muramidase and Protein-Aggregation--Pathological

Protein aggregation into amyloid fibrils is linked to multiple disorders. The understanding of how natively non-harmful proteins convert to these highly cytotoxic amyloid aggregates is still not sufficient, with new ideas and hypotheses being presented each year. Recently it has been shown that more than one type of protein aggregates may co-exist in the affected tissue of patients suffering from amyloid-related disorders, sparking the idea that amyloid aggregates formed by one protein may induce another protein's fibrillization. In this work, we examine the effect that lysozyme fibrils have on insulin amyloid aggregation. We show that not only do lysozyme fibrils affect insulin nucleation, but they also alter the mechanism of its aggregation.

Topics: Amyloid; Animals; Chickens; Humans; Insulin; Muramidase; Protein Aggregates; Protein Aggregation, Pathological; Recombinant Proteins

Protein fibrous aggregation is associated with many neurodegenerative diseases including Alzheimer's and Parkinson's diseases. To modulate the process, a number of fibrillation inhibitors have been reported, although their working mechanism remains vague, calling for new means to decipher their interaction. Herein, we identified and characterized a novel inhibitor called Crocein Orange G (COG), which inhibited the nucleation and impeded the protofibril formation, revealed by various experimental approaches as well as molecular docking. In particular, the surface-enhanced Raman spectroscopy (SERS) helps to identify the binding sites and illustrate the interaction mechanism and fibrillation process by using Ag IMNPs as SERS substrate for a label-free detection. Combining with molecular docking, the SERS-based approach provides structural information concerning protein-ligand interaction and protein fibrillation. This study suggests that SERS can be a powerful new means to study the interaction between inhibitors and amyloid proteins and can potentially be a common tool for amyloid research. Strikingly, the SERS signal of COG corresponds very well with the state of protein fibrillation, hinting its function as an amyloid SERS signal amplifier. Therefore, this study provides a new means to monitor and interfere amyloid fibrillation.

Topics: Amyloid; Azo Compounds; Biosensing Techniques; Humans; Insulin; Molecular Docking Simulation; Muramidase; Naphthalenesulfonates; Protein Aggregates; Protein Aggregation, Pathological; Silver; Spectrum Analysis, Raman

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.

Topics: Amyloid; Cytoprotection; Diuretics; Humans; Molecular Docking Simulation; Muramidase; Protein Aggregation, Pathological; Serum Albumin, Human

Many conflicting reports about the involvement of serum amyloid P component (SAP) in amyloid diseases have been presented over the years; SAP is known to be a universal component of amyloid aggregates but it has been suggested that it can both induce and suppress amyloid formation. By using our Drosophila model of systemic lysozyme amyloidosis, SAP has previously been shown to reduce the toxicity induced by the expression of the disease-associated lysozyme variant, F57I, in the Drosophila central nervous system. This study further investigates the involvement of SAP in modulating lysozyme toxicity using histochemistry and spectral analyses on the double transgenic WT and F57I lysozyme flies to probe; i) formation of aggregates, ii) morphological differences of the aggregated lysozyme species formed in the presence or absence of SAP, iii) location of lysozyme and iv) co-localisation of lysozyme and SAP in the fly brain. We found that SAP can counteract the toxicity (measured by the reduction in the median survival time) induced by F57I lysozyme by converting toxic F57I species into less toxic amyloid-like structures, as reflected by the spectral changes that p-FTAA undergoes when bound to lysozyme deposits in F57I-F57I-SAP flies as compared to F57I-F57I flies. Indeed, when SAP was introduced to in vitro lysozyme fibril formation, the endpoint fibrils had enhanced ThT fluorescence intensity as compared to lysozyme fibrils alone. This suggests that a general mechanism for SAP's role in amyloid diseases may be to promote the formation of stable, amyloid-like fibrils, thus decreasing the impact of toxic species formed along the aggregation pathway.

