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Title: Cystatin C and Alzheimer's disease
Author: Williams, Abigail J.
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2014
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Aggregation of amyloid-β in Alzheimer's disease (AD) is modulated in the presence of other amyloidogenic proteins including human cystatin C (hCC), which directly protects neuronal cells from Aβ-induced toxicity and inhibits fibril formation. Determination of the relevant conformations of the interacting Aβ and hCC is a key step to uncovering the molecular mechanism of hCC's activity in AD. A system for the production of recombinant Aβ1-40 has been established and is described here. It is also shown that hCC readily produces stable oligomeric species upon incubation in aggregating conditions, a phenomenon that has not been observed for other members of the cystatin family. Novel structural differences between amyloid fibrils produced by hCC and cystatin B have also been identified using limited proteolysis, indicating that hCC does not retain a monomer-like fold within the fibril and that the N-terminal is disordered and not part of the fibril core. The work presented here shows that hCC inhibits fibril production by Aβ in a dose-dependent manner, instead promoting the production of amorphous aggregates and small assemblies, with 2:1 molar ratios of hCC to Aβ being required for complete inhibition. It is unclear if the assemblies observed are toxic protofibrils or an alternative non-toxic species. A comparison of the inhibitory activity of the monomeric and dimeric forms of hCC was carried out, and indicated that the active region could be the hydrophobic loop involved in protease inhibition. Characterisation of binding by NMR HSQC experiments revealed that no observable complex was being formed between monomeric Aβ and folded monomeric hCC. Taken together these results suggest that hCC is selectively binding to an oligomeric species of Aβ and trapping the peptide in a non-toxic state.
Supervisor: Staniforth, Rosemary Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available