Thioflavin T Fluorescence Research Articles

Thioflavin T indicates mitochondrial membrane potential in mammalian cells

The fluorescent benzothiazole dye thioflavin T (ThT) is widely used as a marker for protein aggregates, most commonly in the context of neurodegenerative disease research and diagnosis. Recently, this same dye was shown to indicate membrane potential in bacteria due to its cationic nature. This finding prompted a question whether ThT fluorescence is linked to the membrane potential in mammalian cells, which would be important for appropriate utilization of ThT in research and diagnosis. Here, we show that ThT localizes into the mitochondria of HeLa cells in a membrane-potential-dependent manner. Specifically, ThT colocalized in cells with the mitochondrial membrane potential indicator tetramethylrhodamine methyl ester (TMRM) and gave similar temporal responses as TMRM to treatment with a protonophore, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP). Additionally, we found that presence of ThT together with exposure to blue light (λ = 405 nm), but neither factor alone, caused depolarization of mitochondrial membrane potential. This additive effect of the concentration and blue light was recapitulated by a mathematical model implementing the potential-dependent distribution of ThT and its effect on mitochondrial membrane potential through photosensitization. These results show that ThT can act as a mitochondrial membrane potential indicator in mammalian cells, when used at low concentrations and with low blue light exposure. However, it causes dissipation of the mitochondrial membrane potential depending additively on its concentrations and blue light exposure. This conclusion motivates a re-evaluation of ThT’s use at micromolar range in live-cell analyses and indicates that this dye can enable future studies on the potential connections between mitochondrial membrane potential dynamics and protein aggregation.

Experimental procedures to investigate fibrillation of proteins

The unwanted phenomenon of protein fibrillation is observed in vivo and during therapeutic protein development in the industry. Protein aggregation is associated with various degenerative disorders and might induce immune-related challenges post-administration of biopharmaceutics. A pipeline for early detection, identification, and removal of pre-formed fibrils is needed to improve the quality, efficacy, and effectiveness of the formulation. Protein fibril formation is accompanied by unfolding, secondary structural changes and the formation of larger aggregates. However, most detection processes come with extensive sample preparation steps and inefficient repeatability, incurring a financial burden on research. The current article summarizes and critically discusses six simple yet powerful methods to detect aggregation phenomena in the line of detecting fibrillar aggregates in heat-induced bovine serum albumin protein. Comparing the native and heat-induced protein samples would provide insights about aggregates. Easy, inexpensive and optimized protocols for detecting the fibrillation of proteins are explained. The procedures mentioned here detected the appearance of β-sheet-rich fibrils in the heat-induced protein sample. The aggregation is characterized by enhanced thioflavin-T fluorescence, alteration in the intrinsic fluorescence, decrease in helicity and subsequent increase in β-sheet and appearance of particles with larger hydrodynamic diameters.•This article summarizes various analytical techniques to easily characterize the fibrillation of proteins.•Various techniques to detect the formation of β-sheet rich structures, changes in the secondary structures and size of aggregates have been discussed.•The stated methodologies are validated on a model protein, albumin.

Cross-Linked α-Synuclein as Inhibitor of Amyloid Formation.

The aggregation and amyloid formation of α-synuclein is associated with Parkinson's disease and other synucleinopathies. In its native, monomeric form α-synuclein is an intrinsically disordered protein represented by highly dynamic conformational ensembles. Inhibition of α-synuclein aggregation using small molecules, peptides, or proteins has been at the center of interest in recent years. Our aim was to explore the effects of cross-linking on the structure and aggregation/amyloid formation properties of α-synuclein. Comparative analysis of available high-resolution amyloid structures and representative structural models and MD trajectory of monomeric α-synuclein revealed that potential cross-links in the monomeric protein are mostly incompatible with the amyloid forms and thus might inhibit fibrillation. Monomeric α-synuclein has been intramolecularly chemically cross-linked under various conditions using different cross-linkers. We determined the location of cross-links and their frequency using mass spectrometry and found that most of them cannot be realized in the amyloid structures. The inhibitory potential of cross-linked proteins has been experimentally investigated using various methods, including thioflavin-T fluorescence and transmission electron microscopy. We found that conformational constraints applied by cross-linking fully blocked α-synuclein amyloid formation. Moreover, DTSSP-cross-linked molecules exhibited an inhibitory effect on the aggregation of unmodified α-synuclein as well.

