Β-sheet Aggregates Research Articles

The Formation Mechanism of Zein Gel Induced by Amyloid-like Fibrils

The preparation of zein gels often leads to unstable product quality due to poor reproducibility, posing substantial challenges for the food industry and biomedical applications. Thus, it is essential to study the gelation mechanism and key factors responsible for gelation. Herein, we report four types of assembled structures from zein solutions in binary ethanol–water solvents with either a high concentration (30% w/v) and a low concentration (5% w/v), namely, spheres, spheres with tangential structures, amyloid-like fibrils, and sub-agglomerates with crystal structures. The tangential structure at the edges of the spheres was first observed by transmission electron microscopy (TEM) at the initial stage, offering a model for studying protein folding and aggregation mechanisms. By observing the gelation processes with TEM for morphology, cryo-TEM to confirm structure preservation, HR-TEM for fine details, and negatively stained TEM to reveal hidden features, along with small-angle X-ray scattering (SAXS), we identified two types of gels: one was mainly network structures formed by sphere–sphere fusion, which was thermoreversible, and the other one was irreversible gel due to the increase in the amount of amyloid-like fibrils. Results show that the existence of thermoreversible zein gels indicates potential effective regulation tools of zein gel properties. In addition, amyloid-like fibrils were first isolated from native zein. During amyloid-like fibril formation, the number of tangential structures increased considerably, which were composed of thinner fibrils. High-resolution TEM showed a substructure with a 0.35 nm interplanar spacing, providing evidence of β-sheet aggregates also as confirmed by SAXS. This unique substructure has not been reported in other edible proteins. Moreover, understanding the fibrillation mechanism of zein provides a basis for controlling and developing novel food structures for food protein function and offers insights into neurodegenerative diseases.

Hydrophobic C-Terminal Peptide Analog Aβ31-41 Protects the Neurons from Aβ-Induced Toxicity.

Spontaneous aggregation of amyloid beta (Aβ) leads to the formation of neurotoxic senile plaque considered as the most crucial event in Alzheimer's disease (AD) progression. Inhibition or disruption of this deadly aggregate formation is one of the most efficient strategies for the development of potential therapeutics, and extensive research is in progress by various research groups. In this direction, the development of a peptide analogous to that of the native Aβ peptide is an attractive strategy. Based on this rationale, β-sheet breakers were developed from the Aβ central hydrophobic core. These peptide derivatives will bind to the full length of the parent Aβ and interfere in self-recognition, thereby preventing the folding of the Aβ peptide into cross β-sheet neurotoxic aggregates. However, this approach is effective in the inhibition of fibrillar aggregation, but this strategy is ineffective in the Aβ neurotoxic oligomer formation. Therefore, an alternative and efficient approach is to use the Aβ peptide analogous to the C-terminal region, which arbitrates fibrillation and oligomerization. Herein, we developed the Aβ C-terminal fragment (ACT-1 to ACT-7) for inhibition of oligomerization as well as fibrillar aggregation. Screening of these seven peptides resulted in an efficient anti-Aβ peptide aggregative agent (ACT-7), which was evaluated by the ThT assay peptide. The ThT assay reveals complete inhibition and showed significant neuroprotection of PC-12-derived neurons from Aβ-induced toxicity and reduced cell apoptosis. Further, analysis using CD and FTIR spectroscopy reveals that the ACT-7 peptide efficiently inhibits the formation of the β-sheet secondary structure content. HR-TEM microscopic analysis confirmed the inhibition of formation. Therefore, the inhibition of β-sheet Aβ fibrillary aggregation by the protease-stable ACT-7 peptide may provide a beneficial effect on AD treatment to control the Aβ aggregates. Finally, we anticipate that our newly designed ACT peptides may also assist as a template molecular scaffold for designing potential anti-AD therapeutics.

Therapeutic potential of Coumarin-polyphenolic acid hybrids in PD: Inhibition of α-Syn aggregation and disaggregation of preformed fibrils, leading to reduced neuronal inclusion formation

This study focuses on the discovery of new potential drugs for treating PD by targeting the aggregation of α-Syn. A series of hybrids combining Coumarin and phenolic acid were designed and synthesized. Four particularly promising compounds were identified, showing strong inhibitory effects with IC50 values ranging from low micromolar to submicromolar concentrations, as low as 0.63 μM. These compounds exhibited a higher binding affinity to α-Syn residues and effectively hindered the entire aggregation process, maintaining the proteostasis conformation of α-Syn and preventing the formation of β-sheet aggregates. This approach holds significant promise for PD prevention. Additionally, these candidate compounds demonstrated the ability to break down preformed α-Syn oligomers and fibrils, resulting in the formation of smaller aggregates and monomers. Moreover, the candidate compounds showed impressive effectiveness in inhibiting α-Syn aggregation within nerve cells, thereby reducing the likelihood of α-Syn inclusion formation resembling Lewy bodies, which highlights their potential for treating PD.