Topics: Amyloid; Amyloidosis; Animals; Animals, Genetically Modified; Disease Models, Animal; Drosophila melanogaster; Drosophila Proteins; Humans; Muramidase; Protein Aggregates; Protein Aggregation, Pathological; Serum Amyloid P-Component

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

Topics: Acids; Amyloid; Amyloidogenic Proteins; Animals; Hydrogen-Ion Concentration; Models, Molecular; Molecular Conformation; Muramidase; Organic Chemicals; Protein Aggregates; Protein Aggregation, Pathological; Spectrum Analysis; Structure-Activity Relationship

Polyamines can induce protein aggregation that can be related to the physiology of the cellular function. Polyamines have been implicated in protein aggregation which may lead to neuropathic and non neuropathic amyloidosis.. Change in the level of polyamine concentration has been associated with ageing and neurodegeneration such as Parkinson's disease, Alzheimer's disease. Lysozyme aggregation in the presence of polyamines leads to non neuropathic amyloidosis. Polyamine analogues can suppress or inhibit protein aggregation suggesting their efficacy against amyloidogenic protein aggregates.. In this study we report the comparative interactions of lysozyme with the polyamine analogue, 1-naphthyl acetyl spermine in comparison with the biogenic polyamines through spectroscopy, calorimetry, imaging and docking techniques. The findings revealed that the affinity of binding varied as spermidine > 1-naphthyl acetyl spermine > spermine. The biogenic polyamines accelerated the rate of fibrillation significantly, whereas the analogue inhibited the rate of fibrillation to a considerable extent. The polyamines bind near the catalytic diad residues viz. Glu35 and Asp52, and in close proximity of Trp62 residue. However, the analogue showed dual nature of interaction where its alkyl amine region bind in same way as the biogenic polyamines bind to the catalytic site, while the naphthyl group makes hydrophobic contacts with Trp62 and Trp63, thereby suggesting its direct influence on fibrillation.. This study, thus, potentiates, the development of a polyamine analogue that can perform as an effective inhibitor targeted towards aggregation of amyloidogenic proteins.

Topics: Amyloid; Amyloidosis; Animals; Avian Proteins; Biogenic Polyamines; Chickens; Hydrophobic and Hydrophilic Interactions; Molecular Docking Simulation; Muramidase; Protein Aggregates; Protein Aggregation, Pathological; Spermidine; Spermine

More than 20 unique diseases such as diabetes, Alzheimer's disease, Parkinson's disease are caused by the abnormal aggregations of pathogenic proteins such as amylin, β-amyloid (Aβ), and α-synuclein. All pathogenic proteins differ from each other in biological function, primary sequences, and morphologies; however, the proteins are toxic when aggregated. Here, we investigated the cellular toxicity of pathogenic or non-pathogenic protein aggregates. In this study, six proteins were selected and they were incubated at acid pH and high temperature. The aggregation kinetic and cellular toxicity of protein species with time were characterized. Three non-pathogenic proteins, bovine serum albumin (BSA), catalase, and pepsin at pH 2 and 65 °C were stable in protein structure and non-toxic at a lower concentration of 1 mg/mL. They formed aggregates at a higher concentration of 20 mg/mL with time and they induced the toxicity in short incubation time points, 10 min and 20 min only and they became non-toxic after 30 min. Other three pathogenic proteins, lysozyme, superoxide dismutase (SOD), and insulin, also produced the aggregates with time and they caused cytotoxicity at both 1 mg/mL and 20 mg/mL after 10 min. TEM images and DSC analysis demonstrated that fibrils or aggregates at 1 mg/mL induced cellular toxicity due to low thermal stability. In DSC data, fibrils or aggregates of pathogenic proteins had low thermal transition compared to fresh samples. The results provide useful information to understand the aggregation and cellular toxicity of pathogenic and non-pathogenic proteins.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid beta-Peptides; Catalase; Cell Line; Diabetes Mellitus; Humans; Insulin; Islet Amyloid Polypeptide; Models, Molecular; Muramidase; Parkinson Disease; Pepsin A; Protein Aggregates; Protein Aggregation, Pathological; Protein Structure, Secondary; Serum Albumin, Bovine; Superoxide Dismutase

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) × 10

Topics: Amyloid; Catechols; Humans; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Models, Chemical; Models, Molecular; Molecular Conformation; Muramidase; Nitriles; Protein Aggregates; Protein Aggregation, Pathological; Spectrum Analysis; Structure-Activity Relationship

