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Protein amyloids have attracted attention for their application as functional amyloid materials because of their strong properties, such as high resistance to chemical or biological degradation, despite their medical issues. Amyloids can be used for various applications by modifying the amyloid surface with functional materials, such as proteins and polymers. In this study, we investigated the effect of polyallylamine (PAA), a functional cationic polymer as a candidate for amyloid modification, on the amyloids formed from amyloid ß (Aß) peptide. It was demonstrated for the first time that PAA can bind to Aß amyloids through fluorescence observations and the quenched emission from the tyrosine at site 10 near the fibrillogenic core. These results suggest that PAA could be used to develop new functional amyloids. However, notably, coating Aß amyloid with PAA could affect conventional amyloid detection assays such as thioflavin T assay and detection using antibodies. Thus, our results also indicate that consideration would be necessary for the analysis of functional amyloids coated with various polymers.

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Alzheimer's disease (AD) is a serious neurodegenerative brain disease that interferes with daily life. The accumulation of beta-amyloid (Aß), along with oxidative stress-inducing neurocellular apoptosis, has been considered one of the causes of AD. Thus, the purpose of this study is to find natural products that can reduce Aß accumulation. The ethanol extract of Metasequoia glyptostroboides Hu & Cheng fruits (Cupressaceae) significantly reduced the aggregation of Aß into oligomers and fibrils determined by Thioflavin T (ThT) assay. The solvent-partitioned ethyl acetate layer was further separated based on the bioassay-guided isolation method combined with the ThT assay. As a result, five compounds were isolated and elucidated as taxoquinone (1), sugiol (2), suginal (3), sandaracopimarinol (4), and sandaracopimaradien-19-ol (5) by comparing NMR data with references. All the compounds significantly reduced the aggregation of Aß and enhanced the disaggregation of pre-formed Aß aggregates in a dose-dependent manner. Furthermore, the inhibition of Aß aggregation by the compounds protected PC12 cells from Aß aggregate-induced toxicity. Among the five compounds, sandaracopimarinol (4) and sandaracopimaradien-19-ol (5) were the most effective. These results suggest that M. glyptostroboides and isolated five compounds have a potential for further study to be developed as anti-AD agents.

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Protein aggregation leads to several human pathologies such as Alzheimer's disease (AD), type 2 diabetes (T2D), Parkinson's disease (PD), etc. Due to the overlap in the mechanisms of type 2 diabetes and brain disorders, common effective pharmacological interventions to treat both T2D and AD is under extensive research. Therefore, major aim of research is to repurpose already established treatment of diabetes to cure AD as well. This study evaluates mechanistic insight into anti-amyloidogenic potential of anti-diabetic drug Vildagliptin (VLD) on human serum albumin fibrillation (HSA) by using biophysical, calorimetric, imaging techniques along with hemolytic assay. Dynamic light scattering (DLS) and Rayleigh light scattering (RLS) results showed presence of few small-sized aggregates in the presence of VLD which are formed by deaccelerating the amyloidogenesis as shown by thioflavin T (ThT) fluorescence and Congo red (CR) binding assay. Further, Isothermal titration calorimetry (ITC), steady state fluorescence quenching, molecular docking results revealed that VLD form complex with amyloid facilitating state of HSA and consequently mask the hydrophobic residues involved in amyloidogenesis as evident from decrease in ANS fluorescence. Differential scanning calorimetry (DSC) results confirm that VLD stabilizes the amyloid facilitating state of HSA. In addition, SEM images demonstrated that VLD alleviates the hemolytic effect induced by fibrils of HSA. This study reports VLD as a potential inhibitor of amyloid fibrillation and provides promising results to repurpose VLD as a drug candidate for the cure of Alzheimer's diseases along with diabetes.

