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Effectively presenting epitopes on immunogens, in order to raise conformationally selective antibodies through active immunization, is a central problem in treating protein misfolding diseases, particularly neurodegenerative diseases such as Alzheimer’s disease or Parkinson’s disease. We seek to selectively target conformations enriched in toxic, oligomeric propagating species while sparing the healthy forms of the protein that are often more abundant. To this end, we computationally modelled scaffolded epitopes in cyclic peptides by inserting/deleting a variable number of flanking glycines (“glycindels”), to best mimic a misfolding-specific conformation of an epitope of α-synuclein enriched in the oligomer ensemble, as characterized by a region most readily disordered and solvent-exposed in a stressed, partially denatured protofibril. We screen and rank the cyclic peptide scaffolds of α-synuclein in silico based on their ensemble overlap properties with the fibril, oligomer-model, and isolated monomer ensembles. We present experimental data of seeded aggregation that supports nucleation rates consistent with computationally predicted cyclic peptide conformational similarity. We also introduce a method for screening against structured off-pathway targets in the human proteome, by selecting scaffolds with minimal conformational similarity between their epitope and the same solvent-exposed primary sequence in structured human proteins. Different cyclic peptide scaffolds with variable numbers of glycines are predicted computationally to have markedly different conformational ensembles. Ensemble comparison and overlap was quantified by the Jensen-Shannon Divergence, and a new measure introduced here—the embedding depth, which determines the extent to which a given ensemble is subsumed by another ensemble, and which may be a more useful measure in developing immunogens that confer conformational-selectivity to an antibody.Graphical TOC Entry

Summary Substantial research efforts have gone into elucidating the role of protein misfolding and self-assembly in the onset and progression of Alzheimer’s disease (AD). Aggregation of the Amyloid-β (Aβ) peptide into insoluble fibrils is closely associated with AD. Here, we use biophysical techniques to study a peptide-based approach to target Aβ amyloid aggregation. A peptide construct, NCAM-PrP, consists of a largely hydrophobic signal sequence linked to a positively charged hexapeptide. The NCAM-PrP peptide inhibits Aβ amyloid formation by forming aggregates which are unavailable for further amyloid aggregation. In a membrane-mimetic environment, Aβ and NCAM-PrP form specific heterooligomeric complexes, which are of lower aggregation states compared to Aβ homooligomers. The Aβ:NCAM-PrP interaction appears to take place on different aggregation states depending on the absence or presence of a membrane-mimicking environment. These insights can be useful for the development of potential future therapeutic strategies targeting Aβ at several aggregation states., Graphical abstract, Highlights • A signal peptide construct, NCAM-PrP, inhibits Aβ peptide amyloid aggregation • Aβ and NCAM-PrP form co-aggregates which are not compatible with amyloid formation • The Aβ and NCAM-PrP interaction occurs both in aqueous solution and in membranes • Co-aggregates formed in solution and in the membrane have different properties, Molecular neuroscience; Structural biology; Biophysics

Graphical abstract, Highlights • Fluorescent tags modify the surface and interactome of amyloid fibrils. • Thermal fluctuations of GFP tags create a size-dependent sieve around a fibril. • High molecular weight proteins are preferentially excluded from Htt-GFP inclusions. • Tags may modify biophysical properties of fibrils and inclusion formation in cells. • Results of studies using tagged proteins should be validated using untagged proteins., The pathological growth of amyloid fibrils in neurons underlies the progression of neurodegenerative diseases including Alzheimer’s and Parkinson’s disease. Fibrils form when soluble monomers oligomerise in the cytoplasm. Their subsequent growth occurs via nucleated polymerization mechanisms involving the free ends of the fibrils augmented by secondary nucleation of new oligomers at their surface. Amyloid fibrils possess a complex interactome with diffusing cytoplasmic proteins that regulates many aspects of their growth, seeding capacity, biochemical activity and transition to pathological inclusions in diseased brains. Changes to their surface are also expected to modify their interactome, pathogenicity and spreading in the brain. Many assays visualise fibril formation, growth and inclusion formation by decorating monomeric proteins with fluorescent tags such as GFP. Recent studies from our group suggest that tags with sizes comparable to the fibril radius may modify the fibril surface accessibility and thus their PTM pattern, interactome and ability to form inclusions. Using coarse-grained molecular simulations of a single alpha synuclein fibril tagged with GFP we find that thermal fluctuations of the tags create a non-monotonic, size-dependent sieve around the fibril that perturbs its interactome with diffusing species. Our results indicate that experiments using tagged and untagged monomers to study the growth and interactome of fibrils should be compared with caution, and the confounding effects of the tags are more complex than a reduction in surface accessibility. The prevalence of fluorescent tags in amyloid fibril growth experiments suggests this has implications beyond the specific alpha synuclein fibrils we model here.

Parkinson’s disease, the second most prevalent neurodegenerative disorder worldwide, is characterized by a progressive loss of dopaminergic neurons in substantia nigra pars compacta, causing motor symptoms. This disorder’s main hallmark is the formation of intraneuronal protein inclusions, named Lewy bodies and neurites. The major component of these arrangements is α-synuclein, an intrinsically disordered and soluble protein that, in pathological conditions, can form toxic and cell-to-cell transmissible amyloid structures. Preventing α-synuclein aggregation has attracted significant effort in the search for a disease-modifying therapy for Parkinson’s disease. Small molecules like SynuClean-D, epigallocatechin gallate, trodusquemine, or anle138b exemplify this therapeutic potential. Here, we describe a subset of compounds containing a single aromatic ring, like dopamine, ZPDm, gallic acid, or entacapone, which act as molecular chaperones against α-synuclein aggregation. The simplicity of their structures contrasts with the complexity of the aggregation process, yet the block efficiently α-synuclein assembly into amyloid fibrils, in many cases, redirecting the reaction towards the formation of non-toxic off-pathway oligomers. Moreover, some of these compounds can disentangle mature α-synuclein amyloid fibrils. Their simple structures allow structure-activity relationship analysis to elucidate the role of different functional groups in the inhibition of α-synuclein aggregation and fibril dismantling, making them informative lead scaffolds for the rational development of efficient drugs.