Non-micellar ganglioside GM1 induces an instantaneous conformational change in Aβ42 leading to the modulation of the peptide amyloid-fibril pathway

Alzheimer's disease is a progressive degenerative condition that mainly affects cognition and memory. Recently, distinct clinical and neuropathological phenotypes have been identified in AD. Studies revealed that structural variation in Aβ fibrillar aggregates correlates with distinct disease phenotypes. Moreover, environmental surroundings, including other biomolecules such as proteins and lipids, have been shown to interact and modulate Aβ aggregation. Model membranes containing ganglioside (GM1) clusters are specifically known to promote Aβ fibrillogenesis. This study unravels the modulatory effect of non-micellar GM1, a glycosphingolipid frequently released from the damaged neuronal membranes, on Aβ42 amyloid fibril formation. Using far-UV circular dichroism experiments, we observed a change in the peptide secondary structure from random-coil to β-turn structures with subsequent generation of predominantly β-sheet-rich species upon interaction with GM1. Thioflavin-T (ThT) fluorescence assays further indicated that GM1 likely interacts with an amyloidogenic Aβ42 intermediate species leading to a possible formation of GM1-modified Aβ42 fibril. Statistically, no significant difference in toxicity to RA-differentiated SH-SY5Y cells was observed between Aβ42 fibrils and GM1-tweaked Aβ42 aggregates. Moreover, GM1-modified Aβ42 aggregates exhibited prion-like properties in catalyzing the amyloid fibril formation of both major isomers of Aβ, Aβ40, and Aβ42.

Inhibition of amyloid β1–42 peptide aggregation by newly designed cyclometallated palladium complexes

Uncontrolled amyloid aggregation is a frequent cause of neurodegenerative disorders such as prions and Alzheimer's disease (AD). As a result, many drug development approaches focus on evaluating novel molecules that can alter self-recognition pathways. Herein, we designed and synthesized the cyclometallated pyrene (Pd-1 and Pd-3) and anthracene (Pd-2) based palladium complexes ([Pd((L1)Cl] Pd-1, [Pd(L2)Cl](Pd-2), and [Pd(L3)Cl] (Pd-3)). This study explores the effect of these complexes on the aggregation, fibrillation, and amyloid formation of bovine serum albumin (BSA) and Aβ1–42 peptide. Several spectroscopic methods were used to characterize all the Pd-complexes, and the molecular structure of Pd-3 was determined by X-ray crystallography. The secondary structures were studied using circular dichroism (CD) and transmission electron microscopy (TEM), while amyloid aggregation and inhibitory activities were investigated using the Thioflavin-T (ThT) fluorescence assay. Molecular docking of the Pd-complex (Pd-3) was done using fibril (PDB: 2BEG) and monomeric (PDB: 1IYT) peptides using Auto-dock Vina. As a result, the hydrogen bonding and hydrophobic interaction between the aromatic rings of the Pd-complexes and the amino acids of amyloid-β peptides significantly reduced the production of ordered β-sheets of amyloid fibrils and protein aggregation in the presence of Pd-2 and Pd-3 complexes.

A G-triplex and G-quadruplex concatemer-enhanced fluorescence probe coupled with hybridization chain reaction for ultrasensitive aptasensing of ochratoxin A

Ochratoxin A (OTA), a typical mycotoxin contaminant found in various agricultural products and foods, poses a serious threat to human health. In this study, an aptasensor based on a novel fluorescence probe comprising a G-rich DNA sequence (G43) and thioflavin T (ThT) was designed via hybridization chain reaction (HCR) for the ultrasensitive detection of OTA. G43 is a concatemer of G-quadruplex and G-triplex (a G-quadruplex-like structure with one G-quartet removed), which can drastically enhance the fluorescence intensity of ThT. For this strategy to work, the OTA aptamer is pro-locked in a hairpin structure, denoted “hairpin-locked aptamer” (HL-Apt). OTA binds to HL-Apt, opens the hairpin structure, releases the trigger sequence, and initiates the HCR reaction to form a long DNA duplex and numerous side chains. The side chains combine entirely with the complementary DNA and liberate the pro-locked G43 DNA, dramatically enhancing the intensity of the ThT fluorescence signal. The fluorescence intensity correlates linearly with the OTA concentration between 0.02 and 2.00 ng mL−1, and the method has a detection limit of 0.008 ng mL−1. The developed aptasensor was used to detect OTA in foodstuffs with satisfactory results.

