Effect of plasmonic excitation on mature insulin amyloid fibrils.

Interactions between amyloid protein structures and nanomaterials have been extensively studied to develop effective inhibitors of amyloid aggregation. Limited investigations are reported on the impact of nanoparticles on mature fibrils. In this work, gold nanoparticles are used as photothermal agents to alter insulin fibrils. To this end, gold colloids bearing a negatively charged capping shell, with an average diameter of 14 nm and a plasmon resonance maximum at 520 nm are synthesized. The effects on mature insulin fibril morphology and structure upon plasmonic excitation of the nanoparticles-fibril samples have been monitored by spectroscopic and microscopic methods. The obtained data indicate that an effective destruction of the amyloid aggregates occur upon irradiation of the plasmonic nanoparticles, allowing the development of emerging strategies to alter the structure of amyloid fibrils.

N-Acetylcysteine-Loaded Magnetic Nanoparticles for Magnetic Resonance Imaging.

Acute respiratory distress syndrome (ARDS) is a life-threatening condition characterized by the rapid onset of lung inflammation Therefore, monitoring the spatial distribution of the drug directly administered to heterogeneously damaged lungs is desirable. In this work, we focus on optimizing the drug N-acetylcysteine (NAC) adsorption on poly-l-lysine-modified magnetic nanoparticles (PLLMNPs) to monitor the drug spatial distribution in the lungs using magnetic resonance imaging (MRI) techniques. The physicochemical characterizations of the samples were conducted in terms of morphology, particle size distributions, surface charge, and magnetic properties followed by the thermogravimetric quantification of NAC coating and cytotoxicity experiments. The sample with the theoretical NAC loading concentration of 0.25 mg/mL was selected as an optimum due to the hydrodynamic nanoparticle size of 154 nm, the surface charge of +32 mV, good stability, and no cytotoxicity. Finally, MRI relaxometry confirmed the suitability of the sample to study the spatial distribution of the drug in vivo using MRI protocols. We showed the prevailing transverse relaxation with high transverse relaxivity values and a high r2(*)/r1 ratio, causing visible hypointensity in the final MRI signal. Furthermore, NAC adsorption significantly affects the relaxation properties of PLLMNPs, which can help monitor drug release in vitro/in vivo.

Modulation of Insulin Amyloid Fibrillization in Imidazolium-Based Ionic Liquids with Hofmeister Series Anions.

Amyloid fibrils have immense potential to become the basis of modern biomaterials. The formation of amyloid fibrils in vitro strongly depends on the solvent properties. Ionic liquids (ILs), alternative solvents with tunable properties, have been shown to modulate amyloid fibrillization. In this work, we studied the impact of five ILs with 1-ethyl-3-methylimidazolium cation [EMIM+] and anions of Hofmeisterseries hydrogen sulfate [HSO4-], acetate [AC-], chloride [Cl-], nitrate [NO3-], and tetrafluoroborate [BF4-] on the kinetics of insulin fibrillization and morphology, and the structure of insulin fibrils when applying fluorescence spectroscopy, AFM and ATR-FTIR spectroscopy. We found that the studied ILs were able to speed up the fibrillization process in an anion- and IL-concentration-dependent manner. At an IL concentration of 100 mM, the efficiency of the anions at promoting insulin amyloid fibrillization followed the reverse Hofmeister series, indicating the direct binding of ions with the protein surface. At a concentration of 25 mM, fibrils with different morphologies were formed, yet with similar secondary structure content. Moreover, no correlation with the Hofmeister ranking was detected for kinetics parameters. IL with the kosmotropic strongly hydrated [HSO4-] anion induced the formation of large amyloid fibril clusters, while the other kosmotropic anion [AC-] along with [Cl-] led to the formation of fibrils with similar needle-like morphologies to those formed in the IL-free solvent. The presence of the ILs with the chaotropic anions [NO3-] and [BF4-] resulted in longer laterally associated fibrils. The effect of the selected ILs was driven by a sensitive balance and interplay between specific protein-ion and ion-water interactions and non-specific long-range electrostatic shielding.

Design and synthesis of novel carbohydrate-amino acid hybrids and their antioxidant and anti-ß-amyloid aggregation activity.