Senile plaques are well-known hallmarks of Alzheimer's Diseases (AD). However, drugs targeting tangles of the protein tau and plaques of β-amyloid have no significant effect on disease progression, and the studies on the underlying mechanism of AD remain in high demand. Growing evidence supports the protective role of senile plaques in local inflammation driven by S100A9. We herein demonstrate that oleic acid (OA) micelles interact with hen egg white lysozyme (HEWL) and promote its amyloid formation. Consequently, SH-SY5Y cell line and mouse neural stem cells are rescued from OA toxicity by co-aggregation of OA and HEWL. Using atomic force microscopy in combination with fluorescence microscopy, we revealed that HEWL forms round-shaped aggregates in the presence of OA micelles instead of protofibrils of HEWL alone. These HEWL amyloids act as a sink for toxic OA micelles and their co-aggregate form large clumps, suggesting a protective function in amyloid and OA cytotoxicity.

Topics: Alzheimer Disease; Amyloid; Animals; Cell Line; Chickens; Humans; Micelles; Muramidase; Oleic Acid; Protein Aggregation, Pathological; Rats

Increasing evidence proposed that amyloid deposition by proteins play a crucial role in an array of neurotoxic and degenerative disorders like Parkinson's disease, systemic amyloidosis etc, that could be controlled by anti-aggregation methodologies which either inhibit or disaggregate such toxic aggregates. The present work targets the amyloid inhibiting and disaggregating potential of promethazine (PRM) against human insulin (HI) and human lysozyme (HL) fibrillogenesis. Biophysical techniques like Rayleigh scattering measurements (RLS), Thioflavin T (ThT) and 8-Anilinonaphthalene-1-sulfonic acid (ANS) fluorescence measurement, circular dichroism (CD) and dynamic light scattering (DLS) measurements illustrated the inhibitory action of PRM. The half maximal inhibitory concentration (IC

Topics: Amyloid; Amyloidogenic Proteins; Amyloidosis; Anilino Naphthalenesulfonates; Benzothiazoles; Circular Dichroism; Dynamic Light Scattering; Fluorescence; Humans; Insulin; Muramidase; Parkinson Disease; Promethazine; Protein Aggregates; Protein Aggregation, Pathological; Thiazoles

Aggregation of amyloid β-proteins (Aβ) mediated by metal ions such as Zn

Topics: Amyloid beta-Peptides; Cell Line; Cell Survival; Humans; Imino Acids; Muramidase; Protein Aggregation, Pathological; Zinc

The 129-residue lysozyme has been shown to form amyloid fibrils in vitro. While methylene blue (MB), a compound in the phenothiazinium family, has been shown to dissemble tau fibril formation, its anti-fibrillogenic effect has not been thoroughly characterized in other proteins/peptides. This study examines the effects of MB on the in vitro fibrillogenesis of lysozyme at pH 2.0 and 55 °C. Our results demonstrated that, upon 7-day incubation, the plateau ThT fluorescence of the sample was found to be ~8.69% or ~2.98% of the control when the molar ratio of lysozyme to MB was at 1:1.11 or 1:3.33, respectively, indicating that the inhibitory potency of MB against lysozyme fibrillogenesis is positively correlated with its concentration. We also found that MB is able to destabilize the preformed lysozyme fibrils. Moreover, molecular docking and molecular dynamics simulations results revealed that MB's mechanism of fibril formation inhibition may be triggered by binding with lysozyme's aggregation-prone region. Results reported here provide solid support for MB's effect on amyloid fibrillogenesis. We believe the additional insights gained herein may pave way to the discovery of other small molecules that may have similar action toward amyloid fibril formation and its associated diseases.

Topics: Amyloid; Amyloidosis; Methylene Blue; Molecular Conformation; Molecular Docking Simulation; Molecular Dynamics Simulation; Muramidase; Protein Aggregates; Protein Aggregation, Pathological; Protein Binding; Spectrum Analysis; Structure-Activity Relationship

Protein aggregation is a hallmark of several neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. It has been shown that lysine residues play a key role in the formation of these aggregates. Thus, the ability to disrupt aggregate formation by covalently modifying lysine residues could lead to the discovery of therapeutically relevant antiamyloidogenesis compounds. Herein, we demonstrate that an ortho-iminoquinone (IQ) can be utilized to inhibit amyloid aggregation. Using alpha-synuclein and Aβ