Amyloid cross-seeding and amyloid inhibition are two different research subjects being studied separately for different pathological purposes, in which amyloid cross-seeding targets to study the co-aggregation of different amyloid proteins and potential molecular links between different neurodegenerative diseases, while amyloid inhibition aims to design different molecules for preventing amyloid aggregation. While both amyloid cross-seeding and amyloid inhibition are critical for better understanding the pathological causes of different neurodegenerative diseases including Parkinson disease (PD) and Type 2 diabetes (T2D), less efforts have been made to reconcile the two phenomena. Herein, we proposed a new preventive strategy to demonstrate (i) the cross-seeding of octapeptide TKEQVTNV from α-synuclein (associated with PD) with hIAPP (associated with T2D) and (ii) the cross-seeding-promoted hIAPP fibrillization and cross-seeding-reduced hIAPP toxicity. Collective results confirmed that TKEQVTNV can indeed cross-seed with hIAPP monomers and oligomers, not protofibrils, to form β-structure-rich fibrils and to accelerate hIAPP fibrillization. Moreover, such cross-seeding-induced promotion effect by TKEQVTNV also rescued the pancreatic cells from hIAPP-induced cytotoxicity by increasing cell viability and reducing cell apoptosis simultaneously. This work provides a new angle to discover amyloid fragments and use them as amyloid modulators (inhibitors or promotors) to interfere with amyloid aggregation of other amyloid proteins, as well as sequence/structure basis to explore the amyloid cross-seeding between different amyloid proteins that may help explain a potential molecular talk between different neurodegenerative diseases. This article is protected by copyright. All rights reserved.

Amyloid fibrils are structurally heterogeneous protein aggregates that are implicated in a wide range of neurodegenerative and other proteopathic diseases. These fibrils exist in a variety of different tertiary and higher-level structures, and this exhibited polymorphism greatly complicates any structural study of amyloid fibrils. In this work, we demonstrate a method of using polarization-resolved microscopy to directly observe the structural heterogeneity of individual amyloid fibrils using amyloid-bound fluorophores. We formulate a mathematical quantity, helical anisotropy, which utilizes the polarized emission of amyloid-bound fluorophores to report on the local structure of individual fibrils. Using this method, we show how model amyloid fibrils generated from short peptides exhibit diverse structural properties both between different fibrils and within a single fibril, in a manner that is replicated for fibrils assembled from longer proteins. Our method represents an accessible and easily adaptable technique by which polymorphism in the structure of amyloid fibrils can be probed. Additionally, the methodology we describe here can be easily extended to the study of other fibrillar and otherwise ordered supramolecular structures.

Controlling oriented crystallization is key to producing bonelike composite materials with a well-organized structure. However, producing this type of composite material using synthetic biopolymers as scaffolds is challenging. Inspired by the molecular structure of collagen-I, a collagenlike peptide─(Pro-Hyp-Gly)10 (POG10)─was designed to produce self-assembled fibrils that resemble the structure of collagen-I fibrils. In addition, the oriented mineralization of HAP crystals is formed in the fibrils that reproduces a bonelike material similar to collagen-I fibril mineralization. Unlike collagen-I fibrils, POG10 fibrils do not contain gap spaces. The molecular simulation results indicate that in addition to space confinement, the molecular field generated by POG10 can also confine the orientation of HAP, enriching our understanding of physical confinement and shedding light on the design of synthetic biopolymer scaffolds for bonelike material fabrication.

Anti-Aβ therapy has dominated clinical trials for the prevention and treatment of Alzheimer's disease (AD). However, suppressing Aβ aggregation and disintegrating mature fibrils simultaneously remains a great challenge. In this work, we developed a new strategy using a charged tubular supramolecule (CTS) with pillar[5]arene as the backbone and modifying amino and carboxyl groups at the tubular terminals (noted as CTS-A, CTS-A/C, and CTS-C, respectively) to suppress Aβ fibrillation for the first time. According to the spectroscopic and microscopic characterizations, Aβ40 fibrillation can be efficiently suppressed by CTS-A in a very low inhibitor:peptide (I:P) molar ratio (1:10). A greatly alleviated cytotoxic effect of Aβ peptides after the inhibition or disaggregation process is further disclosed. The well-organized supramolecular structure drives multivalent interaction and gains enhanced efficiency on amyloid fibrillar modulation. These results open a new path for the design of supramolecules in the application of AD treatment.

Pathological aggregation of amyloid polypeptides is associated with numerous degenerative diseases. Preventing aggregation and clearing amyloid deposits are considered as promising strategies against amyloidosis. With the capacity of crossing the blood-brain barrier and good biocompatibility, the hydroxylated single-walled carbon nanotube (SWCNT-OH) has been shown with excellent anti-amyloid properties. Here, we systematically studied the SWCNT-OH effects on the fibrillization of the β2m21–31 peptides utilizing all-atom discrete molecular dynamics (DMD) simulation. Our results demonstrated the isolated β2m21–31 peptides first nucleated into unstructured oligomers followed by coil-to-sheet conformational conversions in oligomers with at least six peptides. The elongation and lateral surfaces of the preformed β-sheet could catalyze the other unstructured monomers and small oligomers converted into β-sheet formations via dock-lock fibril growth and secondary nucleation processes. Eventually, the β2m21–31 peptides would self-assemble into well-ordered cross-β structures. Regardless of isolated monomers or well-defined cross-β assemblies, the β2m21–31 would attach on the surfaces of SWCNT-OH adopting unstructured formations indicating the SWCNT-OH not only inhibited the fibrillization of β2m21–31 but also destroyed pre-formed proto-fibrils. Overall, our study displays a complete picture of the fibrillization mechanism of β2m21–31 and the amyloid inhibitory mechanism of SWCNT-OH, offering new insight into the de-novo design of anti-amyloid inhibitors.

Collagen XIV is poorly characterized in the body, and the current knowledge of its function in the cornea is limited. The aim of the current study was to elucidate the role(s) of collagen XIV in regulating corneal stromal structure and function. Analysis of collagen XIV expression, temporal and spatial, was performed at different postnatal days (Ps) in wild-type C57BL/6 mouse corneal stromas and after injury. Conventional collagen XIV null mice were used to inquire the roles that collagen XIV plays in fibrillogenesis, fibril packing, and tissue mechanics. Fibril assembly and packing as well as stromal organization were evaluated using transmission electron microscopy and second harmonic generation microscopy. Atomic force microscopy was used to assess stromal stiffness. Col14a1 mRNA expression was present at P4 to P10 and decreased at P30. No immunoreactivity was noted at P150. Abnormal collagen fibril assembly with a shift toward larger-diameter fibrils and increased interfibrillar spacing in the absence of collagen XIV was found. Second harmonic generation microscopy showed impaired fibrillogenesis in the collagen XIV null stroma. Mechanical testing suggested that collagen XIV confers stiffness to stromal tissue. Expression of collagen XIV is up-regulated following injury. This study indicates that collagen XIV plays a regulatory role in corneal development and in the function of the adult cornea. The expression of collagen XIV is recapitulated during wound healing.

