The aggregation of misfolded proteins into amyloid fibrils is implicated in the pathogenesis of several human degenerative diseases, including Alzheimer’s, Parkinson’s and Type II diabetes. Links between the deposition of amyloid fibrils and the progression of these diseases are poorly understood, with much of the current research focused on monomer misfolding and subsequent assembly of oligomers and mature fibrils. This project examines the formation of human apolipoprotein (apo) C-II amyloid fibrils, with a focus on the stability and reversibility of amyloid fibril assembly. / The initial stages of the project were to develop a model for apoC-II amyloid fibril formation. This was achieved by analysis of the concentration dependent kinetics of apoC-II amyloid fibril formation, and correlation of these data with the final size distribution of the fibrils, determined by sedimentation velocity experiments. On the basis of these studies, a new reversible model for apoC-II amyloid fibril formation is proposed that includes fibril breaking and re-joining as integral parts of the assembly mechanism. The model was tested by rigorous experimentation, with antibody-labelling transmission electron microscopy providing direct evidence for spontaneous fibril breaking and re-joining. / The development of this model for apoC-II fibril assembly provided the foundation for experiments to investigate factors that promote, inhibit or reverse amyloid fibril formation. Factors that were considered include a molecular chaperone protein, αB-crystallin, and a chemical modification, methionine oxidation. Investigations on the effect of αB-crystallin revealed that the inhibition of apoC-II fibril formation occurs by two distinct mechanisms: transient interaction with monomer preventing oligomerisation, and binding to mature fibrils, which inhibits fibril elongation. Studies on the effect of methionine oxidation on apoC-II fibril formation showed that both the assembly and stability of the fibrils was affected by this modification. ApoC-II contains two methionine residues (Met-9 and Met-60), and upon oxidation of these residues fibril formation was inhibited. In addition, the treatment of pre-formed fibrils with hydrogen peroxide caused dissociation of the fibrils via the oxidation of Met-60, located with the fibril core structural region. The final chapter details the development of antibodies that specifically recognise the conformation of apoC-II amyloid fibrils, which provide the foundation for future studies to examine the role that apoC-II amyloid fibrils play in disease. / Overall, this thesis reveals the dynamic and reversible nature of amyloid fibril formation. New insight is also obtained of the general stability of amyloid fibrils and the processes that may regulate their formation, persistence and disease pathogenesis in vivo.
| Identifer | oai:union.ndltd.org:ADTP/245695 |
| Date | January 2009 |
| Creators | Binger, Katrina Jean |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
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