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The autophagy-lysosomal pathway is impaired in many neurodegenerative diseases characterized by protein aggregation, but the link between aggregation and lysosomal dysfunction remains poorly understood. Here, we combine cryo-electron tomography, proteomics, and cell biology studies to investigate the effects of protein aggregates in primary neurons. We use artificial amyloid-like β-sheet proteins (β proteins) to focus on the gain-of-function aspect of aggregation. These proteins form fibrillar aggregates and cause neurotoxicity. We show that late stages of autophagy are impaired by the aggregates, resulting in lysosomal alterations reminiscent of lysosomal storage disorders. Mechanistically, β proteins interact with and sequester AP-3 μ1, a subunit of the AP-3 adaptor complex involved in protein trafficking to lysosomal organelles. This leads to destabilization of the AP-3 complex, missorting of AP-3 cargo, and lysosomal defects. Restoring AP-3μ1 expression ameliorates neurotoxicity caused by β proteins. Altogether, our results highlight the link between protein aggregation, lysosomal impairments, and neurotoxicity., (© 2021 Riera-Tur et al.)

The loss of crystallins solubility with aging and the formation of amyloid-like aggregates is considered the hallmark characteristic of cataract pathology. The present study was carried out to assess the effect of temperature on the soluble lens protein and the formation of protein aggregates with typical amyloid characteristics. The soluble fraction of lens proteins was subjected for heat treatment in the range of 40-60 °C, and the nature of protein aggregates was assessed by using Congo red (CR), thioflavin T (ThT), and 8-anilinonaphthalene-1-sulfonic acid (ANS) binding assays, circular dichroism (CD), Fourier-transform infrared (FT-IR) spectroscopy, and transmission electron microscopy (TEM). The heat-treated protein samples displayed a substantial bathochromic shift (≈15 nm) in the CR's absorption maximum (λ max ) and increased ThT and ANS binding. The heat treatment of lens soluble proteins results in the formation of nontoxic, β-sheet rich, non-fibrillar, protein aggregates similar to the structures evident in the insoluble fraction of proteins isolated from the cataractous lens. The data obtained from the present study suggest that the exposure of soluble lens proteins to elevated temperature leads to the formation of non-fibrillar aggregates, establishing the role of amyloid in the heat-induced augmentation of cataracts pathology., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The development and testing of new antimicrobial peptides (AMPs) represent an important milestone toward the development of new antimicrobial drugs that can inhibit the growth of pathogens and multidrug-resistant microorganisms such as Pseudomonas aeruginosa, Gram-negative bacteria. Most AMPs achieve these goals through mechanisms that disrupt the normal permeability of the cell membrane, which ultimately leads to the death of the pathogenic cell. Here, we developed a unique combination of a membrane penetrating peptide and peptides prone to amyloidogenesis to create hybrid peptide: "cell penetrating peptide + linker + amyloidogenic peptide". We evaluated the antimicrobial effects of two peptides that were developed from sequences with different propensities for amyloid formation. Among the two hybrid peptides, one was found with antibacterial activity comparable to antibiotic gentamicin sulfate. Our peptides showed no toxicity to eukaryotic cells. In addition, we evaluated the effect on the antimicrobial properties of amino acid substitutions in the non-amyloidogenic region of peptides. We compared the results with data on the predicted secondary structure, hydrophobicity, and antimicrobial properties of the original and modified peptides. In conclusion, our study demonstrates the promise of hybrid peptides based on amyloidogenic regions of the ribosomal S1 protein for the development of new antimicrobial drugs against P. aeruginosa .

This review will focus on the process of amyloid-type protein aggregation. Amyloid fibrils are an important hallmark of protein misfolding diseases and therefore have been investigated for decades. Only recently, however, atomic or near-atomic resolution structures have been elucidated from various in vitro and ex vivo obtained fibrils. In parallel, the process of fibril formation has been studied in vitro under highly artificial but comparatively reproducible conditions. The review starts with a summary of what is known and speculated from artificial in vitro amyloid-type protein aggregation experiments. A partially hypothetic fibril selection model will be described that may be suitable to explain why amyloid fibrils look the way they do, in particular, why at least all so far reported high resolution cryo-electron microscopy obtained fibril structures are in register, parallel, cross-β-sheet fibrils that mostly consist of two protofilaments twisted around each other. An intrinsic feature of the model is the prion-like nature of all amyloid assemblies. Transferring the model from the in vitro point of view to the in vivo situation is not straightforward, highly hypothetic, and leaves many open questions that need to be addressed in the future.

Accumulation of ubiquitin-positive, tau- and α-synuclein-negative intracellular inclusions of TDP-43 in the central nervous system represents the major hallmark correlated to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Such inclusions have variably been described as amorphous aggregates or more structured deposits having amyloid properties. Here we have purified full-length TDP-43 (FL TDP-43) and its C-terminal domain (Ct TDP-43) to investigate the morphological, structural and tinctorial features of aggregates formed in vitro by them at pH 7.4 and 37 °C. AFM images indicate that both protein variants show a tendency to form filaments. Moreover, we show that both FL TDP-43 and Ct TDP-43 filaments possess a largely disordered secondary structure, as ascertained by far-UV circular dichroism and Fourier transform infra-red spectroscopy, do not bind Congo red and induce a very weak increase of thioflavin T fluorescence, indicating the absence of a clear amyloid-like signature.