Brain accumulation of amyloid-β (Aβ) peptides (resulting from a disrupted balance between biosynthesis and clearance) occurs during the progression of Alzheimer's disease (AD). Aβ peptides have diverse posttranslational modifications (PTMs) that variously modulate Aβ aggregation into fibrils, but understanding the mechanistic roles of PTMs in these processes remains a challenge. Here, we chemically synthesized three homogeneously modified isoforms of Aβ (1-42) peptides bearing Tyr10 O-glycosylation, an unusual PTM initially identified from the cerebrospinal fluid samples of AD patients. We discovered that O-glycans significantly affect both the aggregation and degradation of Aβ42. By combining cryo-EM and various biochemical assays, we demonstrate that a Galβ1-3GalNAc modification redirects Aβ42 to form a new fibril polymorphic structure that is less stable and more vulnerable to Aβ-degrading enzymes (e.g., insulin-degrading enzyme). Thus, beyond showing how particular O-glycosylation modifications affect Aβ42 aggregation at the molecular level, our study provides powerful experimental tools to support further investigations about how PTMs affect Aβ42 fibril aggregation and AD-related neurotoxicity.

Intrafibrillar mineralization of type I collagen fibrils is of great significance in dental remineralization, which is the key of caries prevention and treatment. Herein, two substances that have the remineralization ability, carboxylated polyamidoamine dendrimer (PAMAM-COOH) and nano-sized amorphous calcium phosphate (n-ACP) were combined to synthesize a novel nanomaterial, carboxylated polyamidoamine dendrimer/amorphous calcium phosphate nanocomposite (PAMAM-COOH/ACP). This article aims to evaluate the remineralization effect of PAMAM-COOH/ACP of dentin type I collagen fibrils in vitro. Fluorescence labeling technique was innovatively used to observe and evaluate the remineralization effect. PAMAM-COOH/ACP showed superior remineralization ability of human dentin type I collagen fibrils, especially the intrafibrillar remineralization. Therefore, the novel nanomaterial PAMAM-COOH/ACP is promising to prevent and treat various diseases caused by dentin demineralization and to improve various dental materials.

Emerging experimental evidence suggests tau pathology spreads between neuroanatomically connected brain regions in a prion-like manner in Alzheimer's disease (AD). Tau seeding, the ability of prion-like tau to recruit and misfold naïve tau to generate new seeds, is detected early in human AD brains before the development of major tau pathology. Many antitumour drugs have been reported to confer protection against neurodegeneration, supporting the repurposing of approved and experimental or investigational oncology drugs for AD therapy. In this study, we evaluated whether antitumour drugs that abrogate the generation of seed-competent aggregates of tau Repeat 3 (R3) domain peptides can prevent tau seeding and toxicity in Tau-RD P301S FRET Biosensor cells and Caenorhabditis elegans. We demonstrate that drugs that interact with the N-terminal VQIVYK or the C-terminal region housing the Cys322 prevent R3 dimerisation, abolishing the generation of prion-like R3 seeds. Preformed R3 seeds (fibrils) capped with, or R3 seeds formed in the presence of VQIVYK- or Cys322-targeting drugs have a reduced potency to cause aggregation of naïve tau in biosensor cells and protect worms from aggregate toxicity. These findings indicate that VQIVYK- or Cys322-targeting drugs may act as prophylactic agents against tau seeding.

Aggregation of the tau protein plays a central role in several neurodegenerative diseases collectively known as tauopathies, including Alzheimer’s and Parkinson’s disease. Tau misfolds into fibrillar beta sheet structures that constitute the paired helical filaments found in Neurofibrillary tangles. It is known that there can be significant structural heterogeneities in tau aggregates associated with different diseases. However, while structures of mature fibrils have been studied, the structural distributions in early stage tau aggregates is not well understood. In the present study, we use AFM-IR to investigate nanoscale spectra of individual tau fibrils at different stages of aggregation and demonstrate the presence of multiple fibrillar polymorphs that exhibit different secondary structures. We further show that mature fibrils contain significant amounts of antiparallel beta sheets. Our results are the very first application of nanoscale infrared spectroscopy to tau aggregates and underscore the promise of spatially resolved infrared spectroscopy for investigating protein aggregation.

The progression of Alzheimer’s disease (AD) is characterized with the deposition and aggregation of β-amyloid (Aβ). Visualizing Aβ aggregates at high spatial resolution is beneficial for AD diagnosis and treatment. Herein, we designed a new molecule by conjugating corannulene (Cor) with rhodamine B isothiocyanate (Rhb), namely Cor-Rhb, for the nanoscopic imaging and modulating Aβ peptide fibrillation. The low duty cycle, high photon output and sufficient switching cycles enable Cor-Rhb suitable for localization-based nanoscopic fluorescence imaging. We find that Cor-Rhb can inhibit Aβ peptides fibrillization and interact directly with mature fibrils, triggering their disaggregation under light illumination. Noticeably reduced Aβ-mediated cytotoxicity after the addition of Cor-Rhb is also confirmed. These explorations suggest that Cor-Rhb displays great potential as a multifunctional therapeutic agent against amyloid-related diseases, and may largely facilitate a variety of super-resolution based biological applications.

