ANS Binding Assay Research Articles

Quercetin-Loaded Nanocarriers as Effective Inhibitors for Copper Metal Ion-Induced γD-Crystallin Aggregation.

Cataract is one of the leading causes of blindness worldwide. Till date, the only solution for cataracts is surgery, which is a resource-intensive solution. A much simpler solution is to find a potential drug that could inhibit aggregation. It is well established that nonamyloid aggregates of eye lens protein result in cataract. γD-Crystallin, a thermodynamically stable protein, is one of the most abundant proteins in the core of the eye lens and is found to aggregate under stress conditions, leading to the cataract. It has also been found that in cataractous lens, the concentration of metals like copper is elevated significantly as compared to healthy eye lens, suggesting their role in inducing aggregation. In our present study, aggregation of γD-Crystallin was carried out in the presence of Cu (II). Using techniques like turbidity assay, CD spectroscopy, ANS binding assay, and microscopic studies like TEM, it could be confirmed that protein aggregates in the presence of Cu (II) and the nature of aggregates is amorphous. Various polyphenols were tested to suppress aggregation of the protein. Quercetin was observed to be the most efficient. To overcome the problems associated with the delivery of polyphenols, such as solubility and bioavailability, quercetin was encapsulated in two types of nanocarriers. Their characterization was done using TEM, DLS, and other techniques. The potency of quercetin-loaded CS-TPP/CS-PLGA NPs as inhibitors of γD-Crystallin aggregation was confirmed by various experiments.

Biophysical insight into the binding mechanism of epigallocatechin-3-gallate and cholecalciferol to albumin and its preventive effect against AGEs formation: An in vitro and in silico approach

Advanced glycation end products (AGEs) are produced non-enzymatically through the process of glycation. Increased AGEs production has been linked to several diseases including polycystic ovary syndrome (PCOS). PCOS contributes to the development of secondary comorbidities, such as diabetes, cardiovascular complications, infertility, etc. Consequently, research is going on AGEs-inhibiting phytochemicals for their potential to remediate and impede the progression of hyperglycaemia associated disorders. In this study human serum albumin is used as a model protein, as albumin is predominantly present in follicular fluid. This article focusses on the interaction and antiglycating potential of (−)-Epigallocatechin-3-gallate (EGCG) and vitamin D in combination using various techniques. The formation of the HSA-EGCG and HSA-vitamin D complex was confirmed by UV and fluorescence spectroscopy. Thermodynamic analysis verified the spontaneity of reaction, and presence of hydrogen bonds and van der Waals interactions. FRET confirms high possibility of energy transfer. Cumulative antiglycation resulted in almost 60 % prevention in AGEs formation, decreased alterations at lysine and arginine, and reduced protein carbonylation. Secondary and tertiary structural changes were analysed by circular dichroism, Raman spectroscopy and ANS binding assay. Type and size of aggregates were confirmed by Rayleigh and dynamic light scattering, ThT fluorescence, SEM and SDS-PAGE. Effect on cellular redox status, DNA integrity and cytotoxicity was analysed in lymphocytes using dichlorofluorescein (DCFH-DA), DAPI and MTT assay which depicted an enhancement in antioxidant level by cumulative treatment. These findings indicate that EGCG and vitamin D binds strongly to HSA and have antiglycation ability which enhances upon synergism.

Endotoxin accelerates insulin amyloid formation and inactivates insulin signal transduction

