Subsequent Fibril Formation Research Articles

Bifunctional Inhibitor Lentinan Inhibits Fibrillogenesis of Amyloid-β Protein and α-Synuclein and Alleviates Their Cytotoxicity: In Vitro and In Vivo Studies.

Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most common neurodegenerative diseases in the world. Misfolding of β-amyloid (Aβ) and α-synuclein (α-syn) and subsequent fibril formation are closely associated with the pathogenesis of AD and PD, respectively. Lentinan is a natural product commonly used in medicine and dietary supplements. It has potential antitumor, anti-inflammatory, and antiviral effects, but the underlying mechanism of its action on AD and PD remains unclear. In this study, lentinan inhibited the formation of Aβ and α-syn fibers in a dose-dependent manner and disrupted their mature fibers. Lentinan inhibited the conversion of Aβ and α-syn conformations to β-sheet-rich conformations. Additionally, lentinan protected Caenorhabditis elegans against damage caused by the accumulation of Aβ and α-syn aggregation and prolonged their lifespan. Notably, the beneficial effects of lentinan in AD and PD mice were also demonstrated, including ameliorating the cognitive and memory impairments in AD mice and behavioral deficits in PD mice. Finally, molecular interactions between lentinan and Aβ/α-syn pentamers were also explored using molecular docking.

Cross-Linked Amyloid β-Protein Oligomers: A Missing Link in Alzheimer’s Disease Pathology?

Amyloid β-protein (Aβ) oligomers are broadly viewed as the proximate mediators of toxicity in Alzheimer's disease (AD). Recent studies, however, provide substantial evidence that Aβ is involved in protection and repair of the central nervous system whereby Aβ oligomer and subsequent fibril formation are integral to its normal antimicrobial and antiviral function. These developments raise a question of what exactly makes Aβ oligomers toxic in the context of AD. This Perspective describes a paradigm shift in the search for toxic Aβ oligomer species that involves oxidative-stress-induced stabilization of Aβ oligomers via cross-linking and reviews most recent research elucidating structural aspects of cross-linked Aβ oligomers and potential inhibition of their toxicity.

FT-IR versus EC-QCL spectroscopy for biopharmaceutical quality assessment with focus on insulin\u2014total protein assay and secondary structure analysis using attenuated total reflection

For the quality control of biopharmaceutical products, which contain proteins as the most important active ingredients, shelf life may be limited due to inappropriate storage conditions or mechanical stress. For insulins as representatives of life-saving pharmaceuticals, analytical methods are needed, which are providing additional information than obtained by assays for total protein quantification. Despite sophisticated formulations, the chemical stability may be challenged by temperatures deviating from recommended conditions or shear rate exposure under storage, leading to misfolding, nucleation, and subsequent fibril formation, accompanied by a decrease in bioactivity. A reliable method for insulin quantification and determination of secondary structure changes has been developed by attenuated total reflection (ATR) Fourier-transform infrared spectroscopy of insulin formulations by a silver halide fiber-coupled diamond probe with subsequent dry-film preparation. A special emphasis has been placed on the protein amide I band evaluation, for which spectral band analysis provides unique information on secondary structure fractions for intact and misfolded insulins. Quantitative measurements are possible down to concentrations of less than 0.5 mg/ml, whereas the dry-film preparation delivers high signal-to-noise ratios due to the prior water evaporation, thus allowing a reliable determination of secondary structure information.Graphical abstract

Molecular Details of a Salt Bridge and Its Role in Insulin Fibrillation by NMR and Raman Spectroscopic Analysis.

Insulin, a simple polypeptide hormone with huge biological importance, has long been known to self-assemble in vitro and form amyloid-like fibrillar aggregates. Utilizing high-resolution NMR, Raman spectroscopy, and computational analysis, we demonstrate that the fluctuation of the carboxyl terminal (C-ter) residues of the insulin B-chain plays a key role in the growth phase of insulin aggregation. By comparing the insulin sourced from bovine, human, and the modified glargine (GI), we observed reduced aggregation propensity in the GI variant, resulting from two additional Arg residues at its C-ter. NMR analysis showed atomic contacts and residue-specific interactions, particularly the salt bridge and H-bond formed among the C-ter residues Arg31B, Lys29B, and Glu4A. These inter-residue interactions were reflected in strong nuclear Overhauser effects among Arg31BδH-Glu4AδH and Lys29BδHs-Glu4AδH in GI, as well as the associated downfield chemical shift of several A-chain amino terminal (N-ter) residues. The two additional Arg residues of GI, Arg31B and Arg32B, enhanced the stability of the GI native structure by strengthening the Arg31B, Lys29B, and Glu4A salt bridge, thus reducing extensive thermal distortion and fluctuation of the terminal residues. The high stability of the salt bridge retards tertiary collapse, a crucial biochemical event for oligomerization and subsequent fibril formation. Circular dichroism and Raman spectroscopic measurement also suggest slow structural distortion in the early phase of the aggregation of GI because of the restricted mobility of the C-ter residues as explained by NMR. In addition, the structural and dynamic parameters derived from molecular dynamics simulations of insulin variants highlight the role of residue-specific contacts in aggregation and amyloid-like fibril formation.

