Non-fibrillar Aggregates Research Articles

Minimalist Adjuvant-Free Nano-Vaccine Based on Antigen Self-Assembled Amyloid-Like Fibrils to Induce Potent Immune Response.

The development of cancer vaccines is at the forefront of cancer immunotherapy. Most existing strategies to induce an efficient anti-tumor immune response rely on molecular adjuvants and the incorporation of complex synthetic vectors into vaccine formulations. In contrast, this study introduces a one-step engineering technique to assemble the model antigen, Ovalbumin (OVA), into amyloid aggregates, leveraging biomimetic folding and aggregation to create non-fibrillar OVA globular aggregates and OVA amyloid-like fibrils as single-component, adjuvant-free vaccines. Notably, the OVA amyloid-like fibrils induced stronger immune responses compared to the native form, as evidenced by robust humoral immune reactions and the establishment of immune memory. These enhanced responses can be attributed to the self-adjuvant effect of the unique assembled structure, which preserves antigenic epitopes, improves antigen stability, facilitates antigen internalization, prolongs retention at the injection site, enhances antigen trafficking to the lymphoid organs, and promotes increased secretion of antibodies and cytokines. Furthermore, the efficacy of the vaccine was validated in a high OVA-expressing tumor model, demonstrating the potential of OVA amyloid-like fibrils as an effective vaccine for cancer immunoprevention. This minimalist self-adjuvant vaccine strategy holds promising implications for cancer immunotherapy and can inform the design of other protein antigen-based vaccines.

Neuroprotective effect against amyloidogenic transthyretin aggregates - Induced cytotoxicity on human neuroblastoma cell by phenolic-rich Centella asiatica extract

Transthyretin (ATTR) amyloidosis is a progressive and life-threatening neurodegenerative disease caused by aggregation of the plasma transport protein, transthyretin, for which treatment is rare and cure unavailable. Centella asiatica is a small edible herb with a long history of neurological application in ethnomedicine. This work investigated whether hydrophilic extract of C. asiatica (CAB) could suppress the toxic effects of transthyretin amyloid aggregate (TTRa) in cell model derived from the same in vivo target. TTRa was prepared via thermal-induced aggregation. Chemical cross-linking and Tricine-SDS-PAGE, Thioflavin-T fluorescence, and TEM analyses confirmed that TTRa matched the profile of TTRL55P nonfibrillar amyloid aggregates. PrestoBlue cell viability assay revealed that exposure of IMR-32 human neuroblastoma cells to TTRa (2–8 μM) resulted in significant cytotoxicity. Conversely, exposure of IMR-32 cells to CAB did not adversely affect their viability. In addition, when IMR-32 cells were co-treated with TTRa and varied concentrations of CAB, the toxic effect of TTRa was significantly (p < 0.01) inhibited dose-dependently. The extract was found to possess potent radical scavenging effects, and quantitative RP-HPLC analysis showed that asiaticoside and phenolics were its main components. The cytoprotective effect against TTRa, antioxidant property, and good safety profile collectively suggest that CAB could be applied in the development of nutraceuticals or therapeutics against transthyretin amyloidosis.

Zinc and pH modulate the ability of insulin to inhibit aggregation of islet amyloid polypeptide

Aggregation of the human islet amyloid polypeptide (hIAPP) contributes to the development and progression of Type 2 Diabetes (T2D). hIAPP aggregates within a few hours at few micromolar concentration in vitro but exists at millimolar concentrations in vivo. Natively occurring inhibitors of hIAPP aggregation might therefore provide a model for drug design against amyloid formation associated with T2D. Here, we describe the combined ability of low pH, zinc, and insulin to inhibit hIAPP fibrillation. Insulin dose-dependently slows hIAPP aggregation near neutral pH but had less effect on the aggregation kinetics at acidic pH. We determine that insulin alters hIAPP aggregation in two manners. First, insulin diverts the aggregation pathway to large nonfibrillar aggregates with ThT-positive molecular structure, rather than to amyloid fibrils. Second, soluble insulin suppresses hIAPP dimer formation, which is an important early aggregation event. Further, we observe that zinc significantly modulates the inhibition of hIAPP aggregation by insulin. We hypothesize that this effect arose from controlling the oligomeric state of insulin and show that hIAPP interacts more strongly with monomeric than oligomeric insulin.