Oligomeric intermediates on the pathway of amyloid fibrillation are suspected as the main cytotoxins responsible for amyloid-related pathogenicity. As they appear to be a part of the lag phase of amyloid fibrillation when analyzed using standard methods such as Thioflavin T (ThT) fluorescence, a more sensitive method is needed for their detection. Here we apply Fourier transform infrared spectroscopy (FTIR) in attenuated total reflectance (ATR) mode for fast and cheap analysis of destabilized hen-egg-white lysozyme solution and detection of oligomer intermediates of amyloid fibrillation. Standard methods of protein aggregation analysis- Thioflavin T (ThT) fluorescence, atomic force microscopy (AFM), and 8-anilinonaphthalene-1-sulphonic acid (ANS) fluorescence were applied and compared to FTIR spectroscopy data. Results show the great potential of FTIR for both, qualitative and quantitative monitoring of oligomer formation based on the secondary structure changes. While oligomer intermediates do not induce significant changes in ThT fluorescence, their secondary structure changes were very prominent. Normalization of specific Amide I region peak intensities by using Amide II peak intensity as an internal standard provides an opportunity to use FTIR spectroscopy for both qualitative and quantitative analysis of biological samples and detection of potentially toxic oligomers, as well as for screening of efficiency of fibrillation procedures.

The misfolding and aggregation of polypeptide chains into β-sheet-rich amyloid fibrils is associated with a wide range of neurodegenerative diseases. Growing evidence indicates that the oligomeric intermediates populated in the early stages of amyloid formation rather than the mature fibrils are responsible for the cytotoxicity and pathology and are potentially therapeutic targets. However, due to the low-populated, transient, and heterogeneous nature of amyloid oligomers, they are hard to characterize by conventional bulk methods. The development of single molecule approaches provides a powerful toolkit for investigating these oligomeric intermediates as well as the complex process of amyloid aggregation at molecular resolution. In this review, we present an overview of recent progress in characterizing the oligomerization of amyloid proteins by single molecule fluorescence techniques, including single-molecule Förster resonance energy transfer (smFRET), fluorescence correlation spectroscopy (FCS), single-molecule photobleaching and super-resolution optical imaging. We discuss how these techniques have been applied to investigate the different aspects of amyloid oligomers and facilitate understanding of the mechanism of amyloid aggregation.

Artemia cysts are the essential food product for industrial larviculture of fishes. The cyst shell protects the artemia embryo from mechanical damage, ultraviolet light, excessive water loss, thermal variation and anoxia condition. However, the underlying mechanism of such environmental protection is largely unclear. The embryonic cuticle of cyst shell mainly constitutes chitin and proteins. Absence of cyst shell proteins compromises embryo survival. In literature, there are few examples of functional amyloids where proteins adapt amyloid-like structures and act as protective covering. We hypothesized that the proteins from the embryonic cuticle of artemia cyst shell may have amyloid-like properties. Using FTIR and CD analysis, we found that proteins in embryonic cuticle have high β-sheet secondary structures. Embryonic cuticles displayed high Congo red binding affinity and stained samples showed apple-green birefringence under polarized light, confirming the presence of amyloid-like structures. Amyloid structures have a tendency to propagate and cause amyloidosis. However, feeding of amyloid rich embryonic cuticles to zebrafish did not show any signs of discomfort or morbidity and amyloid deposition. Taken together, the study reveals that amyloid-like structures are present in embryonic cuticle of artemia cyst and their consumption does not induce amyloidosis in zebrafish.

Systemic light chain (AL) amyloidosis is a fatal protein misfolding disease in which excessive secretion, misfolding, and subsequent aggregation of free antibody light chains eventually lead to deposition of amyloid plaques in various organs. Patient-specific mutations in the antibody V L domain are closely linked to the disease, but the molecular mechanisms by which certain mutations induce misfolding and amyloid aggregation of antibody domains are still poorly understood. Here, we compare a patient V L domain with its nonamyloidogenic germline counterpart and show that, out of the five mutations present, two of them strongly destabilize the protein and induce amyloid fibril formation. Surprisingly, the decisive, disease-causing mutations are located in the highly variable complementarity determining regions (CDRs) but exhibit a strong impact on the dynamics of conserved core regions of the patient V L domain. This effect seems to be based on a deviation from the canonical CDR structures of CDR2 and CDR3 induced by the substitutions. The amyloid-driving mutations are not necessarily involved in propagating fibril formation by providing specific side chain interactions within the fibril structure. Rather, they destabilize the V L domain in a specific way, increasing the dynamics of framework regions, which can then change their conformation to form the fibril core. These findings reveal unexpected influences of CDR-framework interactions on antibody architecture, stability, and amyloid propensity., Competing Interests: Conflict of interest The authors declare no conflict of interest., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The aggregation of β-crystallins in the human eye lens constitutes a critical step during the development of cataract. We anticipated that the presence of Aggregation-Prone Regions (APRs) in their primary structure, which might be responsible for conformational change required for the self-assembly. To examine the presence of APRs, we systematically analyzed the primary structures of β-crystallins. Out of seven subtypes, the βB1-crystallin found to possess the highest aggregation score with 9 APRs in its primary structure. To confirm the amyloidogenic nature of these newly identified APRs, we further studied the aggregation behavior of one of the APRs spanning from 174 to 180 residues ( 174 LWVYGFS 180 ) of βB1-crystallin, which is referred as βB1 (174-180) . Under in vitro conditions, the synthetic analogue of βB1 (174-180) peptide formed visible aggregates and displayed high Congo red (CR) bathochromic shift, Thioflavin T (ThT) binding and fibrilar morphology under transmission electron microscopy, which are the typical characteristics of amyloids. Further, the aggregated βB1 (174-180) was found to induce aggregation of the soluble fraction of proteins isolated from the human cataractous lens. This observation suggests that the presence of APRs in βB1-crystallin might be serving as one of the intrinsic supplementary factors responsible for constitutive aggregation behavior of βB1-crystallin and development of cataract., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Amyloidogenic protein assembly into insoluble fibrillar aggregates is linked with several neurodegenerative disorders, such as Alzheimer's or Parkinson's disease, affecting millions of people worldwide. The search for a potential anti-amyloid drug has led to the discovery of hundreds of compounds, none of which have passed all clinical trials. Gallic acid has been shown to both modulate factors leading to the onset of neurodegenerative disorders, as well as directly inhibit amyloid formation. However, the conditions under which this effect is seen could lead to oxidation of this polyphenol, likely changing its properties. Here we examine the effect of gallic acid and its oxidised form on the aggregation of a model amyloidogenic protein-insulin at low pH conditions. We show a vastly higher inhibitory potential of the oxidised form, as well as an alteration in the aggregation pathway, leading to the formation of a specific fibril conformation.

