Principal Supervisor: Dr. Paul Wilson, Department of Chemistry
Co-supervisor: Professor Thomas P. Davis, Monash Institute of Pharmaceutical Sciences
PhD project title: Investigating amyloidogenesis and amyloid-protein interactions and their effect on amyloid structure and properties
University of Registration: University of Warwick
Type 2 diabetes mellitus (T2D) affects approx. 10% of the global adult population and is an expanding economic and healthcare burden.1 Though the burden can be mitigated by healthier lifestyles, T2D is predicted to become one of the top 10 leading causes of death by 2030.2 The onset of T2D can be correlated to increased cell death and dysfunction of pancreatic β-cells, located within the islets of Langerhans.3 Human islet amyloid polypeptide (IAPP, Amylin), is a 37-residue peptide co-secreted with insulin by pancreatic β-cells that contributes to glycemic control. However, there is also a strong correlation between to IAPP and β-cell death. The monomeric peptide is highly amyloidogenic, aggregating in a concentration dependent manner to form insoluble amyloids and plaques that have been detected in 90% of T2D patients, implicating erroneous secretion with the development and progression of the disease.4
Amylin fibrillates rapidly in aqueous media, in vitro.5 Consequently, the fibrillation process and precise amyloid structure-property relationships are not well characterised compared to amyloids with slower fibrillation processes, e.g. amyloid-β (Alzheimer’s disease). There is evidence to suggest that both the mature amyloids and soluble oligomeric intermediates contribute to the loss of β-cell viability in vitro.6 However, in the human system, all amyloids, will be exposed to complex mixtures of proteins in the intra- and/or extracellular milieu. This is likely to result in the formation of an amyloid-protein corona, supported by hydrogen-bonding, electrostatic and Van Der Waals interactions between the parent amyloid and proteins, which will affect the structural organisation of the amyloid and contribute the final properties. This hypothesis is justified by the formation of protein coronae on (in)organic nanoparticles in biological media/systems, which continues to be well studied and has been shown to determine their subsequent biological interactions and properties.7
Amylin contains a Cys2-Cys7 disulfide bridge, which is not involved in the fibrillation process. The reactivity of the disulfide bond makes it an ideal target for bioconjugation and a number of strategies exist that enable modification with a range of thiol reactive reagents.8 During this investigation (Fig. 1), such modifications will be used to investigate;
The effect of disulfide bond modification on amylin fibrillation and the resulting amyloid structure to answer the question; can we chemically modulate/tune amyloid structure?
The formation and constitution of the amyloid-protein corona by incubating mature amyloids with fetal bovine serum (FBS), as a model, and human plasma (initial protocols for this have been developed using model systems).
The relationships and trends between the structures of the amyloids formed their physiological properties in vitro (cell viability) and in/ex vivo (biodistribution, etc.).
Figure 1. (top) Preliminary work has shown that amylin (IAPP) spontaneously fibrillates to give amyloids. The TEM images show the structure of mature amylin amyloids which changes upon incubation with proteins α-lactalbumin and lysozyme. The changes in structure can be correlated to changes in the properties (toxicity). (bottom) The proposed work in which amylin will be chemically (star) modified and the impact on fibrillation and the formation of a protein corona will be investigated.
- Mendis, S., Global status report on noncommunicable diseases 2014; World Health Organisation, Geneva, 2014
- Mathers, C. D., Loncar, D., PLoS Med. (2006) 3, 2011
- Leahy, J. L., Arch. Med. Res. (2005) 36, 197
- Zhang, S., et al., Proc. Natl. Acad. Sci. U. S. A. (2013) 110, 2798
- Knowles, T. P. J., et al., Nat. Rev. Mol. Cell Biol. (2014) 15, 384
- Sakagashira, S., et al., Am. J. Pathol. (2000) 157, 2101
- C. D. Walkey, W. C. Chan, Chem. Soc. Rev., 2012, 41, 2780–2799
- T. Weil et al., Chem. Eur. J. 2016, 22, 17112
BBSRC Strategic Research Priority: Bioenergy and Industrial biotechnology, Molecules, Cells and Systems
Techniques that will be undertaken during the project:
Peptide modification and fibrillation will require the use of fluorescence spectroscopy, circular dichroism (CD), quartz-crystal microbalance (QCM) and transition electron microscopy (TEM).
Amyloid-protein corona formation and identification will require the use and development of QCM and ultracentrifugation methods whilst the constituent proteins mixtures will be identified using LC-MS and LC-MALDI-ToF-MS. This will generate large data sets that will need to be handled and analysed statistically.
Physiological properties will be assessed using cell viability assays and established in/ex vivo animal models (MIPS) with the required institutional ethical approval.
Contact: Dr. Paul Wilson, Department of Chemistry