Study On The Aggregation And Membrane Damage Features Of Amyloid Peptide By D-amino Acid Substitution

Amyloid peptides or proteins are soluble under normal physiological conditions and participate in the regulation of physiological balance in human body.However,they could misfold under pathological conditions,resulting in aggregate deposition on the cell surface.This has been associated with a variety of diseases,including type 2diabetes,Alzheimer’s disease and Parkinson’s disease.Although different diseases have their corresponding kinds of amyloid peptides,these peptides with different amino acid sequences show similar aggregation properties and activity in membrane damage,suggesting that these amyloid peptides may share a common mechanism underlying the aggregation and toxicity.The damage of phospholipid membrane by oligomeric intermediates formed in the initial stage of amyloid aggregation is suggested to be one of various possible mechanisms of toxicity of amyloid polypeptides to cells.However,it has been debated which structural component is significant for the activity of oligomers in the destruction of phospholipid membrane.In addition toα-helix conformation,α-sheet conformation is also believed to be an intermediate conformation that could induce cytotoxicity and mediate the fibrillar assembly of amyloid polypeptides.However,α-sheet structure is extremely unstable and difficult to be observed under normal conditions,leading to the lack of evidence for the existence ofα-sheet conformational intermediate,and thus a little knowledge about it.To investigate the role ofα-sheet structure in inducing fibrillar aggregation and membrane destruction of amyloid polypeptide,we used human islet amyloid polypeptide（hIAPP）as an object in this study.Human IAPP is synthesized and secreted by isletβ-cells and participates in the regulation of physiological homeostasis in human body.Amyloid aggregation of hIAPP leads to apoptosis ofβ-cells,and thus is associated with type 2 diabetes.We selected hIAPP_18-27fragment and replaced part of left-handed amino acid（L）residues by right-handed amino acid（D）residues.A stableα-sheet structure can be obtained by an alternating substitution of L-amino acid residues by D-amino acid residues in the peptide under certain conditions.We investigated the formation ofα-sheet structure and its role in the aggregation and membrane destruction of hIAPP using several kinds of D-amino acid modified hIAPP_18-27short peptides.The research contents and main results include the following three aspects:1.We used the experiments of ThT fluorescence,NMR,CD,ATR-FTIR,TEM etc.to characterize the structure of hIAPP_18-27peptide isomer with an alternating arrangement of D-and L-amino acid residues under different conditions,investigate the fibrillar aggregation of the decapeptide and its transfer from anα-sheet structure to aβ-sheet structure in the aggregation process.We obtained the experimental evidence of the conversion between the two structures and observed for the first time the formation ofα-sheet structured fibers in aqueous solution.We found that water molecules play an important role in stabilizingα-sheet structured fibers.2.We studied the effects of both all-L hIAPP_18-27and D,L-alternating hIAPP_18-27isomer on the aggregation of hIAPP and the ability of hIAPP to damage the lipid membrane using ThT fluorescence,CD,TEM,NMR and dye leakage fluorescence experiments.The results showed that the D,L-alternating peptide in anα-sheet structure is more effective in promoting hIAPP aggregation and decreasing the ability of hIAPP to damage POPC:POPG 4:1 membrane,compared with its all-L isomer in a random coil structure.The D,L-alternating decapeptide could induce the core region of hIAPP to form transientα-sheet conformation,by which it hinders the interaction between hIAPP and phospholipid membrane more effectively.3.We performed an alternating substitution of the D-amino acids for the L-amino acids in the hydrophilic N-terminal region,hydrophobic C-terminal region and the whole region of hIAPP_18-27decapeptide,respectively,used these peptide isomers to prepare different kinds of oligomers and studied the differences of these oligomers in the ability to damage POPC:POPG 4:1 membrane.We examined the morphology,structure,surface hydrophobicity and structural compactness of these oligomers,and monitored the changes in aggregation structure and aggregation rate of the oligomers during the interaction with phospholipid membrane.We found that the extension of the peptide chain in the hydrophobic region enhances the surface hydrophobicity of oligomers and their ability to impair the phospholipid membrane,while the extension of the peptide chain only in the hydrophilic region enhances the aggregation propensity of oligomers.Our results suggest that a positive relation between the surface hydrophobicity and the disruptive ability could be established only for the oligomers with flexible peptide chains and loose packing.