Tooth enamel is formed in an extracellular environment. Amelogenin, the major component in the protein matrix of tooth enamel during the developing stage, could assemble into high molecular weight structures, regulating enamel formation. However, the molecular structure of amelogenin protein assembly at the functional state is still elusive. In this work, we found that amelogenin is able to induce calcium phosphate minerals into hydroxyapatite (HAP) structure in vitro at pH 6.0. Assessed using X-ray diffraction (XRD) and 31 P solid-state NMR (SSNMR) evidence, the formed HAP mimics natural enamel closely. The structure of amelogenin protein assembly coexisting with the HAP was also studied using atomic force microscopy (AFM), transmission electron microscopy (TEM) and XRD, indicating the β-amyloid structure of the protein. SSNMR was proven to be an important tool in detecting both the rigid and dynamic components of the protein assembly in the sample, and the core sequence 18 EVLTPLKWYQSI 29 was identified as the major segment contributing to the β-sheet secondary structure. Our research suggests an amyloid structure may be an important factor in controlling HAP formation at the right pH conditions with the help of other structural components in the protein assembly.

In this report, we describe a near-infrared fluorescent probe called quinaldine red (QR) which lights up the β-sheet structure of amyloid fibrils. The photochemical and biophysical properties of QR along with other canonical amyloid probes in the presence of protein fibrils were investigated by using fluorescence spectroscopy, confocal fluorescent microscopy and isothermal titration calorimetry. Moreover, the binding sites and interaction mode between QR and insulin fibrils were calculated based on molecule docking. Among these amyloid probes, QR showed several advantages including strong supramolecular force, near-infrared emission, high sensitivity and resistance to bleaching. A linear response of the fluorescence intensity of QR towards fibril samples in the presence of sera was visualized in the range of 1-30 μM, with the limit of detection (LOD) of 2.31 μM. The recovery and relative standard deviation (RSD) of the proposed method for the determination of protein fibrils was 90.4%-99.2% and 3.05%-3.47%, respectively. Finally, QR can be fluorescently lighted up when meeting the aberrant protein aggregates of pathogenic mice. We recommend QR as a novel and excellent alternative tool for monitoring conformational transition of amyloid proteins., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Prion-like proteins form multivalent assemblies and phase separate into membraneless organelles. Heterogeneous ribonucleoprotein D-like (hnRNPDL) is a RNA-processing prion-like protein with three alternative splicing (AS) isoforms, which lack none, one, or both of its two disordered domains. It has been suggested that AS might regulate the assembly properties of RNA-processing proteins by controlling the incorporation of multivalent disordered regions in the isoforms. This, in turn, would modulate their activity in the downstream splicing program. Here, we demonstrate that AS controls the phase separation of hnRNPDL, as well as the size and dynamics of its nuclear complexes, its nucleus-cytoplasm shuttling, and amyloidogenicity. Mutation of the highly conserved D378 in the disordered C-terminal prion-like domain of hnRNPDL causes limb-girdle muscular dystrophy 1G. We show that D378H/N disease mutations impact hnRNPDL assembly properties, accelerating aggregation and dramatically reducing the protein solubility in the muscle of Drosophila, suggesting a genetic loss-of-function mechanism for this muscular disorder., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