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Membrane-mediated assembly has been well characterised for toxic amyloid species such as the amyloid-ß peptide implicated in Alzheimer's disease. However, little is known about the membrane-mediated assembly of functional-amyloid forming peptides, recently identified as a natural storage state for neuropeptide hormones in vivo. Here, we study the aggregation of somatostatin-14 (SST-14) co-incubated with model lipid membranes. Atomic force microscopy (AFM) studies confirmed that nanofibrils formed in the presence of various lipid membranes display reduced fibrillogenesis and promote the formation of non-fibrillar oligomers. Both circular dichroism (CD) and intrinsic tryptophan fluorescence studies confirmed interaction between the peptide and the lipid bilayer; this interaction appears to drive changes in membrane-mediated aggregation kinetics. We show that both the surface charge of the membrane and chain packing drive changes in the electrostatic and hydrophobic interactions between the peptide and the membrane, and hence the rate of assembly. The similarities in the effect of the lipid membrane on aggregation of functional amyloids and the more well studied toxic amyloids suggest strong aggregation modifying lipid bilayer interactions are a ubiquitous feature of all amyloid fibrils and highlight the need for further investigation as to why this leads to toxicity in some systems and not others.

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Islet amyloid polypeptide (IAPP) is a neuroendocrine hormone from pancreatic ß-cells. Misfolded, aggregated IAPP is believed to be toxic to islet cells and amyloid deposits in the pancreas are pathological hallmarks of type 2 diabetes. Rapid fibrillization of this peptide makes it difficult to study in its soluble form, impeding a better understanding of its role. In this study, a variety of popular pretreatment methods were tested for their ability to delay aggregation of IAPP, including solutions of hexafluoroisopropanol, sodium hydroxide, hydrochloric acid, phosphate buffered saline, ammonium hydroxide, as well as tris buffer at different pH and containing either calcium (II), zinc (II), or iron (II). Aggregation was assessed using the thioflavin T fluorescence assay as well as by transmission electron microscopy. Tris buffer at pH 8.1 containing Zn(II) was found to have the best balance of temporary inhibition of aggregation and biological relevance.

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Plaques formed by abnormal accumulation of amyloid ß-peptide (Aß) lead to onset of Alzheimer's disease (AD). Pharmacological treatments do not reduce Aß aggregation neither restore learning and memory. Noninvasive techniques have emerged as an alternative to treat AD, such as stimulation with electromagnetic fields (EMF) that decrease Aß deposition and reverses cognitive impairment in AD mice, even though some studies showed side effects on parallel magnetic fields stimulation. As a new approach of magnetic field (MF) stimulation, vortex magnetic fields (VMF) have been tested inducing a random movement of charged biomolecules in cells, promoting cell viability and apparently safer than parallel magnetic fields. In this study we demonstrate the effect of VMF on Aß aggregation. The experimental strategy includes, i) design and construction of a coil capable to induce VMF, ii) evaluation of VMF stimulation on Aß peptide induced-fibrils-formation, iii) evaluation of VMF stimulation on SH-SY5Y neuroblastoma cell line in the presence of Aß peptide. We demonstrated for the first time that Aß aggregation exposed to VMF during 24 h decreased ~ 86% of Aß fibril formation compared to control. Likewise, VMF stimulation reduced Aß fibrils-cytotoxicity and increase SH-SY5Y cell viability. These data establish the basis for future investigation that involve VMF as inhibitor of Aß-pathology and indicate the therapeutic potential of VMF for AD treatment.

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A simple NMR method to analyze the data obtained by NMR titration experiment of amyloid formation inhibitors against uniformly 15N-labeled amyloid-ß 1-42 peptide (Aß(1-42)) was described. By using solution nuclear magnetic resonance (NMR) measurement, the simplest method for monitoring the effects of Aß fibrilization inhibitors is the NMR chemical shift perturbation (CSP) experiment using 15N-labeled Aß(1-42). However, the flexible and dynamic nature of Aß(1-42) monomer may hamper the interpretation of CSP data. Here we introduced principal component analysis (PCA) for visualizing and analyzing NMR data of Aß(1-42) in the presence of amyloid inhibitors including high concentration osmolytes. We measured 1H-15N 2D spectra of Aß(1-42) at various temperatures as well as of Aß(1-42) with several inhibitors, and subjected all the data to PCA (PCA-HSQC). The PCA diagram succeeded in differentiating the various amyloid inhibitors, including epigallocatechin gallate (EGCg), rosmarinic acid (RA) and curcumin (CUR) from high concentration osmolytes. We hypothesized that the CSPs reflected the conformational equilibrium of intrinsically disordered Aß(1-42) induced by weak inhibitor binding rather than the specific molecular interactions.