Herein we report the synthesis of new furanoid sugar amino acids and thioureas, prepared by coupling aromatic amino acids and dipeptides with isothiocyanato- functionalized ribofuranose ring. Since carbohydrate-derived structures possess many biological activities, synthesized compounds were evaluated as anti-amyloid and antioxidant agents. The anti-amyloid activity of the studied compounds was evaluated based on their potential to destroy amyloid fibrils of intrinsically disordered Aß40 peptide and globular hen egg-white (HEW) lysozyme. The destructive efficiency of the compounds differed between the studied peptides. While the destruction activity of the compounds on the HEW lysozyme amyloid fibrils was negligible, the effect on Aß40 amyloid fibrils was significantly higher. Furanoid sugar α-amino acid 1 and its dipeptide derivatives 8 (Trp-Trp) and 11 (Trp-Tyr) were the most potent anti-Aß fibrils compounds. The antioxidant properties of synthesized compounds were estimated by three complementary in vitro assays (DPPH, ABTS, and FRAP). The ABTS assay was the most sensitive for assessing the radical scavenging activity of all tested compounds compared to the DPPH test. Significant antioxidant activity was detected for compounds in the group of aromatic amino acids depending on the present amino acid, with the highest activity in the case of dipeptides 11 and 12 containing the Tyr and Trp moiety. Regarding the FRAP assay, the best reducing antioxidant potential revealed Trp-containing compounds 5, 10, and 12.

Green tea leaf constituents inhibit the formation of lysozyme amyloid aggregates: An effect of mutual interactions.

Amyloidoses represent a group of pathological conditions characterized by amyloid fibrils accumulating in the form of deposits in intra- or extracellular space, leading to tissue damage. The lysozyme from hen egg-white (HEWL) is often used as a universal model protein to study the anti-amyloid effects of small molecules. The in vitro anti-amyloid activity and mutual interactions of green tea leaf constituents: (-)-epigallocatechin gallate (EGCG), (-)-epicatechin (EC), gallic acid (GA), caffeine (CF) and their equimolar mixtures were studied. The inhibition of HEWL amyloid aggregation was monitored by a Thioflavin T fluorescence assay and atomic force microscopy (AFM). The interactions of the analyzed molecules with HEWL were interpreted by ATR-FTIR and protein-small ligand docking studies. EGCG was the only substance efficiently inhibiting amyloid formation (IC50 âˆ¼193 µM), slowing the aggregation process, reducing the number of fibrils and partially stabilizing the secondary structure of HEWL. Compared to EGCG alone, EGCG-containing mixtures displayed lower overall anti-amyloid efficacy. The decrease in efficiency results from (a) the spatial interference of GA, CF and EC with EGCG while binding to HEWL, (b) the propensity of CF to form a less active adduct with EGCG, which participates in interactions with HEWL in parallel with pure EGCG. This study confirms the importance of interaction studies, revealing the possible antagonistic behavior of molecules when combined.

The influence of cations on α-lactalbumin amyloid aggregation.

There is limited knowledge regarding α-lactalbumin amyloid aggregation and its mechanism. We examined the formation of α-lactalbumin amyloid fibrils (α-LAF) in the presence of cations (Mg²⁺, Ca²⁺, Na⁺, K⁺, NH₄⁺, and Cs⁺) in the form of chloride salts at two concentrations. We have shown that studied cations affect the conformation of α-lactalbumin, the kinetics of its amyloid formation, morphology, and secondary structure of α-LAF in a different manner. The higher salts concentration significantly accelerated the aggregation process. Both salt concentrations stabilized α-lactalbumin's secondary structure. However, the presence of divalent cations resulted in shorter fibrils with less ß-sheet content. Moreover, strongly hydrated Mg²⁺ significantly altered α-lactalbumin's tertiary structure, followed by Na⁺, NH₄⁺, K⁺, and weakly hydrated Cs⁺. On the other hand, Ca²⁺, despite being also strongly hydrated, stabilized the tertiary structure, supposedly due to its high affinity towards α-lactalbumin. Yet, Ca²⁺ was not able to inhibit α-lactalbumin amyloid aggregation.

Effect of 1-Ethyl-3-methylimidazolium Tetrafluoroborate and Acetate Ionic Liquids on Stability and Amyloid Aggregation of Lysozyme.

