The Effect of Site-Specific Mutations on hIAPP Fibril Polymorphism: A Cryo-EM Study Towards Potential Inhibitors

Abstract

The development of amyloid deposits in tissues, as a consequence of the self-assembly of misfolded proteins, is a critical pathological feature in a range of diseases, including neurodegenerative disorders and metabolic conditions such as diabetes. A comprehensive understanding of the mechanisms that drive this protein aggregation is essential for developing strategies capable of intervening in these disease processes and aiding in the creation of novel therapeutic approaches.

This thesis focuses on the aggregation of the human islet amyloid polypeptide (hIAPP), which plays a significant role in the progression of type 2 diabetes through the degeneration of the insulin-producing β-cells. The molecular basis of β-cell death associated with hIAPP aggregation remains poorly understood. One significant challenge is the rapid and intricate nature of amyloid fibril formation by hIAPP in vitro. Additionally, the application of traditional structural biology techniques to analyze this process proves difficult due to the structural complexity of the amyloid aggregates.

The environment of hIAPP has a strong effect on the aggregation, which has been investigated through papers I and III using ThT kinetics study and cryo-EM. The results indicated that buffers played a huge part in influencing the fibrils kinetics, distribution of fibrils on cryo-EM grids, and especially the fibril polymorphism.

In paper II, site-specific mutation has been carried out to evaluate the effect of proline on fibril formation. With the same proline mutation performed on three different sites, the results showed drastic changes in the kinetics. The molecular investigation using cryo-EM has shown that each mutant formed distinct polymorphic structures that were different from those published for wild-type IAPP. The results deliver insights into how the aggregation of hIAPP can be influenced through mutations.

Overall, this thesis contributes to our understanding of hIAPP aggregation in vitro, influenced by both mutations and co-aggregation. The insights gained from these studies are instrumental in advancing the development of therapeutic strategies aimed at disrupting or inhibiting amyloid aggregation pathways in a range of diseases, including type 2 diabetes.