Evolution of large Aβ16-22 aggregates at atomic details and potential of mean force associated to peptide unbinding and fragmentation events.

Atomic characterization of large nonfibrillar aggregates of amyloid polypeptides cannot be determined by experimental means. Starting from β-rich aggregates of Y and elongated topologies predicted by coarse-grained simulations and consisting of more than 100 Aβ16-22 peptides, we performed atomistic molecular dynamics (MD), replica exchange with solute scaling (REST2), and umbrella sampling simulations using the CHARMM36m force field in explicit solvent. Here, we explored the dynamics within 3 μs, the free energy landscape, and the potential of mean force associated with either the unbinding of one single peptide in different configurations within the aggregate or fragmentation events of a large number of peptides. Within the time scale of MD and REST2, we find that the aggregates experience slow global conformational plasticity, and remain essentially random coil though we observe slow beta-strand structuring with a dominance of antiparallel beta-sheets over parallel beta-sheets. Enhanced REST2 simulation is able to capture fragmentation events, and the free energy of fragmentation of a large block of peptides is found to be similar to the free energy associated with fibril depolymerization by one chain for longer Aβ sequences.

Assessing p53 aggregates over a lifetime: Studies on young, adult, and aged mice.

p53 is a tumor-suppressing transcription factor critical to cellular health and function. The importance of intact p53 has been well established over the forty-plus years since its discovery. However, the role of amyloid-like aggregation in p53-related pathologies remains a significant knowledge gap. A growing body of knowledge over the last two decades has connected several mutations prevalent in cancer to the amyloid-like aggregation of p53 into both cross beta-sheet stacked fibrils and non-fibrillar aggregates. Here, non-fibrillar aggregates encompass all non-native p53-p53 (homomeric) complexes currently off-pathway to highly structured low-energy endpoint fibrils. p53 aggregation has reasonably been considered a cancer-centric phenomenon; however, it is unknown whether fibrillar-like structures form throughout the organismal lifespan, analogous to amyloid plaques. Furthermore, it is unclear if these structural states are observable as end-point or transient intermediates, including those either on or off-pathway to fibrillization. Notably, it has been shown that tau aggregates in the frontal cortex of Alzheimer's disease (AD) patients and AD mouse models contained aggregated p53, suggesting p53 aggregation may be more prevalent in aging-related diseases than previously thought. Here, I use immunoprecipitation to isolate p53 from young, adult, and aged mice and compare the aggregation state and propensity using native-page, fluorescence spectroscopy, and electron microscopy. Further, I will compare these data to the aggregation state typically observed in immortalized cell lines. I hypothesize that in mice, WT and mutant p53 may form aggregates over time, although those observed with mutant p53 will appear more fibrillar than WT, and may be distinct from those observed in immortalized cell lines. Together, these experiments help broaden our understanding of p53 aggregation related to age and health in vitro and in vivo.

Small soluble α-synuclein aggregates are the toxic species in Parkinson’s disease

Soluble α-synuclein aggregates varying in size, structure, and morphology have been closely linked to neuronal death in Parkinson’s disease. However, the heterogeneity of different co-existing aggregate species makes it hard to isolate and study their individual toxic properties. Here, we show a reliable non-perturbative method to separate a heterogeneous mixture of protein aggregates by size. We find that aggregates of wild-type α-synuclein smaller than 200 nm in length, formed during an in vitro aggregation reaction, cause inflammation and permeabilization of single-liposome membranes and that larger aggregates are less toxic. Studying soluble aggregates extracted from post-mortem human brains also reveals that these aggregates are similar in size and structure to the smaller aggregates formed in aggregation reactions in the test tube. Furthermore, we find that the soluble aggregates present in Parkinson’s disease brains are smaller, largely less than 100 nm, and more inflammatory compared to the larger aggregates present in control brains. This study suggests that the small non-fibrillar α-synuclein aggregates are the critical species driving neuroinflammation and disease progression.

