Many neurodegenerative disorders are characterised by protein misfolding and subsequent amyloid fibrillisation and deposition. Amyloid-beta peptide (Aβ) was found to be the main constituent of the extracellular amyloid plaques of Alzheimer’s disease (AD) in the 1980s. What triggers amyloid formation or inhibits it are of particular interest. This thesis focuses on the effect of endogenous proteins and molecules on amyloid fibrillisation. In Chapter 3, I show that at physiological micromolar levels found in the cerebrospinal fluid, human serum albumin inhibits the rate of Aβ fibrillisation. Indeed in vitro the amount of amyloid fibres generated directly correlates to the proportion of Aβ not competitively bound to albumin. Albumin binds cholesterol and fatty acids in vivo, both of which have been linked with an increased risk of developing AD. In Chapter 4, I show Aβ competes with these molecules for albumin binding, so disrupting albumin’s ability to inhibit Aβ fibrillisation. My observations suggest a significant role for albumin regulating Aβ fibril growth. Albumin also binds Cu2+ in vivo with a tight picomolar affinity. Animal models suggest disrupted Cu2+ homeostasis potentiate AD phenotype. In Chapter 5, I show that regardless of Aβ alloform or fibrillisation stage, the affinity for Cu2+ is in the ~20 picomolar range but weaker than albumin. Circular Dichroism spectroscopy in the visible region (Vis-CD) is a powerful technique to study metal-protein interactions. In Chapter 6, I develop a set of empirical rules that relates the appearance of particular Vis-CD spectral features to the conformation of the complex. These rules are used to gain insight into Cu2+-protein complexes in Prion disease and Parkinson’s disease. I show the N-terminal amino group of cellular prion protein (PrPC) has a tighter affinity for Cu2+ than the individual octa-repeat binding sites present within PrPC and show for the first time that Cu2+ loads on to the N-terminal amino group before the single octa-repeat binding sites. I determine the affinity of Cu2+ for model N-terminal peptides of alpha-synuclein of Parkinson’s disease and show that side- chain coordination stabilises the complex and increases the affinity for copper compared to main-chain coordination only. This thesis highlights the importance of overlapping interactions with endogenous proteins and molecules in the development of neurodegenerative disease. Indeed, the amyloid protein binding partners studied here are all co-localised at the synapse thus future in vitro studies of neurodegenerative disease should consider the complex nature of interactions possible in situ.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:667466 |
| Date | January 2015 |
| Creators | Stanyon, Helen Felicity |
| Publisher | Queen Mary, University of London |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://qmro.qmul.ac.uk/xmlui/handle/123456789/9119 |
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