The potential of Rhein's aromatic amines for Parkinson's disease prevention and treatment: α-Synuclein aggregation inhibition and disaggregation of preformed fibers

The aggregation of α-Syn is a pivotal mechanism in Parkinson's disease (PD). Effectively maintaining α-Syn proteostasis involves both inhibiting its aggregation and promoting disaggregation. In this study, we developed a series of aromatic amide derivatives based on Rhein. Two of these compounds, 4,5-dihydroxy-N-(3-hydroxyphenyl)-9,10-dioxo-9,10-dihydroanthracene-2-carboxamide (a5) and 4,5-dihydroxy-N-(2-hydroxy-4-chlorophenyl)-9,10-dioxo-9,10-dihydroanthracene-2-carboxamide (a8), exhibited good binding affinities to α-Syn residues, demonstrating promising inhibitory activity against α-Syn aggregation in vitro, with low IC50 values (1.35 and 1.08 μM, respectivly). These inhibitors acted throughout the entire aggregation process by stabilizing α-Syn's conformation and preventing the formation of β-sheet aggregates. They also effectively disassembled preformed α-Syn oligomers and fibrils. Preliminary mechanistic insights indicated that they bound to the specific domain within fibrils, inducing fibril instability, collapse, and the formation of smaller aggregates and monomeric α-Syn units. This research underscores the therapeutic potential of Rhein's aromatic amides in targeting α-Syn aggregation for PD treatment and suggests broader applications in managing and preventing neurodegenerative diseases.

Membrane-induced tau amyloid fibrils

The intrinsically disordered protein tau aggregates into β-sheet amyloid fibrils that spread in human brains afflicted with Alzheimer’s disease and other neurodegenerative diseases. Tau interaction with lipid membranes might play a role in the formation and spreading of these pathological aggregates. Here we investigate the conformation and assembly of membrane-induced tau aggregates using solid-state NMR and transmission electron microscopy. A tau construct that encompasses the microtubule-binding repeats and a proline-rich domain is reconstituted into cholesterol-containing phospholipid membranes. 2D 13C-13C correlation spectra indicate that tau converted from a random coil to a β-sheet conformation over weeks. Small unilamellar vesicles (SUVs) cause different equilibrium conformations from large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs). Importantly, SUV-bound tau developed long fibrils that exhibit the characteristic β-sheet chemical shifts of Tyr310 in heparin-fibrillized tau. In comparison, LUVs and MLVs do not induce fibrils but cause different β-sheet aggregates. Lipid-protein correlation spectra indicate that these tau aggregates reside at the membrane-water interface, without inserting into the middle of the lipid bilayer. Removal of cholesterol from the SUVs abolished the fibrils, indicating that both membrane curvature and cholesterol are required for tau fibril formation. These results have implications for how lipid membranes might nucleate tau aggregates.

A peptide rich in glycine-serine-alanine repeats ameliorates Alzheimer-type neurodegeneration.

Repeated amino acid sequences in proteins are widely found, and the glycine-serine-alanine repeat is an element with a general propensity to form β-sheet aggregates as found in key pathological factors, in several neurodegenerative diseases. Such properties of this repeat may guide development of disease-modifying therapies for neurodegenerative disease. However, details of its role and underlying mechanism(s) remain largely unknown. Actions of specific glycine-serine-alanine repeat peptides (SNPs), especially SNP-9, on Alzheimer's disease (AD)-like abnormalities were evaluated in transgenic mice and Caenorhabditis elegans, andin rat and cell models. Entry of SNPs into the brain, SNP activity in neuronal cells and peptide entry into cells were analysed in vivo and in vitro. Cell-free systems and the yeast two-hybrid system were also used to explore possible targets of SNP-9, and interactions of potential targets with SNP-9 were confirmed in cell-based systems. We first identified SNP-9 as a potent neuroprotective peptide with the activity to decrease oligomeric amyloid β (Aβ) via co-assembling with the toxic Aβ oligomer to form hetero-oligomers. Also, calcyclin-binding protein was found to act as a SNP-9-binding protein, by screening of a human brain cDNA library. Such binding showed that SNP-9 could regulate the abnormal hyperphosphorylation of tau via calcyclin-bindingprotein. Our study provides a foundation for development of SNPs, especially SNP-9, as potential therapeutic interventions for AD. We propose SNP-9 as a potential therapeutic agent for the treatment of AD.