Topics: alpha-Synuclein; Amyloid beta-Peptides; Animals; Catechin; Cell Survival; Cells, Cultured; Chickens; Dopaminergic Neurons; HEK293 Cells; Humans; Lysine; Methionine; Mice; Micrococcus luteus; Microtubule-Associated Proteins; Muramidase; Neuroprotective Agents; Oxidation-Reduction; Peptide Fragments; Protein Aggregation, Pathological; Quinones; Tyrosine 3-Monooxygenase

A number of human diseases, including Alzheimer's and Parkinson's have been linked to amyloid formation. To search for an anti-amyloidogenic product, alkaloid enriched extract from borage leaves was examined for anti-amyloidogenic activity using Hen Egg White Lysozyme (HEWL) as a model protein. After isolation of the plant extract using rHPLC, only one fraction indicated a significant bioactivity. TEM analysis confirmed a remarkable reduction of amyloid fibrils in the presence of the bioactive fraction. To identify the effective substance in the fraction, mass spectrometry, FTIR, and NMR were performed. Our analyses determined that the bioactive compound as 1-acetyl-19,21-epoxy-15,16-dimethoxyaspidospermidine-17-ol, a derivative of aspidospermine. To investigate the mechanism of the inhibition, ANS binding, intrinsic fluorescence, and amide I content were performed in the presence of the bioactive compound. All the results confirmed the role of the compound in assisting the proper folding of the protein. In addition, molecular docking indicated the aspidospermine derivative binds the amyloidogenic region of the protein. Our results show that the alkaloid extracted from borage leaves reduces protein aggregation mediating through structural elements of the protein, promoting the correct folding of lysozyme. Since a number of aspidospermine compounds have been shown to possess potent antimalarial activities, the action of compound identified in the present study suggests a possible link between protein aggregation and aspidospermine drugs.

Topics: Amyloid; Antimalarials; Borago; Indole Alkaloids; Molecular Docking Simulation; Muramidase; Plant Extracts; Plant Leaves; Protein Aggregation, Pathological; Protein Folding; Quinolines

Protein amyloid aggregation is an important pathological feature of a group of different degenerative human diseases called amyloidosis. We tested effect of two phospholipids, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) on amyloid aggregation of hen egg white (HEW) lysozyme in vitro.. Effect of phospholipids was investigated using spectroscopic techniques (fluorescence and CD spectroscopy), atomic force microscopy and image analysis.. Phospholipids DMPC and DHPC are able dose-dependently inhibit lysozyme fibril formation. The length of the phospholipid tails and different structural arrangement of the phospholipid molecules affect inhibitory activity; long-chain DMPC inhibits fibrillization more efficiently. Interestingly, interference of DMPC with lysozyme amyloid fibrils has no effect on their morphology or amount.. Phospholipid molecules have significant effect on lysozyme amyloid fibrillization. We suggest that inhibitory activity is due to the interference of phospholipids with lysozyme leading to the blocking of the intermolecular protein interactions important for formation of the cross-β structure within the core of the fibrils. The higher inhibitory activity of DMPC is probably due to adsorption of protein molecules on the liposome surfaces which caused decrease of species needed for fibrillization. Interaction of the phospholipids with formed fibrils is not sufficient enough to interrupt the bonds in β-sheets which are required for destroying of amyloid fibrils.. The obtained results contribute to a better understanding of the effect of phospholipids on amyloid fibrillization of the lysozyme. The data suggest that DMPC and DHPC phospholipids represent agents able to modulate lysozyme amyloid aggregation.

Topics: Amyloid; Amyloidogenic Proteins; Amyloidosis; Animals; Chickens; Dimyristoylphosphatidylcholine; Humans; Microscopy, Atomic Force; Muramidase; Phosphatidylcholines; Phospholipid Ethers; Phospholipids; Phosphorylcholine; Protein Aggregation, Pathological

Topics: Amino Acid Sequence; Amyloid beta-Peptides; Animals; Benzothiazoles; Brain; Insulin; Mice; Mice, Transgenic; Muramidase; Peptide Termination Factors; Prion Proteins; Protein Aggregates; Protein Aggregation, Pathological; Protein Conformation, beta-Strand; Protein Folding; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Serum Albumin; Spectrometry, Fluorescence; Thiazoles

