Structural Investigation of Amylin Oligomers Associated to Type-2 Diabetes

The accumulation of particular proteins into long fibrillar aggregates known as amyloids is a common feature of many devastating aging-related pathologies. In type II diabetes mellitus, the main constituent of these aggregates is Islet Amyloid Polypeptide (IAPP, also known as amylin). Like many other amyloidogenic proteins, the aggregation of IAPP has been linked to cellular dysfunction and death. However, the mechanism by which IAPP aggregates form and how this aggregation is linked to cell death remain mysterious. To help reduce this gap, we propose to characterize the oligomeric intermediates of human-IAPP formed in solution, in presence of metals (such as zinc and copper), and in lipid-membrane via three specific aims. 1) In Aim 1, we propose to characterize the intermediates formed by human-IAPP at atomic resolution by NMR spectroscopy. The identified oligomeric intermediates will be tested for cell toxicity and the structural models derived from NMR constraints will be used to evaluate the mechanism and efficiency of amyloid inhibitors. 2) Since a possible genetic link between zinc regulation and type II diabetes has been discovered, we will characterize zinc-IAPP adducts by cell toxicity, NMR and other biophysical experiments in Aim 2. Mutants of oxidized and reduced forms of human-IAPP will be used to probe the metal binding sites, and isothermal titration experiments will be used to measure the metal binding affinities to different amyloid species. In addition, the non-fibril forming and non-toxic rat-IAPP and pramlintide (trade name symlin approved by FDA for use by both type 1 and type 2 diabetic patients) will be used as controls. 3) To gain insight into the lipid-membrane assisted hIAPP aggregation and the mechanism by which hIAPP disrupts the lipid-membrane, we propose to characterize the role of lipid membrane by a variety of biophysical techniques (including high-speed atomic force microscopy), and stabilize hIAPP oligomeric intermediates using lipid-nanodisc technology and solve the high-resolution structure of oligomers by a combination of solid-state and solution NMR techniques. These high-resolution structures will aid in the development of drugs to stop beta-cell death.