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For the first time, the effect of two novel designed pentapeptides on amyloid growth of human insulin using combined biophysical, microscopic, cell viability and computational approaches. Collective experimental data from ThT, ANS, and TEM demonstrate that in spite of having contrasting features, both peptides can effectively inhibit amyloid formation by prolonging lag phase, slowing down aggregation rate, and reducing final fibril formation (up to 84.26% and 85.24% by P1 and P7 respectively). Although pure amyloid caused profound cellular toxicity in SH-SY5Y neuronal cells, amyloid formed in the presence of peptides showed much reduced cellular toxicity. Such an inhibitory effect can be attributed to interference with the structural transition of insulin toward ß-sheet structure by peptides. Furthermore, molecular dynamic simulations confirm that peptide preferentially binds to nearby region which is more prone to form aggregates that consequently disrupts self-assembly into amyloid fibrils (P1 and P7 possess inhibition constant value of 0.000183 and 0.000216 nm, respectively), supporting our experimental observations. This study underscores the information about the sequence based inhibition mechanism of peptides that might dictate their inhibition or modulation capacity, which might be helpful in designing anti-amyloid therapeutics.

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Protein misfolding and deposition of aggregated proteins inside as well as outside of the cells have been associated with several neurotoxic and neurodegenerative disorders like Alzheimer's, Parkinson's and familial amyloid polyneuropathy etc. that could be controlled by anti-aggregation methodologies employing either inhibition or disaggregation of toxic aggregates. Also, the Alzheimer's disease develops in later life is somehow related to the high mid-life blood pressure. Therefore the present work targets the amyloid inhibiting potential of diuretics (a class of antihypertensive drugs) - Indapamide (INDP) and Hydrochlorothiazide (HCTZ) against human serum albumin (HSA) and human lysozyme (HL) fibrillogenesis. The effect of both INDP and HCTZ on the kinetics of amyloid formation of HSA and HL was illustrated and various biophysical techniques like Thioflavin T (ThT) and 8-Anilinonaphthalene-1-sulfonic acid (ANS) fluorescence measurement, Congo red measurements and circular dichroism (CD) measurements depicted the inhibitory action of both INDP and HCTZ in a dose dependent manner. Transmission Electronic Microscopy (TEM) confirmed the absence of fibrillar structures when HSA and HL were co-incubated with INDP and HCTZ. In addition, molecular docking results revealed that both the drugs interacts with HSA and HL through hydrophobic interactions as well as hydrogen bonding, and also showed non-hemolytic activity on human RBCs demonstrated by the Hemolytic assay. Thus, both INDP and HCTZ could be propitious as a therapeutic agent and aid in the cure of amyloid related diseases.

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Studying the aggregation of amyloid proteins like α-synuclein in vitro is a convenient and popular tool to gain kinetic insights into aggregation as well as to study factors (e.g., aggregation inhibitors) that influence it. These aggregation assays typically make use of the fluorescence dye Thioflavin T as a sensitive fluorescence reporter of amyloid fibril formation and are conducted in a plate-reader-based format, permitting the simultaneous screening of multiple samples and conditions. However, aggregation assays are generally prone to poor reproducibility due to the stochastic nature of fibril nucleation and the multiplicity of modulating factors. Here we present a simple and reproducible protocol to study the aggregation of α-synuclein in a plate-reader based assay.

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Inhibition of fibrillation process and disaggregation of mature fibrils using small peptide are the promising remedial strategies to combat neurodegenerative diseases. However, designing peptide-based drugs to target ß-sheet-rich amyloid has been a major challenge. The current work describes, for the first time, the amyloid inhibitory potential of the two short peptides (selected on the basis of predisposition of their amino acid residues toward ß-sheet formation) using combination of biophysical, imaging methods, and docking approaches. Results showed that peptides employed different mechanisms to inhibit the amyloid fibrillation. Furthermore, they were also effective in blocking the amyloid fibrillation pathway. In contrary to the insulin fibrillar mesh, significantly less fibrillar species appeared in the presence of peptides, as confirmed by transmission electron microscopy. Circular dichroism analysis indicated that although peptides did not stabilize the native state of insulin, they inhibited amyloid aggregation by reducing the formation of ß-sheet rich structures. Hemolytic assay revealed the non-hemolytic nature of the species formed when insulin was co-incubated with the peptides. Therefore, despite the inherent potential to form ß-sheet structure, these peptides inhibited the amyloid formation and potentially can be used as therapeutics for the treatment of amyloid-related diseases.

