Study On The Interactions Between Human Islet Amyloid Polypeptide And Lipid Membranes

The misfolding of normal soluble protein into highly ordered aggregates can trigger a series of diseases. Type 2 diabetes(T2DM), Alzheimer's disease(AD), Parkinson's disease(PD) and other neurodegenerative diseases are related to the misfolding of this kind of proteins. Although these proteins have their own amino acid sequence and spatial structure, they all can misfold into insoluble amyloid deposit from soluble protein. Amyloid diseases caused by this kind of proteins are likely to have a similar pathogenic mechanism. The characteristic of the patients with T2 DM is that human islet amyloid polypeptide(h IAPP) misfolds into fiber deposited on islet ?-cell with ?-sheet structure and results in death of ?-cell. Human IAPP is composed of 37 amino acid residues, and co-secrets with insulin by pancreatic islet ?-cells. The interaction of the peptide with the membrane is the key step in the pathogenic process of hIAPP. Therefore, it is important to understand the mechanism of the interaction between h IAPP and membrane for the inhibition of hIAPP aggregation and development of the anti-disease drugs. The interaction between h IAPP and membrane depends on many environmental factors because of the complex celluar environment. In addition, cholesterol is a significant component in the cellular membranes and may have an important effect on the aggregation behaviors and toxic mechanism of the amyloid peptides. In this thesis, we study the aggregation of h IAPP at phospholipid membranes and the effects of chondroitin sulfate A and bovine serum albumin on the aggregation of h IAPP at phospholipid membranes. We also study the effects of cholesterol on the interactions between the N-terminal region of h IAPP and phospholipid membranes. The main works in this thesis are summarized below:1. We selected zwitterionicl POPC, anionic POPG and their mixture at a ratio of 3:7 of POPG:POPC as the model membranes to study the effects of chondroitin sulfate A(CSA) and bovine serum albumin(BSA) on the fibrillation behaviors of human islet amyloid polypeptide(h IAPP) in the phospholipid membrane environment using the experimental methods of ThT fluorescence assay, circular dichroism, atomic force microscope, and 31P-NMR. The existence of the two cellular components of CSA and BSA provides a heterogeneous membrane environment for hIAPP, which results in the fibrillation of hIAPP different from the occurrence in a homogeneous bulk solution. In bulk solution, CSA is a promoter and BSA is an inhibitor to the fibril formation of hIAPP. However, at POPC membrane, CSA has an extra promotion impact on the fibrillation of h IAPP because of the specific interaction of CSA and POPC. At the POPG-containing membranes(POPG and POPC/POPG),the inhibiting efficiency of BSA on h IAPP fibrillation is suppressed because of the specific interaction of the peptide with anionic POPG component. Contrary to a homogeneous environment, a heterogeneous environment is likely to be more close to the real environment of h IAPP. Therefore, the study performed in a more complex environmental system including lipid membrane and the components in the extracelluar matrix may provide more useful information for understanding the mechanism of hIAPP aggregation.2. The N-terminal region is the key domain in the interaction between hIAPP and lipid membrane. The study found that, although h IAPP has a strong tendency to aggregation, h IAPP1-19 is different from the full length h IAPP. Human IAPP1-19 does not aggregate into long fiber either in aqueous solution or at phospholipid membranes,. Although h IAPP1-19 has a weak aggregation tendency, it has the same cytotoxicity as full-length h IAPP. A triad of residues composed of R11, F15 and V17 in the 1-19 regions constitutes an inversed cholesterol recognition amino-acid consensus(CARC). The CARC, perticularly F15, may involve in the peptide-cholesterol binding. We choose the 1-19 peptide as the structure model of h IAPP interacting with membrane and synthesize a mutant of F15L(h IAPP1-19/F15L) as well to study the interaction of h IAPP1-19 with DPPC membrane and the role of the CARC in the interaction mainly using differential scanning calorimetry, 31P-NMR, 1H-NMR, CD and leakage assays. We find that cholesterol forms lipid rafts in DPPC membrane, by which it promotes the binding of hIAPP1-19 with membrane, induces a structural conversion of the peptide from a random coil to an ?-helix, facilitates the formation of toxic oligomers, and destroys the membrane more severely. In this processes, F15 involves the binding to cholesterol.3. We also selecte h IAPP1-19 and h IAPP1-19/F15 L as the research objects to study the interactions between the peptides and DOPC membrane, a lipid bilayer with more flexible chains than DPPC membrane, and the effects of cholesterol on the interactions using 31P-NMR, 1H-NMR titration experiments, CD and leakage experiments. We find that cholesterol can also promote the binding of hIAPP1-19 with DOPC membrane, resulting in greater permeability of the membrane. However, compared with the result obtained in DPPC membrane system, the binding of h IAPP1-19 with DOPC membrane is weaker, likely because cholesterol could not form the lipid rafts in the flexible DOPC membrane.