Aims and objectivesThe aim of this study is to discuss the influence of endotoxin on insulin amyloid formation, to provide guidance for therapeutic insulin preparation and storage. Materials and methodsThe ThT and ANS binding assays were applied to characterize the dynamics curve of insulin amyloid formation with the presence or absence of endotoxin. The morphological structures of intermediate and mature insulin fibrils were observed with SEM and TEM. Secondary structural changes of insulin during fibriliation were examined with CD, FTIR and Raman spectral analysis. The cytotoxic effects of oligomeric and amyloidogenic insulin aggregates were detected using a cck-8 cell viability assay kit. The influence of endotoxin on insulin efficacy was analyzed by monitoring the activation of insulin signal transduction. Key findingsThT analysis showed that endotoxin, regardless of species, accelerated insulin fibrils formation in a dose-dependent manner, as observed with a shorter lag phase. ANS binding assay demonstrated endotoxin provoked the exposure of insulin hydrophobic patches. The results of SEM and TEM data displayed that endotoxin drove insulin to cluster into dense and viscous form, with thicker and stronger filaments. Based on CD, FTIR and Raman spectra, endotoxin promoted the transition of α-helix to random coil and β-strand secondary structures during insulin aggregation. Insulins in both oligomeric and amyloidogenic forms were cytotoxic to HepG2 cells, with the former being more severe. Finally, the efficacy of endotoxin treated insulin obviously decreased. SignificanceOur studies revealed that endotoxin disrupts the structural integrity of insulin and promotes its amyloidosis. These findings offered theoretical guidance for insulin storage and safe utilization, as well as pointing up a new direction for insulin resistance research.

Mechanistic insight into differential interactions of iron oxide nanoparticles with native, glycated albumin and their effect on erythrocytes parameters

Nanoparticles and protein bioconjugates have been studied for multiple biomedical applications. We sought to investigate the interaction and structural modifications of bovine serum albumin (BSA) with iron oxide nanoparticles (IONPs). The IONPs were green synthesized using E. crassipes aqueous leaf extract following characterization using transmission electron microscopy, energy dispersive X-ray analysis and X-ray diffraction. Two different concentrations of native/glycated albumin (0.5 and 1.5 mg/ml) with IONPs were allowed to interact for 1 h at 37 °C. Glycation markers, protein modification markers, cellular antioxidant, and hemolysis studies showed structural modifications and conformational changes in albumin due to the presence of IONPs. UV–visible absorbance resulted in hyperchromic and bathochromic effects of IONPs-BSA conjugates. Fluorescence measurements of tyrosine, tryptophan, advanced glycated end products, and ANS binding assay were promising and quenching effects proved IONPs-BSA conjugate formation. In FTIR of BSA-IONPs, transmittance was increased in amide A and B bands while decreased in amide I and II bands. In summary, native PAGE, HPLC, and FTIR analysis displayed a differential behaviour of IONPs with native and glycated BSA. These results provided an understanding of the interaction and structural modifications of glycated and native BSA which may provide fundamental repercussions in future studies.

Elucidating the binding and inhibitory potential of p-coumaric acid against amyloid fibrillation and their cytotoxicity: Biophysical and docking analysis

P-Coumaric acid (p-CA) is a plant metabolite with anti-inflammatory and antioxidant effects. Due to its therapeutic potential, p-CA has attracted much attention from the scientific community lately. Oxidative stress, amyloid formation, and impaired proteasomal degradation are hallmarks of neurodegenerative diseases like Alzheimer's (AD) and are targets for developing therapeutics against such conditions. Here, we have investigated the anti-amyloidogenic properties of p-coumaric acid on hen egg white lysozyme (HEWL). Heat, pH, and agitation (55 °C, pH 2.0, 600 rpm) stress were used to induce amyloid formation in lysozyme. The aggregates characterization was done by turbidity, Rayleigh light scattering (RLS), and thioflavin-T (ThT) assays. Moreover, ANS (1-anilino naphthalene sulphate) binding assay and circular dichroism (CD) were employed to unveil protein hydrophobicity and secondary structure perturbation, respectively. Lysozyme demonstrated increased hydrophobicity and transition of α-helix to β-sheet under aggregating conditions. Moreover, co-incubation of lysozyme with p-coumaric acid attenuates the process of amyloid in a concentration dependent manner. At 50 and 200 μM concentrations of p-coumaric acid, lysozyme retained its native-like folded structure. Cytotoxicity protection on human SK-N-SH neuroblastoma cell line was also observed using MTT assay and phase contrast microscopy. In addition, transmission electron microscopy (TEM) reaffirms the fibrillar nature of lysozyme aggregates and their attenuation by p-coumaric acid. The steady state fluorescence revealed that the mode of fluorescence quenching for the HEWL-p-coumaric acid interaction is static rather than dynamic. Moderate strength of binding in order of 104 M−1 exists between HEWL and p-coumaric acid. Thermodynamic parameters (∆H and ∆S) obtained from van't Hoff plot suggested spontaneous reaction with hydrophobic interaction. A slight micro-environmental change in HEWL around Tyr residue was observed during the binding process with the help of synchronous fluorescence. Molecular docking analysis reported the involvement of amino acid residues (TRP63, LEU75, ASP101, LYS97) to form a complex between HEWL-p-coumaric acid. The observed anti-amyloidogenic and inherent antioxidative properties of p-coumaric acid could be helpful to design a neuroprotective agent.