Amyloid fibrils draw attention as potential novel biomaterials due to their high stability, strength, elasticity or resistance against degradation. Therefore, the controlled and fast fibrillization process is of great interest, which raises the demand for effective tools capable of regulating amyloid fibrillization. Ionic liquids (ILs) were identified as effective modulators of amyloid aggregation. The present work is focused on the study of the effect of 1-ethyl-3-methyl imidazolium-based ILs with kosmotropic anion acetate (EMIM-ac) and chaotropic cation tetrafluoroborate (EMIM-BF4) on the kinetics of lysozyme amyloid aggregation and morphology of formed fibrils using fluorescence and CD spectroscopy, differential scanning calorimetry, AFM with statistical image analysis and docking calculations. We have found that both ILs decrease the thermal stability of lysozyme and significantly accelerate amyloid fibrillization in a dose-dependent manner at concentrations of 0.5%, 1% and 5% (v/v) in conditions and time-frames when no fibrils are formed in ILs-free solvent. The effect of EMIM-BF4 is more prominent than EMIM-ac due to the different specific interactions of the anionic part with the protein surface. Although both ILs induced formation of amyloid fibrils with typical needle-like morphology, a higher variability of fibril morphology consisting of a different number of intertwining protofilaments was identified for EMIM-BF4.

Extracts from Chinese herbs with anti-amyloid and neuroprotective activities.

Many Chinese herbs are well known for their neuroprotective and anti-oxidant properties. Extracts of Salvia miltiorrhiza and Anemarrhenae asphodeloides, tanshinone IIA (tanIIA), salvianolic acid B (Sal B) and sarsasapogenin (ML-1), were selected to study their dissociation potential towards Aß42 peptide fibrils and neuroprotective effect on cells. Moreover, derivatives of sarsasapogenin (ML-2, ML-3 and ML-4) have been prepared by the addition of modified carbamate moiety. TanIIA and Sal B have shown to possess a strong ability to dissociate Aß42 fibrils. The dissociation potential of ML-1 increased upon the introduction of carbamate moiety with N-heterocycles. In silico data revealed that derivatives ML-4 and Sal B interact with Aß42 regions responsible for fibril stabilization through hydrogen bonds. Contrary, tanIIA binds close to a central hydrophobic region, which may lead to destabilization of fibrils. Sarsasapogenin derivative ML-2 decreased nitride oxide production, and derivative ML-4 enhanced the growth of neurites. The reported data highlight the possibility of using active compounds to design novel treatment agents for Alzheimer's disease.

Inhibition behavior of Sennoside A and Sennoside C on amyloid fibrillation of human lysozyme and its possible mechanism.

Amyloid proteins were recognized as the crucial cause of many senile diseases. In this study, the inhibitory effects of Sennoside A (SA) and Sennoside C (SC) on amyloid fibrillation were evaluated by the combination of biophysical approaches and molecular docking tool using human lysozyme (HL) as amyloid-forming model. The results of thioflavin-T (ThT), 8-anilino-1-naphthalenesulfonic acid (ANS) and congo red (CR) assays indicated that both SA and SC could inhibit the amyloid fibrillation of HL in a dose-dependent manner. The IC50 value of SA and SC on HL fibrillation was 200.09 µM and 186.20 µM, respectively. These findings were further verified by transmission electron microscopy (TEM) and atomic force microscopy (AFM), which showed that the addition of SA or SC could sharply reduce the amyloid fibrillation of HL. Additionally, the interactions of HL with SA and SC were investigated by steady-state fluorescence spectra and molecular docking studies. The results suggested that both SA and SC could bind to the binding pocket of HL and form a stable complex mainly via hydrogen bonds, van-der-Waals forces and hydrophobic interactions. In conclusion, our experiments revealed that both SA and SC can significantly inhibit amyloid fibrillation of HL.

Tacrine - Benzothiazoles: Novel class of potential multitarget anti-Alzheimers drugs dealing with cholinergic, amyloid and mitochondrial systems.