The RNA-binding protein fused in sarcoma (FUS) forms physiological granules and pathological fibrils, which facilitate RNA functions and cause neurodegenerative diseases, respectively. Phosphorylation at Ser/Thr residues may regulate the functional assembly of FUS and prevent pathological aggregation in cells. However, the low-complexity nature of the FUS sequence makes it challenging to characterize how phosphorylation of specific sites within the core amyloid-forming segment affects aggregation. Taking advantage of the recently solved molecular structures of the fibrillar core of the FUS low-complexity (FUS-LC) domain, we systematically investigated the aggregation of repeated segments within the core. We identified a segment with a strong amyloid-forming tendency that induced the aggregation of FUS-LC domain in phase-separated liquid droplets and further seeded the aggregation of full-length FUS. The aggregation propensity and seeding ability of this amyloid-forming segment were modulated by site-specific phosphorylation. Solid-state nuclear magnetic resonance (NMR) spectroscopy and computational modeling implied that site-specific phosphorylation at Ser61 plays key roles in FUS assembly by disrupting both intra- and intermolecular interactions that maintain the amyloid core structure., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Aggregation of amyloidogenic proteins is an abnormal biological process implicated in neurodegenerative disorders. Whereas the aggregation process of amyloid-forming proteins has been studied extensively, the mechanism of aggregate removal is poorly understood. We recently demonstrated that proteasomes could fragment filamentous aggregates into smaller entities, restricting aggregate size [1]. Here, we show in vitro that UBE2W can modify the N-terminus of both α-synuclein and a tau tetra-repeat domain with a single ubiquitin. We demonstrate that an engineered N-terminal ubiquitin modification changes the aggregation process of both proteins, resulting in the formation of structurally distinct aggregates. Single-molecule approaches further reveal that the proteasome can target soluble oligomers assembled from ubiquitin-modified proteins independently of its peptidase activity, consistent with our recently reported fibril-fragmenting activity. Based on these results, we propose that proteasomes are able to target oligomers assembled from N-terminally ubiquitinated proteins. Our data suggest a possible disassembly mechanism by which N-terminal ubiquitination and the proteasome may together impede aggregate formation., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

In dialysis patients, the protein β2-microglobulin (β2m) forms amyloid fibrils in a condition known as dialysis-related amyloidosis. To understand the early stages of the amyloid assembly process, we have used native electrospray ionization (ESI) together with ion mobility mass spectrometry (IM-MS) to study soluble preamyloid oligomers. ESI-IM-MS reveals the presence of multiple conformers for the dimer, tetramer, and hexamer that precede the Cu(II)-induced amyloid assembly process, results which are distinct from β2m oligomers formed at low pH. Experimental and computational results indicate that the predominant dimer is a Cu(II)-bound structure with an antiparallel side-by-side configuration. In contrast, tetramers exist in solution in both Cu(II)-bound and Cu(II)-free forms. Selective depletion of Cu(II)-bound species results in two primary conformers-one that is compact and another that is more expanded. Molecular modeling and molecular dynamics simulations identify models for these two tetrameric conformers with unique interactions and interfaces that enthalpically compensate for the loss of Cu(II). Unlike with other amyloid systems in which conformational heterogeneity is often associated with different amyloid morphologies or off-pathway events, conformational heterogeneity in the tetramer seems to be a necessary aspect of Cu(II)-induced amyloid formation by β2m. Moreover, the Cu(II)-free models represent a new advance in our understanding of Cu(II) release in Cu(II)-induced amyloid formation, laying a foundation for further mechanistic studies as well as development of new inhibition strategies., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The adsorption of amyloidogenic peptides, amyloid beta 1-40 (Aβ 1-40 ), alpha-synuclein (α-syn), and beta 2 microglobulin (β2m), was attempted over the surface of nano-gold colloidal particles, ranging from d = 10 to 100 nm in diameter ( d ). The spectroscopic inspection between pH 2 and pH 12 successfully extracted the critical pH point (pH o ) at which the color change of the amyloidogenic peptide-coated nano-gold colloids occurred due to aggregation of the nano-gold colloids. The change in surface property caused by the degree of peptide coverage was hypothesized to reflect the ΔpH o , which is the difference in pH o between bare gold colloids and peptide coated gold colloids. The coverage ratio (Θ) for all amyloidogenic peptides over gold colloid of different sizes was extracted by assuming Θ = 0 at ΔpH o = 0. Remarkably, Θ was found to have a nano-gold colloidal size dependence, however, this nano-size dependence was not simply correlated with d . The geometric analysis and simulation of reproducing Θ was conducted by assuming a prolate shape of all amyloidogenic peptides. The simulation concluded that a spiking-out orientation of a prolate was required in order to reproduce the extracted Θ. The involvement of a secondary layer was suggested; this secondary layer was considered to be due to the networking of the peptides. An extracted average distance of networking between adjacent gold colloids supports the binding of peptides as if they are "entangled" and enclosed in an interfacial distance that was found to be approximately 2 nm. The complex nano-size dependence of Θ was explained by available spacing between adjacent prolates. When the secondary layer was formed, Aβ 1-40 and α-syn possessed a higher affinity to a partially negative nano-gold colloidal surface. However, β2m peptides tend to interact with each other. This difference was explained by the difference in partial charge distribution over a monomer. Both Aβ 1-40 and α-syn are considered to have a partial charge (especially δ+) distribution centering around the prolate axis. The β2m, however, possesses a distorted charge distribution. For a lower Θ (i.e., Θ <0.5), a prolate was assumed to conduct a gyration motion, maintaining the spiking-out orientation to fill in the unoccupied space with a tilting angle ranging between 5° and 58° depending on the nano-scale and peptide coated to the gold colloid.