Amyposomes, a nanotechnological chaperone with anti-amyloidogenic activity

Aim The effect of liposomes bi-functionalized with phosphatidic acid and with a synthetic peptide derived from human apolipoprotein E has been evaluated on the aggregation features of different amyloidogenic proteins: human Amyloid β1–40 (Aβ1–40), transthyretin (TTR) variant S52P, human β2microglobulin (β2m) variants ΔN6 and D76N, Serum Amyloid A (SAA). Methods The formation of fibrillar aggregates of the proteins was investigated by ThioflavinT fluorescence assay and validated by Atomic Force Microscopy. Results The results show that liposomes are preventing the transition of non-aggregated forms to the fibrillar state, with stronger effects on Aβ1–40, β2m ΔN6 and SAA. Liposomes also induce disaggregation of the amyloid aggregates of all the proteins investigated, with stronger effects on Aβ1–40, β2 D76N and TTR. SPR assays show that liposomes bind Aβ1–40 and SAA aggregates with high affinity (KD in the nanomolar range) whereas binding to TTR aggregates showed a lower affinity (KD in the micromolar range). Aggregates of β2m variants showed both high and low affinity binding sites. Computed Structural analysis of protein fibrillar aggregates and considerations on the multidentate features of liposomes allow to speculate a common mechanism of action, based on binding the β-stranded peptide regions responsible for the amyloid formation. Conclusion Thus, multifunctional liposomes perform as pharmacological chaperones with anti-amyloidogenic activity, with a promising potential for the treatment of a number of protein-misfolding diseases. Key message Amyloidosis is a group of diseases, each due to a specific protein misfolding. Anti-amyloidogenic nanoparticles have been gaining the utmost importance as a potential treatment for protein misfolding disorders. Liposomes bi-functionalized with phosphatidic acid and with a synthetic peptide derived from human apolipoprotein E showed anti-amyloidogenic activity.

Manganese Ion-Induced Amyloid Fibrillation Kinetics of Hen Egg White-Lysozyme in Thermal and Acidic Conditions.

As manganese ions (Mn2+) are identified as an environmental risk factor for neurodegenerative diseases, uncovering their action mechanism on protein amyloid fibril formation is crucial for related disease treatments. Herein, we performed a combined study of Raman spectroscopy, atomic force microscopy (AFM), thioflavin T (ThT) fluorescence, and UV-vis absorption spectroscopy assays, in which the distinctive effect of Mn2+ on the amyloid fibrillation kinetics of hen egg white-lysozyme (HEWL) was clarified at the molecular level. With thermal and acid treatments, the unfolding of protein tertiary structures is efficiently accelerated by Mn2+ to form oligomers, as indicated by two Raman markers for the Trp residues on protein side chains: the FWHM at 759 cm-1 and the I1340/I1360 ratio. Meanwhile, the inconsistent evolutionary kinetics of the two indicators, as well as AFM images and UV-vis absorption spectroscopy assays, validate the tendency of Mn2+ toward the formation of amorphous aggregates instead of amyloid fibrils. Moreover, Mn2+ plays an accelerator role in the secondary structure transition from α-helix to organized β-sheet structures, as indicated by the N-Cα-C intensity at 933 cm-1 and the amide I position of Raman spectroscopy and ThT fluorescence assays. Notably, the more significant promotion effect of Mn2+ on the formation of amorphous aggregates provides credible clues to understand the fact that excess exposure to manganese is associated with neurological diseases.

Suppression of TDP-43 aggregation by artificial peptide binder targeting to its low complexity domain

TAR DNA-binding protein 43 (TDP-43), aggregation prone protein, is a potential target of drug discovery for amyotrophic lateral sclerosis. The molecular binders, targeting the disordered low complexity domain (LCD) relevant to the aggregation, may suppress the aggregation. Recently, Kamagata et al. developed a rational design of peptide binders targeting intrinsically disordered proteins based on contact energies between residue pairs. In this study, we designed 18 producible peptide binder candidates to TDP-43 LCD by using this method. Fluorescence anisotropy titration and surface plasmon resonance assays demonstrated that one of the designed peptides bound to TDP-43 LCD at 30 μM. Thioflavin-T fluorescence and sedimentation assays showed that the peptide binder suppressed the aggregation of TDP-43. In summary, this study highlights the potential applicability of peptide binder design for aggregation prone proteins.