Atomistic Insights into the Inhibitory Mechanism of Tyrosine Phosphorylation against the Aggregation of Human Tau Fragment PHF6.

Abnormal aggregation of the microtubule-associated protein tau into intracellular fibrillary inclusions is characterized as the hallmark of tauopathies, including Alzheimer's disease and chronic traumatic encephalopathy. The hexapeptide 306VQIVYK311 (PHF6) of R3 plays an important role in the aggregation of tau. Recent experimental studies reported that phosphorylation of residue tyrosine 310 (Y310) could decrease the propensity of PHF6 to form fibrils and inhibit tau aggregation. However, the underlying inhibitory mechanism is not well understood. In this work, we systematically investigated the influences of phosphorylation on the conformational ensembles and oligomerization dynamics of PHF6 by performing extensive all-atom molecular dynamics (MD) simulations. Our replica exchange MD simulations demonstrate that Y310 phosphorylation could effectively suppress the formation of β-structure and shift PHF6 oligomers toward coil-rich aggregates. The interaction analyses show that hydrogen bonding and hydrophobic interactions among PHF6 peptides, as well as Y310-Y310 π-π stacking and I308-Y310 CH-π interactions, are weakened by phosphorylation. Additional microsecond MD simulations show that Y310 phosphorylation could inhibit the oligomerization of PHF6 by preventing the formation of large β-sheet oligomers and multi-layer β-sheet aggregates. This study provides mechanistic insights into the phosphorylation-inhibited tau aggregation, which may be helpful for the in-depth understanding of the pathogenesis of tauopathies.

Design of aggregation-induced emission-active fluorogen-based nanoparticles for imaging and scavenging Alzheimer's β-amyloid by photo-oxygenation.

Photo-oxygenation has emerged as an effective modality for scavenging Alzheimer's amyloid-β (Aβ) plaques. However, limitations of the current photo-oxidants, such as low Aβ-targeting and single functionality, hinder the scavenging of Aβ plaques via photo-oxygenation. Herein, based on an aggregation-induced emission (AIE)-active fluorogen (named TPMD), we designed AIE photo-oxidant nanoparticles (T-LD NPs) for Aβ imaging, inhibition, and disaggregation. The T-LD NPs were prepared by the assembly of hydrophobic TPMD with an Aβ-targeting peptide (LPPFD, L) conjugated amphiphilic polymer (DSPE-PEG). Such T-LD NPs could specifically label Aβ plaques for image-guided therapy. Under laser irradiation, T-LD NPs generated a plethora of reactive oxygen species (ROS), including 1O2, ˙OH, and O2˙-, to oxygenate Aβ species, leading to the potent inhibition of Aβ fibrillization, and significant alleviation of Aβ-mediated neurotoxicity (36% to 10% at 20 μg mL-1). Notably, T-LD NPs could rapidly disaggregate mature Aβ fibrils into fractured β-sheet rich aggregates via photo-oxygenation, resulting in alleviated cytotoxicity. In vivo studies revealed that the photo-activated T-LD NPs scavenged amyloid plaques in the transgenic C. elegans strain CL2006 and extended the lifespan by 4 days. Taken together, this multifunctional T-LD NP integrated Aβ-targeting, near-infrared fluorescence imaging, and photo-oxygenation, provides a new strategy for the development of multifunctional AIE photo-oxidants for the treatment of Alzheimer's disease.

Compact Luminol Chemiluminophores for In Vivo Detection and Imaging of β-Sheet Protein Aggregates.