Flavonoids are polyphenol compounds abundantly found in plants and reported to have an inhibitory effect on amyloid fibrillation. The number and position of hydroxyl groups, as well as the arrangement of flavonoids rings, may influence their inhibitory effects. In this study, we investigate the effect of structural characteristics of flavonoids on amyloid fibril formation. For this purpose, five compounds (i.e., biochanin A, daidzein, quercetin, chrysin and fisetin) were selected that represent a variety in the number and position of their hydroxyl groups. The inhibitory effect of these flavonoids on the amyloid fibril formation of apo-carbonic anhydrase (apo-BCA), as a model protein, was evaluated using thioflavin T and transmission electron microscopy. The results showed that fisetin possessed the most significant inhibitory effect. Interestingly, upon apo-BCA acetylation, none of the tested flavonoids could inhibit the fibrillation process, which indicates that the interactions of these compounds with the amine groups of lysine residues could be somewhat important.

In order to investigate the early phase of the amyloid formation by the short amyloidogenic octapeptide sequence ('NFGAILSS') derived from IAPP, we carried out a 100ns all-atom molecular dynamics (MD) simulations of systems that contain 27 peptides and over 30,000 water molecules. The large-scale calculations were performed for the wild type sequence and seven alanine-scanned sequences using AMBER 8.0 on RIKEN's special purpose MD-GRAPE3 supercomputer, using the all-atom point charge force field ff99, which do not favor β-structures. Large peptide clusters (size 18-26 mers) were observed for all simulations, and our calculations indicated that isoleucine at position 5 played important role in the formation of β-rich clusters. In the oligomeric state, the wild type and the S7A sequences had the highest β-structure content (~14%), as calculated by DSSP, in line with experimental observations, whereas I5A and G3A had the highest helical content (~20%). Importantly, the β-structure preferences of wild type IAPP originate from its association into clusters and are not intrinsic to its sequence. Altogether, the results of this first large-scale, multi-peptide all-atom molecular dynamics simulation appear to provide insights into the mechanism of amyloidogenic and non-amyloidogenic oligomers that mainly corroborate previous experimental observations.

Curcumin is a natural product with multiple biological activities and numerous potential therapeutic applications. In present study, the influence of curcumin and its degradation products (DPs) on the amyloid aggregation of Tau protein and the related PHF6 peptide were investigated. We provided experimental/theoretical evidence for suppressing effects of the compounds on the amyloid formation using far-UV CD as well as AFM, XRD and docking techniques and showed that the parent curcumin displayed stronger inhibition effect against Tau fibril aggregation. The obtained results suggest that the curcumin/DPs binding sites on the Tau molecule are likely to be the same, and provide a good structural basis to explain the efficient aggregation suppressing behavior of the curcumin, compared to the DPs. So, developing more stable curcumin nanoparticle formulations with improved curcumin bioavailability are of great importance. Curcumin's multi-functionality is also highly significant for the therapeutic application of this natural compound against various human diseases., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Amyloid fibrils are polymers formed by proteins under specific conditions and in many cases they are related to pathogenesis, such as Parkinson's and Alzheimer's diseases. Their hallmark is the presence of a β-sheet structure. High resolution structural data on these systems as well as information gathered from multiple complementary analytical techniques is needed, from both a fundamental and a pharmaceutical perspective. Here, a previously reported de novo designed, pH-switchable coiled coil-based peptide that undergoes structural transitions resulting in fibril formation under physiological conditions has been exhaustively characterized by transmission electron microscopy (TEM), cryo-TEM, atomic force microscopy (AFM), wide-angle X-ray scattering (WAXS) and solid-state NMR (ssNMR). Overall, a unique 2-dimensional carpet-like assembly composed of large coexisiting ribbon-like, tubular and funnel-like structures with a clearly resolved protofilament substructure is observed. Whereas electron microscopy and scattering data point somewhat more to a hairpin model of β-fibrils, ssNMR data obtained from samples with selectively labelled peptides are in agreement with both, hairpin structures and linear arrangements., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Amyloid-like aggregation of natural proteins or polypeptides is an important process involved in many human diseases as well as some normal biological functions. Plenty of works have been done on this ubiquitous phenomenon, but the molecular mechanism of amyloid-like aggregation has not been fully understood yet. In this study, we showed that a series of designer bolaamphiphilic peptides could undergo amyloid-like aggregation even though they didn't possess typical β-sheet secondary structure. Through systematic amino acid substitution, we found that for the self-assembling ability, the number and species of amino acid in hydrophobic section could be variable as long as enough hydrophobic interaction is provided, while different polar amino acids as the hydrophilic heads could change the self-assembling nanostructures with their aggregating behaviors affected by pH value change. Based on these results, novel self-assembling models and aggregating mechanisms were proposed, which might provide new insight into the molecular basis of amyloid-like aggregation., (Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.)

