Structural Studies of Cystatin B Amyloid Fibre and Oligomer

Amyloid fibres are characteristic of over 25 degenerative human diseases including Alzheimer’s and Parkinson’s disease. Amyloid fibres are insoluble, highly stable, ordered cross-β sheet structures, which form as a result of conformational change and aggregation in a range of unrelated soluble proteins and peptides. High molecular weight oligomers, potentially on-pathway to amyloid formation, have been posited as the toxicity agent in amyloid-associated disease. Heterogeneity of amyloid fibres and oligomers precludes the use of standard structural biology techniques. The work presented utilised recombinant human cystatin B as a model system for structural analysis of amyloid fibres and oligomers, particularly for the structurally homologous human cystatin C which directly causes a form of amyloid angiopathy. Limited proteolysis of cystatin B amyloid fibre shows that the core structure consists maximally of residues 27-80 out of a total 98. Electron microscopy techniques, including mass per unit length measurements, reveal an average fibre width of 8.6 nm and formation of four fibre classes, composed of 4, 8 and 16 molecules per 4 -strand rise. These data are incompatible with the previous native-like model, therefore a new working fibre model is proposed where native β-strands 2 and 3 are extended into a single strand with adjacent β-strands 4 and 5 forming the other half of a β-sheet arc, which is stacked in parallel, perpendicular to the fibre axis. Stable proteinase-K resistant G4R mutant cystatin B oligomers were generated and preliminary characterisation presented, with identification of five structural classes ranging in diameter from 8-88 nm. Furthermore, investigation of a potential amyloid therapeutic in Salvia plant extracts was carried out with dye binding kinetic assays and electron microscopy. The work herein primarily constrains a new non-native β-sheet cystatin B amyloid fibre model and has initiated new avenues of research into cystatin oligomer structure and novel therapeutic agents.