Protein aggregation has been found in a wide range of neurodegenerative protein-misfolding diseases. The demand for in vivo technologies to identify protein aggregation is at the leading edge for the pathogenic study, diagnostic development, and therapeutic intervention of these devastating disorders. Herein, we report a series of luminol analogues to construct a facile chemiluminescence (CL)-based approach for in vivo detection and imaging of β-sheet protein aggregates. The synthesized compounds exhibited a distinct chemiluminescent response with long emission wavelengths toward reactive oxygen species under physiological conditions and displayed signal amplification in the presence of β-sheet protein aggregates, including α-synuclein, β-amyloid, and tau. Among them, CyLumi-3 was further evaluated as a chemiluminescent probe in preclinical models. By intravenous administration into the model mice via the tail vein, in vivo CL imaging noninvasively detected the specific CL of the probe targeting the α-synuclein aggregates in the brains of living mice. Based on its structural characteristics, CyLumi-3 can readily interact with α-synuclein aggregates with significantly enhanced fluorescence and can identify α-synuclein aggregates in vivo via distinctive CL amplification, which could pave the way for a more comprehensive understanding of protein aggregation in preclinical studies and would provide new hints for developing small-molecule chemiluminophores for protein aggregates.

Probing the origin of prion protein misfolding via reconstruction of ancestral proteins.

Prion diseases are fatal neurodegenerative diseases caused by pathogenic misfolding of the prion protein, PrP. They are transmissible between hosts, and sometimes between different species, as with transmission of bovine spongiform encephalopathy to humans. Although PrP is found in a wide range of vertebrates, prion diseases are seen only in certain mammals, suggesting that infectious misfolding was a recent evolutionary development. To explore when PrP acquired the ability to misfold infectiously, we reconstructed the sequences of ancestral versions of PrP from the last common primate, primate-rodent, artiodactyl, placental, bird, and amniote. Recombinant ancestral PrPs were then tested for their ability to form β-sheet aggregates, either spontaneously or when seeded with infectious prion strains from human, cervid, or rodent species. The ability to aggregate developed after the oldest ancestor (last common amniote), and aggregation capabilities diverged along evolutionary pathways consistent with modern-day susceptibilities. Ancestral bird PrP could not be seeded with modern-day prions, just as modern-day birds are resistant to prion disease. Computational modeling of structures suggested that differences in helix 2 could account for the resistance of ancestral bird PrP to seeding. Interestingly, ancestral primate PrP could be converted by all prion seeds, including both human and cervid prions, raising the possibility that species descended from an ancestral primate have retained the susceptibility to conversion by cervid prions. More generally, the results suggest that susceptibility to prion disease emerged prior to ~100 million years ago, with placental mammals possibly being generally susceptible to disease.

Characterization of the Conformations of Amyloid Beta 42 in Solution That May Mediate Its Initial Hydrophobic Aggregation.

Intrinsically disordered peptides, such as amyloid β42 (Aβ42), lack a well-defined structure in solution. Aβ42 can undergo abnormal aggregation and amyloidogenesis in the brain, forming fibrillar plaques, a hallmark of Alzheimer's disease. The insoluble fibrillar forms of Aβ42 exhibit well-defined, cross β-sheet structures at the molecular level and are less toxic than the soluble, intermediate disordered oligomeric forms. However, the mechanism of initial interaction of monomers and subsequent oligomerization is not well understood. The structural disorder of Aβ42 adds to the challenges of determining the structural properties of its monomers, making it difficult to understand the underlying molecular mechanism of pathogenic aggregation. Certain regions of Aβ42 are known to exhibit helical propensity in different physiological conditions. NMR spectroscopy has shown that the Aβ42 monomer at lower pH can adopt an α-helical conformation and as the pH is increased, the peptide switches to β-sheet conformation and aggregation occurs. CD spectroscopy studies of aggregation have shown the presence of an initial spike in the amount of α-helical content at the start of aggregation. Such an increase in α-helical content suggests a mechanism wherein the peptide can expose critical non-polar residues for interaction, leading to hydrophobic aggregation with other interacting peptides. We have used molecular dynamics simulations to characterize in detail the conformational landscape of monomeric Aβ42 in solution to identify molecular properties that may mediate the early stages of oligomerization. We hypothesized that conformations with α-helical structure have a higher probability of initiating aggregation because they increase the hydrophobicity of the peptide. Although random coil conformations were found to be the most dominant, as expected, α-helical conformations are thermodynamically accessible, more so than β-sheet conformations. Importantly, for the first time α-helical conformations are observed to increase the exposure of aromatic and hydrophobic residues to the aqueous solvent, favoring their hydrophobically driven interaction with other monomers to initiate aggregation. These findings constitute a first step toward characterizing the mechanism of formation of disordered, low-order oligomers of Aβ42.