Zinc Induced Aβ16 Aggregation Modeled by Molecular Dynamics.

It is widely accepted that the addition of zinc leads to the formation of neurotoxic nonfibrillar aggregates of beta-amyloid peptides Aβ40 and Aβ42 and at the same time destabilizes amyloid fibrils. However, the mechanism of the effect of zinc on beta-amyloid is not fully understood. In this study, a fast zinc-induced aggregation of Aβ16 (as compared to a system without zinc) via the formation of Aβ16 dimers with one zinc ion coordinated in the metal-binding site 11EVHH14, followed by their polymerization, has been studied by molecular dynamics. The best aggregation was shown by the system composed of Aβ16 dimers bound by one zinc ion, with no additional zinc in solution. The presence of Aβ16 dimers was a major condition, sufficient for fast aggregation into larger complexes. It has been shown that the addition of zinc to a system with already formed dimers does not substantially affect the characteristics and rate of aggregation. At the same time, an excessive concentration of zinc at the early stages of the formation of conglomerates can negatively affect aggregation, since in systems where zinc ions occupied the 11EVHH14 coordination center and the His6 residue of every Aβ16 monomer, the aggregation proceeded more slowly and the resulting complexes were not as large as in the zinc-free Aβ system. Thus, this study has shown that the formation of Aβ16 dimers bound through zinc ions at the 11EVHH14 sites of the peptides plays an important role in the formation of neurotoxic non-fibrillar aggregates of beta-amyloid peptide Aβ16. The best energetically favorable structure has been obtained for the complex of two Aβ16 dimers with two zinc ions.

Effect of Metal Ions on the Intrinsic Blue Fluorescence Property and Morphology of Aromatic Amino Acid Self-Assembly.

Metal ions are known to strongly bind with different proteins and peptides, resulting in alteration of their different physicochemical properties. In this work, we investigate the effect of metal ions of different nuclear charges and sizes on the intrinsic blue luminescence of the self-assembled structures formed by aromatic amino acids, namely, phenylalanine and tryptophan, using spectroscopic and imaging techniques. The study reveals that the intrinsic blue fluorescence of amino acid assemblies is influenced by metal ions and the pH of the medium. The metal ions with a higher charge to radius ratio promote clusterization which results in the enhancement of the intrinsic fluorescence, an effect known as "clusteroluminescence" of the amino acids aggregates. The imaging study reveals that metal ions with a higher charge to size ratio inhibit the large fibrillation of aromatic amino acids by promoting the formation of small nonfibrillar aggregates through increased hydrophobicity in the medium. The nanoaggregates are assumed to be responsible for the enhancement in the blue "clusteroluminescence".

Expression of the human molecular chaperone domain Bri2 BRICHOS on a gram per liter scale with an E. coli fed-batch culture

BackgroundThe human Bri2 BRICHOS domain inhibits amyloid formation and toxicity and could be used as a therapeutic agent against amyloid diseases. For translation into clinical use, large quantities of correctly folded recombinant human (rh) Bri2 BRICHOS are required. To increase the expression and solubility levels of rh Bri2 BRICHOS it was fused to NT*, a solubility tag derived from the N-terminal domain of a spider silk protein, which significantly increases expression levels and solubility of target proteins. To increase the expression levels even further and reach the g/L range, which is a prerequisite for an economical production on an industrial scale, we developed a fed-batch expression protocol for Escherichia coli.ResultsA fed-batch production method for NT*-Bri2 BRICHOS was set up and systematically optimized. This gradual improvement resulted in expression levels of up to 18.8 g/L. Following expression, NT*-Bri2 BRICHOS was purified by chromatographic methods to a final yield of up to 6.5 g/L. After removal of the NT*-tag and separation into different oligomeric species, activity assays verified that different assembly states of the fed-batch produced rh Bri2 BRICHOS have the same ability to inhibit fibrillar and non-fibrillar protein aggregation as the reference protein isolated from shake flask cultures.ConclusionsThe protocol developed in this work allows the production of large quantities of rh Bri2 BRICHOS using the solubility enhancing NT*-tag as a fusion partner, which is required to effectively conduct pre-clinical research.