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Amyloid beta 1-40 peptide was specifically isolated and analyzed from human plasma spiked with amyloid beta using a combined method of biotinylated anti-amyloid beta antibody binding to membrane-immobilized avidin (immunoaffinity membrane) and matrix-assisted laser desorption /ionization time-of-flight mass spectrometry (MALDI-TOF MS). A solution of 10 µL containing 13.6 ng to 2.9 µg of amyloid beta peptide was examined in this method. After the isolated amyloid beta peptide from the spiked human plasma containing 2.9 µg of amyloid beta peptide was incubated in the presence of trifluoroacetic acid, fibrillization of the peptides was observed using a thioflavin T assay. Furthermore, an immunoaffinity membrane present on the inner wall of a tube (diameter 2 mm) captured the amyloid beta peptide from the spiked human plasma. Our results indicate that the combination of the immunoaffinity membrane procedure and MALDI-TOF MS can be used to capture and analyze the target antigens such as amyloid beta in micro-spaces.

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We here describe the amyloid fibrils promoting behavior of curcumin, which ability to inhibit amyloid fibrillization of several globular proteins is well documented. Transmission electron microscopy (TEM), 90° light scattering (RLS), thioflavine T (ThT) and Congo red (CR) binding studies demonstrated that both F (pH3.4) and E (pH1.8) isomers of human serum albumin (HSA) in the absence and presence of curcumin initially converted into amorphous aggregates. Interestingly, only the sample containing F isomer preincubated with curcumin formed fibrils on incubation for longer period. We also found that curcumin strongly bind to the F isomer, alter its secondary, tertiary structures and thermal stability. We conclude that the conversion of intermediate states into amorphous aggregate to fibrils is dictated by its conformation. This study provides unique insights into ligand-controlled HSA aggregation pathway and should provide a useful model system to study both amorphous and the fibrillar aggregation of multidomain proteins.

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Interaction of small molecule inhibitors with protein aggregates has been studied extensively, but how these inhibitors modulate aggregation kinetic parameters is little understood. In this work, we investigated the ability of two potential aggregation inhibiting drugs, curcumin and kaempferol, to control the kinetic parameters of aggregation reaction. Using thioflavin T fluorescence and static light scattering, the kinetic parameters such as amplitude, elongation rate constant and lag time of guanidine hydrochloride-induced aggregation reactions of hen egg white lysozyme were studied. We observed a contrasting effect of inhibitors on the kinetic parameters when aggregation reactions were measured by these two probes. The interactions of these inhibitors with hen egg white lysozyme were investigated using fluorescence quench titration method and molecular dynamics simulations coupled with binding free energy calculations. We conclude that both the inhibitors prolong nucleation of amyloid aggregation through binding to region of the protein which is known to form the core of the protein fibril, but once the nucleus is formed the rate of elongation is not affected by the inhibitors. This work would provide insight into the mechanism of aggregation inhibition by these potential drug molecules.

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Aggregated α-synuclein is one of the main components of the pathological Lewy bodies associated with Parkinson's disease (PD). Many other proteins, including chaperones such as Hsp90 and Hsp70, have been found co-localized with Lewy bodies and the expression levels of Hsp90 have been found to be increased in brains of PD patients. Although the role of Hsp70 in the aggregation of α-synuclein has been extensively studied, relatively little is known about the effect of Hsp90 on this process. Here, we have investigated if Hsp90 can prevent the aggregation of the A53T pathological mutant of α-synuclein in vitro. A detailed study using many biophysical methods has revealed that Hsp90 prevents α-synuclein from aggregating in an ATP-independent manner and that it forms a strong complex with the transiently populated toxic oligomeric α-synuclein species formed along the aggregation pathway. We have also shown that, upon forming a complex with Hsp90, the oligomers are rendered harmless and nontoxic to cells. Thus, we have clear evidence that Hsp90 is likely to play an important role on these processes in vivo.

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