Biophysical Characterization of Polymorphic Amyloid and Lipid Aggregation Associated with Type 2 Diabetes

Abstract

Type 2 Diabetes (T2D) is an existing and emerging threat to global health. While treatments exist to manage symptoms, no cure has been developed due to uncertainty in the molecular basis of T2D. It is known that a crucial component of T2D is amyloid aggregate formation by the human islet amyloid polypeptide (hIAPP) and that progression of T2D is likely mediated by toxic intermediate aggregates of hIAPP. Moreover, interactions between hIAPP and lipid membranes are proposed to facilitate toxicity. Motivated by this, work to characterize the nature of physiological hIAPP aggregation inhibitors, oligomeric intermediates, and lipid interactions is described here. Collectively, the results emphasize the heterogeneity and polymorphism of hIAPP aggregates and suggest research directions for identifying the disease-relevant hIAPP species and mechanisms of toxicity, which will guide development of drugs that target the root cause of T2D. Physiological inhibitors of hIAPP aggregation might provide a model for drug design against amyloid formation associated with T2D, so I first described the combined ability of low pH, zinc, and insulin to inhibit hIAPP fibrillation. Insulin dose-dependently slowed hIAPP aggregation near neutral pH but had less effect on the aggregation kinetics at acidic pH. I determined that insulin altered hIAPP aggregation in two manners. Insulin diverted the aggregation pathway to large nonfibrillar aggregates with ThT-positive molecular structure, rather than to amyloid fibrils, and soluble insulin suppressed hIAPP dimer formation, which is an important early aggregation event. Further, we observed that zinc significantly modulated the inhibition of hIAPP aggregation by insulin. I hypothesized that this effect arose from controlling the oligomeric state of insulin and showed that hIAPP interacted more strongly with monomeric than oligomeric insulin. Next, structural studies of oligomeric hIAPP have been hampered by heterogeneity and poor stability in standard aqueous conditions. A novel methodology for producing stable, on-pathway oligomers of hIAPP was developed using the mechanical forces associated with magic angle spinning (MAS). The species were a heterogeneous mixture of globular and short rod-like species with significant β-sheet content and the capability of seeding hIAPP fibrillation. MAS NMR was used todemonstrate that the nature of the species was sensitive to sample conditions including peptide concentration, ionic strength, and buffer. The methodology should be suitable for studies of other aggregating systems. Lastly, hIAPP interacts strongly with anionic phospholipids which are present in the inner leaflet of cell membranes. But hIAPP interactions with gangliosides, the primary anionic lipid in outer leaflets, have not been extensively studied, so a suite of biophysical tools was used to investigate the role of three gangliosides, GM1, GM3, and GD3, in hIAPP aggregation. The gangliosides both promoted and inhibited hIAPP aggregation, depending on the ratio between lipid and peptide. GD3 most effectively promoted aggregation, and hIAPP adopted more β-sheet structure in the presence of GD3 than GM1 or GM3. Moreover, the gangliosides induced formation of polymorphic hIAPP aggregates, and hIAPP altered the aggregation behavior of the lipids, suggesting possible mechanisms for hIAPP-associated toxicity mediated by gangliosides. For further investigations of membrane interactions of hIAPP and other membrane-binding proteins, a novel lipid nanodisc system with a saponin belt was developed and its suitability for use in NMR-based protein structural studies was demonstrated. Additionally, magnetically aligned nanodiscs were demonstrated to enable the measurement of 17O residual quadrupolar couplings for investigations of molecular structure by NMR.