Heat treatment of soluble proteins isolated from human cataract lens leads to the formation of non-fibrillar amyloid-like protein aggregates

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 (λmax) 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.

Glycosylation of a Nonfibrillizing Appendage Alters the Self-Assembly Pathway of a Synthetic β-Sheet Fibrillizing Peptide.

Owing to their biocompatibility and biodegradability, short synthetic peptides that self-assemble into elongated β-sheet fibers (i.e., peptide nanofibers) are widely used to create biomaterials for diverse medical and biotechnology applications. Glycosylation, which is a common protein post-translational modification, is gaining interest for creating peptide nanofibers that can mimic the function of natural carbohydrate-modified proteins. Recent reports have shown that glycosylation can disrupt the fibrillization of natural amyloid-forming peptides. Here, using transmission electron microscopy, fluorescence microscopy, and thioflavin T spectroscopy, we show that glycosylation at a site external to the fibrillization domain can alter the self-assembly pathway of a synthetic fibrillizing peptide, NSGSGQQKFQFQFEQQ (NQ11). Specifically, an NQ11 variant modified with N-linked N-acetylglucosamine, N(GlcNAc)SGSG-Q11 (GQ11), formed β-sheet nanofibers more slowly than NQ11 in deionized water (pH 5.8), which correlated to the tendency of GQ11 to form a combination of short fibrils and nonfibrillar aggregates, whereas NQ11 formed extended nanofibers. Acidic phosphate buffer slowed the rate of GQ11 fibrillization and altered the morphology of the structures formed yet had no effect on NQ11 fibrillization rate or morphology. The buffer ionic strength had no effect on the fibrillization rate of either peptide, while the diphosphate anion had a similar effect on the rate of fibrillization of both peptides. Collectively, these data demonstrate that a glycan moiety located external to the β-sheet fibrillizing domain can alter the pH-dependent self-assembly pathway of a synthetic peptide, leading to significant changes in the fibril mass and morphology of the structures formed. These observations add to the understanding of the effect of glycosylation on peptide self-assembly and should guide future efforts to develop biomaterials from synthetic β-sheet fibrillizing glycopeptides.

11C-PiB and 124I-Antibody PET Provide Differing Estimates of Brain Amyloid-β After Therapeutic Intervention.

PET imaging of amyloid-β (Aβ) has become an important component of Alzheimer disease diagnosis. 11C-Pittsburgh compound B (11C-PiB) and analogs bind to fibrillar Aβ. However, levels of nonfibrillar, soluble, aggregates of Aβ appear more dynamic during disease progression and more affected by Aβ-reducing treatments. The aim of this study was to compare an antibody-based PET ligand targeting nonfibrillar Aβ with 11C-PiB after β-secretase (BACE-1) inhibition in 2 Alzheimer disease mouse models at an advanced stage of Aβ pathology. Methods: Transgenic ArcSwe mice (16 mo old) were treated with the BACE-1 inhibitor NB-360 for 2 mo, whereas another group was kept as controls. A third group was analyzed at the age of 16 mo as a baseline. Mice were PET-scanned with 11C-PiB to measure Aβ plaque load followed by a scan with the bispecific radioligand 124I-RmAb158-scFv8D3 to investigate nonfibrillar aggregates of Aβ. The same study design was then applied to another mouse model, AppNL-G-F In this case, NB-360 treatment was initiated at the age of 8 mo and animals were scanned with 11C-PiB-PET and 125I-RmAb158-scFv8D3 SPECT. Brain tissue was isolated after scanning, and Aβ levels were assessed. Results: 124I-RmAb158-scFv8D3 concentrations measured with PET in hippocampus and thalamus of NB-360-treated ArcSwe mice were similar to those observed in baseline animals and significantly lower than concentrations observed in same-age untreated controls. Reduced 125I-RmAb158-scFv8D3 retention was also observed with SPECT in hippocampus, cortex, and cerebellum of NB-360-treated AppNL-G-F mice. Radioligand invivo concentrations corresponded to postmortem brain tissue analysis of soluble Aβ aggregates. For both models, mice treated with NB-360 did not display a reduced 11C-PiB signal compared with untreated controls, and further, both NB-360 and control mice tended, although not reaching significance, to show higher 11C-PiB signal than the baseline groups. Conclusion: This study demonstrated the ability of an antibody-based radioligand to detect changes in brain Aβ levels after anti-Aβ therapy in ArcSwe and AppNL-G-F mice with pronounced Aβ pathology. In contrast, the decreased Aβ levels could not be quantified with 11C-PiB PET, suggesting that these ligands detect different pools of Aβ.

