ABSTRACT
We investigated the effects of nanoparticles (NPs) on the structure and activity of hen egg-white lysozyme (HEWL) using CeO2 and ZnO NPs. Our results showed that CeO2 NPs triggered the transition of lysozyme secondary structure from α-helix to ß-sheet. CeO2 NPs also induced the hydrophobic region of lysozyme to become exposed to the solvent. In contrast, the secondary structure content and hydrophobic region of lysozyme were only slightly changed in the case of ZnO NPs. In addition, the activity of the lysozyme was observed to decrease upon adsorption on CeO2 NPs, whereas the effect of ZnO NPs on activity was negligible. The glutaraldehyde crosslinking results indicated that the percentage of the dimeric form of lysozyme was greatly enhanced by the addition of both NPs. Furthermore, the adsorption capacity, degree of favorability of adsorption, and surface heterogeneity for CeO2 NPs were found to be greater than those on ZnO NPs. Given that CeO2 NPs exhibit a higher surface area/mass than ZnO NPs, the surface concentration of lysozyme on CeO2 NPs was lower than that on ZnO NPs. This result suggested that more direct interactions were involved between CeO2 NPs and lysozyme, thereby leading to a more significant effect. Moreover, higher surface curvatures may also cause destruction of lysozyme's structure and thus affect its activity. In addition, taking into account the surface properties and protein properties, the Toth adsorption model along with the generated site energy distribution was further used to exaplain the difference between the results (e.g., structure, stability, and activity) of lysozyme adsorption on CeO2 and ZnO NPs. The results reported here may aid in better understanding the beneficial or harmful impacts of nanoparticles on the biological systems.
Subject(s)
Cerium/chemistry , Metal Nanoparticles/chemistry , Muramidase/chemistry , Zinc Oxide/chemistry , Adsorption , Animals , Binding Sites , Chickens , Circular Dichroism , Cross-Linking Reagents/chemistry , Dose-Response Relationship, Drug , Glutaral/chemistry , Isoelectric Point , Microscopy, Electron, Scanning , Microscopy, Electron, Transmission , Protein Isoforms/chemistry , Protein Structure, Secondary , Protein Structure, Tertiary , Solvents/chemistry , Surface PropertiesABSTRACT
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
The fibrillation of hen egg-white lysozyme (HEWL) in the absence and presence of simple, unstructured D,L-lysine-co-glycine (D,L-Lys-co-gly) and D,L-lysine-co-L-phenylalanine (D,L-Lys-co-Phe) copolypeptides was studied by using a variety of analytical techniques. The attenuating and decelerating effects on fibrillation are significantly dependent on the polypeptide concentration and the composition ratios in the polypeptide chain. Interestingly, D,L-Lys-co-gly and D,L-Lys-co-Phe copolypeptides with the same composition ratio have comparable attenuating effects on fibrillation. The copolypeptide with highest molar fraction of glycine residue exhibits the strongest suppression of HEWL fibrillation. The copolypeptide has the highest hydrophobic interacting capacity due to the more molar ratio of apolar monomer in the polymer backbone. The major driving forces for the association of HEWL and copolypeptides are likely to be hydrogen bonding and hydrophobic interactions, and these interactions reduce the concentration of free protein in solution available to proceed to fibrillation, leading to the increase of lag time and attenuation of fibrillation. The results of this work may contribute to the understanding of the molecular factors affecting amyloid fibrillation and the molecular mechanism(s) of the interactions between the unstructured polypeptides and the amyloid proteins.