Glycation induced advanced glycation end products (AGEs) of proteins formed as a result of Maillard reaction is currently at the heart of a number of pathological conditions. The formation of chemically stable AGEs can permanently alter protein structure and function; hence can serve as an implication in long term complications. Cystatins with high amyloidogenic inclination are implicated in various diseases including cancer and neurodegenerative conditions. The aggregates of cystatin purified from caprine brain have been studied on addition of glucose and ribose using UV absorption, fluorescence emission, circular dichroism (CD) spectroscopy and transmission electron microscopy (TEM). In the present study AGEs have been monitored and characterized. CBC was incubated for varying time intervals up to 41days in the presence of 17 and 100mM each of glucose and ribose. Ribose at both the concentrations was found to be more potent glycating agent as compared to glucose at these concentrations which is evident from UV and fluorescence spectroscopic studies. Altered intrinsic and high ANS fluorescence for 100mM and 17mM sugar concentrations respectively, suggested the existence of molten globule state of CBC. Glycated CBC as AGEs and aggregates were observed on day 27 and 41 respectively. Formation of AGEs was confirmed by employing AGEs specific fluorescence studies. CBC aggregates confirmed the presence of β-sheet structure as shown by far-UV CD, dye binding assay and transmission electron microscopy (TEM). Current study is of immense importance as cystatin is a potential candidate of amyloidogenic tendency and a potent endogenous regulator of thiol proteases; hence serves to be an attractive model to study amyloidogenesis of brain cysteine protease inhibitor., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The diphenyl-pyrazole compound anle138b is a known inhibitor of oligomeric aggregate formation in vitro and in vivo. Therefore, anle138b is considered a promising drug candidate to beneficially interfere with neurodegenerative processes causing devastating pathologies in humans. The atomistic details of the aggregation inhibition mechanism, however, are to date unknown since the ensemble of small nonfibrillar aggregates is structurally heterogeneous and inaccessible to direct structural characterization. Here, we set out to elucidate anle138b's mode of action using all-atom molecular dynamics simulations on the multi-microsecond time scale. By comparing simulations of dimeric to tetrameric aggregates from fragments of four amyloidogenic proteins (Aβ, hTau40, hIAPP, and Sup35N) in the presence and absence of anle138b, we show that the compound reduces the overall number of intermolecular hydrogen bonds, disfavors the sampling of the aggregated state, and remodels the conformational distributions within the small oligomeric peptide aggregates. Most notably, anle138b preferentially interacts with the disordered structure ensemble via its pyrazole moiety, thereby effectively blocking interpeptide main chain interactions and impeding the spontaneous formation of ordered β-sheet structures, in particular those with out-of-register antiparallel β-strands. The structurally very similar compound anle234b was previously identified as inactive by in vitro experiments. Here, we show that anle234b has no significant effect on the aggregation process in terms of reducing the β-structure content. Moreover, we demonstrate that the hydrogen bonding capabilities are autoinhibited due to steric effects imposed by the molecular geometry of anle234b and thereby indirectly confirm the proposed inhibitory mechanism of anle138b. We anticipate that the prominent binding of anle138b to partially disordered and dynamical aggregate structures is a generic basis for anle138b's ability to suppress toxic oligomer formation in a wide range of amyloidogenic peptides and proteins.

Amyloid fibril formation is associated with numerous neurodegenerative diseases. To elucidate the mechanism of fibril formation, the thioflavin T (ThT) fluorescence assay is widely used. ThT is a fluorescent dye that selectively binds to amyloid fibrils and exhibits fluorescence enhancement, which enables quantitative analysis of the fibril formation process. However, the detailed binding mechanism has remained unclear. Here we acquire real-time profiles of fibril formation of superoxide dismutase 1 (SOD1) using high-sensitivity Rheo-NMR spectroscopy and detect weak and strong interactions between ThT and SOD1 fibrils in a time-dependent manner. Real-time information on the interaction between ThT and fibrils will contribute to the understanding of the binding mechanism of ThT to fibrils. In addition, our method provides an alternative way to analyze fibril formation., Competing Interests: The authors declare no conflict of interest.

We describe a novel approach to produce conformational monoclonal antibodies selected to specifically react with the β-sheet secondary structure of pathological oligomeric conformers, characteristic of many neurodegenerative diseases. Contrary to past and current efforts, we utilize a mammalian non-self-antigen as an immunogen. The small, non-self peptide selected was covalently polymerized with glutaraldehyde until it reached a high β-sheet secondary structure content, and species between 10-100kDa that are immunogenic, stable and soluble (p13Bri). Inoculation of p13Bri in mice elicited antibodies to the peptide and the β-sheet secondary structure conformation. Hybridomas were produced and clones selected for their reactivity with at least two different oligomeric conformers from Alzheimer's, Parkinson and/or Prion diseases. The resulting conformational monoclonals are able to detect pathological oligomeric forms in different human neurodegenerative diseases by ELISA, immunohistochemistry and immunoblots. This technological approach may be useful to develop tools for detection, monitoring and treatment of multiple misfolding disorders.