Natural Compound from Olive Oil Inhibits S100A9 Amyloid Formation and Cytotoxicity: Implications for Preventing Alzheimer's Disease.

Polyphenolic compounds in the Mediterranean diet have received increasing attention due to their protective properties in amyloid neurodegenerative and many other diseases. Here, we have demonstrated for the first time that polyphenol oleuropein aglycone (OleA), which is the most abundant compound in olive oil, has multiple potencies for the inhibition of amyloid self-assembly of pro-inflammatory protein S100A9 and the mitigation of the damaging effect of its amyloids on neuroblastoma SH-SY5Y cells. OleA directly interacts with both native and fibrillar S100A9 as shown by intrinsic fluorescence and molecular dynamic simulation. OleA prevents S100A9 amyloid oligomerization as shown using amyloid oligomer-specific antibodies and cross-β-sheet formation detected by circular dichroism. It decreases the length of amyloid fibrils measured by atomic force microscopy (AFM) as well as reduces the effective rate of amyloid growth and the overall amyloid load as derived from the kinetic analysis of amyloid formation. OleA disintegrates already preformed fibrils of S100A9, converting them into nonfibrillar and nontoxic aggregates as revealed by amyloid thioflavin-T dye binding, AFM, and cytotoxicity assays. At the cellular level, OleA targets S100A9 amyloids already at the membranes as shown by immunofluorescence and fluorescence resonance energy transfer, significantly reducing the amyloid accumulation in GM1 ganglioside containing membrane rafts. OleA increases overall cell viability when neuroblastoma cells are subjected to the amyloid load and alleviates amyloid-induced intracellular rise of reactive oxidative species and free Ca2+. Since S100A9 is both a pro-inflammatory and amyloidogenic protein, OleA may effectively mitigate the pathological consequences of the S100A9-dependent amyloid-neuroinflammatory cascade as well as provide protection from neurodegeneration, if used within the Mediterranean diet as a potential preventive measure.

Potential involvement of environmental triggers in protein aggregation with mercuric chloride as a model

Heavy metal based toxicity has a direct relation with the perturbation of protein structure. We have investigated the progressive unfolding of ovalbumin, in the presence of increasing concentration mercury (0–6.25 μM) using different spectroscopic techniques. Formation of amorphous aggregate has been observed at the physiological pH. Initial addition of HgCl2 resulted in the association of monomers to oligomers that proceeded to non-fibrillar aggregates on further addition. The sigmoidal curve obtained from the Stern-Volmer plot clearly divided into three stage transition. A strong lag phase is observed indicating the time dependence for the association of competent monomers. The second stage was resolved into non-cooperative binding. These results match very well with the data from atomic force microscopy and the free energy change observed in the regions. Raman spectroscopic studies indicated toxic antiparallel β-sheets structure. Time dependent atomic force microscopy study revealed the off-pathway nature of amorphous aggregates. At molten globular state, similar quenching behaviour is observed. The atomic force microscopy images clearly indicate at pH 2.2 the initiation of fibril formation occurs at lower concentration of HgCl2 itself. Our results revealed the conformation switch of ovalbumin upon the contact of an environmental toxin and its possible way of toxicity.

