Study Of The Effects Of Divalent Metal Ions On The Ability Of Human Islet Amyloid Polypeptide Oligomers To Damage The Lipid Membrane

Many degenerative diseases and metabolic diseases are closely related to the misfolding of amyloid peptides/proteins and the resulting amyloid deposition at the cells.The amyloid deposits are rich inβ-sheet fibrillar aggregates.The fibrillation process of human amyloid polypeptide（hIAPP）on the pancreaticβcells is closely associated with the pathology of type II diabetes mellitus（T2DM）,and the bioavailable divalent metal ions（such as calcium,zinc,and copper ions）have an important effect on the fibrillar aggregation and cytotoxicity of hIAPP.The imbalance of the divalent metal ions in the human body can aggravate T2DM.However,there is still a lack of understanding of how divalent metal ions affect the toxicity of hIAPP to the cells.It has been found in the studies of the cytotoxic mechanism of various amyloid peptides and proteins that the oligomeric intermediates formed in the early stage of the fibrillar aggregation process are the main toxic substances,while monomers and mature fibers are non-toxic.Therefore,studying the effects of divalent metal ions on the ability of hIAPP oligomers to destroy phospholipid membranes has important scientific significance for understanding the mechanism of hIAPP toxicity.In this thesis,we constructed a model membrane using POPC/POPG 4:1 lipid system and studied the effects of divalent calcium,zinc,and copper ions on the aggregation properties of the oligomers constructed by both hIAPP_18-27and hIAPP and the ability of the oligomers to damage the phospholipid membrane.The thesis includes two aspects of research works:1.We selected the peptide fragment from the core region of hIAPP(hIAPP_18-27)and prepared four species of oligomers using the short peptide in the absence and presence of Ca（Ⅱ）,Zn（Ⅱ）and Cu（Ⅱ）ions,respectively,at a peptide-to-metal ratio of1:1.we characterized the morphologies and assembly structures of these species of oligomers,tested for the surface hydrophobicity of the oligomers and their ability to destroy POPC/POPG 4:1 vesicles through various experimental methods including TEM,AFM,DLS,ThT dye fluorescence,CD,ANS probe fluorescence,acrylamide fluorescence quenching,calcein leakage experiments.The results showed that the binding to the metal ions leads to a reduction in the ability of peptide oligomers to damage the lipid membrane.The roles of Zn（Ⅱ）and Cu（Ⅱ）in reducing the disruptive ability of oligomers are obvious,while the role of Ca（Ⅱ）is rather small.The binding to the metal ions also caused a decrease in the hydrophobic exposure of the oligomers,and the order of the decrease in the hydrophobic exposure of these species of oligomers was consistent with that of the decrease in the ability of these oligomers to damage the lipid membrane.2.We detected the effects of these metal ions on the aggregation property of the full-length hIAPP oligomers and the activity of the oligomers in the membrane disruption using the same experimental methods mentioned above.We found that the influence of Ca（Ⅱ）on the membrane damage of hIAPP oligomers is small,while the influences of Zn（Ⅱ）and Cu（Ⅱ）are much larger,and the influence of Zn（Ⅱ）and Cu（Ⅱ）is related to the hIAPP/metal ion ratio.Moreover,compared with the effect of hIAPP oligomers in the absence of metal ion,the membrane damage induced by hIAPP oligomers in the presence of Zn（Ⅱ）and Cu（Ⅱ）ions is alleviated at a molar ratio of hIAPP to metal ion of 1:0.33,and the membrane damage is aggravated at a molar ratio of hIAPP to metal ion of 1:1.However,the surface hydrophobicity of hIAPP oligomers is reduced in the presence of Zn（Ⅱ）and Cu（Ⅱ）either at 1:0.33 or 1:1.The difference in the disruptive activities of hIAPP oligomers observed at a higher and lower concentrations of Zn（Ⅱ）and Cu（Ⅱ）is attributed to a competitive effect between an increase in the amount of hIAPP oligomers promoted by binding to the metal ions and a decrease in the disruptive ability of metal-associated oligomer individuals.At the peptide-to-metal ratio of 1:1,the increase in the number of oligomers may play a predominant role in the activity of hIAPP oligomers to damage the lipid membrane,while the reduction in the disruptive ability of metal-associated oligomer individuals may play a predominant role in the membrane damage of hIAPP oligomers at the peptide-to-metal ratio of 1:0.33.