Peptides derived from apolipoprotein A-I (apoA-I), the main component of high-density lipoprotein (HDL), constitute the main component of amyloid deposits that colocalize with atherosclerotic plaques. Here we investigate the molecular details of full-length, lipid-deprived apoA-I after assembly into insoluble aggregates under physiologically relevant conditions known to induce aggregation in vitro. Unmodified apoA-I is shown to remain soluble at pH 7 for at least 3 days, retaining its native α-helical-rich structure. Upon acidification to pH 4, apoA-I rapidly assembles into insoluble nonfibrillar aggregates lacking the characteristic cross-β features of amyloid. In the presence of heparin, the rate and thioflavin T responsiveness of the aggregates formed at pH 4 increase and short amyloid-like fibrils are observed, which give rise to amyloid-characteristic X-ray reflections at 4.7 and 10 Å. Solid-state nuclear magnetic resonance (SSNMR) and synchrotron radiation circular dichroism spectroscopy of fibrils formed in the presence of heparin show they retain some α-helical characteristics together with new β-sheet structures. Interestingly, SSNMR indicates a similar molecular structure of aggregates formed in the absence of heparin at pH 6 after oxidation of the three methionine residues, although their morphology is rather different from that of the heparin-derived fibrils. We propose a model for apoA-I aggregation in which perturbations of a four-helix bundle-like structure, induced by interactions of heparin or methionine oxidation, cause the partially helical N-terminal residues to disengage from the remaining, intact helices, thereby allowing self-assembly via β-strand associations.

There are different insulin analogues with various pharmacokinetic characteristics, such as, rapid-acting, long-acting, or intermediate-acting analogues. Since insulin tends to form amyloid aggregates, it is of particular interest to measure characteristic times of formation of amyloid aggregates and compare those to action times for insulin and its analogues. For the study we have chosen one of the insulin analogues - insulin Lispro, which is a fast acting insulin analog. It is usually thought of amyloid aggregation as a nucleation-dependent process. We have estimated the size of the primary nucleus to be one monomer and the size of the secondary nucleus to be around zero in both insulin and Lispro insulin aggregation processes. The main structural element of insulin and Lispro insulin amyloid fibrils is a rounded ring oligomer of about 6-7 nm in diameter, about 2-3 nm in height and about 2 nm in diameter of the hole. Fibrils of several μm in length are produced due to interaction of such oligomers. The packing of ring oligomers in fibrils differs because of the difference in their orderliness. Though the initial stages of fibril formation (monomer, oligomer) are similar, the further process depends on the unique sequence of each peptide. Namely the sequence affects the final morphology of mature amyloids. These observations allow us to conclude that formation of fibrils by short peptides occurs via and by means of oligomer ring structures. Such an important issue as the nature of polymorphism of insulin amyloid fibrils has been settled by us. The role of early oligomeric aggregates in such processes as nucleation and aggregation of amyloid fibrils has been examined.

Intrinsically Disordered Proteins (IDPs) are fundamental actors of biological processes. Their inherent plasticity facilitates very specialized tasks in cell regulation and signalling, and their malfunction is linked to severe pathologies. Understanding the functional role of disorder requires the structural characterization of IDPs and the complexes they form. Small-angle Scattering of X-rays (SAXS) and Neutrons (SANS) have notably contributed to this structural understanding. In this review we summarize the most relevant developments in the field of SAS studies of disordered proteins. Emphasis is given to ensemble methods and how SAS data can be combined with computational approaches or other biophysical information such as NMR. The unique capabilities of SAS enable its application to extremely challenging disordered systems such as low-complexity regions, amyloidogenic proteins and transient biomolecular complexes. This reinforces the fundamental role of SAS in the structural and dynamic characterization of this elusive family of proteins.

We performed a comparative study of the process of amyloid formation by short homologous peptides with a substitution of aspartate for glutamate in position 2 - VDSWNVLVAG (AspNB) and VESWNVLVAG (GluNB) - with unblocked termini. Peptide AspNB (residues 166-175) corresponded to the predicted amyloidogenic region of the protein glucantransferase Bgl2 from the Saccharomyces cerevisiae cell wall. The process of amyloid formation was monitored by fluorescence spectroscopy (FS), electron microscopy (EM), tandem mass spectrometry (TMS), and X-ray diffraction (XD) methods. The experimental study at pH3.0 revealed formation of amyloid fibrils with similar morphology for both peptides. Moreover, we found that the morphology of fibrils made of untreated ammonia peptide is not mentioned in the literature. This morphology resembles snakes lying side by side in the form of a wave without intertwining. Irrespective of the way of the peptide preparation, the rate of fibril formation is higher for AspNB than for GluNB. However, preliminary treatment with ammonia highly affected fibril morphology especially for AspNB. Such treatment allowed us to obtain a lag period during the process of amyloid formation. It showed that the process was nucleation-dependent. With or without treatment, amyloid fibrils consisted of ring-like oligomers with the diameter of about 6nm packed either directly ring-to-ring or ring-on-ring with a slight shift. We also proposed the molecular structure of amyloid fibrils for two studied peptides., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Amyloid aggregates display striking features of detergent stability and self-seeding. Human serum albumin (HSA), a preferred drug-carrier molecule, can also aggregate in vitro. So far, key amyloid properties of stability against ionic detergents and self-seeding, are unclear for HSA aggregates. Precautions against amyloid contamination would be required if HSA aggregates were self-seeding. Here, we show that HSA aggregates display detergent sarkosyl stability and have self-seeding potential. HSA dimer is preferable for clinical applications due to its longer retention in circulation and lesser oedema owing to its larger molecular size. Here, HSA was homodimerized via free cysteine-34, without any potentially immunogenic cross-linkers that are usually pre-requisite for homodimerization. Alike the monomer, HSA dimers also aggregated as amyloid, necessitating precautions while using for therapeutics., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