Influence of cadmium ion on denaturation kinetics of hen egg white-lysozyme under thermal and acidic conditions

To study the influence of Cd(II) ions on denaturation kinetics of hen egg white lysozyme (HEWL) under thermal and acidic conditions, spontaneous Raman spectroscopy in conjunction with Thioflavin-T fluorescence, AFM imaging, far-UV circular dichroism spectroscopy, and transmittance assays was conducted. Four distinctive Raman spectral markers for protein tertiary and secondary structures were recorded to follow the kinetics of conformational transformation. Through comparing variations of these markers in the presence or absence of Cd(II) ions, Cd(II) ions show an ability to efficiently accelerate the disruption of tertiary structure, and meanwhile, to promote the direct formation of organized β-sheets from the uncoiling of α-helices by skipping intermediate random coils. More significantly, with the action of Cd(II) ions, the initially resulting oligomers with disordered structures tend to assemble into aggregates with random structures like gels more than amyloid fibrils, along with a so-called “off-pathway” denaturation pathway. Our results advance the in-depth understanding of corresponding ion-specific effects.

An efficient DNAzyme-based DNA scaffold for label-free and sensitive bacterial pathogen detection

Even though it is very important, it is still rather difficult to detect minuscule levels of the bacterial pathogen in clinical practice, such as samples from dental implants. We construct here an efficient scaffold for label-free and sensitive Staphylococcus aureus (S. aureus) detection. The precise recognition of target bacteria by the detection scaffold leads to the self-assembly of Chain i and DNAzyme based cleavage of Chain iii. In detail, active DNAzyme conformation is formed based on the hybridization of Chain iii and Chain ii, and a nicking site is generated in Chain iii, making it possible to form a self-primer in Chain i. With the assistance of DNA polymerase, a single-strand DNA chain is added to the 3′ terminal of Chain i, in which process the bacteria is released for the complex to bind with a next detection scaffold, forming a signal recycle. Following DNAzyme-based cleavage, the liberated sequences unroll MB and release G-rich sequences that can specifically bind with the fluorescent dye Thioflavin T (ThT), initiating ThT's fluorescence signal production. The approach demonstrates a wide detection range of 102 CFU/mL and 106 CFU/mL with a low limit of detection of 45 CFU/mL based on the developed detection scaffold, offering good prospects in the diagnosis of bacterial illnesses.

Effect of Antihypertensive Drug (Chlorothiazide) on Fibrillation of Lysozyme: A Combined Spectroscopy, Microscopy, and Computational Study.

Amyloid fibrils abnormally accumulate together in the human body under certain conditions, which can result in lethal conditions. Thus, blocking this aggregation may prevent or treat this disease. Chlorothiazide (CTZ) is a diuretic and is used to treat hypertension. Several previous studies suggest that diuretics prevent amyloid-related diseases and reduce amyloid aggregation. Thus, in this study we examine the effects of CTZ on hen egg white lysozyme (HEWL) aggregation using spectroscopic, docking, and microscopic approaches. Our results showed that under protein misfolding conditions of 55 °C, pH 2.0, and 600 rpm agitation, HEWL aggregated as evidenced by the increased turbidity and Rayleigh light scattering (RLS). Furthermore, thioflavin-T, as well as trans electron microscope (TEM) analysis confirmed the formation of amyloid structures. An anti-aggregation effect of CTZ is observed on HEWL aggregations. Circular dichroism (CD), TEM, and Thioflavin-T fluorescence show that both CTZ concentrations reduce the formation of amyloid fibrils as compared to fibrillated. The turbidity, RLS, and ANS fluorescence increase with CTZ increasing. This increase is attributed to the formation of a soluble aggregation. As evidenced by CD analysis, there was no significant difference in α-helix content and β-sheet content between at 10 µM CTZ and 100 µM. A TEM analysis of HEWL coincubated with CTZ at different concentrations validated all the above-mentioned results. The TEM results show that CTZ induces morphological changes in the typical structure of amyloid fibrils. The steady-state quenching study demonstrated that CTZ and HEWL bind spontaneously via hydrophobic interactions. HEWL-CTZ also interacts dynamically with changes in the environment surrounding tryptophan. Computational results revealed the binding of CTZ to ILE98, GLN57, ASP52, TRP108, TRP63, TRP63, ILE58, and ALA107 residues in HEWL via hydrophobic interactions and hydrogen bonds with a binding energy of -6.58 kcal mol-1. We suggest that at 10 µM and 100 μM, CTZ binds to the aggregation-prone region (APR) of HEWL and stabilizes it, thus preventing aggregation. Based on these findings, we can conclude that CTZ has antiamyloidogenic activity and can prevent fibril aggregation.