Human lysozyme is homologous in the three-dimensional structure to hen lysozyme and the latter is commonly used to understand folding and amyloid aggregation pathway of the former. The fibrillation of the two proteins is known to occur via partial unfolding. A work dedicated to comparing the aggregation-prone conformations and their subsequent conversion into amyloid-like fibrils in an identical condition is not available. This has provided an opportunity to compare the fibrillation behaviors of the two homologous proteins under identical solution condition. In this work, we have shown that the temperature-induced unfolding of the two proteins at pH 1.5 occurred via a three states process. We found that temperature-unfolded states of the two proteins differ in contents of residual secondary and tertiary structures. The temperature-unfolded states of both proteins rapidly converted into well-defined amyloid-like fibrils on stirring at 230 RPM. We further observed that the kinetic parameters, lag time (t

Topics: Amyloid; Animals; Chickens; Circular Dichroism; Humans; Hydrogen-Ion Concentration; Kinetics; Microscopy, Electron, Transmission; Models, Molecular; Muramidase; Protein Aggregation, Pathological; Protein Structure, Secondary; Protein Unfolding; Solutions; Temperature

Protein misfolding and aggregation have been associated with several human diseases such as Alzheimer's, Parkinson's and familial amyloid polyneuropathy etc. In this study, anti-fibrillation activity of vitamin k3 and its effect on the kinetics of amyloid formation of hen egg white lysozyme (HEWL) and Aβ-42 peptide were investigated. Here, in combination with Thioflavin T (ThT) fluorescence assay, circular dichroism (CD), transmission electron microscopy and cell cytotoxicity assay, we demonstrated that vitamin k3 significantly inhibits fibril formation as well as the inhibitory effect is dose dependent manner. Our experimental studies inferred that vitamin k3 exert its neuro protective effect against amyloid induced cytotoxicity through concerted pathway, modifying the aggregation formation towards formation of nontoxic aggregates. Molecular docking demonstrated that vitamin k3 mediated inhibition of HEWL and Aβ-42 fibrillogenesis may be initiated by interacting with proteolytic resistant and aggregation prone regions respectively. This work would provide an insight into the mechanism of protein aggregation inhibition by vitamin k3; pave the way for discovery of other small molecules that may exert similar effect against amyloid formation and its associated neurodegenerative diseases.

Topics: Amyloid beta-Peptides; Animals; Cell Line, Tumor; Cell Survival; Chickens; Humans; Muramidase; Protein Aggregation, Pathological; Vitamin K 3

Lysozyme amyloidosis is a hereditary disease in which mutations in the gene coding for lysozyme leads to misfolding and consequently accumulation of amyloid material. To improve understanding of the processes involved we expressed human wild type (WT) lysozyme and the disease-associated variant F57I in the central nervous system (CNS) of a Drosophila melanogaster model of lysozyme amyloidosis, with and without co-expression of serum amyloid p component (SAP). SAP is known to be a universal constituent of amyloid deposits and to associate with lysozyme fibrils. There are clear indications that SAP may play an important role in lysozyme amyloidosis, which requires further elucidation. We found that flies expressing the amyloidogenic variant F57I in the CNS have a shorter lifespan than flies expressing WT lysozyme. We also identified apoptotic cells in the brains of F57I flies demonstrating that the flies' neurological functions are impaired when F57I is expressed in the nerve cells. However, co-expression of SAP in the CNS prevented cell death and restored the F57I flies' lifespan. Thus, SAP has the apparent ability to protect nerve cells from damage caused by F57I. Furthermore, it was found that co-expression of SAP prevented accumulation of insoluble forms of lysozyme in both WT- and F57I-expressing flies. Our findings suggest that the F57I mutation affects the aggregation process of lysozyme resulting in the formation of cytotoxic species and that SAP is able to prevent cell death in the F57I flies by preventing accumulation of toxic F57I structures.