Structural and mechanistic insights into modulation of α-Synuclein fibril formation by aloin and emodin

α-Synuclein (α-Syn) aggregation/fibrillation is a leading cause of neuronal death and is one of the major pathogenic factors involved in the progression of Parkinson's' disease (PD). Against this backdrop, discovering new molecules as inhibitors or modulators of α-Syn aggregation/fibrillation is a subject of enormous research. In this study, we have shown modulation, disaggregation, and neuroprotective potential of aloin and emodin against α-Syn aggregation/fibrillation. Thioflavin T (ThT) fluorescence assay showed an increase in lag phase from (51.14 ± 2) h to (68.58 ± 2) h and (74.14 ± 3) h in the presence of aloin and emodin respectively. ANS binding assay represents a modulatory effect of these molecules on hydrophobicity which is crucial for aggregates/fibril formation. NMR spectroscopy and tyrosine quenching studies reveal the binding of aloin/emodin with monomeric α-Syn. TEM and DLS micrographs illustrate the attenuating effect of aloin/emodin against the development of large aggregates/fibrils. Our seeding experiments suggest aloin/emodin generate seeding incompetent oligomers that direct the off-pathway aggregation/fibrillation. Also, aloin/emodin capably reduces the fibrils-induced cytotoxicity and disassembles the preexisting amyloid fibrils. These findings provide deep insight into the modulatory mechanism of α-Syn aggregation/fibrillation in the presence of aloin and emodin, thereby suggesting their potential roles as promising therapeutic molecules against aggregation/fibrillation related disorders.

Mechanistic In Vitro Dissection of the Inhibition of Amyloid Fibrillation by n-Acetylneuraminic Acid: Plausible Implication in Therapeutics for Neurodegenerative Disorders.

A variety of neurodegenerative disorders including Parkinson's disease are due to fibrillation in amyloidogenic proteins. The development of therapeutics for these disorders is a topic of extensive research as effective treatments are still unavailable. The present study establishes that n-acetylneuraminic acid (Neu5ac) inhibits the amyloid fibrillation of hen egg-white lysozyme (HEWL) and α-synuclein (SYN), as observed using various biophysical techniques and cellular assays. Neu5ac inhibits the amyloid formation in both proteins, as suggested from the reduction in the ThT fluorescence and remnant structures in transmission electron microscopy micrographs observed in its presence. In HEWL fibrillation, Neu5ac decreases the hydrophobicity and resists the transition of the α-helix to a β-sheet, as observed by an ANS binding assay, circular dichroism (CD) spectra, and Fourier transform infrared measurements, respectively. Neu5ac stabilizes the states that facilitate the amyloid formation in HEWL and SYN, as demonstrated by an enhanced intrinsic fluorescence in its presence, which is further confirmed by an increase in Tm obtained from differential scanning calorimetry thermograms and an increase in the near-UV CD signal for HEWL with Neu5ac. However, the increase in stability is not a manifestation of Neu5ac binding to amyloid facilitating (partially folded or native) states of both proteins, as verified by isothermal titration calorimetry and fluorescence binding measurements. Besides, Neu5ac also attenuates the cytotoxicity of amyloid fibrils, as evaluated by a cell toxicity assay. These findings provide mechanistic insights into the Neu5ac action against amyloid fibrillation and may establish it as a plausible inhibitor molecule against neurodegenerative disorders.

