Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.759576
Title: Factors determining the cytotoxic nature of pathogenic amyloid proteins
Author: Vadukul, Devkee M.
ISNI:       0000 0004 7431 6104
Awarding Body: University of Sussex
Current Institution: University of Sussex
Date of Award: 2018
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Abstract:
Amyloid proteins feature in neurodegenerative diseases and functionally throughout many organisms. Furthermore, due to their structural properties, amyloid proteins have been developed as materials in biotechnology. This raises the question of what makes disease-related amyloid proteins toxic. β-amyloid 1-42 (Aβ42) is a self-assembling protein that goes through many structural changes before forming the extracellular plaques characteristic of Alzheimer's disease. We have studied the conformational changes of the Aβ42 peptide over time by combining a range of biophysical approaches including circular dichroism, and Thioflavin T fluorescence with Transmission Electron Microscopy. Aβ42 assembly is compared to a novel, rationally-designed, assembly-resistant Aβ42 peptide variant (vAβ42), as well as the two main Aβ42 controls, Aβ reversed (Aβ42-1)and Aβ scrambled (AβS). The vAβ42 differs in sequence by only two amino acids, however, does not self-assemble or form β-sheet structures, unlike Aβ42-1and AβS which both display a high propensity to form amyloid. All three variants of Aβ42 were non-toxic in primary hippocampal cultures, highlighting the importance of primary sequence in determining the toxic nature of an amyloid protein. Furthermore, the structure andtoxicity of the naturally functional amyloid protein, GNNQQNY,and the designedfunctional amyloid peptide, FEFKFEFKK (F9), have also been characterised. These show immediate assembly into mature fibrils, do not form intermediary species and are not cytotoxic. Together, this data suggests the ability to form oligomers and the time spent in this conformation is a requirement of amyloid toxicity. To further investigate the link between size, conformation and toxicity, we compared the cytotoxicity and internalisation of oligomeric, fibrillar and sonicated fibres of Aβ42 in primary hippocampal neurons using immunolabelling and live cell imaging. As expected, the oligomeric Aβ42 was highly neurotoxic in hippocampal cultures, however fibrillar and sonicated fibrils did not have the same effect. Finally, the necessity of internalisation in mediating cytotoxicity was investigated and showed a certain threshold of intracellular accumulation must be met to induce cytotoxicity. Overall, our data suggests primary sequence, the resultant self-assembly and intermediary species formed, and intracellular accumulation are vital in determining the pathogenic properties of amyloid proteins.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.759576  DOI: Not available
Keywords: QD0431 Proteins, peptides, amino acids, etc.
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