Studies On The Interactions Between IAPP And Lipid Membranes And The Inhibition Against The Fibrillation Of IAPP

Alzheimer’s disease,amyotrophic lateral sclerosis,type II diabetes mellitus(T2DM)and other series of diseases are related to the misfolding of proteins.These diseases are characterized by the soluble proteins misfolding into intracellular or extracellular insoluble amyloid deposits.Although proteins related to these diseases have various amino acid sequences,they have similar mechanisms of aggregation and cytotoxicity caused by membrane damage.Islet amyloid polypeptide(IAPP)composed of 37 amino acid residues is the main component of amyloid deposits in the pancreas of patients affected by T2 DM.In normal physiological condition,IAPP adopts a random coil structure,however,aggregates to form β-sheet-rich fibrils under pathological condition.The amyloid deposits of IAPP in the β-cells lead to mass loss and dysfunction of the b-cells,whichis assumed to be an important cause of T2 DM.Series of studies have shown that it is the soluble oligomeric intermediates during the process of aggregation rather than the mature amyloid fibers are the major components that cause membrane disruption and cytotoxicity in vitro and vivo.Therefore,the study on the interaction of IAPP with membrane and the relationship between the structures of IAPP oligomers and their membrane damage abilities are important for understanding the aggregation and cytotoxicity mechanisms of IAPP.Furthermore,preventing h IAPP fibrillogenesis has been a primary strategy in development of drugs treating for T2 DM.In this thesis,we study the interactions between IAPP and lipid membranes and the inhibition against the fibrillation of IAPP.The main works in this paper are summarized as follows:1.As the oligomeric intermediates of h IAPP were heterogeneous and transient in solution,we used rat amylin(rIAPP)that has an extremelylowpropensity of aggregation as a model peptide to study the effects of different structures of peptide oligomers on the lipid membrane.We prepared rIAPP oligomers with varying structures,sizes and morphologies by various treatment methods,explored their potencies in membrane disruption and mechanism under the activity usingthe lipids POPC and POPG at a molar ratio of 4:1 as the model membranes.Our results show that the disruptive potencies of rIAPP oligomers depend on the size and hydrophobic exposure of the assemblies.The oligomeric species with sizes belowa limit(~50 nm in diameter)are more disruptive than those with sizes above the limit.The correlation between hydrophobic exposure and disruptive effect is valid for the oligomers with sizes smaller than the abovementioned limit,but not applicable for the oligomers larger than the size limit.2.The N-terminal sequence of IAPP contains a triad of amino-acid residues composed of R11,F15 and V17 that accord with the amino acid sequence of the inverse cholesterol recognition amino-acid consensus(CARC).The CARC sequence may play a role inmediate IAPP interacting with cholesterol,by which the disruptive effect of the peptide on the membrane is modulated by cholesterol.We used rIAPP(instead of hIAPP because of its rapidfibrillation)and three variants of rIAPP including R11 A,F15L and R18H(H is the residue at position 18 in hIAPP)to study the interactions between the peptides and DPPC lipid system and explored the effects of cholesterol on the peptide-membrane interactions.Our results demonstrate thatalthough the presence of cholesterol deteriorates the damage of the membrane by all the peptides,the peptide-membrane interactions are rather weak(may be transient)and the mechanisms underlying the interactions may be different for different types of peptides.Both rIAPP and R18 H variant preferentially partition into the cholesterol-rich domain of the membrane and decrease the interactions between cholesterol molecules.However,rIAPP displaysgreater influence on the distribution of cholesterol in the membrane.The F15 L and R11 A substitutions decrease the recognition to cholesterol,leading tomore peptide molecules partition into the cholesterol-poor domain of the membrane.Different degrees of membrane damage induced by the peptides may be associated with different interactions of these peptides with cholesterol.3.Increasing studiesuse graphene oxide(GO)as an inhibitor to prevent the fibrillation of amyloid peptides in recent years in term of its unique two-dimensional structure,highly hydrophobic surface,well dispersed and easily modified properties.In this study,we prepared GO-PEI complex by modification of polyetherimide(PEI)on GO using covalent binding method.The results of experiments show that GO-PEI is more stable in buffer and more efficient in inhibiting hIAPP fibril formation than GO.GO-PEI inhibits hIAPP fibrillation with the most efficiency as it is added at the initial stage of hIAPP aggregation,and less effectively when added in the growing period of h IAPP aggregation,but the inhibitor disables to disrupt the matured hIAPP fibrils.