Probing Medin Monomer Structure and its Amyloid Nucleation Using 13C-Direct Detection NMR in Combination with Structural Bioinformatics

Aortic medial amyloid is the most prevalent amyloid found to date, but remarkably little is known about it. It is characterised by aberrant deposition of a 5.4 kDa protein called medin within the medial layer of large arteries. Here we employ a combined approach of ab initio protein modelling and 13C-direct detection NMR to generate a model for soluble monomeric medin comprising a stable core of three β-strands and shorter more labile strands at the termini. Molecular dynamics simulations suggested that detachment of the short, C-terminal β-strand from the soluble fold exposes key amyloidogenic regions as a potential site of nucleation enabling dimerisation and subsequent fibril formation. This mechanism resembles models proposed for several other amyloidogenic proteins suggesting that despite variations in sequence and protomer structure these proteins may share a common pathway for amyloid nucleation and subsequent protofibril and fibril formation.

Fibronectin unfolded by adherent but not suspended platelets: An in vitro explanation for its dual role in haemostasis

Fibronectin (FN), a dimeric adhesive glycoprotein, which is present both in plasma and the extracellular matrix can interact with platelets and thus contribute to platelet adhesion and aggregation. It has been shown that FN can decrease platelet aggregation but enhance platelet adhesion, suggesting a dual role of FN in haemostasis. The prevalent function(s) of FN may be determined by its fibril form. To explore the suggested dual role of this adhesive protein for haemostasis in further detail, we now tested for any differences of adherent and suspended platelets with regard to their effect to unfold and assemble FN upon interaction. Platelet aggregation and adhesion assays were performed using washed platelets in the presence of exogenous FN. Addition of plasma FN reduced platelet aggregation in response to collagen or PMA by 50% or 25% but enhanced platelet adhesion onto immobilized collagen, as compared to control experiments. Analyses by fluorescence resonance energy transfer (FRET) demonstrated that adherent platelets but not suspended platelets were capable of unfolding FN during 3h incubation. Fluorescence microscopy and deoxycholate (DOC) solubility assays demonstrated that FN fibrils formed only on the surfaces of adherent platelets. In addition, platelets adherent onto FN revealed a significantly higher activity of specific Src phosphorylation (pY418) than platelets in suspension. These data suggest (1) that the function of FN in haemostasis is prevalent to its assembly, unfolding and subsequent fibril formation on the surface of adherent platelets and (2) that outside-in signaling contributes to the interaction of platelets and FN.

Evaluation of polyphenols as possible therapeutics for amyloidoses: Comparative analysis of Kaempferol and Catechin

Several mammalian proteins fold abnormally under non physiological conditions, to form pathological deposits that are associated with many degenerative diseases. In vitro variation of solvent conditions and pH can lead to partial unfolding and subsequent fibril formation. In the present study, we examined the effects of low pH on goat brain cystatin (GBC) with a focus on amyloid fibril formation. The results demonstrate that GBC can form amyloid like fibrils at pH 3.0. Moreover this study is aimed at exploring the inhibitory activity of polyphenols, Kaempferol (KM) and Catechin (CA) against the fibrillation of GBC. Using fluorescence spectroscopic analysis with Thioflavin T, CD and electron microscopic studies, anti-fibrillation effects of polyphenols, KM and CA were analyzed. The study also revealed that KM and CA produced a concentration dependent anti-fibrillogenic effects with KM producing more pronounced effect compared to CA. The study proposed a mechanistic approach assuming structural constraints and specific aromatic interactions of polyphenols with β sheets of GBC fibrils.

Initiation of assembly of tau(273-284) and its ΔK280 mutant: an experimental and computational study

The microtubule associated protein tau is essential for the development and maintenance of the nervous system. Tau dysfunction is associated with a class of diseases called tauopathies, in which tau is found in an aggregated form. This paper focuses on a small aggregating fragment of tau, (273)GKVQIINKKLDL(284), encompassing the (PHF6*) region that plays a central role in tau aggregation. Using a combination of simulations and experiments, we probe the self-assembly of this peptide, with an emphasis on characterizing the early steps of aggregation. Ion-mobility mass spectrometry experiments provide a size distribution of early oligomers, TEM studies provide a time course of aggregation, and enhanced sampling molecular dynamics simulations provide atomistically detailed structural information about this intrinsically disordered peptide. Our studies indicate that a point mutation, as well the addition of heparin, lead to a shift in the conformations populated by the earliest oligomers, affecting the kinetics of subsequent fibril formation as well as the morphology of the resulting aggregates. In particular, a mutant associated with a K280 deletion (a mutation that causes a heritable form of neurodegeneration/dementia in the context of full length tau) is seen to aggregate more readily than its wild-type counterpart. Simulations and experiment reveal that the ΔK280 mutant peptide adopts extended conformations to a greater extent than the wild-type peptide, facilitating aggregation through the pre-structuring of the peptide into a fibril-competent structure.