A series of tacrine - benzothiazole hybrids incorporate inhibitors of acetylcholinesterase (AChE), amyloid ß (Aß) aggregation and mitochondrial enzyme ABAD, whose interaction with Aß leads to mitochondrial dysfunction, into a single molecule. In vitro, several of 25 final compounds exerted excellent anti-AChE properties and interesting capabilities to block Aß aggregation. The best derivative of the series could be considered 10w that was found to be highly potent and selective towards AChE with the IC50 value in nanomolar range. Moreover, the same drug candidate exerted absolutely the best results of the series against ABAD, decreasing its activity by 23% at 100 µM concentration. Regarding the cytotoxicity profile of highlighted compound, it roughly matched that of its parent compound - 6-chlorotacrine. Finally, 10w was forwarded for in vivo scopolamine-induced amnesia experiment consisting of Morris Water Maze test, where it demonstrated mild procognitive effect. Taking into account all in vitro and in vivo data, highlighted derivative 10w could be considered as the lead structure worthy of further investigation.

MIRRAGGE - Minimum Information Required for Reproducible AGGregation Experiments.

Reports on phase separation and amyloid formation for multiple proteins and aggregation-prone peptides are recurrently used to explore the molecular mechanisms associated with several human diseases. The information conveyed by these reports can be used directly in translational investigation, e.g., for the design of better drug screening strategies, or be compiled in databases for benchmarking novel aggregation-predicting algorithms. Given that minute protocol variations determine different outcomes of protein aggregation assays, there is a strong urge for standardized descriptions of the different types of aggregates and the detailed methods used in their production. In an attempt to address this need, we assembled the Minimum Information Required for Reproducible Aggregation Experiments (MIRRAGGE) guidelines, considering first-principles and the established literature on protein self-assembly and aggregation. This consensus information aims to cover the major and subtle determinants of experimental reproducibility while avoiding excessive technical details that are of limited practical interest for non-specialized users. The MIRRAGGE table (template available in Supplementary Information) is useful as a guide for the design of new studies and as a checklist during submission of experimental reports for publication. Full disclosure of relevant information also enables other researchers to reproduce results correctly and facilitates systematic data deposition into curated databases.

Investigating the effect of sugar-terminated nanoparticles on amyloid fibrillogenesis of ß-lactoglobulin.

In vivo tissue deposition of fibrillar protein aggregates is the cause of several degenerative diseases. Evidence suggests that interfering with the pathology-associated amyloid fibrillogenesis by inhibitory molecules is envisaged as the primary therapeutic strategy. Amyloid fibril formation of proteins has been demonstrated to be influenced by nanoparticles/nanomaterials. As compared with their molecular form counterpart, this work examined the effect of sucrose-terminated nanoparticles on the in vitro amyloid fibrillogenesis and structural properties of ß-lactoglobulin at pH 2.0 and 80 °C. ThT binding and electron microscopy results demonstrated that sucrose-terminated nanoparticles were able to suppress ß-lactoglobulin fibrillogenesis in a concentration-dependent fashion. Importantly, sucrose-terminated nanoparticles showed better ß-lactoglobulin fibril-inhibiting ability than sucrose molecules. ANS fluorescence and right-angle light scattering results showed reduced solvent exposure and decreased aggregation, respectively, in the ß-lactoglobulin samples upon treatment with sucrose-terminated nanoparticles. Moreover, fluorescence quenching analyses revealed that the static quenching mechanism and formation of a non-fluorescent fluorophore-nanoparticle complex are involved in the nanoparticle-ß-lactoglobulin interaction. We believe that the results from this study may suggest that the nanoparticle form of biocompatible sugar-related osmolytes may serve as effective inhibiting/suppressing agents toward protein fibrillogenesis.

Destabilization effect of imidazolium cation-Hofmeister anion salts on cytochrome c.

We have analyzed an effect of imidazolium cation-Hofmeister anion salts on stability of basic horse heart cytochrome c (cyt c) at pH4.5 (net charge +17). The effect of salts consisting of imidazolium cations, 1-ethyl-3-methylimidazolium (EMIm+) and 1-butyl-3-methylimidazolium (BMIm+), and five anions: chloride, bromide, iodide, nitrate, and thiocyanate on thermal and pH stability of cyt c was compared with the effect of corresponding sodium salts. Correlation between parameter of dTtrs/d [ion] (Ttrs; thermal midpoints) with surface tension changes of solvent in the presence of both imidazolium and sodium salts implies direct interaction between ions and proteins. Surprisingly, the imidazolium salts have more pronounced destabilization effect on highly positively charged cyt c than the corresponding sodium counterparts. Our analysis suggests the direct interaction of imidazolium cations with polypeptide chain, in analogy to guanidium cation, but the destabilization effect is significantly strengthened by decreased surface tension of imidazolium salt solvents. Comparison of an effect of imidazolium and sodium salts on acidic and alkaline transitions and to thermal transition of cyt c implies a role of hydrophobic interaction between imidazolium cation and polypeptide chain.

