Regulation Of Arginine-Cysteine-Modified Gold Clusters On The Fibrillation Of Aβ And HIAPP

So far,more than 20 diseases have been identified as protein conformation diseases by clinical medicine,including neurodegenerative diseases represented by Alzheimer’s disease（AD）and localized disease represented by type 2 diabetes（T2D）.All protein conformational diseases have a common pathological feature—the presence of misfolding and abnormal fibrosis of one or more proteins/polypeptides,finally forming insoluble fibrous deposition plaques in the body.The most common ones includeβ-amyloid（Aβ）in AD and human islet amyloid polypeptide（hIAPP）in T2D.The inhibition of misfolding and abnormal fibrosis of pathogenic proteins can only prevent or delay the progress of the corresponding protein conformational diseases,and there is no treatment for those patients whose pathogenic proteins have been misfolded and formed fibrous deposition.Therefore,the development of new drug materials that simultaneously inhibit the misfolding and fibrosis of two pathogenic proteins and has a disassembly effect on the pathogenic protein fibers is a hot spot of research in the biomedical field at present,which has great challenges and cutting-edge significance.In addition,since the onset of such diseases is closely related to oxidative stress,it is also very important to develop protein fibrosis inhibitory materials with good antioxidant properties and good bioavailability.Based on our previous work,we found that gold nanoclusters（AuNCs）modified by different ligands have excellent inhibitory effects on Aβand hIAPP fibrosis respectively,so we try to optimize the AuNCs ligands to achieve the above goals.The specific research content is as follows:（1）Using the dipeptides CR and RC（consists of arginine（R）and cysteine（C））as ligands,two clusters—CR-AuNCs and RC-AuNCs were prepared by one-pot method.The prepared CR-AuNCs and RC-AuNCs were characterized by DLC,DLS,XPS and other characterization methods.It was found that the particle sizes of the two AuNCs were below 2 nm,and CR and RC are linked to the gold nucleus via Au-S.Cytotoxicity experiments proved that both AuNCs had no cytotoxicity in the concentration range of0-100 mg·L^-1,showing excellent biocompatibility.Subsequently,two types of AuNCs were added to the Aβ₄₀and hIAPP solutions for fibrosis inhibition experiments.It was found that CR-AuNCs and RC-AuNCs inhibited Aβ₄₀ fibrosis in a dose-dependent manner,and can achieve complete inhibition at 50 mg·L^-1 and 25 mg·L^-1 respectively,showing that both AuNCs have excellent inhibitory effect on Aβ₄₀ fibrosis.However,neither CR-AuNCs nor RC-AuNCs can completely inhibit fiber growth for hIAPP,and only show a dose-dependent delay in fiber growth.The effects of CR-AuNCs and RC-AuNCs on the repair of protein-induced damaged cells were explored.The results showed that both AuNCs could alleviate their cytotoxicity by inhibiting Aβ₄₀ fibrosis.However,neither AuNCs could relieve the cytotoxicity caused by hIAPP fibrosis.（2）RC-AuNCs with better inhibitory effect were further selected for Aβ₄₀ fiber disassembly experiment.Th T fluorescent labeling assay,AFM and DLS were also used to monitor the disassembly process of mature Aβ₄₀ fiber.It was found that the disassembly process was also dose-dependent.25 mg·L^-1 RC-AuNCs can dissolve mature Aβ₄₀ fibers into non-cytotoxic nano-scale cluster-protein complexes.Real-time in situ CD spectrum monitoring of secondary structure changes of Aβ₄₀ fibers during disassembly revealed that the conformation of Aβ₄₀ aggregates changed fromβ-sheet toα-helix and random during fiber disassembly,which reversed the transition from random andα-helix toβ-sheet during Aβ₄₀ misfolding and fibrosis.Further Aβ₄₀ fiber damaged cell model experiments showed that RC-AuNCs can effectively alleviate or even eliminate the cytotoxicity caused by Aβ₄₀ fibrosis aggregation by dismantling mature fibers.（3）Based on the previous work,the natural extract ferulic acid（Fumalic acid,FA）was used to modify CR-AuNCs to obtain FA-CR-AuNCs,and FA-CR-AuNCs were used in the inhibition experiment of Aβfibrosis.It was found that FA-CR-AuNCs showed a dose-dependent inhibitory effect on Aβ₄₀ protein fibrosis.CD spectroscopy showed that FA-CR-AuNCs can inhibit the transition of Aβ₄₀ protein from unfolded state toβ-folded.Cell model experiments show that FA-CR-AuNCs can reduce the cytotoxicity caused by this process by inhibiting Aβ₄₀ protein fibrosis.At the same time,the total antioxidant capacity of FA-CR-AuNCs（T-AOC）was tested,and it was found that the antioxidant capacity of FA-CR-AuNCs was greatly improved compared with CR-AuNCs,reaching 1/3 of glutathione,indicating that it has potential of anti-oxidative stress.These results indicate that FA-CR-AuNCs can not only inhibit the misfolding and abnormal fibrosis of the pathogenic protein Aβ,but also have a certain antioxidant capacity,and have the potential to prevent AD from multiple targets and multiple pathways.To sum up,aim at the major challenges of AD,this thesis successfully prepared a gold cluster RC-AuNCs with the ability to dissolve mature Aβ₄₀ fibers,and FA-CR-AuNCs with the inhibitory effect on pathogenic proteins Aβ₄₀.This thesis not only accumulated basic data for the development of AuNCs as new drugs for protein conformational disease,but also provided new insights for the development of drugs for protein conformational diseases.