Study On The Interaction Between Human Islet Amyloid Polypeptide And Cholesterol

Alzheimer’s disease,Parkinson’s disease,type 2 diabetes（T2DM）,and other age-related degenerative diseases are associated with protein misfolding and resulting accumulation of insoluble protein deposits which are composed ofβ-sheet-rich fibrous aggregates.Human islet amyloid polypeptide（hIAPP）contains 37 amino acid residues,usually secreted together with insulin by isletβ-cells.The aggregation of IAPP on the surface of isletβ-cells and the process of forming amyloid plaques are closely related to the pathology of type II diabetes.Cholesterol is a key component of the cell membrane and is involved in the formation of lipid rafts in the membrane.Elevated levels of cholesterol inβ-cells are found in patients with T2DM,which may be associated with dysfunction ofβ-cells in insulin secretion.However,we have little knowledge about how the aggregation of hIAPP at membrane surface and resulting damage to membrane are related with cholesterol.The N-terminal 11-17 region of hIAPP(R₁₁LANF₁₅LV₁₇)contains a motif comprising of triad R11-F15-V17,which fulfils a“reverse cholesterol recognition amino acid consensus sequence（CARC）”with fomular（K/R）-X_1-5-（Y/F）-X_1-5-（L/V）usually found in transmembrane proteins.This promotes us to think whether cholesterol regulates the interaction between hIAPP and lipid membrane,whether the three key residues making up of CARC motif mediate the specific recognition of hIAPP to cholesterol,and how the compositions of lipid membranes affect the interactions with hIAPP.For this purpose,we explored the cholesterol-sensing role of F15 in the interaction of hIAPP and hIAPP_1-19 with various compositions of lipids including DOPC,DPPC and DOPC/DPPC using NMR,CD,ThT fluorescence and dye leakage assays.We show that both hIAPP and hIAPP₁–₁₉ are more potent in the disruption to the membranes with cholesterol than they are in the disruption to the membranes without cholesterol.A substitution of F15 by leucine affects the binding and disruption of the peptides to the membranes slightly in the absence of cholesterol,but decreases the activities largely in the presence of cholesterol.F15 also plays a role in accelerating fibrillar assembly of hIAPP,but the function is independent of cholesterol in nature.Our results suggest that F15 plays a key role in the cholesterol-sensing binding and disruption of hIAPP to the PC membranes and the distribution of cholesterol in the membranes has an influence on the disruptive activity of hIAPP.We further study the binding and disruption of hIAPP_1-19 to the membranes as well as the role of cholesterol therein using the membrane systems with the lipid compositions closer to the biomembranes.We substituted three frame residues in CARC motif by Leu or Ala separately and explored the interactions of these variants with the lipid membranes in the absence and presence of cholesterol.We also used two-dimensional NMR spectroscopy to study possible mode of hIAPP_1-19-cholesterol interaction in a membrane-mimic composed of detergents.Our results demonstrate that of the three CARC frame residues,the aromatic residue F15 is the most important residue in mediating the interaction of hIAPP_1-19 with cholesterol.The basic residue R11 also plays a considerable role in the interaction between hIAPP_1-19 and cholesterol,while the mediating role of the hydrophobic residue V17 is very small.The specific interaction between hIAPP_1-19 and cholesterol promotes the binding of the peptide to the lipid membrane and aggravates the membrane damage by the peptide.We found the difference between the interactions of hIAPP_1-19 and its F15L varant with cholesterol by 2D NMR spectroscopy.