Alzheimer's disease (AD) is the most prevalent and devastating neurodegenerative disease occurred in the elderly. One of the pathogenic hallmarks is senile plaques composed of amyloid-β (Aβ) fibrils. Single mutations resided in Aβ were found in familial AD (FAD) patients that have early onset of the disease. The molecular details and properties of each FAD Aβ variants are still elusive. Here, we employed collective spectroscopic techniques to examine the properties of various Aβ40 fibrils. We generated fibrils of wild type (WT) and three FAD mutants on residue E22 including E22G, E22K, and E22Q. We monitored fibril formation by thioflavin T (ThT) assay, examined secondary structure by Fourier transform infrared and far-UV circular dichroism spectroscopy, imaged fibril morphology by transmission electron microscopy, and evaluated ThT-binding kinetics. In the thermal experiments, we found E22K fibrils resisted to high temperature and retained significant β-sheet content than the others. E22K fibril seeds after high-temperature treatment still possess the seeding property, whereas WT fibril seeds are disturbed after the treatment. Therefore, in this study we demonstrated the mutation at E22K increases the thermal stability and seeding function of amyloid fibrils.

The effects of varying the concentration of pentasodium triphosphate (PP) emulsifying salt [0, 0.6, 1.2, 1.5, and 1.8%, plus 0.9% of a mixture of citric acid (CA) and disodium phosphate (DSP) to adjust cheese pH to 5.85] on rheological, textural, physicochemical, and microstructural properties were studied in a processed cheese model system containing ~20% micellar casein concentrate, ~20% sunflower oil, and ~59% water. Special emphasis was placed on the unique casein fibrils recently described in a comparable processed cheese model system. Our results show that during processing (90°C, 17.37 rpm over 270 min) the apparent viscosity increased more and faster for formulations containing higher concentrations of PP, in analogy to the so-called creaming reaction, a general thickening of the molten cheese mass with prolonged processing. We found that 1.2% PP (plus 0.9% CA-DSP) appeared to be the threshold for the creaming reaction to take place. With increasing PP concentrations, cheese hardness increased in a sigmoidal fashion, and insoluble (protein-bound) calcium concentration decreased exponentially. Light micrographs of samples taken at the end of processing indicated initially large and dense casein aggregates within the matrix that disappeared with higher levels of PP, in parallel with the development of a finer emulsion. With transmission electron microscopy analysis on the same samples, the highly complex restructuring of the casein matrix was evident; casein fibrils had formed de novo at the periphery of the loosening casein aggregates. With higher levels of PP, amorphous areas were observed in place of the dense casein aggregates that appeared progressively void of protein, whereas fibril concentration increased throughout the rest of the matrix. Fibrils progressively attached to the surface of fat globules, thereby emulsifying them. Reverse-phase HPLC analysis of insoluble and soluble fractions indicated κ-casein to be the most likely constituent of the newly formed fibrils. The results of this study suggest that PP induced a concentration-dependent dissociation of caseins (through increased calcium chelation) and further led to their spatial separation. In essence, their chaperone activity was hindered, which resulted in amorphous aggregation on the one hand and fibril formation on the other.

Background: The coexistence of magnetite within protein aggregates in the brain is a typical pathologic feature of Alzheimer’s disease (AD), and the formation of amyloid-β (Aβ) plaques induces critical impairment of cognitive function. Objective: This study aimed to investigate the therapeutic effect of proton stimulation (PS) targeting plaque magnetite in the transgenic AD mouse brain. Methods: A proton transmission beam was applied to the whole mouse brain at a single entrance dose of 2 or 4 Gy to test the effect of disruption of magnetite-containing Aβ plaques by electron emission from magnetite. The reduction in Aβ plaque burden and the cognitive function of the PS-treated mouse group were assayed by histochemical analysis and memory tests, respectively. Aβ-magnetite and Aβ fibrils were treated with PS to investigate the breakdown of the amyloid protein matrix. Results: Single PS induced a 48–87%reduction in both the amyloid plaque burden and ferrous-containing magnetite level in the early-onset AD mouse brain while saving normal tissue. The overall Aβ plaque burden (68–82%) and (94–97%) hippocampal magnetite levels were reduced in late onset AD mice that showed improvements in cognitive function after PS compared with untreated AD mice (p

Biological deconstruction of polymer materials gains efficiency from the spatiotemporally coordinated action of enzymes with synergetic function in polymer chain depolymerization. To perpetuate enzyme synergy on a solid substrate undergoing deconstruction, the overall attack must alternate between focusing the individual enzymes locally and dissipating them again to other surface sites. Natural cellulases working as multienzyme complexes assembled on a scaffold protein (the cellulosome) maximize the effect of local concentration yet restrain the dispersion of individual enzymes. Here, with evidence from real-time atomic force microscopy to track nanoscale deconstruction of single cellulose fibers, we show that the cellulosome forces the fiber degradation into the transversal direction, to produce smaller fragments from multiple local attacks ("cuts"). Noncomplexed enzymes, as in fungal cellulases or obtained by dissociating the cellulosome, release the confining force so that fiber degradation proceeds laterally, observed as directed ablation of surface fibrils and leading to whole fiber "thinning". Processive cellulases that are enabled to freely disperse evoke the lateral degradation and determine its efficiency. Our results suggest that among natural cellulases, the dispersed enzymes are more generally and globally effective in depolymerization, while the cellulosome represents a specialized, fiber-fragmenting machinery.

Staphylococcus epidermidis are gram-positive bacteria that form a biofilm around implanted devices and develop an infection into a chronic state. Recently, it has been revealed that microvesicles have important roles in biofilm formation and intercellular communication among bacteria. However, biofilm formation of Staphylococcus epidermidis, and its relation to microvesicle secretion, is poorly understood because of the difficulty required to preserve the delicate water-rich morphology of biofilm for high-resolution observations. Here, we successfully imaged the microvesicles secreted from Staphylococcus epidermidis and the subsequent process of their integration into biofilm using liquid-phase imaging using atmospheric scanning electron microscopy (ASEM). In the biofilm, cells were connected by nanotube-like structures attached by microvesicles, and surrounded by extracellular polymeric substances. Cells cultured in the ASEM specimen holder were aldehyde-fixed and stained using positively charged nanogold labelling and/or using National Center for Microscopy and Imaging Research method. The samples immersed in aqueous radical scavenger glucose buffer were imaged by the inverted SEM of ASEM. Information regarding the morphologies of microvesicles, nanotube-like fibrils, and biofilm formed by Staphylococcus epidermidis is expected to be useful to elucidate the biological mechanism of biofilm formation and to develop a medicine against biofilms and their associated infections.