Peptide-based inhibitors of amyloid beta aggregation and toxicity.

Neurotoxic aggregates of amyloid beta (Aβ) peptide contribute to the etiology of Alzheimer's disease. Aβ1-42 forms oligomeric structures that undergo further aggregation into protofibrils and fibrils. Previous work has shown that seven overlapping fragments of Aβ1-42 can disrupt the process of fibril formation, possibly through intercalation into the growing aggregate. The potential neuroprotective properties of these fragments were not directly examined. In this project, Aβ1-42 was allowed to aggregate in aqueous buffer for 2, 4, or 24 h in the absence and presence of an Aβ fragment, at 1:2 Aβ1-42/fragment molar ratio. Neuronal PC12 cells were then incubated with the samples for 50 h before conducting a cell viability assay. Thioflavin-T fluorescence assays to detect the fibrillar form of Aβ1-42 were also conducted. There was an inverse correlation between the time span of Aβ1-42 aggregation and cellular toxicity, with the 2 h sample exhibiting the greatest level of toxicity. Aβ fragments displayed varying effects on the inhibition of Aβ1-42 fibrillogenesis and suppression of its toxicity. Three fragments completely neutralized the toxicity of the 2 h aggregate, while two others conferred weak cellular protection against Aβ1-42 toxicity. One fragment enhanced Aβ1-42 toxicity at all stages of aggregation. Every fragment disrupted fibril formation to some extent, but there was no obvious correlation between the inhibition of Aβ1-42 aggregation and the inhibition of cellular toxicity. This suggests that fragment intercalation into the growing fibril may block further aggregation but not the toxicity of oligomeric Aβ1-42. Fragments that block both Aβ1-42 aggregation and toxicity represent potential targets for therapeutic development.

Development of a novel fluorescence assay for studying lipid bilayer perturbation induced by amyloidogenic peptides using cell plasma membrane vesicles

Numerous pathophysiological conditions are associated with the misfolding and aggregation of proteins into insoluble amyloid fibrils. The mechanisms by which this process leads to cellular dysfunction remain elusive, though several hypotheses point toward the perturbation of the cell plasma membrane by pre-fibrillar intermediates and/or amyloid growth. However, current models to study membrane perturbations are largely limited to synthetic lipid vesicles and most of experimental approaches cannot be transposed to complex cell-derived plasma membrane systems. Herein, vesicles originating from the plasma membrane of erythrocytes and β-pancreatic cells were used to study the perturbations induced by an amyloidogenic peptide, the islet amyloid polypeptide (IAPP). These biologically relevant lipid vesicles displayed a characteristic clustering in the presence of the amyloidogenic peptide, which was able to rupture membranes. By exploiting Förster resonance energy transfer (FRET), a rapid, simple, and potentially high-throughput assay to detect membrane perturbations of intact mammalian cell plasma membrane vesicles was implemented. The FRET kinetics of membrane perturbations closely correlated with the kinetics of thioflavin-T fluorescence associated with amyloid formation. This novel kinetics assay expands the toolbox available to study amyloid-associated membrane damage, bridging the gap between synthetic lipid vesicles and living cells.

Molecular interactions of resveratrol with Aβ 42 peptide and fibril during in-vitro Aβ 42 aggregation

