ABSTRACT
Carnosine, a common dipeptide in mammals, has previously been shown to dissemble alpha-crystallin amyloid fibrils. To date, the dipeptide's anti-fibrillogensis effect has not been thoroughly characterized in other proteins. For a more complete understanding of carnosine's mechanism of action in amyloid fibril inhibition, we have investigated the effect of the dipeptide on lysozyme fibril formation and induced cytotoxicity in human neuroblastoma SH-SY5Y cells. Our study demonstrates a positive correlation between the concentration and inhibitory effect of carnosine against lysozyme fibril formation. Molecular docking results show carnosine's mechanism of fibrillogenesis inhibition may be initiated by binding with the aggregation-prone region of the protein. The dipeptide attenuates the amyloid fibril-induced cytotoxicity of human neuronal cells by reducing both apoptotic and necrotic cell deaths. Our study provides solid support for carnosine's amyloid fibril inhibitory property and its effect against fibril-induced cytotoxicity in SH-SY5Y cells. The additional insights gained herein may pave way to the discovery of other small molecules that may exert similar effects against amyloid fibril formation and its associated neurodegenerative diseases.
Subject(s)
Amyloid/antagonists & inhibitors , Avian Proteins/toxicity , Carnosine/pharmacology , Muramidase/toxicity , Neurons/drug effects , Amyloid/agonists , Amyloid/chemistry , Animals , Apoptosis/drug effects , Avian Proteins/antagonists & inhibitors , Avian Proteins/chemistry , Binding Sites , Carnosine/chemistry , Cell Line, Tumor , Chickens , Humans , Molecular Docking Simulation , Muramidase/antagonists & inhibitors , Muramidase/chemistry , Neurons/cytology , Neurons/metabolism , Protein BindingABSTRACT
BACKGROUND: More than twenty-seven human proteins can fold abnormally to form amyloid deposits associated with a number of degenerative diseases. The research reported here is aimed at exploring the connection between curcumin's thermostability and its inhibitory activity toward the amyloid fibrillation of hen egg-white lysozyme (HEWL). METHODS: ThT fluorescence spectroscopy, equilibrium thermal denaturation analysis, and transmission electron microscopy were employed for structural characterization. MTT reduction and flow cytometric analyses were used to examine cell viability. RESULTS AND CONCLUSION: The addition of thermally pre-treated curcumin was found to attenuate the formation of HEWL fibrils and the observed fibrillation inhibition was dependent upon the pre-incubation temperature of curcumin. Our results also demonstrated that the cytotoxic effects of fibrillar HEWL species on PC 12 and SH-SY5Y cells were decreased and negatively correlated with curcumin's thermostability. Next, an enhanced stability of HEWL was perceived upon the addition of curcumin pre-incubated at lower temperature. Furthermore, we found that the alteration of curcumin's thermostability was associated with its inhibitory potency against HEWL fibrillation. GENERAL SIGNIFICANCE: We believe that the results from this research may contribute to the development of effective therapeutics for amyloidoses.
Subject(s)
Amyloid/antagonists & inhibitors , Curcumin/pharmacology , Muramidase/pharmacology , Amyloidosis/drug therapy , Animals , Cell Survival/drug effects , Curcumin/chemistry , Flow Cytometry , Muramidase/chemistry , PC12 Cells , Protein Folding , Rats , Spectrophotometry, Ultraviolet , Temperature , ThermodynamicsABSTRACT
This work examines the effects of L-arginine (L-Arg) on the aggregation and amyloid fibrillation of bovine serum albumin (BSA). We demonstrate that L-Arg dose-dependently reduces thioflavin T (ThT) fluorescence of BSA within the L-Arg concentration range used (0-1.4 M). However, as revealed by electron microscopy, size exclusion chromatography, and dynamic light scattering results, L-Arg does not prevent amyloid-like fibril formation by BSA. We conclude that L-Arg competes against ThT for binding sites on BSA amyloid-like fibrils, leading to biased results in ThT fluorescence measurements. Moreover, the use of ThT fluorescence assay to screen for potential inhibitors against amyloid fibrillation can give misleading results.