Glycine-rich regions feature prominently in intrinsically disordered regions (IDRs) of proteins that drive phase separation and the regulated formation of membraneless biomolecular condensates. Interestingly, the Gly-rich IDRs seldom feature poly-Gly tracts. The protein fused in sarcoma (FUS) is an exception. This protein includes two 10-residue poly-Gly tracts within the prion-like domain (PLD) and at the interface between the PLD and the RNA binding domain. Poly-Gly tracts are known to be highly insoluble, being potent drivers of self-assembly into solid-like fibrils. Given that the internal concentrations of FUS and FUS-like molecules cross the high micromolar and even millimolar range within condensates, we reasoned that the intrinsic insolubility of poly-Gly tracts might be germane to emergent fluid-to-solid transitions within condensates. To assess this possibility, we characterized the concentration-dependent self-assembly for three non-overlapping 25-residue Gly-rich peptides derived from FUS. Two of the three peptides feature 10-residue poly-Gly tracts. These peptides form either long fibrils based on twisted ribbon-like structures or self-supporting gels based on physical cross-links of fibrils. Conversely, the peptide with similar Gly contents but lacking a poly-Gly tract does not form fibrils or gels. Instead, it remains soluble across a wide range of concentrations. Our findings highlight the ability of poly-Gly tracts within IDRs that drive phase separation to undergo self-assembly. We propose that these tracts are likely to contribute to nucleation of fibrillar solids within dense condensates formed by FUS.

The aggregation of human alpha-synuclein (hαS) is pivotally implicated in the development of most types of synucleinopathies. Molecules that can inhibit or reverse the aggregation process of amyloidogenic proteins have potential therapeutic value. The anti-aggregating activity of multiple carotenoid compounds has been reported over the past decades against a growing list of amyloidogenic polypeptides. Here, we aimed to determine whether crocin, the main carotenoid glycoside component of saffron, would inhibit hαS aggregation or could disassemble its preformed fibrils. By employing a series of biochemical and biophysical techniques, crocin was exhibited to inhibit hαS fibrillation in a dose-dependent fashion by stabilizing very early aggregation intermediates in off-pathway non-toxic conformations with little β-sheet content. We also observed that crocin at high concentrations could efficiently destabilize mature fibrils and disassemble them into seeding-incompetent intermediates by altering their β-sheet conformation and reshaping their structure. Our atomistic molecular dynamics (MD) simulations demonstrated that crocin molecules bind to both the non amyloid-β component (NAC) region and C-terminal domain of hαS. These interactions could thereby stabilize the autoinhibitory conformation of the protein and prevent it from adopting aggregation-prone structures. MD simulations further suggested that ligand molecules prefer to reside longitudinally along the fibril axis onto the edges of the inter-protofilament interface where they establish hydrogen and hydrophobic bonds with steric zipper stabilizing residues. These interactions turned out to destabilize hαS fibrils by altering the interstrand twist angles, increasing the rigidity of the fibril core, and elevating its radius of gyration. Our findings suggest the potential pharmaceutical implication of crocin in synucleinopathies.

The use of wildtype recombinant alpha-synuclein preformed fibrils (aSyn PFFs) to induce endogenous alpha-synuclein to form pathological phosphorylation and trigger neurodegeneration is a popular model for studying Parkinson’s disease (PD) biology and testing therapeutic strategies. The strengths of this model lie in its ability to recapitulate the phosphorylation/aggregation of aSyn and nigrostriatal degeneration seen in PD, as well as its suitability for studying the progressive nature of PD and the spread of aSyn pathology. Although the model is commonly used and has been adopted by many labs, variability in observed phenotypes exists. Here we provide summaries of the study design and reported phenotypes from published reports characterizing the aSyn PFF in vivo model in rodents following injection into the brain, gut, muscle, vein, peritoneum, and eye. These summaries are designed to facilitate an introduction to the use of aSyn PFFs to generate a rodent model of PD—highlighting phenotypes observed in papers that set out to thoroughly characterize the model. This information will hopefully improve the understanding of this model and clarify when the aSyn PFF model may be an appropriate choice for one’s research.

This paper reports the self-assembly of the fibrillar network in a concentrated solution of macromolecules with an amphiphilic structure of repeating units. The investigation of amphiphilic homopolymers and alternating copolymers with the linear and cyclic topologies, the solution with different polymer concentrations and solvent qualities, allows us to conclude that the ability to form a fibrillar gel with branched fibrils and regular subchain thickness is inherent for macromolecules with the solvophobic backbone and solvophilic pendants. The elements of the gel structure, such as the mesh size and fibrillar thickness, the number of cross-links, and their functionality, can be tuned and customized according to the requirements of their application. The results could be helpful for the directed design of the synthetic analogue of the relevant extracellular matrix, in tissue engineering, for fibrotic disease treatment and cell encapsulation.

Amyloid beta (Aβ of Alzheimer's disease) and α-synuclein (α-Syn of Parkinson's disease) form large fibrils. Evidence is increasing however that much smaller oligomers are more toxic and that these oligomers can form transmembrane ion channels. We have proposed previously that Aβ42 oligomers, annular protofibrils, and ion channels adopt concentric β-barrel molecular structures. Here we extend that hypothesis to the superfamily of α, β, and γ-synucleins. Our models of numerous Synuclein oligomers, annular protofibrils, tubular protofibrils, lipoproteins, and ion channels were developed to be consistent with sizes, shapes, molecular weights, and secondary structures of assemblies as determined by EM and other studies. The models have the following features: 1) all subunits have identical structures and interactions; 2) they are consistent with conventional β-barrel theory; 3) the distance between walls of adjacent β-barrels is between 0.6 and 1.2 nm; 4) hydrogen bonds, salt bridges, interactions among aromatic side-chains, burial and tight packing of hydrophobic side-chains, and aqueous solvent exposure of hydrophilic side-chains are relatively optimal; and 5) residues that are identical among distantly related homologous proteins cluster in the interior of most oligomers whereas residues that are hypervariable are exposed on protein surfaces. Atomic scale models of some assemblies were developed. This article is protected by copyright. All rights reserved.

