glu(-)-Arg+)3 interactions are important in fibril formation, their lateral separations must be large enough to insure that they act independently.

Unraveling the factors that control the conformation of molecular chains is of great interest both for understanding the shape of biological molecular strands and for designing artificial ones that adopt desired forms. Thus, a variety of artificial folding codons have been identified that enforce the formation, among others, of helices, strands, and loops, the major emphasis being on the shape of the foldamer. We report herein the synthesis and study of a family of foldamers and macrocycles based on the 1,8-naphthyridine and pyrimidine units, whose internal cavity is large enough to accommodate ionic substrates, and focus on the impact of guest binding within a cylindrical environment. Interestingly, the binding event within these large oligomers is translated to the outside of the receptors and affects the interaction of the overall complexes with the outside world. For instance, alkali cations bind to the one-turn helices and macrocycles to promote fibril formation and aggregation. Also, polyammonium su...

Phenylazo benzenesulfonamides were designed and synthesized as beta-amyloid (Abeta40) fibril assembly inhibitors, and evaluated for inhibition of Abeta40 aggregation and neurotoxicity using rat cortical neurons. Compound 2 (LB-152) was the most potent compound in this study, and the para-NMe(2) group on the end of the phenylazo moiety may play an important role in preventing Abeta40 fibril formation. LB-152 provides a new lead for further development of potential beta-amyloid aggregation inhibitors to treat AD.

Proteins adopt an amazing array of sequence-dependent structures that enable them to perform the many chemical functions critical to life. Over the past decade, however, it has become clear that many different protein sequences can also form misfolded, insoluble aggregates known as amyloid fibrils, with common structural elements. Amyloid fibrils appear to be involved in a number of diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, prion diseases, and type II diabetes (1). Even ordinary globular proteins (e.g., myoglobin) can form amyloid fibrils under certain conditions (2), suggesting that fibril formation is a previously unappreciated general property of many proteins. Thus, it is of fundamental interest to understand how so many different protein sequences can adopt this alternative structure and it is of medical interest to understand and control disease-related amyloid formation. Knowledge of the three-dimensional structure of amyloid fibrils is critical for understanding the mechanism of fibrillogenesis and for design of possible inhibitors. Unfortunately, amyloid fibrils are noncrystalline and insoluble, and therefore are not amenable to x-ray crystallography and solution NMR, the classic tools of structural biology. In a recent issue of PNAS, Petkova et al. (3) report a structural model for Alzheimer's β-amyloid fibrils deduced primarily from solid-state NMR experiments. This work provides a significant step forward in understanding β-amyloid formation and showcases the power of solid-state NMR for obtaining structural information on important but challenging biomolecules.