Subject(s)
Arginine/chemistry , Serum Albumin, Bovine/chemistry , Thiazoles/chemistry , Animals , Benzothiazoles , Binding Sites , Cattle , Fluorescence , Microscopy, Electron, Transmission , Protein Binding , Protein Conformation , Serum Albumin, Bovine/ultrastructureABSTRACT
At least 25 human proteins can fold abnormally to form pathological deposits that are associated with several degenerative diseases. Despite extensive investigation on amyloid fibrillation, the detailed molecular mechanisms remain rather elusive and there are currently no effective cures for treating these amyloid diseases. The present study examined the effects of dithiothreitol on the fibrillation of hen egg-white lysozyme (HEWL). Our results revealed that the fibrillation of hen lysozyme was significantly inhibited by reduced dithiothreitol (DTT(red)) while oxidized dithiothreitol (DTT(ox)) had no anti-aggregating activity. Effective inhibitory activity against hen lysozyme fibrillation was observed only when DTT(red) was added within 8 days of incubation. Our results showed that the initial addition of DTT(red) interacted with HEWL, leading to a loss in conformational stability. It was concluded from our findings that DTT(red)-induced attenuation of HEWL fibrillation may be associated with disulfide disruption and extensive structural unfolding of HEWL. Our data may contribute to rational design of effective therapeutic strategies for amyloid diseases.
Subject(s)
Amyloid/metabolism , Avian Proteins/metabolism , Central Nervous System Agents/pharmacology , Dithiothreitol/pharmacology , Egg Proteins/metabolism , Muramidase/metabolism , Protein Multimerization/drug effects , Animals , Central Nervous System Agents/chemistry , Chickens , Dithiothreitol/chemistry , Egg White , Female , Hydrophobic and Hydrophilic Interactions , Kinetics , Oxidation-Reduction , Protein Conformation/drug effects , Protein Folding/drug effects , Protein Stability/drug effects , Thermodynamics , Urea/metabolismABSTRACT
Amyloid fibrils have been associated with at least 25 different degenerative diseases. The 51-residue polypeptide hormone insulin, which is associated with type II diabetes, has been shown to self-assemble to form amyloid fibrils in vitro. With bovine insulin as a model, the research presented here explores the effects of two amphiphilic surfactants (1,2-dihexanoyl-sn-glycero-3-phosphocholine (di-C7-PC) and 1,2-diheptanoyl-sn-glycero-3-phosphocholine (di-C7-PC)) on the in vitro fibrillation process of bovine insulin at pH 2.0 and 55 degrees C. We demonstrated that insulin fibrillation may be inhibited by both surfactants in a dose-dependent fashion. The best inhibition of fibril formation is observed when insulin is incubated with 4mM di-C7-PC. Moreover, the addition of either surfactant at the concentrations studied attenuated insulin fibril-induced cytotoxicity in both PC12 and SH-SY5Y cell lines. The results from this work may contribute to the understanding of the molecular factors affecting amyloid fibrillation and the molecular mechanism(s) of the interactions between the membrane and amyloid proteins.
