Structure, interactions and applications of functional amyloids

Abstract

Amyloids are β-sheet rich, fibrillar, protein assemblies that have been traditionally associated with neurodegenerative conditions such as Alzheimer’s and Parkinson’s diseases. However, these protein structures have also been identified in nature, where they serve functions ranging from surface coatings on fungal aerial structures, to signalling complexes in host immunity responses. These assemblies, now known as functional amyloids, are increasingly being recognised as the predominant form of amyloid in nature.

One type of human functional amyloid mediates a form of programmed cell death, known as necroptosis, which functions under conditions where other cell death mechanisms are inhibited or impaired - as is the case during certain viral infections. In response to this evolutionary pressure, Herpesviruses have adapted mechanisms to inhibit necroptosis activity. This was first identified in the murine cytomegalovirus which encodes a protein, M45, which can engage in interactions with the host adapter proteins to disrupt cell death signalling. The work presented in this thesis provides important structural insights into the mechanisms underlying the assembly of this viral amyloid, characterised through solution nuclear magnetic resonance (NMR), and the final M45 amyloid fold, revealed by electron cryo-microscopy (cryo-EM). The structure of M45 amyloid fibrils aids our understanding of the molecular basis for viral inhibition of necroptosis. Additionally, functional amyloids produced by filamentous fungi, known as hydrophobin rodlets, have been suggested as prime candidates for biomaterial applications in industry and medicine. The work presented in this thesis provides the first step in assessment of the biocompatibility of hydrophobin monolayers and establishes conditions for the preparation of hydrophobin samples for structural characterisation by cryo-EM. Together, these methods will facilitate the further development and safe application of hydrophobins.