We evaluate the effects of the new Dentine matrix protein 1 (DMP-1) biomimetic system composed of phosphorylated polyamidoamine dendrimer (PAMAM-PO3H2) and carboxylated polyamidoamine dendrimer (PAMAM-COOH) on the mineralization of type I collagen fibrils. PAMAM-PO3H2 and PAMAM-COOH were observed to have the ability to induce internal and external mineralization of type I collagen fibrils in vitro through non-classical mineralization crystallization pathway, which has become a hopeful biomimetic system of biomimetic remineralization and demineralization of dentin type I collagen fibrils and has great potential in inducing biomimetic remineralization of demineralized dentin.

openaire: EC/H2020/788489/EU//BioELCell Funding Information: We thank Dr. Markus Bacher for his support with NMR measurements. This work was performed in part at the Materials Characterization and Fabrication Platform at The University of Melbourne. The authors thank the financial support from the Austrian Biorefinery Centre Tulln (ABCT), the Academy of Finland (Project #311255, “WTF-Click-Nano”), and the H2020-ERC-2017-Advanced Grant “BioELCell” (788489). This research was funded in whole, or in part, by the Austrian Science Fund (FWF) (J4356). Publisher Copyright: © 2021 American Chemical Society. All rights reserved. Selective surface modification of biobased fibers affords effective individualization and functionalization into nanomaterials, as exemplified by the TEMPO-mediated oxidation. However, such a route leads to changes of the native surface chemistry, affecting interparticle interactions and limiting the development of potential supermaterials. Here we introduce a methodology to extract elementary cellulose fibrils by treatment of biomass with N-succinylimidazole, achieving regioselective surface modification of C6-OH, which can be reverted using mild post-treatments. No polymer degradation, cross-linking, nor changes in crystallinity occur under the mild processing conditions, yielding cellulose nanofibrils bearing carboxyl moieties, which can be removed by saponification. The latter offers a significant opportunity in the reconstitution of the chemical and structural interfaces associated with the native states. Consequently, 3D structuring of native elementary cellulose nanofibrils is made possible with the same supramolecular features as the biosynthesized fibers, which is required to unlock the full potential of cellulose as a sustainable building block.

Funder: EC | EC Seventh Framework Programm | FP7 Ideas: European Research Council (FP7-IDEAS-ERC - Specific Programme: "Ideas" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013)); doi: https://doi.org/10.13039/100011199, Funder: EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council); doi: https://doi.org/10.13039/100010663, Molecular chaperones contribute to the maintenance of cellular protein homoeostasis through assisting de novo protein folding and preventing amyloid formation. Chaperones of the Hsp70 family can further disaggregate otherwise irreversible aggregate species such as α-synuclein fibrils, which accumulate in Parkinson’s disease. However, the mechanisms and kinetics of this key functionality are only partially understood. Here, we combine microfluidic measurements with chemical kinetics to study α-synuclein disaggregation. We show that Hsc70 together with its co-chaperones DnaJB1 and Apg2 can completely reverse α-synuclein aggregation back to its soluble monomeric state. This reaction proceeds through first-order kinetics where monomer units are removed directly from the fibril ends with little contribution from intermediate fibril fragmentation steps. These findings extend our mechanistic understanding of the role of chaperones in the suppression of amyloid proliferation and in aggregate clearance, and inform on possibilities and limitations of this strategy in the development of therapeutics against synucleinopathies.

Parkinson's disease (PD) is linked to the aberrant self-assembly of the amyloid protein, α-synuclein (αS), where αS monomers aggregate to form oligomers and fibrils. Out of the three conformers, αS oligomers are the major toxic agents in PD, while αS fibrils may work as a reservoir for toxic oligomeric conformers. Thus, compounds that inhibit aggregation of αS monomers and disaggregate αS oligomers and fibrils may serve as therapeutic agents against PD. In this regard, resveratrol and its synthetic derivatives (e.g., AM17, which contains a copper ion-selective ionophoric motif) have previously been examined for their inhibitory effects on aggregation of amyloid proteins, such as the β-amyloid peptide implicated in Alzheimer's disease. In the current study, we employed an array of experimental tools, such as Thioflavin T fluorescence, transmission electron microscopy, immuno-dot blot assays, SDS- and native-PAGE, and circular dichroism, to determine the impact of AM17 and resveratrol on αS aggregation. To the best of our knowledge, we show for the first time that AM17 not only inhibits aggregation of αS monomers but also disaggregates αS oligomers and fibrils, independent of the copper ions. Similar αS aggregation inhibitory effects were observed with resveratrol only in the presence of the copper ion. The present study supports the high promise of applicability of AM17 as an effective amyloid aggregation inhibitor for various conformers and protein sequences.

Self-assembly of disordered amyloid-beta (Aβ) peptides results in highly ordered amyloid fibrils. The structural information of the early-stage events and also in the presence of inhibitors is of great significance. It is challenging to acquire due to the nature of the amyloids and experimental constraints. Here, we demonstrate the cascade of aggregation (early to late) of the Aβ25-35 peptide in the absence and presence of carvedilol, a nonselective β-adrenergic receptor blocker. The aggregation process of Aβ25-35 peptide is monitored using Thioflavin T (ThT) fluorescence, dynamic light scattering (DLS), circular dichroism (CD), Raman spectroscopic techniques, and imaging experiments. We find that the Aβ25-35 peptide undergoes an early-stage (3-6 h) helical intermediate formation across the fibrillation pathway using CD and Raman measurements. Carvedilol obstructs the helical intermediate formation of Aβ25-35 peptide resulting in inhibition. CD spectra and deconvolution of the Raman bands suggest the β-sheet formation (24-100 h) in the absence of carvedilol. Spectroscopic results indicate a disordered structure for the peptide in the presence of carvedilol (24-100 h). Electron microscopy (EM) shows the formation of polymorphic fibrils for the peptide alone and non-amyloidal aggregates in the presence of carvedilol. Molecular docking study suggests that the plausible mode of interaction with carvedilol involves the C-terminal residues of the peptide.