Topics: Amyloidosis; Animals; Animals, Genetically Modified; Apoptosis; Central Nervous System; Disease Models, Animal; Drosophila melanogaster; Gene Expression; Humans; Longevity; Muramidase; Mutation; Neurons; Plaque, Amyloid; Protective Factors; Protein Aggregation, Pathological; Serum Amyloid P-Component; Transgenes

Amyloid-associated diseases, such Alzheimer's, Huntington's, Parkinson's, and type II diabetes, are related to protein misfolding and aggregation. Herein, the time evolution of scattered light intensity, hydrophobic properties, and conformational changes during fibrillation processes of rHL solutions at 55 °C and pH 2.0 were used to monitor the aggregation process of recombinant human lysozyme (rHL). Dynamic light scattering (DLS), thioflavin T (ThT) fluorescence, and surface tension (ST) at the air-water interface were used to analyze the hydrophobic properties of pre-amyloid aggregates involved in the fibrillation process of rHL to find a correlation between the hydrophobic character of oligomers, protofibrils and amyloid aggregates with the gain in cross-β-sheet structure, depending on the increase in the incubation periods. The ability of the different aggregates of rHL isolated during the fibrillation process to be adsorbed at the air-water interface can provide important information about the hydrophobic properties of the protein, which can be related to changes in the secondary structure of rHL, resulting in cytotoxic or non-cytotoxic species. Thus, we evaluated the cytotoxic effect of oligomers, protofibrils and amyloid fibrils on the cell line ARPE-19 using the MTT reduction test. The more cytotoxic protein species arose after a 600-min incubation time, suggesting that the hydrophobic character of pre-amyloid fibrils, in addition to the high prevalence of the cross-β-sheet conformation, can become toxic for the cell line ARPE-19.

Topics: Amyloid; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Humans; Hydrophobic and Hydrophilic Interactions; Muramidase; Particle Size; Protein Aggregation, Pathological; Recombinant Proteins; Structure-Activity Relationship; Surface Properties

In vitro fibrillation of hen egg white lysozyme (HEWL) causes complete reduction of Cu(II) to Cu(I) at pH 7. Here in the present article, we have shown the presence of both Cu(II) and Cu(I) at pH 11 during fibrillation of HEWL using electron paramagnetic resonance and Raman spectroscopy. Our results suggest the existence of a partially reducing environment during fibrillation of hen egg white lysozyme at pH 11. The fibrillation process is governed by the pH of the solution and maximum fibrillation is found to occur at pH 11. Fibrils formed in the absence of Cu(II) were also found to cause significant hemolysis of RBC.

Topics: Amyloid; Animals; Copper; Egg White; Female; Hydrogen-Ion Concentration; Kinetics; Models, Molecular; Muramidase; Nuclear Magnetic Resonance, Biomolecular; Oxidation-Reduction; Protein Aggregates; Protein Aggregation, Pathological; Protein Conformation; Protein Multimerization; Spectrum Analysis, Raman

Numerous studies demonstrate that natural polyphenols can inhibit amyloid formation and disrupt preformed amyloid fibrils. In the present study, the fibril-disruptive effects of epigallocatechin-3-gallate (EGCG) were examined using lysozyme as a model protein. The results indicated that EGCG dose dependently inhibited lysozyme fibrillation and modified the peptide chains with quinonoid moieties under acidic conditions, as measured by ThT fluorescence, transmission electron microscopy, and an NBT-staining assay. Moreover, EGCG transformed the preformed lysozyme fibrils to amorphous aggregates through quinopeptide formation. The thiol blocker, N-ethylmaleimide, inhibited the disruptive effect of EGCG on preformed fibrils, suggesting that thiol groups are the binding sites for EGCG. We propose that the formation of quinone intermediates via oxidation and subsequent binding to lysozyme chains are the main processes driving the inhibition of amyloid formation and disruption of preformed fibrils by EGCG. The information presented in this study may provide fresh insight into the link between the antioxidant capacity and anti-amyloid activity of polyphenols.

