Amylin Structure, Aggregation and Pancreatic β Cell Toxicity

In most type 2 diabetes patients, amyloid plaques have been found juxtaposed with membranes of pancreatic β-cells. These plaques are composed of amyloid fibrils of the 37 residue endocrine hormone amylin and cause distinct changes in cell membrane morphology associated with the destruction of β-cells. Research is still ongoing to identify the toxic species involved and the mechanisms by which mature fibrils or oligomers cause cytotoxicity. The projects undertaken were designed to study the molecular structural features of amylin, mechanism of amyloid aggregation and, to develop cytotoxicity inhibitors. We determined the structure of human amylin bound to SDS micelles using NMR. In this membrane mimetic environment amylin adopts an α- helix structure for residues 5-28, with a kink between residues 18 and 22. The last nine C-terminal residues are unfolded. Paramagnetic spin probe data suggest the segment between residues 5 and 17 is within the hydrophobic lipid environment while residues 20-29 remain at the lipid-solvent interface. Experimental techniques such as total internal reflection fluorescence microscopy, fluorescence assays and cytotoxicity assays were undertaken to study fibrillization and the effect of designed cytotoxicity inhibitors. Microscopy experiments demonstrate three types of aggregate growth processes: globular seed formation, unidirectional fibril elongation and fibril thickening. The three aggregation processes are suggestive of secondary nucleation mechanisms. Lastly, three amylin peptide analogs were designed to inhibit fibrillization via electrostatic charge repulsion. The three analogs fibrillize poorly on their own and inhibit fibril elongation of WT-amylin at physiological salt concentrations. The most effective inhibitor Arg-1, inhibits amylin fibrillization and cytotoxicity with a potency comparable to other types of fibrillization inhibitors described in the literature