Insight into the Gel Properties of Antarctic Krill and Pacific White Shrimp Surimi Gels and the Feasibility of Polysaccharides as Texture Enhancers of Antarctic Krill Surimi Gels.

Antarctic krill is a potential and attractive resource for consumption. However, most Antarctic krill meat is used to produce primary products with low commercial value, with few highly processed products. This study aimed to evaluate and improve the gelling properties of Antarctic krill surimi, with Pacific white shrimp surimi as control. Compared with Pacific white shrimp surimi, the lower β-sheet content and protein aggregation degree had a severe impact on the formation of the gel network of Antarctic krill surimi, which resulted in weaker breaking force, gel strength, and viscoelasticity (p < 0.05). Moreover, water retention capacity and molecular forces had a positive effect on the stability of the gel matrix of shrimp surimi. Thus, the high α-helix/β-sheet ratio, weak intermolecular interactions, and low level of protein network cross-linkage were the main reasons for the poor quality of Antarctic krill surimi. On this basis, the effects of six polysaccharides on the texture properties of Antarctic krill surimi were studied. Chitosan, konjac glucomannan, sodium carboxyl methyl cellulose, and waxy maize starch resulted in no significant improvement in the texture properties of Antarctic krill surimi (p > 0.05). However, the addition of ι-carrageenan (2%) or κ-carrageenan (1~2%) is an effective way to improve the texture properties of Antarctic krill surimi (p < 0.05). These findings will contribute to the development of reconstituted Antarctic krill surimi products with high nutritional quality and the promotion of deep-processing products of Antarctic krill meat.

Coagulansin-A improves spatial memory in 5xFAD Tg mice by targeting Nrf-2/NF-κB and Bcl-2 pathway

The present study was designed to investigate the underlying molecular signaling of Coagulansin-A (Coag-A) as a therapeutic agent against Alzheimer’s disease (AD). Preliminarily, it exhibited a neuroprotective effect against H2O2-induced oxidative stress in HT-22 cells. The in vivo studies were performed by administering Coag-A (0.1, 1, and 10 mg/kg) intraperitoneally to 5xFAD transgenic (Tg) mouse model. Coag-A (10 mg/kg) significantly attenuated the cognitive decline compared to Tg mice group in the shallow water maze (SWM) and Y-maze test paradigms. The anti-aggregation potential of Coag-A was determined by performing Fourier transform-infrared (FT-IR) spectroscopy and differential scanning calorimeter (DSC) analysis in the prefrontal cortex (PFC) and hippocampal (HC) regions of mice brain. The FT-IR spectra demonstrated the inhibition of amyloid beta (Aβ) through a decrease in β-sheet aggregation, along with the inhibition of changes in the lipids, proteins, and phospholipids. The DSC analysis displayed a low-temperature exotherm associated with the reversible process of aggregation of soluble protein fractions prior to denaturation. Furthermore, Coag-A treatment displayed a regular density of granule cells in H&E stained sections, along with a reduced amyloid load and PAS-positive granules in all the regions of interest in mice brain. The real-time polymerase chain reaction (q-PCR), western blot and immunohistochemical (IHC) analysis demonstrated antioxidant, anti-inflammatory, and anti-apoptotic effect of Coag-A by enhancing the expression of nuclear factor erythroid-2-related factor (Nrf-2) and reducing nuclear factor kappa B (NF-κB) and Bax protein expression. In addition, Coag-A significantly increased the antioxidant enzymes and proteins level, along with a reduced pro-inflammatory cytokines production.

Cannabidiol protects against Alzheimer's disease in C. elegans via ROS scavenging activity of its phenolic hydroxyl groups

