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Mechanisms of amyloid fibril-mediated toxicity

Tipping, Kevin William (2014) Mechanisms of amyloid fibril-mediated toxicity. PhD thesis, University of Leeds.

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Abstract

Amyloid diseases are a group of protein misfolding disorders characterised by the formation of highly ordered filamentous assemblies known as amyloid fibrils. Soluble aggregation intermediates whose formation precedes that of mature fibrils are commonly considered the major source of toxicity in amyloid diseases. Oligomer toxicity has often led to mature amyloid fibrils being referred to as inert end products of aggregation. Recent evidence, however, has shown that fibrils themselves are capable of facilitating toxicity by a variety of mechanisms. One such protein, β2-microglobulin (β2m), has been shown to form amyloid fibrils that bind to liposome membranes causing deformations and disruption in a pH dependent manner. In this thesis, the in vitro mechanisms of β2m fibril-induced membrane disruption are explored to elucidate why reducing the pH from 7.4 to 6.4 leads to an enhancement in fibril membrane disruption. A combination of chemical kinetics, NMR and liposome dye-release assays show that at both pH values, membrane disruption is mediated through the shedding of soluble species induced upon diluting fibrils formed at low pH into either buffer. Fibril depolymerisation at pH 6.4 leads to the persistence of membrane-active non-native species, whereas depolymerisation at pH 7.4 is driven rapidly to membrane-inactive native monomer. Further analysis reveals that these non-native species are structurally disordered, spherical particles that display significant surface-exposed hydrophobicity. The observed pH-dependent formation of oligomers shed during depolymerisation is likely to play an important role in mediating cellular effects upon incubation with fibrils. Consistent with this, chemical cross-linking of fibrils and co-incubating fibrils with Hsp70-1A prevents the depolymerisation of β2m fibrils at both pH 7.4 and 6.4 in vitro and reduces metabolic defects associated with β2m fibril depolymerisation. The results suggest that kinetically stabilising fibrils to prevent molecular shedding could be a means of helping to remedy amyloid-associated toxicity.

Item Type: Thesis (PhD)
Academic Units: The University of Leeds > University of Leeds Research Centres and Institutes > Astbury Centre for Structural Molecular Biology (Leeds)
The University of Leeds > Faculty of Biological Sciences (Leeds) > Institute for Molecular and Cellular Biology (Leeds)
Identification Number/EthosID: uk.bl.ethos.638889
Depositing User: Mr Kevin W Tipping
Date Deposited: 26 Feb 2015 11:52
Last Modified: 06 Oct 2016 14:42
URI: http://etheses.whiterose.ac.uk/id/eprint/7689

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