Topics: Amyloid; Animals; Catechin; Molecular Structure; Muramidase; Peptides; Polyphenols; Protein Aggregation, Pathological

Clinical application of curcumin for Alzheimer's disease treatment is severely limited with regard to its poor bioavailability, high rate of metabolism, and instability under neutral condition. In the current study, we designed three compounds in which the diketone moiety of curcumin was replaced by cyclohexanone. In these compounds, the linker length of the molecules was optimal; and substitution of dioxolane for hydroxyl groups on compound 3 should prevent metabolic inactivation. The inhibitory effect of the compounds was investigated against hen egg white lysozyme (HEWL) fibrillation using AFM (atomic force microscope), ThT (thioflavin T) and MTT assay. We found that all three compounds were able to inhibit HEWL aggregation in a dose-dependent manner and inhibit the cytotoxic activity of aggregated HEWL. Docking results demonstrated that the compounds could bind into lysozyme and occupy the whole active site groove. In conclusion, we present chemical analogs of curcumin with various modifications in the spacer and the phenolic rings as improved inhibitors of amyloid aggregation.

Topics: Amyloid; Cell Line, Tumor; Cell Survival; Curcumin; Humans; Microscopy, Atomic Force; Models, Molecular; Molecular Conformation; Molecular Structure; Muramidase; Protein Aggregation, Pathological; Protein Binding; Spectrophotometry, Ultraviolet

An increasing number of studies conducted under in vitro and in vivo conditions, have concluded that polyphenols, compounds frequently occurring in many herbs with antioxidant properties, prevent and reverse amyloid fibril formation. However, the mechanisms by which these natural products modulate the protein aggregation process are poorly understood. Herein, a range of techniques including thioflavin T (ThT) and ANS fluorescence assays, electron microscopy and circular dichroism have been employed to determine the efficacy of rosmarinic acid (RA) and resveratrol (Res) on the inhibition/reversion of fibrillogenesis and hindering cytotoxicity induced by protofibrils and amyloid fibrils of hen egg white lysozyme (HEWL). Results demonstrated that both polyphenols effectively inhibit fibrillogenesis and destabilize preformed fibrils of HEWL in a concentration-dependent manner. Cytotoxicity protection on PC12 cells was also observed using the MTT assay, ROS production assay, and phase-contrast microscopy. It is suggested that the mechanism underlying the inhibitory effects of RA and Res is to prevent hydrophobic interactions between HEWL amyloidogenic prefibrillar species, although additional studies is needed to elucidate the detailed mechanisms involved. A combination of antioxidative and anti-amyloidogenic properties of these molecules may provide them with the described neuroprotective capacities.

Topics: Amyloid; Animals; Antioxidants; Cell Shape; Cell Survival; Cinnamates; Depsides; Drug Evaluation, Preclinical; Hydrophobic and Hydrophilic Interactions; Inhibitory Concentration 50; Muramidase; PC12 Cells; Protein Aggregation, Pathological; Protein Stability; Protein Structure, Secondary; Rats; Resveratrol; Rosmarinic Acid; Stilbenes

Protein aggregation with the concomitant formation of amyloid fibrils is related to several neurodegenerative diseases, but also to non-neuropathic amyloidogenic diseases and non-neurophatic systemic amyloidosis. Lysozyme is the protein involved in the latter, and it is widely used as a model system to study the mechanisms underlying fibril formation and its inhibition. Several phenolic compounds have been reported as inhibitors of fibril formation. However, the anti-aggregating capacity of other heteroaromatic compounds has not been studied in any depth. We have screened the capacity of eleven different hydroxypyridines to affect the acid-induced fibrillization of hen lysozyme. Although most of the tested hydroxypyridines alter the fibrillation kinetics of HEWL, only 3-hydroxy-2-methylpyridine, 3-hydroxy-6-methylpyridine and 3-hydroxy-2,6-dimethylpyridine completely abolish fibril formation. Different biophysical techniques and several theoretical approaches are combined to elucidate their mechanism of action. O-methylated 3-hydroxypyridines bind non-cooperatively to two distinct but amyloidogenic regions of monomeric lysozyme. This stabilises the protein structure, as evidenced by enhanced thermal stability, and results in the inhibition of the conformational transition that precedes fibril assembly. Our results point to o-methylated 3-hydroxypyridines as a promising molecular scaffold for the future development of novel fibrillization inhibitors.