Recent discoveries have implicated the potential of Cannabidiol (CBD) in the prevention of Alzheimer's disease (AD). However, how CBD affects such neurodegenerative disorders remains unclear. Herein, Caenorhabditis elegans (C. elegans) was used as the model organism to elucidate the mechanism by which CBD ameliorates AD in vivo. CBD was found to alleviate the progression of Aβ-induced AD but not tau protein-induced AD or α-syn-induced Parkinson's disease. CBD inhibited the aggregation of Aβ in C. elegans. However, CBD failed to prevent the formation of β-sheet aggregation in vitro. Moreover, CBD was found to scavenge reactive oxygen species (ROS) in vivo without inducing the overexpression of antioxidative genes. In addition, CBD treatment enhanced the worm resistance to oxidative stress, which was independent of the classical transcription factors DAF-16 and SKN-1. These results supported that the in vivo antioxidative activity of CBD was most likely due to its intrinsic antioxidative property. Furthermore, the phenolic hydroxyl groups of CBD were found to be critical for scavenging ROS in vitro and in vivo, alleviating the aggregation of Aβ in vivo, and ameliorating Aβ-associated neurotoxicity. These studies show that CBD protects against AD in C. elegans via the ROS scavenging activity of its phenolic hydroxyl groups, which provides insight for further structure-activity relationship studies of CBD as an AD therapeutic.

Label-Free Infrared Spectroscopic Imaging Reveals Heterogeneity of β-Sheet Aggregates in Alzheimer's Disease.

The aggregation of the amyloid beta (Aβ) protein into plaques is a pathological feature of Alzheimer's disease (AD). While amyloid aggregates have been extensively studied in vitro, their structural aspects and associated chemistry in the brain are not fully understood. In this report, we demonstrate, using infrared spectroscopic imaging, that Aβ plaques exhibit significant heterogeneities in terms of their secondary structure and phospholipid content. We show that the capabilities of discrete frequency infrared imaging (DFIR) are ideally suited for characterization of amyloid deposits in brain tissues and employ DFIR to identify nonplaque β-sheet aggregates distributed throughout brain tissues. We further demonstrate that phospholipid-rich β-sheet deposits exist outside of plaques in all diseased tissues, indicating their potential clinical significance. This is the very first application of DFIR toward a characterization of protein aggregates in an AD brain and provides a rapid, label-free approach that allows us to uncover β-sheet heterogeneities in the AD, which may be significant for targeted therapeutic strategies in the future.

Amyloid-like aggregation of bovine serum albumin at physiological temperature induced by cross-seeding effect of HEWL amyloid aggregates

BSA can form amyloid-like aggregates in vitro at 65 °C. Heterologous amyloid can proposedly cross-seed other protein's aggregation, however, general mechanisms and driving conditions remain to be vividly elucidated. Here, we examined if pre-formed HEWL amyloid can cross-seed the aggregation of BSA at physiological temperature, 37 °C, and whether the efficacy depends on the BSA conformation. We find that at pH 3.0, 37 °C where BSA manifests exposure of abundant hydrophobic patches, HEWL amyloid efficiently drives BSA into ThT-positive, sarkosyl-resistant, β-sheet rich amyloid-like aggregates exhibiting fibrils in TEM. On the contrary, HEWL amyloid fails to cross-seed the BSA aggregation at pH 7.0, 37 °C where BSA has largely internalized hydrophobic patches. Strikingly, human lysozyme amyloid could also cross-seed human serum albumin aggregation at pH 3.0, 37 °C. Thus, heterologous amyloid cross-seeding can help overcome the energy-barrier for aggregation of other proteins that, for any reason, may have perturbed and promiscuous structural conformation at physiological temperatures.

Imaging protein aggregates in the serum and cerebrospinal fluid in Parkinson's disease.

Aggregation of α-synuclein plays a key role in the development of Parkinson’s disease. Soluble aggregates are present not only within human brain but also the CSF and blood. Characterizing the aggregates present in these biofluids may provide insights into disease mechanisms and also have potential for aiding diagnosis.We used two optical single-molecule imaging methods called aptamer DNA-PAINT and single-aggregate confocal fluorescence, together with high-resolution atomic force microscopy for specific detection and characterization of individual aggregates with intermolecular β-sheet structure, present in the CSF and serum of 15 early stage Parkinson’s disease patients compared to 10 healthy age-matched controls.We found aggregates ranging in size from 20 nm to 200 nm, in both CSF and serum. There was a difference in aggregate size distribution between Parkinson’s disease and control groups with a significantly increased number of larger aggregates (longer than 150 nm) in the serum of patients with Parkinson’s disease. To determine the chemical composition of the aggregates, we performed aptamer DNA-PAINT on serum following α-synuclein and amyloid-β immunodepletion in an independent cohort of 11 patients with early stage Parkinson’s disease and 10 control subjects. β-Sheet aggregates in the serum of Parkinson’s disease patients were found to consist of, on average, 50% α-synuclein and 50% amyloid-β in contrast to 30% α-synuclein and 70% amyloid-β in control serum [the differences in the proportion of these aggregates were statistically significant between diseased and control groups (P = 1.7 × 10−5 for each species)]. The ratio of the number of β-sheet α-synuclein aggregates to β-sheet amyloid-β aggregates in serum extracted using our super-resolution method discriminated Parkinson’s disease cases from controls with an accuracy of 98.2% (AUC = 98.2%, P = 4.3 × 10−5).Our data suggest that studying the protein aggregates present in serum can provide information about the disruption of protein homeostasis occurring in Parkinson’s disease and warrants further investigation as a potential biomarker of disease.