Site-specific single point mutation by anthranilic acid in hIAPP8-37 enhances anti-amyloidogenic activity.

Amylin or hIAPP, together with insulin, plays a significant role in glucose metabolism. However, it undergoes β-sheet rich amyloid formation associated with pancreatic β-cell dysfunction leading to type-2 diabetes (T2D). Recent studies suggest that restricting β-sheet formation in it may halt amyloid formation, which may limit the risk for the disease. Several peptide-based inhibitors have been reported to prevent aggregation. However, most of them have limitations, including low binding efficiency, active only at higher doses, poor solubility, and proteolytic degradation. Insertion of non-coded amino acids renders proteolytically stable peptides. We incorporated a structurally rigid β-amino acid, Anthranilic acid (Ant), at different sites within the central hydrophobic region of hIAPP and developed two singly mutated hIAPP8–37 peptidomimetics. These peptidomimetics inhibited the amyloid formation of hIAPP substantially even at low concentration, as evident from in vitro ThT, CD, FT-IR, TEM, and Congo red staining birefringence results. These peptidomimetics also disrupted the preformed aggregates formed by hIAPP into non-toxic species. These β-amino acid-based peptidomimetics can be an attractive scaffold for therapeutic design towards T2D or other protein misfolding diseases.

Cobalt(III) Schiff base complexes stabilize non-fibrillar amyloid-β aggregates with reduced toxicity

The aggregation of amyloid-β (Aβ) is believed to be foundational to the pathogenesis of Alzheimer's disease (AD). In vitro aggregation kinetics have been shown to correlate with rates of disease progression in both AD patients and animal models, thus proving to be a useful metric for testing Aβ-targeted therapeutics. Here we present evidence of cobalt(III) Schiff base complex ([Co(acetylacetonate)(NH3)2]Cl; Co(III)-sb) modulation of Aβ aggregation kinetics by a variety of complementary techniques. These include Thioflavin T (ThT) fluorescence, circular dichroism (CD) spectroscopy, transmission electron microscopy (TEM), and atomic force microscopy (AFM). Our data was fitted to kinetic rate laws using a mathematical model developed by Knowles et al. in order to extract mechanistic information about the effect of Co(III)-sb on aggregation kinetics. Our analysis revealed that Co(III)-sb alters Aβ aggregation by decreasing the polymerization rate and increasing the nucleation rate, suggesting that Co(III)-sb causes Aβ to rapidly stabilize oligomeric species with reduced elongation into mature fibrils. This result was corroborated by TEM and AFM of Aβ aggregates in vitro. We also demonstrate that Aβ aggregate mixtures produced in the presence of Co(III)-sb exhibit decreased cytotoxicity compared to untreated samples.

Recombinant Bri3 BRICHOS domain is a molecular chaperone with effect against amyloid formation and non-fibrillar protein aggregation

Molecular chaperones assist proteins in achieving a functional structure and prevent them from misfolding into aggregates, including disease-associated deposits. The BRICHOS domain from familial dementia associated protein Bri2 (or ITM2B) probably chaperones its specific proprotein region with high β-sheet propensity during biosynthesis. Recently, Bri2 BRICHOS activity was found to extend to other amyloidogenic, fibril forming peptides, in particular, Alzheimer’s disease associated amyloid-β peptide, as well as to amorphous aggregate forming proteins. However, the biological functions of the central nervous system specific homologue Bri3 BRICHOS are still to be elucidated. Here we give a detailed characterisation of the recombinant human (rh) Bri3 BRICHOS domain and compare its structural and functional properties with rh Bri2 BRICHOS. The results show that rh Bri3 BRICHOS forms more and larger oligomers, somewhat more efficiently prevents non-fibrillar protein aggregation, and less efficiently reduces Aβ42 fibril formation compared to rh Bri2 BRICHOS. This suggests that Bri2 and Bri3 BRICHOS have overlapping molecular mechanisms and that their apparently different tissue expression and processing may result in different physiological functions.