Disrupting putative N-glycosylation site N17 in lipase Lip11 of Yarrowia lipolytica yielded a catalytically efficient and thermostable variant accompanying conformational changes

Lip11 gene from oleaginous yeast Yarrowia lipolytica MSR80 was recombinantly expressed in Pichia pastoris X33. Native secretion signal present in its sequence resulted in 92 % expression in comparison to α-secretion factor which resulted to 900 U/L in the extracellular broth. Catalytic triad in Lip11, like most lipases, was formed by serine, histidine, and aspartate residues. While point mutation disrupting putative glycosylation site (N389) present towards the C-terminus ruinously effected its stability and catalytic activity, disruption of the first putative glycosylation site (N17) located towards the N-terminus presented interesting insights. Mutation resulted in a variant N1 exhibiting higher thermal and acid stability; a t1/2 of 198 min was obtained at 50 °C and it retained almost 80 % activity following incubation at pH 3. Catalytic efficiency was improved by 2.7 fold and a 10 °C rise in temperature optima was accompanied by higher relative activity in acidic range. Thermal stability corresponded to convoying structural modifications in the tertiary structure, findings of fluorescence spectroscopy suggested. Thermal fluorescence studies revealed a Tm of 65 °C for both Lip11 and N1 and λmax of Lip11 exhibited a blue shift upon refolding while no shift in the λmax of N1 was observed. A resilient tertiary structure which could fold back to its native confirmation upon thermal denaturation and increase in surface-exposed hydrophobic residues as revealed by ANS binding assay summed up to thermal stability of N1. Furthermore, circular dichroism data disclosed an alternate ratio of alpha-helices and beta-sheets; respective values changed from 36 % and 8%–27% and 19 %. Following mutation, substrate specificity remained unaffected and similar to native protein, N1 showed activation in presence of organic solvents and most divalent cations.

Modulation of α-synuclein fibrillation by plant metabolites, daidzein, fisetin and scopoletin under physiological conditions

The aggregation of α-synuclein is linked to neurological disorders, and of these, Parkinson's disease (PD) is among the most widely studied. In this background, we have investigated here the effects of three α, β-unsaturated carbonyl based plant metabolites, daidzein, fisetin and scopoletin on α-Syn aggregation. The ThT and light scattering kinetics studies establish that these compounds have ability to inhibit α-Syn fibrillation to different extents; this is confirmed by TEM studies. It is pertinent to note here that daidzein and scopoletin have been predicted to be able to cross the blood brain barrier. ANS binding assays demonstrate that the compounds interfere in the hydrophobic interactions. The tyrosine quenching, molecular docking and MD simulation studies showed that the compounds bind with α-Syn and provide structural rigidity which delays onset of structural transitions, which is confirmed by CD spectroscopy. The results obtained here throw light on the mechanisms underlying inhibition of α-Syn fibrillation by these compounds. Thus, the current work has significant therapeutic implications for identifying plant based potent therapeutic molecules for PD and other synucleinopathies, an area which needs extensive exploration.

Elevated temperatures accelerate the formation of toxic amyloid fibrils of hen egg‐white lysozyme

The formation of amyloid fibrils is critical for neurodegenerative diseases. Some physiochemical conditions can promote the conversion of proteins from soluble globular shapes into insoluble well‐organized amyloid fibrils. The aim of this study was to investigate the effect of temperatures on amyloid fibrils formation in vitro using the protein model of hen egg‐white lysozyme (HEWL). The HEWL fibrils were prepared at temperatures of 37, 45, 50 and 57°C in glycine solution of pH 2.2. Under transmission electron microscopy, we found the well‐organized HEWL amyloid fibrils at temperatures of 45, 50 and 57°C after 10 days of incubation. Thioflavin T and Congo red florescence assays confirmed that the formation and growth of HEWL fibrils displayed a temperature‐dependent increase, and 57°C produced the most amounts. Meanwhile, the surface hydrophobicity of aggregates was greatly increased by ANS binding assay, and β‐sheet contents by circular dichroism analysis were increased by 17.8%, 22.0% and 34.9%, respectively. Furthermore, the HEWL fibrils formed at 57°C caused significant cytotoxicity in SH‐SY5Y cells after 48 hr exposure, and the cell viability determined by MTT assay was decreased, with 81.35 ± 0.29% for 1 μM, 61.45 ± 2.62% for 2 μM, and 11.58 ± 0.39% (p < .01) for 3 μM. Nuclear staining results also confirmed the apoptosis features. These results suggest that the elevated temperatures could accelerate protein unfolding of the native structure and formation of toxic amyloid fibrils, which can improve understanding the mechanisms of the unfolding and misfolding process of prion protein.