The aggregation of peptides into amyloid fibrils is associated with several diseases, including Alzheimer’s and Parkinson’s disease. Because hydrophobic interactions often play an important role in amyloid formation, the presence of various hydrophobic or amphiphilic molecules, such as lipids, may influence the aggregation process. We have studied the effect of a fatty acid, linoleic acid, on the fibrillation process of the amyloid-forming model peptide NACore (GAVVTGVTAVA). NACore is a peptide fragment spanning residue 68–78 of the protein α-synuclein involved in Parkinson’s disease. Based primarily on circular dichroism measurements, we found that even a very small amount of linoleic acid can substantially inhibit the fibrillation of NACore. This inhibitory effect manifests itself through a prolongation of the lag phase of the peptide fibrillation. The effect is greatest when the fatty acid is present from the beginning of the process together with the monomeric peptide. Cryogenic transmission electron microscopy revealed the presence of nonfibrillar clusters among NACore fibrils formed in the presence of linoleic acid. We argue that the observed inhibitory effect on fibrillation is due to co-association of peptide oligomers and fatty acid aggregates at the early stage of the process. An important aspect of this mechanism is that it is nonmonomeric peptide structures that associate with the fatty acid aggregates. Similar mechanisms of action could be relevant in amyloid formation occurring in vivo, where the aggregation takes place in a lipid-rich environment.

The bi-thiophene-vinylene-benzothiazole (bTVBT4) ligand developed for Alzheimers disease (AD)-specific detection of amyloid tau has been studied by a combination of several theoretical methods and experimental spectroscopies. With reference to the cryo-EM tau structure of the tau protofilament (Nature 2017, 547, 185), a periodic model system of the fibril was created, and the interactions between this fibril and bTVBT4 were studied with nonbiased molecular dynamics simulations. Several binding sites and binding modes were identified and analyzed, and the results for the most prevailing fibril site and ligand modes are presented. A key validation of the simulation work is provided by the favorable comparison of the theoretical and experimental absorption spectra of bTVBT4 in solution and bound to the protein. It is conclusively shown that the ligand-protein binding occurs at the hydrophobic pocket defined by the residues Ile360, Thr361, and His362. This binding site is not accessible in the Picks disease (PiD) fold, and fluorescence imaging of bTVBT4-stained brain tissue samples from patients diagnosed with AD and PiD provides strong support for the proposed tau binding site. Funding Agencies|European CommissionEuropean CommissionEuropean Commission Joint Research Centre [765739]; Swedish Research CouncilSwedish Research CouncilEuropean Commission [2018-4343, 2016-07213]; Swedish e-Science Research Centre (SeRC); German Research FoundationGerman Research Foundation (DFG) [KO 5423/1-1]; U.S. National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [UO1NS110437]

Recently, the collagen-keratin (CK) composites have received much attention for the purpose of biomedical applications due to the intrinsic biocompatibility and biodegradability of these two proteins. However, few studies have reported the CK composites developed by the self-assembly approach and the influence of the keratin on the collagen self-assembly in vitro was still unknown. In this study, the keratin nanoparticles (KNPs) were successfully prepared by the reduction method, and we focused on investigating the effect of the varying concentrations of KNPs on the mechanism of the fibrillogenesis process of collagen. The intermolecular interaction between the two proteins revealed by the ultraviolet spectroscopy, Fourier transform-infrared (FT-IR) spectroscopy and circular dichromatic (CD) spectroscopy showed that KNPs would interact with the collagen, and keratin significantly influenced the hydrogen bonding interaction existed in collagen molecules. The SEM images exhibited the formation of exquisite fibrillar networks after incorporating the KNPs into collagen, and it was conspicuous that the KNPs could uniformly distribute on the surface of collagen fibrils via electrostatic interaction, for both of the two proteins possessed many charged moieties. In addition, the AFM images confirmed the presence of the characteristic D-periodicity of collagen fibrils, indicating that the introduction of KNPs did not disrupt the self-assembly nature of the native collagen. The cell adhesion, proliferation and migration experiments on the CK fibrils were also performed in this study. The results demonstrated that the CK composites showed a better cellular affinity compared with the collagen, thus it might be a promising candidate for the biomedical applications.

Biosynthesis of bacterial cellulose (BC) in cylindrical oxygen permeable molds allows the production of hollow tubular structures of increasing interest for biomedical applications (artificial blood vessels, ureters, urethra, trachea, esophagus, etc.). In the current contribution a simple set-up is used to obtain BC tubes of predefined dimensions; and the effects of fermentation time on the water holding capacity, nanofibrils network architecture, specific surface area, chemical purity, thermal stability, mechanical properties, and cell adhesion, proliferation and migration of BC tubes are systematically analysed for the first time. The results reported highlight the role of culture time on key properties of the BC tubes produced, with significant differences arising from the denser and more compact fibril arrangements generated at longer fermentation intervals.

Amyloid proteins can aggregate into insoluble fibrils and form amyloid deposits in the human brain, which is the hallmark of many neurodegenerative diseases. Promising strategies toward pathological amyloid proteins and deposition include investigating inhibitors that can disrupt amyloid aggregation or induce misfolding protein degradation. In this review, recent progress of peptide-based inhibitors, including amyloid sequence–derived inhibitors, designed peptides, and peptide mimics, is highlighted. Based on the increased understanding of peptide design and precise amyloid structures, these peptides exhibit advanced inhibitory activities against fibrous aggregation as well as enhanced druggability.

Alzheimer's disease is characterized by the accumulation of amyloid beta (Aβ) and Tau aggregates in the brain, which induces various pathological events resulting in neurodegeneration. There have been continuous efforts to develop modulators of the Aβ and Tau aggregation process to halt or modify disease progression. A few small-molecule-based inhibitors that target both Aβ and Tau pathology have been reported. Here, we report the screening of a targeted library of small molecules to modulate Aβ and Tau aggregation together with their in vitro, in silico and cellular studies. In vitro ThT fluorescence assay, dot blot assay, gel electrophoresis and transmission electron microscopy (TEM) results have shown that thiophene-based lead molecules effectively modulate Aβ aggregation and inhibit Tau aggregation. In silico studies performed by employing molecular docking, molecular dynamics and binding-free energy calculations have helped in understanding the mechanism of interaction of the lead thiophene compounds with Aβ and Tau fibril targets. In cellulo studies revealed that the lead candidate is biocompatible and effectively ameliorates neuronal cells from Aβ and Tau-mediated amyloid toxicity.

