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Cystatin C and Alzheimer's Disease

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Cystatin C and Alzheimer's Disease Cystatin C and Alzheimer’s Disease Abigail J. Williams A thesis submitted for the degree of Doctor of Philosophy September 2014 Department of Molecular Biology and Biotechnology University of Sheffield Abstract 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. Acknowledgements First and foremost I must thank my supervisor Rosie Staniforth who has been wonderful, both for offering me this PhD in the first place and for all her guidance, support and encouragement throughout the last four years. I would also like to thank Jon Waltho, Mike Williamson and Jeremy Craven for all their help and guidance, as well as my advisors Neil Hunter and Stuart Wilson. Andrea Hounslow has been amazing, assisting me with the NMR experiments and analysis, and also thank you to Simon Thorpe for help with the MS. Thanks to Svet Tzokov for all of his help with EM and his constant reassurance that I hadn’t broken the microscope. Thank you also to the cystatin group, particularly Pete, Amy and Sirwan. I’m sorry for filling the office with ducks and owls and lobsters. Pete has been simply awesome, full of knowledge and ideas and always around for a chat. Amy and Sirwan have made D07 such an enjoyable place to work for the last two years, with lots of fun and laughter (and celebrity gossip!). Much of my research has been based on the findings of Adham and Emma, for which I am very grateful. The NMR group have been great; it would be impossible to name everybody (and I would undoubtedly forget someone), but thank you all so much for the friendships, discussions and advice. Cake Friday definitely brightens up the week, and there have been some pretty impressive concoctions (forever immortalised in the brilliant NMR cookbook). I have met many amazing people since moving to Sheffield, but special thanks must go to Wednesday Club, to Sophie, Simon, Katie, Sarah, Suze and Lucy (and Larry of course!). Thank you for keeping me sane, for our many hours spent in the Uni Arms, and the greatest farm trip ever. I’m so glad I met you all, and I definitely wouldn’t have made it through without you. Also thank you to George for his words of encouragement and sense of perspective over the last few weeks of writing up. Massive thanks to the fabulous Hannah and Simon for many awesome weekends of cocktails and culture, and for reminding me that there is a life outside of science. Thank you to my wonderful family, to Mum, Dad and Elle for all your love and support, it means so much to me and I am truly grateful. Last but not least, thank you to Simon for moving to Sheffield, for putting up with me and for always believing in me. I couldn’t have done it without you. Table of Contents Chapter One: Introduction .................................................................................................... 1 1.1. Amyloid ....................................................................................................................................... 1 1.1.1. The Importance of Protein Folding ............................................................................. 3 1.1.2. Conversion to an Amyloid-forming Competent State ........................................... 6 1.1.3. Mechanism of Fibril Formation .................................................................................... 7 1.1.4. High Molecular Weight Oligomers ........................................................................... 11 1.1.5. Fibril Structure ................................................................................................................. 13 1.1.6. Therapeutics ..................................................................................................................... 23 1.1.7. Natural regulation of amyloid formation in vivo ................................................... 24 1.2. Amyloidogenic Disease ........................................................................................................ 26 1.2.1. Cytotoxicity ...................................................................................................................... 27 1.2.2. Alzheimer’s Disease ...................................................................................................... 29 1.2.3. Cerebral Amyloid Angiopathies ................................................................................. 31 1.2.4. Amyloid-β ......................................................................................................................... 32 1.3. Cystatins .................................................................................................................................... 34 1.3.1. Cystatin C .......................................................................................................................... 35 1.3.2. Biological role of hCC .................................................................................................. 36 1.3.3. Inhibition Mechanism .................................................................................................... 37 1.3.4. Structure of hCC ............................................................................................................. 37 1.3.5. L68Q Variant of hCC .................................................................................................... 40 1.3.6. Cystatins and Disease .................................................................................................... 40 1.4. Interactions with Aβ ............................................................................................................... 41 1.4.1. Albumin ............................................................................................................................ 42 1.4.2. Amyloidogenic Proteins ............................................................................................... 43 1.5. Overview of Thesis ................................................................................................................ 50 Chapter Two: Materials and Methods ............................................................................. 51 2.1. Buffers and Reagents ............................................................................................................. 51 2.2. DNA Manipulation ................................................................................................................. 51 2.2.1. Expression Vectors ......................................................................................................... 51 2.2.2. Plasmid Extraction ......................................................................................................... 51 2.2.3. Primer Sequences ............................................................................................................ 51 2.2.4. Quantification of DNA .................................................................................................. 52 2.2.5. Competent Cells .............................................................................................................. 52 2.2.5. Site-Directed Mutagenesis ........................................................................................... 53 2.2.6. Transformations .............................................................................................................. 53 2.2.7. DNA Sequencing ............................................................................................................ 53 2.3. Growth Media and Solutions ............................................................................................... 53 2.3.1. Luria-Bertani Media ....................................................................................................... 53 2.3.2. M9 Minimal Media .......................................................................................................
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