A major component of ex vivo amyloid plaques of patients with dialysis-related amyloidosis (DRA) is a cleaved variant of β2-microglobulin (ΔN6) lacking the first six N-terminal residues. Here we perform a computational study on ΔN6, which provides clues to understand the amyloidogenicity of the full-length β2-microglobulin. Contrary to the wild-type form, ΔN6 is able to efficiently nucleate fibrillogenesis in vitro at physiological pH. This behavior is enhanced by a mild acidification of the medium such as that occurring in the synovial fluid of DRA patients. Results reported in this work, based on molecular simulations, indicate that deletion of the N-terminal hexapeptide triggers the formation of an intermediate state for folding and aggregation with an unstructured strand A and a native-like core. Strand A plays a pivotal role in aggregation by acting as a sticky hook in dimer assembly. This study further predicts that the detachment of strand A from the core is maximized at pH 6.2 resulting into higher aggregation efficiency. The structural mapping of the dimerization interface suggests that Tyr10, His13, Phe30 and His84 are hot-spot residues in ΔN6 amyloidogenesis.

Self-assembly of amyloid nanofiber is associated with both functional biological and pathological processes such as those in neurodegenerative diseases. Despite intensive studies, the stochastic nature of the process has made it difficult to elucidate a molecular mechanism for the key amyloid nucleation event. Here we investigated nucleation of the silk-elastin-like peptide (SELP) amyloid using time-lapse lateral force microscopy (LFM). By repeated scanning of a single line on a SELP-coated mica surface, we observed a sudden stepwise height increase. This corresponds to nucleation of an amyloid fiber, which subsequently grew perpendicular to the scanning direction. The lateral force profiles followed either a worm-like chain model or an exponential function, suggesting that the atomic force microscopy (AFM) tip stretches a single or multiple SELP molecules along the scanning direction. The probability of nucleation correlated with the maximum stretching force and extension, implying that stretching of SELP molecules is a key molecular event for amyloid nucleation. The mechanically induced nucleation allows for positional and directional control of amyloid assembly in vitro, which we demonstrate by generating single nanofibers at predetermined nucleation sites.

Recent studies revealed that elk-like S170N/N174T mutation in mouse prion protein (moPrP), which results in an increased rigidity of β2-α2 loop, leads to a prion disease in transgenic mice. Here we characterized the effect of this mutation on biophysical properties of moPrP. Despite similar thermodynamic stabilities of wild type and mutant proteins, the latter was found to have markedly higher propensity to form amyloid fibrils. Importantly, this effect was observed even under fully denaturing conditions, indicating that the increased conversion propensity of the mutant protein is not due to loop rigidity but rather results from greater amyloidogenic potential of the amino acid sequence within the loop region of S170N/N174T moPrP., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

A surprising "upright" fibrilar conformation (with a height of ~2.6 nm) was observed with in situ atomic force microscopy (AFM) for an amyloid-like peptide (NH(2)-VGGAVVAV-COHN(2)) on mica surface, which is very different from its "flat" conformation (with a much smaller height of ~0.9 nm) on the HOPG surface. Our all-atom molecular dynamics (MD) simulations reveal that it is the strong electrostatic interactions between the N-terminus of the peptide and the mica surface that result in an upright conformation and a highly ordered β-stranded structure on mica, with a height of 2.5 ± 0.1 nm, consistent with the AFM experiment. Similarly, our MD simulations show that the same peptides adopt a flat conformation on HOPG surfaces due to the favorable hydrophobic interactions with HOPG. Our simulations also indicate that epitaxial patterns found in mica are preferentially controlled by anisotropic binding sites commensurate with the inherent crystallographic unit cell of the basal substrate.

Bacterial and fungal species produce some of the best-characterized functional amyloids, that is, extracellular fibres that play key roles in mediating adhesion and biofilm formation. Yet, the molecular details underlying their mechanical strength remain poorly understood. Here, we use single-molecule atomic force microscopy to measure the mechanical properties of amyloids formed by Als cell adhesion proteins from the pathogen Candida albicans. We show that stretching Als proteins through their amyloid sequence yields characteristic force signatures corresponding to the mechanical unzipping of β-sheet interactions formed between surface-arrayed Als proteins. The unzipping probability increases with contact time, reflecting the time necessary for optimal inter β-strand associations. These results demonstrate that amyloid interactions provide cohesive strength to a major adhesion protein from a microbial pathogen, thereby strengthening cell adhesion. We suggest that such functional amyloids may represent a generic mechanism for providing mechanical strength to cell adhesion proteins. In nanotechnology, these single-molecule manipulation experiments provide new opportunities to understand the molecular mechanisms driving the cohesion of functional amyloid-based nanostructures.

Amyloid nanofibers derived from hen egg white lysozyme were processed into macroscopic fibers in a wet-spinning process based on interfacial polyion complexation using a polyanionic polysaccharide as cross-linker. As a result of their amyloid nanostructure, the hierarchically self-assembled protein fibers have a stiffness of up to 14 GPa and a tensile strength of up to 326 MPa. Fine-tuning of the polyelectrolytic interactions via pH allows to trigger the release of small molecules, as demonstrated with riboflavin-5'-phophate. The amyloid fibrils, highly oriented within the gellan gum matrix, were mineralized with calcium phosphate, mimicking the fibrolamellar structure of bone. The formed mineral crystals are highly oriented along the nanofibers, thus resulting in a 9-fold increase in fiber stiffness.