Investigating the inhibitory effects of entacapone on amyloid fibril formation of human lysozyme.

The misfolding of soluble protein to amyloid fibers or oligomers leads to cell membrane rupture, cell death, and a variety of amyloid-related diseases. Hence, inhibition of protein fibrillation is an important and promising method to prevent and treat these diseases. In this study, we have investigated the inhibitory effect of entacapone (Ent) on human lysozyme (HL) amyloid fibrillation using a combination of biophysical techniques; Rayleigh scattering (RLS) data indicated that Ent can reduce the aggregation of HL amyloid fibrillation with the inhibition constant (Λ) of (3.0 ± 0.5) × 103 M-1. This finding was further confirmed by thioflavin-T (ThT), 8-Anilino-1-naphthalenesulfonic acid (ANS) fluorescence assays and congo red (CR) binding absorption assays with an IC50 value of 125.89 ± 1.25 µM. Meanwhile, dynamic light scattering (DLS) showed that the size of HL amyloids decreases sharply after Ent treatment. This effect was positively correlated with Ent concentration. Atomic force microscopy (AFM) techniques confirmed that the formation of the fibril decreased significantly when HL was co-incubated with Ent. In addition, steady-state fluorescence spectra and synchronous fluorescence analysis suggested that the formation of stable complexes between Ent and HL contributes to maintain the alpha-helical structure of HL. The molecular docking study revealed that the Ent binds at the active pocket of HL with Glu35, Asp53, Gln58, Trp 64, Ala108 and Trp109 residues via hydrogen bonds, van-der-Waals forces and hydrophobic interactions. The epitope mapping of HL for its interaction with Ent was further elucidated using two-dimensional solution-state nuclear magnetic resonance (NMR) experiments. NMR results showed that the Trp64 and Trp109 of HL plays an important role for binding to Ent, correlating well with our docking result. Thus our study showed the potential of Ent to serve as an effective therapeutic agent for the therapy of amyloid-related diseases.

Amyloid Aggregation of Insulin: An Interaction Study of Green Tea Constituents.

Exogenous insulin, used as a therapeutic agent for diabetes, forms insoluble deposits containing amyloid fibrillar structures near the administration site. We have analyzed the in vitro anti-amyloid activity of four green tea constituents: (-)-epigallocatechin gallate (EGCG), (-)-epicatechin (EC), gallic acid (GA), caffeine (CF), and their equimolar mixtures. Regarding individually tested compounds, only EGCG inhibited the fibrillization process. The individual EC, GA, and CF molecules were ineffective. The presence of EGCG in equimolar combinations with GA, EC, or CF was required for the inhibitory activity of most mixtures. Molecular docking revealed that EGCG interacts with an essential amyloidogenic region of insulin chain B. Individually inactive GA had a potentiating effect on the activity of EGCG. In contrast, EC and CF had a negative impact on the activity of the mixtures. We have observed diverse morphology and the amount of insulin amyloid aggregates formed in the presence of studied compounds. The distinct types of amyloid aggregates created in vitro in the presence of EGCG and other green tea constituents were characterized. Results indicate that the biological activity of individual molecules is not directly applicable to the pooled samples effects prediction.

7-Methoxytacrine and 2-Aminobenzothiazole Heterodimers: Structure-Mechanism Relationship of Amyloid Inhibitors Based on Rational Design.