Inhibitory effect of naturally occurring Ocimum sanctum extract on α-Synuclein aggregation in aqueous solution

α-Synuclein (α-Syn) has a propensity to form amyloids and is one of the major causes of neurodegenerative disorders like Parkinson’s disease (PD). Despite a lot of research, there is no obviating therapy available for PD and related disorders. Therefore, discovering new inhibitors against α-Syn aggregation is a subject of intense research. Against this backdrop, we have investigated the attenuating effect of naturally occurring Ocimum sanctum (OS) against aggregation in aqueous solution using various biochemical and biophysical studies. In this report, Thioflavin (ThT) and Congo red binding assays have shown the hindering effect of OS extract on β-sheet formation in α-Syn, and eventually leading to inhibition of aggregation. ANS binding assay indicated its modulatory effect on hydrophobic patches of α-Syn protein. SDS-PAGE, turbidity measurement, and light scattering assays indicated the role of OS extract in increasing protein solubility. Morphological studies conducted by TEM and DLS suggested that OS extract prevents the formation of large fibrils. This work also extends to reveal the inhibitory and disaggregation effects of OS extract on A53T mutant induced aggregation and pre-existing fibrils respectively. Also, MTT assay implied the protective effect of OS extract on neuroblastoma cells against fibril induced cytotoxicity. Furthermore, docking studies of α-Syn with different molecules of OS extract navigated for putative interacting amino acid residues. Thus, due to the presence of polyphenolic compounds, OS extract binds with α-Syn and inhibits its aggregation. This study implicates that OS extract and associated molecules may become a potential therapeutic intervention against PD and other aggregation related disorders.

Characterization of human serum albumin modified by hair dye component, 4-chloro-1,2-phenylenediamine: Role in protein aggregation, redox biology and cytotoxicity

Hair dyes and their components have been described as mutagenic and carcinogenic. They penetrate the skin, undergo auto-oxidation, yield protein reactive derivatives and modify proteins in an irreversible manner. Investigations into the impact of aromatic diamines on protein structures are expected to have a significant clinical value. This study documents the strong potential of 4-chloro-1, 2-phenylenediamine (4-Cl-o-PDA), a hair dye component, in the modification of human serum albumin (HSA). HSA was modified in vitro by 4-Cl-o-PDA and changes in tertiary and secondary structural characteristics were analyzed by UV–visible, fluorescence and far UV circular dichroism, fourier transform infrared spectroscopical techniques and ANS binding assay. Interaction of 4-Cl-o-PDA and HSA was studied by molecular docking and drug displacement assay. Formation of aggregates and inhibitory effect of antioxidant curcumin was analyzed by ThT binding assay, ThT fluorescence and electron microscopy. Changes in oxidation status of HSA were studied by thiol estimation and NBT assay. Effect of 4-Cl-o-PDA on cellular redox status, DNA integrity and cytotoxicity was analyzed in lymphocytes using dichlorofluorescein method, comet assay and MTT assay. We observed gross structural damage in HSA modified by 4-Cl-o-PDA. Modification led to unmasking of hydrophobic clusters, loss of its alpha helix and an increase in beta strand and unordered structure in HSA. 4-Cl-o-PDA strongly binds at sub-domain IB of HSA. HSA modified by 4-Cl-o-PDA formed amyloid fibrillar aggregates, whose formation was inhibited by antioxidant treatment. Modified HSA showed reduction of free sulfydryl content. 4-Cl-o-PDA-HSA led to the generation of reactive oxygen species in human lymphocytes. Modified HSA caused an increase in the tail length of DNA indicating enhanced single strand DNA breaks in cells. Modified protein was found to be genotoxic and cytotoxic. This works offers insights in the role of hair dye components in the biological world.

