Jack, Kezia; (2024) What makes a prion a prion? Investigating whether template structure or substrate identity determines the formation of an infectious prion or a non-infectious amyloid during in vitro aggregation. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

A prion is a protein-only infectious disease agent, and the relationship between structure and function is central to our understanding of prion diseases. The molecular basis for prion strains is contained within their structure, as there is no variation in primary sequence. For strains to be infectious, templated replication takes place, demonstrating that the structure of the prion is heritable. Templated replication of infectious prions and non-infectious amyloid from PrP could be determined by either the template structure or the environment. My thesis aims to explore the interplay between template and en vironment in prion replication, using a native aggregation assay (NAA) to model seeded growth. I have used fibrils formed from either sponta neously aggregated monomer or purified prions (both murine and hu man) as template, and altered the environment by using different sub strates for aggregation: full length (23-231) or truncated (91-231) PrP monomer. To assess the fidelity of the replication, I have developed a spectral fingerprinting method; a plate-based assay that records and analyses excitation-emission spectra of luminescent conjugated olig othiophene (LCO) dyes and is able to qualitatively report on structure. I have shown that the NAA is not able to fully template prion struc tures, although the structures produced are more similar to genuine prions than fibrils produced under denaturing conditions. The assay is able to support the formation of different structures, so the conditions allow for some degree of templated replication. Results suggest that the interplay between template and environment is complex and varies between systems. My results demonstrate that the NAA is a valuable tool in inves tigating strain adaptation and templating. They demonstrate amyloid growth of human substrate seeded with genuine human prions for the first time, and show evidence for the molecular mechanisms of strain propagation. Spectral fingerprinting has potential to be developed into a rapid and sensitive strain diagnostic tool, replacing current more rudi mentary biochemical tests.

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