Amyloid aggregates are responsible for the development of oxidative stress and neurotoxicity in the brain. Several amino acid residues of amyloid beta (Aβ) 42 establish different molecular interactions to form and stabilize these aggregates, which can be targeted to prevent the aggregation. Resveratrol (Res) has inhibitory properties against Aβ 42 aggregation but studies elucidating its interactions with different residues of Aβ 42 aggregates are scarce. In the present study, we have discerned the molecular interactions of Res with different amino acid residues of Aβ 42 peptide and fibril during in-vitro Aβ 42 aggregation. Inhibitory properties of Res against amyloid aggregation were established through ANS and Thioflavin-T fluorescence assay, congo red assay and CD spectroscopy. The molecular interactions were established through molecular docking and 100 ns molecular dynamics simulations. The hydrophobic regions of Aβ 42 peptide, responsible for the formation of aggregates, were better protected in the presence of Res as indicated by ANS assay. Th-T and congo red assay suggested that Res prevented the formation of cross β-sheet structures. CD spectra analysis revealed that in the presence of Res, the secondary structure content was significantly decreased. MD simulation analysis revealed that Res formed strong molecular interactions with hydrophobic and secondary structure forming amino acid residues, which are involved in the amyloid aggregation and stabilization of aggregates. In conclusion, these interactions might have led to the decline in secondary structure content, formation of unordered nontoxic aggregates and prevention of the formation of sufuranyl free radical.

Versatile lipoprotein-inspired nanocomposites rescue Alzheimer's cognitive dysfunction by promoting Aβ degradation and lessening oxidative stress.

The accumulation of amyloid-β (Aβ) into senile plaques and the resulting continuous oxidative stress are major pathogenic mechanisms in Alzheimer's disease (AD). In this study, we designed a lipoprotein-inspired nanoparticle to facilitate Aβ clearance and alleviate oxidative stress for the treatment of AD. Lipoprotein-like nanocomposites (RLA-rHDL@ANG) were fabricated by assembling reconstituted high density lipoprotein (rHDL) with an apoE-derived peptide (RLA) with Aβ binding and clearance capabilities, and were subsequently camouflaged using reactive oxygen species (ROS)-sensitive DSPE-TK-mPEG2000 and DSPE-TK-PEG3400-ANG with brain penetration as well as ROS scavenging ability. Immunoelectron microscopy, fluorescence colocalization, and enzyme linked immunosorbent assay, together with a thioflavin-T (ThT) fluorescence quantitative test, showed that RLA-rHDL@ANG possessed the ability of high binding affinity to both Aβ monomers and oligomers, and disintegration of pre-formed Aβ aggregates. ROS level monitoring and transmission electron microscopy (TEM) showed that RLA-rHDL@ANG possessed ROS sensitivity and consumption properties. Transcellular assay and in vivo imaging showed that RLA-rHDL@ANG effectively facilitated blood-brain barrier (BBB) penetration and intracerebral accumulation. It promoted the efficient degradation of Aβ by microglia and neurons through lysosomal transport and elimination approaches. Four-week administration of RLA-rHDL@ANG effectively reduced Aβ deposition, decreased the ROS level and improved cognitive functions in AD mice. These findings indicate that multifunctional RLA-rHDL@ANG may serve as a promising and feasible candidate for managing the progression of AD.

The extracellular chaperone clusterin prevents primary and secondary nucleation of an amyloidogenic variant of β2-microglobulin

Amyloid fibril formation by the extracellular protein β2-microglobulin (β2m) and its subsequent accumulation in periarticular tissues have been linked to dialysis-related amyloidosis. A natural variant of human β2m responsible for aggressive systemic amyloidosis contains an aspartate to asparagine mutation at residue 76 (i.e. D76N β2m), which readily forms amyloid fibrils in vitro under physiological conditions. In this study, we examined the role of the extracellular molecular chaperone clusterin in modulating D76N β2m fibril formation in vitro under physiological conditions. The presence of extrinsic charged amino acids modulated D76N β2m fibril formation, implying that electrostatic interactions are involved in the protein’s aggregation. Thioflavin T (ThT) and 1-anilinonaphthalene-8-sulfonate fluorescence assays indicated that clusterin interacts via hydrophobic and electrostatic forces with the monomeric, prefibrillar and fibrillar species of D76N β2m. As a result, clusterin was incorporated into D76N β2m aggregates during the latter’s fibril formation, as indicated by SDS-PAGE of depolymerised fibrils. SYPRO Orange and ThT fluorescence assays suggested that, compared to pure D76N β2m fibrils, those formed in the presence of clusterin are chemically more stable with a reduced ability to act as nucleation seeds. Detailed 15N NMR relaxation studies of mixtures of 15N-labelled β2m with clusterin confirmed that the chaperone interacts transiently and non-specifically with monomeric β2m. Clusterin inhibits both primary and secondary nucleation of D76N β2m fibril formation. In doing so, clusterin binds to D76N β2m fibrils and stabilises them to prevent possible fragmentation. In vivo, the multifaceted chaperone action of clusterin may delay, if not prevent, β2m amyloid proliferation and deposition in tissues.