Tubulin- and ROS-dependent antiproliferative mechanism of a potent analogue of noscapine, N-propargyl noscapine

AimTo rationally-design, synthesize, characterize, biologically evaluate, and to elucidate the anticancer mechanism of action of a novel analogue of noscapine, N-propargyl noscapine (NPN), as a potential drug candidate against triple-negative breast cancer (TNBC). Materials and methodsAfter the synthesis and IR, 1H, 13C NMR and mass spectral characterization of NPN, its antiproliferative efficacy against different cancer cell lines was investigated using Sulforhodamine B assay. Cell cycle progression was analysed using flow cytometry. The drug-tubulin interactions were studied using tryptophan-quenching assay, ANS-binding assay, and colchicine-binding assay. Immunofluorescence imaging was used to examine the effect of NPN on cellular microtubules. Levels of reactive oxygen species (ROS), loss of mitochondrial membrane potential (MMP), and cell death were studied by staining the cells with DCFDA, Rhodamine 123, and acridine orange/ethidium bromide, respectively. Key findingsNPN strongly inhibited the viability (IC50, 1.35 ± 0.2 μM) and clonogenicity (IC50, 0.56 ± 0.06 μM) of the TNBC cell line, MDA-MB-231, with robust G2/M arrest. In vitro, the drug bound to tubulin and disrupted the latter's structural integrity and promoted colchicine binding to tubulin. NPN triggered an unusual form of microtubule disruption in cells, repressed recovery of cold-depolymerized cellular microtubules and suppressed their dynamicity. These effects on microtubules were facilitated by elevated levels of ROS and loss of MMP. SignificanceNPN can be explored further as a chemotherapeutic agent against TNBC.

Scutellarin inhibits the uninduced and metal-induced aggregation of α-Synuclein and disaggregates preformed fibrils: implications for Parkinson's disease.

The aggregation of the protein alpha synuclein (α-Syn), a known contributor in Parkinson's disease (PD) pathogenesis is triggered by transition metal ions through occupational exposure and disrupted metal ion homeostasis. Naturally occurring small molecules such as polyphenols have emerged as promising inhibitors of α-Syn fibrillation and toxicity and could be potential therapeutic agents against PD. Here, using an array of biophysical tools combined with cellular assays, we demonstrate that the novel polyphenolic compound scutellarin efficiently inhibits the uninduced and metal-induced fibrillation of α-Syn by acting at the nucleation stage and stabilizes a partially folded intermediate of α-Syn to form SDS-resistant, higher-order oligomers (∼680 kDa) and also disaggregates preformed fibrils of α-Syn into similar type of higher-order oligomers. ANS binding assay, fluorescence lifetime measurements and cell-toxicity experiments reveal scutellarin-generated oligomers as compact, low hydrophobicity structures with modulated surface properties and significantly reduced cytotoxicity than the fibrillation intermediates of α-Syn control. Fluorescence spectroscopy and isothermal titration calorimetry establish the binding between scutellarin and α-Syn to be non-covalent in nature and of moderate affinity (Ka ∼ 105 M-1). Molecular docking approaches suggest binding of scutellarin to the residues present in the NAC region and C-terminus of monomeric α-Syn and the C-terminal residues of fibrillar α-Syn, demonstrating inhibition of fibrillation upon binding to these residues and possible stabilization of the autoinhibitory conformation of α-Syn. These findings reveal interesting insights into the mechanism of scutellarin action and establish it as an efficient modulator of uninduced as well as metal-induced α-Syn fibrillation and toxicity.