The formation of amyloid fibrils and oligomers is a hallmark of several neurodegenerative disorders, including Alzheimer's disease (AD), and contributes to the disease pathway. To progress our understanding of these diseases at a molecular level, it is crucial to determine the mechanisms and rates of amyloid formation and replication. In the context of AD, the self-replication of aggregates of the Aβ42 peptide by secondary nucleation, leading to the formation of new aggregates on the surfaces of existing ones, is a major source of both new fibrils and smaller toxic oligomeric species. However, the core mechanistic determinants, including the presence of intermediates, as well as the role of heterogeneities in the fibril population, are challenging to determine from bulk aggregation measurements. Here, we obtain such information by monitoring directly the time evolution of individual fibrils by TIRF microscopy. Crucially, essentially all aggregates have the ability to self-replicate via secondary nucleation, and the amplification of the aggregate concentration cannot be explained by a small fraction of "superspreader" fibrils. We observe that secondary nucleation is a catalytic multistep process involving the attachment of soluble species to the fibril surface, followed by conversion/detachment to yield a new fibril in solution. Furthermore, we find that fibrils formed by secondary nucleation resemble the parent fibril population. This detailed level of mechanistic insights into aggregate self-replication is key in the rational design of potential inhibitors of this process.

Several neurodegenerative diseases have been linked to proteins or peptides that are prone to aggregate in different brain regions. Aggregation of amyloid-β (Aβ) peptides is recognized as the main cause of Alzheimer's disease (AD) progression, leading to the formation of toxic Aβ oligomers and amyloid fibrils. The molecular mechanism of Aβ aggregation is complex and still not fully understood. Nanopore technology provides a new way to obtain kinetic and morphological aspects of Aβ aggregation at a single-molecule scale without labeling by detecting the electrochemical signal of the peptides when they pass through the hole. Here, we investigate the influence of nanoscale geometry (conical and bullet-like shape) of a track-etched nanopore pore and the effect of molecular crowding (polyethylene glycol-functionalized pores) on Aβ fibril sensing and analysis. Various Aβ fibril samples that differed by their length were produced by sonication of fibrils obtained in the presence of epigallocatechin gallate. The conical nanopore functionalized with polyethylene glycol (PEG) 5 kDa is suitable for discrimination of the fibril size from relative current blockade. The bullet-like-shaped nanopore enhances the amplitude of the current and increases the dwell time, allowing us to well discern the fibrils. Finally, the nanopore crowded with PEG 20 kDa enhances the relative current blockade and increases the dwell time; however, the discrimination is not improved compared to the "bullet-shaped" nanopore.

Light chain (AL) amyloidosis is characterized by deposition of immunoglobulin light chains (LC) as fibrils in target organs. Alongside the full-length protein, abundant LC fragments are always present in AL deposits. Herein, by combining gel-based and mass spectrometry analyses, we identified and compared the fragmentation sites of amyloid LCs from multiple organs of an AL λ amyloidosis patient (AL-55). The positions pinpointed here in kidney and subcutaneous fat, alongside those previously detected in heart of the same patient, were aligned and mapped on the LC's dimeric and fibrillar states. All tissues contain fragmented LCs along with the full-length protein; the fragment pattern is coincident across organs, although microheterogeneity exists. Multiple cleavage positions were detected; some are shared, whereas some are organ-specific, likely due to a complex of proteases. Cleavage sites are concentrated in "proteolysis-prone" regions, common to all tissues. Several proteolytic sites are not accessible on native dimers, while they are compatible with fibrils. Overall, data suggest that the heterogeneous ensemble of LC fragments originates in tissues, and is consistent with digestion of preformed fibrils, or with the hypothesis that initial proteolytic cleavage of the constant domain triggers the amyloidogenic potential of LCs, followed by subsequent proteolytic degradation. This work provides a unique set of molecular data on proteolysis from ex vivo amyloid, which allows discussing hypotheses on role and timing of proteolytic events occurring along amyloid formation and accumulation in AL patients.

The pathological amyloid plaques in Alzheimer's disease (AD) patients contain not only the wild-type β-amyloid (wt-Aβ) peptide sequences but also a variety of post-translationally modified variants. The pyroglutamate-3 Aβ (pyroE3-Aβ), which is generated from its truncated precursors ΔE3-Aβ, shows the highest abundance among all modified Aβ variants. Previous works have shown that pyroE3-Aβ and/or ΔE3-Aβ, compared with the wild-type sequences, led to a more rapid fibrillation process and final fibrils with higher neuronal cytotoxicity levels. However, much less is known about how the formation of pyroE3/ΔE3-Aβ fibrils would affect the amyloid deposition of wt-Aβ peptides, which are the main pathological events in AD. We show in the present work that the pyroE3/ΔE3-Aβ40 fibrils differ significantly from the wt-Aβ40 fibrils in terms of their molecular structures. When added into monomeric wt-Aβ40 peptides, these variant fibrils can cross-seed the formation of wt-Aβ40 fibrils with fibrillation kinetics that are greater than the self-seeded fibrillation of wt-Aβ40. Furthermore, the cross-seeding process modulates the molecular structures of the yielded wt-Aβ40 fibrils, which show similar features as their variant seeds. The cross-seeded fibrillation process also induces higher cytotoxicity levels compared with the self-seeded fibrillation of wt-Aβ40. Overall, our results support the hypothesis that pyroE3 and ΔE3-Aβ40 variants may serve as triggering factors of the pathological amyloid aggregation of wt-Aβ40 and may underlie the pathological significance of pyroE3/ΔE3-Aβ40 variants on the structural polymorphism of Aβ deposits.

Confocal imaging has shown that CELLULOSE SYNTHASE (CESA) particles move through the plasma membrane as they synthesize cellulose. However, the resolution limit of confocal microscopy circumscribes what can be discovered about these tiny biosynthetic machines. Here, we applied Structured Illumination Microscopy (SIM), which improves resolution two-fold over confocal or widefield imaging, to explore the dynamic behaviors of CESA particles in living plant cells. SIM imaging reveals that Arabidopsis thaliana CESA particles are more than twice as dense in the plasma membrane as previously estimated, helping explain the dense arrangement of cellulose observed in new wall layers. CESA particles tracked by SIM display minimal variation in velocity, suggesting coordinated control of CESA catalytic activity within single complexes and that CESA complexes might move steadily in tandem to generate larger cellulose fibrils or bundles. SIM data also reveal that CESA particles vary in their overlaps with microtubule tracks and can complete U-turns without changing speed. CESA track patterns can vary widely between neighboring cells of similar shape, implying that cellulose patterning is not the sole determinant of cellular growth anisotropy. Together, these findings highlight SIM as a powerful tool to advance CESA imaging beyond the resolution limit of conventional light microscopy.