Self‐assembly of amelogenin proteins at the water–oil interface

Self-assembly of amelogenin plays a key role in controlling enamel biomineralization. Recently, we generated self-aligning nanoribbons of amelogenin in water-in-oil emulsions stabilized by the full-length protein (rH174). Here, we tested the hypothesis that the hydrophilic C-terminus is critical for self-assembly of amelogenin into nanoribbons. The self-assembled structures of two amelogenin cleavage products, rH163 and rH146, were compared with structures of rH174 at different pH values and degrees of saturation using atomic force microscopy, electron microscopy, and dynamic light scattering. We observed that the number density of rH174 nanoribbons increased significantly when the initial pH was raised from 4.5 to 5.6. Nanoribbons, as well as unique helical nanostructures, were also readily observed when amelogenin rH146 was used, but showed little tendency for parallel alignment and did not bundle into fibrils like rH174. In contrast, rH163 rarely formed nanoribbons but predominantly assembled into nanospheres under the same conditions. We conclude that the presence of a hydrophilic C-terminus may not be a prerequisite for nanoribbon formation but may be critical for ribbon alignment and subsequent fibril formation. These results highlight the contribution of the hydrophobic domain in the self-assembly of elongated structures of amelogenins. Molecular mechanisms governing these processes based on the formation of reverse micelles are discussed.

Actinidain-hydrolyzed Type I Collagen Reveals a Crucial Amino Acid Sequence in Fibril Formation

We investigated the ability of type I collagen telopeptides to bind neighboring collagen molecules, which is thought to be the initial event in fibrillogenesis. Limited hydrolysis by actinidain protease produced monomeric collagen, which consisted almost entirely of alpha1 and alpha2 chains. As seen with ultrahigh resolution scanning electron microscopy, actinidain-hydrolyzed collagen exhibited unique self-assembly, as if at an intermediate stage, and formed a novel suprastructure characterized by poor fibrillogenesis. Then, the N- and C-terminal sequences of chicken type I collagen hydrolyzed by actinidain or pepsin were determined by Edman degradation and de novo sequence analysis with matrix-assisted laser desorption ionization-tandem time-of-flight mass spectrometry, respectively. In the C-telopeptide region of the alpha1 chain, pepsin cleaved between Asp(1035) and Phe(1036), and actinidain between Gly(1032) and Gly(1033). Thus, the actinidain-hydrolyzed alpha1 chain is shorter at the C terminus by three residues, Gly(1033), Phe(1034), and Asp(1035). In the alpha2 chain, both proteases cleaved between Glu(1030) and Val(1031). We demonstrated that a synthetic nonapeptide mimicking the alpha1 C-terminal sequence including GFD weakly inhibited the self-assembly of pepsin-hydrolyzed collagen, whereas it remarkably accelerated that of actinidain-hydrolyzed collagen. We conclude that the specific GFD sequence of the C-telopeptide of the alpha1 chain plays a crucial role in stipulating collagen suprastructure and in subsequent fibril formation.

3, 4-Dihydroxyphenylacetic Acid (DOPAC) Impairs α-Synuclein Interaction with Lipids

α-Synuclein (α-Syn) is a small intrinsically disordered presynaptic protein known to form insoluble filamen- tous inclusions in Parkinson's disease (PD) and other neurodegenerative disorders. Various catecholamines can inhibit the α-Syn fibrillation in vitro. Recently, non-covalent binding of DOPAC (3,4-dihydroxyphenylacetic acid), a normal product of the dopamine metabolism, was shown to inhibit the fibrillation of α-Syn due to the DOPAC-induced stabilization of the normally transient oligomers thus preventing them from subsequent fibril formation (Zhou, et al. J. Mol. Biol. 2009, 388 (3), 597-610). We are showing here that the interaction of DOPAC with α-Syn decreases the binding affinity of α- Syn to lipids, suggesting that DOPAC might lead to the gain-of-toxicity of α-Syn aggregates and loss-of-function of α- Syn, both of which could be related to progression of PD.

Disulfide bond as a structural determinant of prion protein membrane insertion.