Exploring the Early Stages of the Amyloid Aβ(1-42) Peptide Aggregation Process: An NMR Study.

Alzheimer’s disease (AD) is a neurodegenerative pathology characterized by the presence of neurofibrillary tangles and amyloid plaques, the latter mainly composed of Aβ(1–40) and Aβ(1–42) peptides. The control of the Aβ aggregation process as a therapeutic strategy for AD has prompted the interest to investigate the conformation of the Aβ peptides, taking advantage of computational and experimental techniques. Mixtures composed of systematically different proportions of HFIP and water have been used to monitor, by NMR, the conformational transition of the Aβ(1–42) from soluble α-helical structure to β-sheet aggregates. In the previous studies, 50/50 HFIP/water proportion emerged as the solution condition where the first evident Aβ(1–42) conformational changes occur. In the hypothesis that this solvent reproduces the best condition to catch transitional helical-β-sheet Aβ(1–42) conformations, in this study, we report an extensive NMR conformational analysis of Aβ(1–42) in 50/50 HFIP/water v/v. Aβ(1–42) structure was solved by us, giving evidence that the evolution of Aβ(1–42) peptide from helical to the β-sheet may follow unexpected routes. Molecular dynamics simulations confirm that the structural model we calculated represents a starting condition for amyloid fibrils formation.

Polymorphic SERPINA3-R124C reduces pathogenesis of its wild type by shortening the lifetime of oligomeric Aβ.

Amyloid beta (Aβ) 42 peptide accumulated in Alzheimer disease (AD) patients' brain, often colocalized with serine protease inhibitor family A member 3 (SERPINA3). Being a chaperon, SERPINA3 accelerated Aβ42 fibrillization. While analyzing chaperon activity of human SERPINA3 polymorphisms, we found SERPINA3-R124C played a role in protecting cells from Aβ42 cytotoxicity. SH-SY5Y cells exposed to Aβ42 preincubated with wild-type SERPINA3 (SERPINA3-WT) resulted in extended toxicity leading cell death whereas Aβ42 with SERPINA3-R124C resulted in less cytotoxicity. Transmission electron microscope and thioflavin T assay revealed that SERPINA3-R124C shortened lifetime of small soluble oligomer and maintained β-sheet rich protofibril-like aggregates for longer time compared to that of with SERPINA3-WT. Western blot assay confirmed that SERPINA3-R124C converted Aβ42 mostly into high molecular aggregates. Here, we demonstrate first time that polymorphic SERPINA3 acts as a benign chaperon by modulating the transition states of Aβ42, which may contribute to the reduction of AD risk.

B2bTools: online predictions for protein biophysical features and their conservation.

We provide integrated protein sequence-based predictions via https://bio2byte.be/b2btools/. The aim of our predictions is to identify the biophysical behaviour or features of proteins that are not readily captured by structural biology and/or molecular dynamics approaches. Upload of a FASTA file or text input of a sequence provides integrated predictions from DynaMine backbone and side-chain dynamics, conformational propensities, and derived EFoldMine early folding, DisoMine disorder, and Agmata β-sheet aggregation. These predictions, several of which were previously not available online, capture ‘emergent’ properties of proteins, i.e. the inherent biophysical propensities encoded in their sequence, rather than context-dependent behaviour (e.g. final folded state). In addition, upload of a multiple sequence alignment (MSA) in a variety of formats enables exploration of the biophysical variation observed in homologous proteins. The associated plots indicate the biophysical limits of functionally relevant protein behaviour, with unusual residues flagged by a Gaussian mixture model analysis. The prediction results are available as JSON or CSV files and directly accessible via an API. Online visualisation is available as interactive plots, with brief explanations and tutorial pages included. The server and API employ an email-free token-based system that can be used to anonymously access previously generated results.