Subject(s)
Amyloid/biosynthesis , Insulin/metabolism , Insulin/toxicity , Surface-Active Agents/pharmacology , Amyloid/chemistry , Amyloid/ultrastructure , Amyloidosis/drug therapy , Animals , Cattle , Cell Line , Cell Survival/drug effects , Humans , In Vitro Techniques , Insulin/chemistry , Microscopy, Electron, Transmission , Models, Biological , PC12 Cells , Phosphatidylcholines/pharmacology , Phospholipid Ethers/pharmacology , Protein Multimerization/drug effects , Protein Structure, Secondary/drug effects , RatsABSTRACT
More than twenty different human proteins have been found to fold abnormally resulting in the formation of pathological deposits and several lethal degenerative diseases. Despite extensive investigations on amyloid fibril formation, the detailed molecular mechanism remained rather elusive. The present study is aimed at exploring the effect of the ratio of cysteine and cystine in the buffer on the fibrillation of hen egg-white lysozyme. Our results revealed that the inhibition of lysozyme amyloid formation by cysteine in the redox buffer followed a concentration-dependent fashion. Cystine, the oxidized form of cysteine, nevertheless, did not influence the final level of fibrillation although it lengthened the lag period of fibril formation. Moreover, the effect of the ratio of cysteine to cystine in the buffer on the fibrillogenesis of hen lysozyme was found to be greatly associated with the formation of mixed disulfide derivatives. Finally, a possible reaction mechanism was proposed to explain our experimental results. Our study shows that the concentration of mixed disulfide derivative was inversely correlated with the level of lysozyme fibrillogenesis. The results from this work may aid in comprehending the molecular mechanism(s) of amyloid fibrillogenesis for disulfide bonded proteins and the development of effective therapeutics for amyloidogenic diseases.
Subject(s)
Amyloid/metabolism , Muramidase/metabolism , Amyloid/drug effects , Animals , Buffers , Cysteine/chemistry , Cystine/chemistry , Disulfides/chemistry , Kinetics , Microscopy, Electron, Transmission , Muramidase/drug effects , Oxidation-ReductionABSTRACT
This study examined the effects of glutathione on the fibrillation of hen egg-white lysozyme. We found that the fibrillation of lysozyme was considerably reduced by GSH while no anti-aggregating activity was detected with only GSSG. SDS-PAGE results also revealed that the addition of GSH led to an early occurrence of prominent lysozyme hydrolysis. Moreover, GSH was effective in inhibiting lysozyme fibrillation when GSH was added within 6 days of incubation. We conclude that the attenuation of lysozyme fibrillation is strongly dependent upon the redox environment. Our data may contribute to decipher the molecular mechanism of amyloid fibrillation.
Subject(s)
Amyloid/metabolism , Glutathione/pharmacology , Muramidase/metabolism , Animals , Disulfides/chemistry , Electrophoresis , Hydrophobic and Hydrophilic Interactions , Microscopy, Electron , Muramidase/chemistry , Protein Binding/drug effects , Protein Structure, Secondary/drug effects , Spectrum Analysis , Surface Properties/drug effects , Time FactorsABSTRACT
At least twenty human proteins can fold abnormally to form pathological deposits that are associated with several degenerative diseases. Despite extensive investigation on amyloid fibrillogenesis, its detailed molecular mechanisms remain unknown. This study is aimed at exploring the inhibitory activity of curcumin against the fibrillation of hen lysozyme. We found that the formation of amyloid fibrils at pH 2.0 in vitro was inhibited by curcumin in a dose-dependent manner. Moreover, quenching analysis confirmed the existence of an interaction between curcumin and lysozyme, and Van't Hoff analysis indicated that the curcumin-lysozyme interaction is predominantly governed by Van Der Waals force or hydrogen bonding. Curcumin was also found to acquire disaggregating ability on preformed lysozyme fibrils. Finally, we observed that curcumin pre-incubated at 25 degrees C for at least 7 days inhibited lysozyme fibrillogenesis better than untreated curcumin and the enhanced inhibition against HEWL fibrillation might be attributed to the presence of dimeric species.
Subject(s)
Amyloid/chemistry , Curcumin/chemistry , Muramidase/chemistry , Animals , Hydrogen Bonding , Hydrogen-Ion Concentration , Hydrophobic and Hydrophilic Interactions , Kinetics , Protein BindingABSTRACT
This work examines the inhibitory effect of TCEP on the in vitro fibrillation of hen lysozyme at pH 2. We demonstrate that the inhibition of hen lysozyme fibrillation by TCEP follows a dose-dependent manner. Our data show that the addition of TCEP prevents alpha-to-beta transition and promoted unfolding of lysozyme. Moreover, our findings suggested that the TCEP-induced attenuated fibrillation is associated with disulfide disruption and structural unfolding of HEWL.