Effect of Green Synthesis of Gold Nanoparticles (AuNPs) from Hibiscus sabdariffa on the Aggregation of α-Lactalbumin

Amyloidosis are a hereditary or acquired disorders, which are associated with misfolding of normally soluble, functional peptides and proteins, and their conversion into contentious aggregation can cause amyloid fibrils. These fibrils reduce function of the proteins and with generation of toxic intermediates in the process of self-assembly cause amyloid diseases such as Alzheimer, Parkinson, etc. Nanoparticles are materials with a dimension of 1–100 nm that influence the process of protein fibrillation since nanoparticles mediated by chemical methods have been show side effects, biological methods have been preferred due to its safety for human therapeutic use. In this study, the effect of gold nanoparticle produced from hibiscus on the amyloid formation of α-lactalbumin was evaluated using light scattering absorption, fluorescence (ThT assay, intrinsic fluorescence assay and ANS binding assay), TEM, CD spectroscopy and SDS-PAGE. α -Lactalbumin has been chose as a good sample for study the amyloid formation due to the formation of molten globule state. The results show that AuNPs inhibit formation of amyloid fibrils in reduced α-lactalbumin. This protective effect resulted are due to increase protein adsorption to the surface of the nanoparticles and therefore prevents their structural changes. Nanoparticle by binding to the monomer prevented them from join together and elongation the core of amyloid fibrils. Finally, the results show that biosynthesized AuNPs mediated by hibiscus can be used as effective therapeutic agent for prevention of formation amyloid and treatment of amyloid disease.

Transthyretin Anti-Amyloidogenic and Fibril Disrupting Activities of Bacopa monnieri (L.) Wettst (Brahmi) Extract.

The homotetrameric plasma protein transthyretin (TTR), is responsible for a series of debilitating and often fatal disorders in humans known as transthyretin amyloidosis. Currently, there is no cure for TTR amyloidosis and treatment options are rare. Thus, the identification and development of effective and safe therapeutic agents remain a research imperative. The objective of this study was to determine the effectiveness of Bacopa monnieri extract (BME) in the modulation of TTR amyloidogenesis and disruption of preformed fibrils. Using aggregation assays and transmission electron microscopy, it was found that BME abrogated the formation of human TTR aggregates and mature fibrils but did not dis-aggregate pre-formed fibrils. Through acid-mediated and urea-mediated denaturation assays, it was revealed that BME mitigated the dissociation of folded human TTR and L55P TTR into monomers. ANS binding and glutaraldehyde cross-linking assays showed that BME binds at the thyroxine-binding site and possibly enhanced the quaternary structural stability of native TTR. Together, our results suggest that BME bioactives prevented the formation of TTR fibrils by attenuating the disassembly of tetramers into monomers. These findings open up the possibility of further exploration of BME as a potential resource of valuable anti-TTR amyloidosis therapeutic ingredients.

Natural compound safranal driven inhibition and dis-aggregation of α-synuclein fibrils

Self-assembly of α-synuclein (α-Syn) is linked with a variety of neurodegenerative diseases collectively called as α-synucleiopathies. Therefore, discovering suitable inhibitors for this self-association process of α-Syn is a subject of intense research. In this background, we have demonstrated here that the natural compound, Safranal, delays/inhibits α-Syn fibrillation/aggregation, and we have also characterized its mode of action. The α-Syn fibrillation/aggregation kinetics studies in combination with TEM studies demonstrated that Safranal effectively inhibits α-Syn fibrillation/aggregation. NMR studies revealed that Safranal binds with α-Syn and stabilizes the monomeric protein. ANS fluorescence and CD measurements indicated that Safranal binds to the hydrophobic residues of the protein and causes delay in the formation of β-sheet rich structures which are crucial for the fibrillation to occur. The results obtained from fluorescence quenching, NMR and ANS binding assays, when analysed taking into consideration the molecular structure of Safranal provide valuable insights into the mechanism of inhibition of α-Syn fibrillation/aggregation. We infer that inhibition of α-Syn fibrillation/aggregation is primarily driven by hydrophobic interactions between Safranal and the protein. Further, Safranal is also seen to dis-aggregates pre-formed α-Syn fibrils. These findings implicate that Safranal could become a potent therapeutic intervention in Parkinson's disease and other protein aggregation related disorders.