The formation and accumulation of amyloid aggregates are the phenomena that accompany amyloidoses, which are currently untreatable and include Alzheimer's and Parkinson's diseases, diabetes mellitus, non-neuropathic lysozyme systemic amyloidosis, and others. One of the very promising therapeutic approaches seems to be an inhibition of amyloid formation and/or clearance of amyloid aggregates. Small molecules have a great potential to interfere with amyloid fibrillation of peptides and polypeptides, which can be improved by connection of cyclic structures into single multicyclic molecules and their dimerization. In our study, we focused on heterodimers consisting of 7-methoxytacrine (7-MEOTA) and 2-aminobenzothiazole (BTZ) parent molecules connected by an aliphatic linker. Using in vitro and in silico methods, we investigated the ability of studied compounds to inhibit the amyloid aggregation of hen egg white lysozyme. Heterodimerization led to significant improvement of inhibitory activity compared to that of the parent molecules. The efficiency of the heterodimers varied; the most effective inhibitor contained the longest linker, eight carbons long. We suggest that binding of a heterodimer to a lysozyme blocks the interaction between the ß-domain and C-helix region essential for the formation of amyloid cross-ß structure. Elongation of the linker ultimately enhances the compound's ability to prevent this interaction by allowing the BTZ part of the heterodimer to bind more effectively, increasing the compound's binding affinity, and also by greater steric obstruction. This study represents an important contribution to the recent rational design of potential lead small molecules with anti-amyloid properties, and the heterodimers studied are prospective candidates for the treatment of systemic lysozyme amyloidosis and other amyloid-related diseases.

Targeted inhibition of amyloidogenesis using a non-toxic, serum stable strategically designed cyclic peptide with therapeutic implications.

Amyloidogenic disorders are currently rising as a global health issue, prompting more and more studies dedicated to the development of effective targeted therapeutics. The innate affinity of these amyloidogenic proteins towards the biomembranes adds further complexities to the systems. Our previous studies have shown that biologically active peptides can effectively target amyloidogenesis serving as an efficient therapeutic alternative in several amyloidogenic disorders. The structural uniqueness of the PWWP motif in the de novo designed heptapeptide, KR7 (KPWWPRR-NH2) was demonstrated to target insulin fiber elongation specifically. By working on insulin, an important model system in amyloidogenic studies, we gained several mechanistic insights into the inhibitory actions at the protein-peptide interface. Here, we report a second-generation non-toxic and serum stable cyclic peptide, based primarily on the PWWP motif that resulted in complete inhibition of insulin fibrillation both in the presence and absence of the model membranes. Using both low- and high-resolution spectroscopic techniques, we could delineate the specific mechanism of inhibition, at atomistic resolution. Our studies put forward an effective therapeutic intervention that redirects the default aggregation kinetics towards off-pathway fibrillation. Based on the promising results, this novel cyclic peptide can be considered an excellent lead to design pharmaceutical molecules against amyloidogenesis.

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

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

Amyloid fragments and their toxicity on neural cells.

The formation of amyloid fibrils from soluble proteins is a common form of self-assembly phenomenon that has fundamental connections with biological functions and human diseases. Lysozyme was converted from its soluble native state into highly organized amyloid fibrils. Ultrasonic treatment was used to break amyloid fibrils to fibrillar fragments-seeds. Atomic force microscopy and fluorescence microscopy was employed to characterize the morphology of the amyloid assemblies and neural cells-amyloid complexes. Our results demonstrate that prefibrillar intermediated and their mixture with proteins exhibit toxicity, although native proteins and fibrils appear to have no effect on number of cells. Our findings confirm that innocuous hen lysozyme can be engineered to produce both cytotoxic fibrillar fragments and non-toxic mature amyloid fibrils. Our work further strengthens the claim that amyloid conformation, and not the identity of the protein, is key to cellular toxicity and the underlying specific cell death mechanism.

Amino Acid Functionalized Superparamagnetic Nanoparticles Inhibit Lysozyme Amyloid Fibrillization.

Nanoparticles have great potential to be used in various biomedical applications, including therapy or diagnosis of amyloid-related diseases. The physical and chemical properties of iron oxide superparamagnetic nanoparticles (MNPs) functionalized with different amino acids (AAs), namely, with lysine (Lys), glycine (Gly), or tryptophan (Trp), have been characterized. The cytotoxicity of nanoparticles and their effect on amyloid fibrillization of lysozymes in vitro was also verified. The AA-MNPs under study are nontoxic to human SHSY5Y neuroblastoma cells. Moreover, the AA-MNPs were able to significantly inhibit lysozyme amyloid fibrillization and destroy amyloid fibrils. Kinetic studies revealed that the presence of AA-MNPs affected lysozyme fibrillization, namely, the lag phase and steady-state phase of the growth curves. The most effective activities were observed for Trp-MNPs, which revealed the importance of aromatic rings in the structure of AAs used as coating agents. The obtained results indicate the possible application of these AA-MNPs in the treatment of amyloid diseases associated with lysozyme or other amyloidogenic proteins.