The Kinetics of Amyloid Fibril Formation by de Novo Protein Albebetin and Its Mutant Variants.

Engineering of amyloid structures is one of the new perspective areas of protein engineering. Studying the process of amyloid formation can help find ways to manage it in the interests of medicine and biotechnology. One of the promising candidates for the structural basis of artificial functional amyloid fibrils is albebetin (ABB), an artificial protein engineered under the leadership of O.B. Ptitsyn. Various aspects of the amyloid formation of this protein and some methods for controlling this process are investigated in this paper. Four stages of amyloid fibrils formation by this protein from the first non-fibrillar aggregates to mature fibrils and large micron-sized complexes have been described in detail. Dependence of albebetin amyloids formation on external conditions and some mutations also have been described. The introduction of similar point mutations in the two structurally identical α-β-β motifs of ABB lead to different amiloidogenesis kinetics. The inhibitory effect of a disulfide bond and high pH on amyloid fibrils formation, that can be used to control this process, was shown. The results of this work are a good basis for the further design and use of ABB-based amyloid constructs.

Inhibition of lysozyme aggregation and cellular toxicity by organic acids at acidic and physiological pH conditions

The misfolding of proteins can lead to fibrillar and non-fibrillar deposits that are the hallmark of numerous human diseases. Inhibition of protein aggregation is considered as a promising strategy for the prevention of such diseases. Here we induced the fibrillar and non-fibrillar aggregates of hen egg white lysozyme (HEWL) at acidic (pH 3) and physiological (pH 7.4) environments. HEWL formed non-fibrillar aggregates rapidly at pH 7.4, whereas fibrillar HEWL aggregates were formed slowly at pH 3. Both fibrillar and non-fibrillar aggregates had cytotoxic effects on PC12 cells. Next, four organic acids, succinic acid, maleic acid, tartaric acid and citric acid, were tested for their inhibition potencies against fibrillar and non-fibrillar HEWL species. The four inhibitors were found to prevent the aggregation of HEWL at pH 7.4 with a reduction rate of over 95% as compared with the reduction rate of 42–58% for HEWL aggregation at pH 3. Other biophysical and computational analyses reveal that the candidate inhibitors have higher inhibition efficacy against HEWL monomers incubated at pH 7.4 than at pH 3. These results emphasize the importance of validating the newly identified aggregation drugs against different aggregate species, which would enhance the understanding of small molecules-induced protein aggregation inhibition.

Small molecule-mediated co-assembly of amyloid-β oligomers reduces neurotoxicity through promoting non-fibrillar aggregation.

Amyloid-β (Aβ) oligomers, particularly low molecular weight (LMW) oligomers, rather than fibrils, contribute very significantly to the onset and progression of Alzheimer's Disease (AD). However, due to the inherent heterogeneity and metastability of oligomers, most of the conventional anti-oligomer therapies have indirectly modulated oligomers' toxicity through manipulating Aβ self-assembly to reduce oligomer levels, which are prone to suffering from the risk of regenerating toxic oligomers from the products of modulation. To circumvent this disadvantage, we demonstrate, for the first time, rational design of rigid pincer-like scaffold-based small molecules with blood–brain barrier permeability that specifically co-assemble with LMW Aβ oligomers through directly binding to the exposed hydrophobic regions of oligomers to form non-fibrillar, degradable, non-toxic co-aggregates. As a proof of concept, treatment with a europium complex (EC) in such a structural mode can rescue Aβ-mediated dysfunction in C. elegans models of AD in vivo. This small molecule-mediated oligomer co-assembly strategy offers an efficient approach for AD treatment.