BBSRC EASTBIO DTP - Optimising a prion diagnostic assay

Prion diseases are neurodegenerative disorders of humans and animals that involve the misfolding and subsequent deposition of prion protein (PrP) in the brain, CNS and peripheral lymphoid tissues of infected individuals. The BSE epidemic demonstrated the devastating consequences that TSE diseases can have to the agricultural industry and the resulting disease (vCJD) in the UK human populace remains a major cause for concern. Additionally, novel forms of prion disease in animals threaten food security – prominent examples include chronic wasting disease in deer, which is currently in epidemic proportions in the US, atypical forms of BSE and scrapie and other prionopathies. If we are effectively to combat existing and future prion epidemics in humans and/or animals, then access to cutting edge diagnostic tools is an absolute prerequisite. Over recent years there has been much research aimed at developing non-invasive, pre-mortem and, ideally, pre-clinical diagnostic assays and one of the most promising technologies is termed the quaking-induced conversion (QuIC) assay. The QuIC assay uses recombinant prion protein as a substrate to which portions of a test tissue are added and the mixture is shaken intermittently. If the tissue under test contains “prion seeds” then this will cause the recombinant PrP to fibrillise in a manner that is detectable by an increase in the fluorescence of an amyloid-binding dye.

At present there is inherent variability in the QuIC assay, which currently restricts how useful it might be in future medical or veterinary clinical practice. The largest source of variability appears to relate to inconsistencies in the quality/purity/structure/sequence of the recombinant PrP substrate and this proposal will attempt to determine what those are. Our data are likely to help optimise a non-invasive diagnostic assay that is badly needed by clinicians and veterinarians. The data are also likely to shed significant light on the fundamental mechanisms of prion protein misfolding – the QuIC assay appears to work specifically for prion seeds and understanding those factors that control substrate/species specificity will go a long way to defining whether similar processes go on in vivo.

The PI is a protein biochemist with many years’ experience of investigating prion protein structure. The Co-I has successfully developed a working protocol for QuIC analysis of brain and CSF from patients with suspected CJD. Crucially, we have recently demonstrated that recPrP produced in the PI’s lab is a good substrate for the QuIC assay of the Co-I, which will allow us to vary conditions used in the production of the recPrP whilst monitoring for the ability of this protein to convert in the QuIC assay. Briefly, we will investigate the primary, secondary, tertiary and quaternary structures and molecular compositions of a range of recPrP preparations by a sophisticated and comprehensive combination of biophysical and biochemical tools, including both top-down and bottom up mass spectrometric proteomics experiments, metabolomics, analytical chromatographies and molecular spectroscopies. At the end of our project we will amalgamate all out knowledge into a final recPrP expression protocol that results in an optimised substrate. This project will provide training in several areas of bioscience in which skills are currently lacking, including proteomics and neurodegenerative diseases. The student will also develop skills in protein purification and characterisation, also areas where there is a skills shortage in the UK bioscience community.