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.
Parts of work
1. Dylan Valli, Saik Ann Ooi, Giorgio Scattolini, Himanshu Chaudhary, Alesia A. Tietze, Michał Maj. Improving cryo-EM grids for amyloid fibrils using interface-active solutions and spectator proteins. Manuscript, under revision in Biophysical Journal. 2. Saik Ann Ooi, Dylan Valli, Mikołaj Kuska, Himanshu Chaudhary, Weixiao Yuan Wahlgren, Alesia A. Tietze, Sebastian Westenhoff, Michał Maj. High-resolution Cryo-EM analysis of the fibrils formed by the human Islet Amyloid Polypeptide containing site-specific proline mutations reveals diverse polymorphism and offers new insights for structure-based drug development. Manuscript. 3. Dylan Valli, Saik Ann Ooi, Himanshu Chaudhary, Alesia A. Tietze, Michał Maj. Solution composition regulates the inhibitory effect of rat IAPP on the human IAPP fibril formation. Manuscript.
Degree
Doctor of Philosophy
University
University of Gothenburg. Faculty of Science.
Institution
Department of Chemistry and Molecular Biology ; Institutionen för kemi och molekylärbiologi
Disputation
Torsdagen den 15 feb 2024, kl. 9.00, Korallrevet, Medicinaregatan 7B
Date of defence
2024-02-15
saik.ann.ooi@gu.se
Date
2024-01-25Author
Saik Ann, Ooi
Publication type
Doctoral thesis
ISBN
978-91-8069-595-4 (print)
978-91-8069-596-1 (PDF)
Language
eng