Topics: Amyloid; Animals; Binding Sites; Hydrogen-Ion Concentration; Kinetics; Microscopy, Atomic Force; Models, Molecular; Molecular Docking Simulation; Muramidase; Protein Aggregates; Protein Aggregation, Pathological; Protein Binding; Protein Conformation; Protein Stability; Proteolysis; Pyridines; Thermodynamics

Amyloid fibers are associated with disease but have little chemical reactivity. We investigated the formation and structure of amyloids to identify potential mechanisms for their pathogenic effects. We incubated lysozyme 20 mg/ml at 55C and pH 2.5 in a glycine-HCl buffer and prepared slides on mica substrates for examination by atomic force microscopy. Structures observed early in the aggregation process included monomers, small colloidal aggregates, and amyloid fibers. Amyloid fibers were observed to further self-assemble by two mechanisms. Two or more fibers may merge together laterally to form a single fiber bundle, usually in the form of a helix. Alternatively, fibers may become bound at points where they cross, ultimately forming an apparently irreversible macromolecular network. As the fibers assemble into a continuous network, the colloidal suspension undergoes a transition from a Newtonian fluid into a viscoelastic gel. Addition of salt did not affect fiber formation but inhibits transition of fibers from linear to helical conformation, and accelerates gel formation. Based on our observations, we considered the effects of gel formation on biological transport. Analysis of network geometry indicates that amyloid gels will have negligible effects on diffusion of small molecules, but they prevent movement of colloidal-sized structures. Consequently gel formation within neurons could completely block movement of transport vesicles in neuronal processes. Forced convection of extracellular fluid is essential for the transport of nutrients and metabolic wastes in the brain. Amyloid gel in the extracellular space can essentially halt this convection because of its low permeability. These effects may provide a physical mechanism for the cytotoxicity of chemically inactive amyloid fibers in neurodegenerative disease.

Topics: Amyloid; Amyloidogenic Proteins; Animals; Colloids; Gels; Microscopy, Atomic Force; Microscopy, Electron, Transmission; Muramidase; Protein Aggregation, Pathological; Protein Structure, Secondary; Sodium Chloride; Time Factors

Knowledge of the detailed mechanism by which proteins such as human αB- crystallin and human lysozyme inhibit amyloid beta (Aβ) peptide aggregation is crucial for designing treatment for Alzheimer's disease. Thus, unconstrained, atomistic molecular dynamics simulations in explicit solvent have been performed to characterize the Aβ17-42 assembly in presence of the αB-crystallin core domain and of lysozyme. Simulations reveal that both inhibitor proteins compete with inter-peptide interaction by binding to the peptides during the early stage of aggregation, which is consistent with their inhibitory action reported in experiments. However, the Aβ binding dynamics appear different for each inhibitor. The binding between crystallin and the peptide monomer, dominated by electrostatics, is relatively weak and transient due to the heterogeneous amino acid distribution of the inhibitor surface. The crystallin-bound Aβ oligomers are relatively long-lived, as they form more extensive contact surface with the inhibitor protein. In contrast, a high local density of arginines from lysozyme allows strong binding with Aβ peptide monomers, resulting in stable complexes. Our findings not only illustrate, in atomic detail, how the amyloid inhibitory mechanism of human αB-crystallin, a natural chaperone, is different from that of human lysozyme, but also may aid de novo design of amyloid inhibitors.

Topics: alpha-Crystallin B Chain; Amino Acid Sequence; Amyloid beta-Peptides; Binding Sites; Humans; Molecular Dynamics Simulation; Molecular Sequence Data; Muramidase; Protein Aggregation, Pathological; Protein Binding

The question of how an aggregating protein can influence aggregation of other proteins located in its vicinity is particularly significant because many proteins coexist in cells. We demonstrate in vitro coaggregation and cross-seeding of lysozyme, bovine serum albumin, insulin, and cytochrome c during their amyloid formation. The coaggregation process seems to be more dependent on the temperature-induced intermediate species of these proteins and less dependent on their sequence identities. Because amyloid-linked inclusions and plaques are recognized as multicomponent entities originating from aggregation of the associated protein, these findings may add new insights into the mechanistic understanding of amyloid-related pathologies.

Topics: Amino Acid Sequence; Amyloid; Amyloidosis; Animals; Cattle; Circular Dichroism; Cytochromes c; Humans; Insulin; Kinetics; Microscopy, Electron, Transmission; Molecular Sequence Data; Muramidase; Protein Aggregates; Protein Aggregation, Pathological; Sequence Homology, Amino Acid; Serum Albumin, Bovine; Spectrometry, Fluorescence