Preferential recognition of epitopes on peroxynitrite-modified alpha-2-macroglobulin by circulating autoantibodies in rheumatoid arthritis patients

Autoimmune responses against post-translationally modified antigens are a hallmark of several autoimmune diseases. In this work, we have studied the changes in alpha-2-macroglobulin (α2M) upon modification by peroxynitrite. Furthermore, we have evaluated the immunogenicity of modified α2M in experimental rabbits and rheumatoid arthritis (RA) patients. Peroxynitrite-modified α2M showed disturbed microenvironment and altered aromatic residues under UV and fluorescence studies. Aggregation, reduction in β-sheet content, production of nitrotyrosine and shift in amide I and II bands were observed in the modified α2M by polyacrylamide gel electrophoresis besides CD and FTIR spectroscopic analysis. The exposure of hydrophobic clusters and changes in contact positions were observed in ANS and ThT binding assays. Immunological studies using ELISA showed peroxynitrite-modified α2M as highly immunogenic producing high titre of specific antibodies in immunized rabbits. Cross-reactivity studies revealed the polyspecificity of the elicited antibodies. Direct binding ELISA and competitive inhibition studies confirmed the presence of circulating antibodies in the sera of RA patients having high specificity towards the peroxynitrite-modified α2M as compared to the native α2M. Sera from healthy (normal) human subjects showed lower binding with the native and modified protein. This study confirms that peroxynitrite induces structural modifications in α2M and makes it immunogenic. The presence of neo-antigenic determinants on modified α2M with enhanced binding for circulating autoantibodies in RA patients could offer new possibilities for diagnosis and etiopathology of the disease. Communicated by Ramaswamy H. Sarma

Silver nanoparticle modulates the aggregation of beta-lactoglobulin and induces to form rod-like aggregates

Silver nanoparticles (SNPs) have been increasingly used in medicines and biomaterials as a drug carriers and diagnostic or therapeutic material due to their smaller size, large surface area and cell penetration ability. Here we report the preparation of SNPs of diameter 10 ± 3 nm by using silver nitrate and sodium borohydride and the interaction of synthesized SNPs with our model protein β-lactoglobulin (β-lg) in 10 mM phosphate buffer at pH 7.5 after thermal exposure at 75 °C. Heat exposed β-lg forms amyloidal fibrillar aggregates whereas this protein aggregates adopt rod-like shape instead of fibrillar structure in presence of SNP under the same conditions. Size of the synthesized SNPs is confirmed by UV–Visible spectroscopy, SEM and TEM. Interactions and subsequent formation of molecular assembly of heat stressed β-lg with SNP were investigated using Th-T assay and ANS binding assay, DLS, RLS, CD, FT-IR, SEM, TEM. Docking study parallely also support the experimental findings.

Glycine-rich loop encompassing active site at interface of hexameric M. tuberculosis Eis protein contributes to its structural stability and activity

RvEis is a crucial thermostable hexameric aminoglycoside acetyltransferase of Mycobacterium tuberculosis, overexpression of which confers Kanamycin resistance in clinical strains. The thermostability associated with hexameric RvEis is important for the enhanced intracellular survival of mycobacteria. However, the structural determinants responsible for its thermal stability remain unexplored. In this study, we have assessed the role of glycines of conserved glycine-rich motif (G123GIYG127) present at the oligomeric interface in the hydrophobic core of RvEis in sustenance of its structural stability, oligomerization and functional activity. Substitution of glycines to alanine (G123A/G127A) result in significant decrease in melting temperature (Tm), reduction in the oligomerization with concomitant increase in the monomeric form and higher susceptibility towards the denaturants like GdmCl and urea relative to wild type. G123A/G127A mutant displayed lower catalytic efficiency (kcat/Km) and is completely inactive at 60 °C. ANS binding assay and the complete dissociation of hexameric complex into monomers at lower concentration of urea in G123A/G127A relative to wtRvEis suggests that altered hydrophobic environment could be the reason for its instability. In sum, these results demonstrate the role of G123GIYG127 motif in structural stability and activity of RvEis.