Concentration-dependent effect of Nickel ions on amyloid fibril formation kinetics of hen egg white lysozyme: A Raman spectroscopy study

Nickel, an important transition metal element, is one of the trace elements for human body and has a crucial impact on life and health. Some evidences show the excess exposure to metal ions might be associated with neurological diseases. Herein, we applied Raman spectroscopy to study the Ni(II) ion effect on kinetics of amyloid fibrillation of hen egg white lysozyme (HEWL) in thermal and acidic conditions. Using the well-known Raman indicators for protein tertiary and secondary structures, we monitored and analyzed the concentration effect of Ni(II) ions on the unfolding of tertiary structures and the transformation of secondary structures. The experimental evidence validates the accelerator role of the metal ion in the kinetics. Notably, the additional analysis of the amide I band profile, combined with thioflavin-T fluorescence assays, clearly indicates the inhibitory effect of Ni(II) ions on the formation of amyloid fibrils with organized β-sheets structures. Instead, a more significant promotion influence is affirmed on the assembly into other aggregates with disordered structures. The present results provide rich information about the specific metal-mediated protein fibrillation.

Probing the Tl+-induced structural transformation of G-rich DNA to G-quadruplex with a label‐free thioflavin T-based biosensor

More than 700,000 G-rich sequences in the human genome are able to form G-quadruplex (G4). Since G4 is closely relevant to a variety of essential biological regulatory processes, identification of these G-rich DNAs (G-rich → G4) is thus important for DNA-involved interactions, e.g., metal ions and small molecules. Circular dichroism (CD) and NMR are the dominated tools for studying G4 DNA-based interactions, but suffers from low sensitivity. Herein, with toxic Tl+ as the model inducer, we demonstrated that thioflavin T (ThT), a typical label-free G4 dye, could be explored for highly sensitive probing of Tl+-induced structural transformation of G-rich DNAs to G4. With several model G4-forming DNAs, it was found that the Tl+-induced CD spectra developments correlated well with its fluorescence modulation on ThT (ThT-DNA → ThT-DNA-Tl+). In other words, the Tl+-induced structural change (Free State → G4) could be well probed with the ThT fluorescence change. For typical non-G4-forming DNAs, no appreciable ThT fluorescence modulation was received. To further increase the accuracy of picking up G4-forming DNAs from unknown samples, the photophysical parameters (absorption, fluorescence emission, and fluorescence lifetime) change of ThT before and after Tl+ modulation were integrated to develop a two-step linear discrimination analysis (LDA) protocol, with cross-validation accuracy of over 90%. After picking up G4-forming DNAs from the unknown samples, the Tl+-induced structural transformation of G4 DNAs could be thus evaluated.

Mechanistic insights into monomer level prevention of amyloid aggregation of lysozyme by glycyrrhizic acid

As the primary bioactive compound of glycyrrhiza rhizome, the triterpene glycoside conjugate Glycyrrhizic acid (GA) has demonstrated neuroprotective effects in vivo. This study evaluates the effectiveness of GA as an inhibitor of GuHCl-induced amyloid aggregation of hen egg white lysozyme (HEWL). Fibril formation as measured by Thioflavin-T fluorescence, 900 light scattering, and 8-Anilinonaphthalene-1-sulfonic acid (ANS) fluorescence illustrated ∼90 % prevention of fibrils at [GA]/[HEWL] ≥2:1. Images of Transmission electron microscopy evidence for the absence of any fibril or amorphous aggregation products. The spectral characteristics of soluble HEWL were in close resemblance to unfolded monomer. Computational and fluorescence investigations performed to analyse GA-HEWL interactions demonstrated slightly higher affinity of GA to unfolded HEWL and aggregation-prone regions. The likely mechanism of monomer level aggregation prevention by GA as dermined by computational, stability, and ANS experiments illustrated that GA modulated the compactness, solvent-accessible surface, and solvent-exposed hydrophobic surfaces of aggregation-prone state of HEWL. Our findings corroborate GA as an effective inhibitor of HEWL amyloid formation. To our knowledge, GA interaction-induced inhibition of aggregation-prone states has not been previously discussed. GA's modulation of aggregation-prone states of disease-related proteins will successfully develop GA as an amyloid inhibitor for clinical trials of amyloidosis and neurodegenerative illnesses.