Conversion of the normal soluble form of prion protein, PrP (PrP(C)), to proteinase K-resistant form (PrP(Sc)) is a common molecular etiology of prion diseases. Proteinase K-resistance is attributed to a drastic conformational change from alpha-helix to beta-sheet and subsequent fibril formation. Compelling evidence suggests that membranes play a role in the conformational conversion of PrP. However, biophysical mechanisms underlying the conformational changes of PrP and membrane binding are still elusive. Recently, we demonstrated that the putative transmembrane domain (TMD; residues 111-135) of Syrian hamster PrP penetrates into the membrane upon the reduction of the conserved disulfide bond of PrP. To understand the mechanism underlying the membrane insertion of the TMD, here we explored changes in conformation and membrane binding abilities of PrP using wild type and cysteine-free mutant. We show that the reduction of the disulfide bond of PrP removes motional restriction of the TMD, which might, in turn, expose the TMD into solvent. The released TMD then penetrates into the membrane. We suggest that the disulfide bond regulates the membrane binding mode of PrP by controlling the motional freedom of the TMD.

Time‐dependent insulin oligomer reaction pathway prior to fibril formation: Cooling and seeding

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.

At Low Concentrations, 3,4-Dihydroxyphenylacetic Acid (DOPAC) Binds Non-Covalently to α-Synuclein and Prevents Its Fibrillation

Several studies have shown that catecholamines can inhibit the fibrillation of α-synuclein (α-Syn), a small presynaptic protein whose aggregation is believed to be a critical step in the etiology of Parkinson's disease and several other neurodegenerative disorders. However, the mechanism of this inhibition is uncertain. We show here that substoichiometric concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC), a normal product of the metabolism of dopamine, can inhibit the fibrillation of α-Syn, due to non-covalent binding of DOPAC to α-Syn monomer. Intriguingly, the presence of α-Syn accelerates the spontaneous oxidation of DOPAC, and the oxidized form of DOPAC (the quinone) is responsible for the fibrillation inhibition. In addition, the presence of DOPAC leads to the oxidation of the methionine residues of α-Syn, probably due to the H 2O 2 production as a by-product of DOPAC oxidation. The lack of fibrillation results from the formation of stable oligomers, which are very similar to those observed transiently at early stages of the α-Syn fibrillation. A possible explanation for this phenomenon is that DOPAC stabilizes the normally transient oligomers and prevents them from subsequent fibril formation. The analysis of the α-Syn Y39W variant suggests that DOPAC binds non-covalently to the same N-terminal region of α-Syn as lipid vesicles, probably in the vicinity of residue 39. In contrast to the compounds with 1,2-dihydroxyphenyl groups (DOPAC and catechol), their 1,4-dihydroxyphenyl isomers (hydroquinone and homogentisic acid) are able to modify α-Syn covalently, probably due to the less steric hindrance in the Michael addition.

Modulation of Self-Association and Subsequent Fibril Formation in an Alanine-Rich Helical Polypeptide

Thermal unfolding, reversible self-association, and irreversible aggregation were investigated for an alanine-rich helical polypeptide, 17-H-6, with sequence [AAAQEAAAAQAAAQAEAAQAAQ] 6. Dynamic light scattering, transmission electron microscopy, and thermal unfolding measurements indicate that 17-H-6 spontaneously and reversibly self-associates at acidic pH and low temperature. The resulting multimers have a compact, globular morphology with an average hydrodynamic radius approximately 10-20 nm and reversibly dissociate to monomers upon an increase to pH 7.4. Both free monomer and 17-H-6 chains within the multimers are alpha-helical and folded at low temperature. Reversible unfolding of the monomer occurs upon heating of solutions at pH 7.4. At pH 2.3, heating first causes incomplete dissociation and unfolding of the constituent chains. Further incubation at elevated temperature induces additional structural and morphological changes and results in fibrils with a beta-sheet 2 degrees structure and a characteristic diameter of 5-10 nm (7 nm mean). The ability to modulate association and aggregation suggests opportunities for this class of polypeptides in nanotechnology and biomedical applications.

Protein crosslinkages: I. Effect of iodination on the time-dependent solubility changes of reconstituted collagen fibrils

It has been shown that the iodination of tropocollagen leads to a more rapid formation of intermoleoular crosslinks of the reconstituted fibrils as judged by the inability to dissolve the fibrils in urea-HOAc solution. Iodination of the tropocollagen without subsequent fibril formation results in the formation of some intramolecular crosslinks as determined by ultracentrifugation at 40° and acrylamide gel electrophoresis and by the melting point and formation of segmented-long-spaced structures of the cooled gelatins derived therefrom. Control experiments and aminoacid analyses have shown that possible oxidation reactions cannot account for the changes. It is postulated that the iodination of histidine and/or tyrosine residues increases the rate of a normally occurring crosslinking reaction.