Townsend, David John and Middleton, David (2016) Molecular level characterisation of apolipoprotein A-I aggregation leading to fibrils comprising of both α-helical and β-sheet structures. PhD thesis, Lancaster University.
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Abstract
Amyloidosis is defined as the misfolding of native proteins into insoluble fibrils that are deposited within tissues and extracellular organs. 30+ structurally and sequentially unrelated proteins have the ability to form amyloid aggregates, all of which contain characteristic features. ApoA-I, the main component in high-density lipoprotein, aggregates and becomes deposited as amyloid, either full-length apoA-I fibrils within atherosclerotic plaques, or N-terminal fragments of mutant apoA-I within organs. The work here aims to further the understanding of conditions that promote the aggregation of apoA-I in vitro, allowing the structural study of aggregated apoA-I at a molecular level. ApoA-I remains soluble at neutral pH, maintaining a predominantly α-helical conformation. Upon acidification to pH 4, apoA-I readily assembles into aggregates that, despite being responsive to the amyloid characteristic ThT dye, do not have the typical amyloid morphology and do not produce XRD diffraction patterns suggestive of β-sheets. The inclusion of heparin, and chemical oxidation of apoA-I methionine residues, in order to mimic physiological conditions, results in an increased ThT response and aggregated material more characteristic of amyloid. Solid-state NMR spectroscopy reveals for the first time that all three aggregation inducing conditions produce aggregates that give rise to cross-peaks corresponding to both α-helical and novel β-sheet structures. This leads to a refinement in the current theory describing apoA-I aggregation. In native apoA-I, the N-terminal 4-helical bundle protects the 3 hot spot regions from self-association into β-sheets. Acidification of apoA-I leads to the destabilisation of this N-terminus, and a conversion of residues 1-90 into β-sheet structures, whilst the C-terminus retains its α-helical structure. EGCG, an inhibitor of Aβ, α-synuclein and huntingtin amyloidosis, is shown here to bind to apoA-I with micro-molar affinity. However, rather than inhibit amyloidosis, EGCG causes a structural rearrangement of the aggregated material, resulting in a reduced α-helical content.