Interaction Of Natural Polyphenolic Compounds With Lysozyme Amyloid Fibrils

Protein or peptide can undergo unfolding and aggregation to produce amyloid fibrils.The fibrils deposit on the interstitial cells,causing cell damage and eventually leading cell to apoptosis. Amyloid fibrils of proteins or peptide fragments have been found to be associated with more than 20 kinds of human diseases,such as familial amyloidosis,Parkinson's disease,Alzheimer's disease,typeⅡdiabetes and a number of systemic amyloidoses.The threat of these diseases on people's health has attracted extensive attentions and numerous studies have been carried out for understanding the molecular mechanism and searching for effective medicinal remedies.However,the mechanism of amyloid fibrils formation and pathology related amyloid-diseases are still kept unclear.Currently, efforts are still focusing,on the elucidation of the amyloidgenic pathways and development of efficacious treatment methods and drugs for amyloid-related diseases.The intermediates of protein or polypeptide assemblies,oligomers and protofibrils,are believed more toxic to cell than the mature fibrils.The therapeutic strategies proposed for the treatment of amyloid-related diseases are mainly based on inhibition of amyloid formation and disruption of fibrillar structures.Oxidative stress has been thought to be the incentive factor and/or the resultant impact on organisms during protein amyloidogenesis.Natural antioxidants in food and traditional herbal medicine has shown a broad pharmacological activity,including inhibiting the formation of amyloid fibrils,changing the structure of fibrils and attenuating the fibrillar cytotoxicity to living cells.Natural polyphenolic compounds,a major category of natural antioxidants,can perform disruption on the amyloid structure of protein fibrils,being considered as potential anti-amyloidogenic reagents.In order to study the impacts of natural polyphenolic compounds on amyloidogenesis.Hen egg white lysozyme,which is referred to as lysozyme in this article,has been selected as an in vitro model for investigation of the interactions between natural polyphenolics and amyloid fibrils. Lysozyme is a kind of small globular protein,of which the structure,function,physical and chemical properties and metabolism have been studied extensively.Investigations showed lysozyme was one of the proteins to be most prone to amyloid fibrillation.Incubation of lysozyme for 10 days under the conditions 65℃and pH2.0 resulted in the formation of amyloid fibrils.ThT and ANS fluorescence analyses showed that the formation of lysozyme fibrils involved the exposure of hydrophobic groups and formation of enriched highly orderedβ-sheet structures.The gold-green birefringence of long and unbranched fibrils fibrils were observed and visualized after CR staining under polarized light microscopy.Bradford assay was used for analyses of fibrillar deposition induced by natural polyphenols. Two typical natural polyphenols,tea catechins and flavonoids,have been tested in this study.Of which epigallocatechin gallate(EGCG) and quercetin have been found to cause strongly conversion of lysozyme fibrils into amorphous aggregations.Other polyphenols catechin(C),epicatechin(EC), and rutin showed weak activity,whereas phenol,vitamin C,α-tocopherol had no effect on the fibrils.Quercetin is one of the plant flavonoids that most widely exist in nature.Experimental results herein showed that quercetin converted efficiently lysozyme fibrils into amorphous aggregates. Therefor further investigations have been performed on the interactions between quercetin and lysozyme amyloid fibrils.The results showed that quercetin disrupted the fibrillar structure of lysozyme.ThT fluorescence analysis showed that the formation of amyloid fibrils involved exposure of interior hydrophobic domains and formation of enriched highly orderedβ-sheet structures.The transformation of the fibrils into amorphous aggregates caused by quercetin was accompanied with a decrease of the affinity of ThT for the fibrils,implicating a decrease of theβ-sheet structure in the amyloids.Amorphous aggregation of the fibrils can be imaged by transmission electron microscope (TEM) and atomic force microscope(AFM).Under the microscopes,fibrils cracked into short fragments,some of the fragments got together into regiments and some others were scattered, indicating substantial structural alterations occurred upon quercetin binding.Amyloid fibrillation of lysozyme was inhibited by incubation of the protein in the presence of quercetin.The signals of ThT and ANS fluorescence kept unchanged in the duration of incubation of lysozyme under an amyloidogeneic condition,in contrast to the growth curve of lysozyme fibrillation in the absence of quercetin,of which the ThT and ANS fluorescence increased along with the incubation.Lysozyme fibrillation is a process involving an increase of the surface hydrophobicity of the fibrillar aggregates.As a consequence the binding affinity of quercetin for the protein was increased. HPLC analysis showed the binding ratio of quercetin to lysozyme increased with the fibril aging,in accordance with the increased capability of quercetin to disrupt the amyloid aggregates upon the protein unfolding.In summary,native lysozyme underwent unfolding and amyloid fibrillation upon incubation of the protein at low pH and high temperature.Amyloid formation was accompanied with increases of surface hydrophobicity of the protein aggregates,the affinity of lysozyme for polyphenols and the cytotoxicity of the fibrillar species.Natural polyphenols,including EGCG and quercetin,have demonstrated strong disruptive effects on lysozyme amyloid fibrils,including converting the fibrils into amorphous aggregates and inhibiting the amyloid formation.Hydrophobic interaction played a crucial role in the interactions between quercetin and amyloid fibrils of lysozyme.The results reported herein can be of significance in the elucidation of interactions between amyloid fibrils and small ligands and in the development of novel remedies for treating amyloid diseases.