Inhibition Of Short Peptide-modified Gold Nanoclusters Against HIAPP Fibrillation And Membrane Damage

Proteins perform physiological functions based on its specific spatial conformation.Abnormal protein conformational transitions,especially misfolding and fibrillation,have been associated with more than twenty human diseases,such as Alzheimer’s disease and Parkinson’s disease.Human islet amyloid polypeptide（hIAPP）consists of 37 amino acid residues,and its misfolding and fibrosis are associated with the pathology of type II diabetes mellitus（T2DM）.Under normal physiological condition,hIAPP is normally present as a monomer and works with glucagon as an insulin chaperone to synergistically regulate glucose levels in vivo.In the pathological state of T2DM,monomeric hIAPP tends to aggregate to form soluble oligomers and eventually insolubleβ-sheet-rich fibers.Previous researches suggested that the cytotoxicity of amyloid proteins/peptides associated with pathogenesis is mainly due to soluble oligomers rather than insoluble fibrils.Therefore,inhibition against misfolding of proteins to form toxic oligomers has emerged as a potential therapeutic method for protein conformation diseases.With the development of nanotechnology in recent years,various nanomaterials are extensively applied in the industries of energy,environment and biomedicine.Gold nanoclusters（Au NCs,d<3 nm）have been attracting interest from biomedical researchers due to their chemical inertness,unique optical properties,good biocompatibility and ability to cross the blood-brain barrier.The features of small size,large specific surface area,and flexibility in surface modification render Au NCs highly potential in inhibiting amyloid aggregation of proteins.The amyloid inhibitors based on functionalized gold nanoclusters can be discovered by screening surface ligands appropriately.In this work,we applied a strategy of modifying gold nanoclusters with the short peptide that recognizes the target amyloid peptide specifically to construct a highly potent nanoscale inhibitor against hIAPP amyloid aggregation and hIAPP induced cytotoxicity.For this purpose,the gold nanoclusters C-HL-8P@Au NCs were synthesized by attaching a short peptide C-HL-8P derived from the core region of hIAPP(hIAPP_18-27)to the surface of gold nanoclusters.Cysteine-modified gold nanoclusters C@Au NCs and glutathione-modified gold nanoclusters GSH@Au NCs were also synthesized as a comparison.The features of the gold nanocluster products,including size distribution,surface charge,UV absorption,etc.,were characterized.The effects of the three kinds of peptide@Au NCs on hIAPP fibrillation and conformational transitions both in the bulk aqueous solution and under lipid membrane（POPC/POPG4:1 LUVs）environment were systematically monitored using thioflavin T fluorescence kinetic experiment,AFM and CD experiments.In addition,the inhibitory potency of the three kinds of peptide@Au NCs on hIAPP-induced membrane damage and cytotoxicity was explored using fluorescent dye（calcein）leakage assay and cytotoxicity assay.Our study revealed that C-HL-8P@Au NCs inhibit the fibrotic aggregation of hIAPP and slow down the conformational transition of hIAPP effectively both in bulk aqueous solution and lipid membrane environment,and reduce the toxicity of hIAPP to INS-1 cells significantly even at lower stoichiometric concentrations.As inhibitors,the inhibitory effect of C-HL-8P@Au NCs was evidently higher than those of C@Au NCs and GSH@Au NCs.The higher inhibition efficiency of C-HL-8P@Au NCs than C@Au NCs and GSH@Au NCs was attributed to the specific recognition between the hydrophobic residues of the ligand and the homologous residues of the hIAPP core region.Our study provides a new idea for the design and development of efficient amyloid peptide inhibitors and new drugs for the treatment of amyloidosis diseases based on gold nanoclusters.