Programme 5: Structural and biochemical features that differentiate prion protein amyloid from prion infectivity

We are trying different ways of detecting misfolded forms of the prion protein (PrP) that are associated with infection and disease. One approach is to understand and reproduce the way the prion replicates in the human body during disease. This will allow us to ‘amplify’ the very small amounts of amyloid and prion in patient samples into large amounts that can be easily detected using conventional laboratory tests. In addition the ability to replicate prions in vitro using simplified systems and synthetic materials may allow detailed structural characterisation of infectious isoforms using techniques such as nuclear magnetic resonance (NMR) spectroscopy.

Using low resolution methods such as protease fragmentation, chaotrope stability, tryptophan fluorescence and Stern-Volmer quenching the aim will be to determine if different prion strains imprint structural constraints onto recombinant PrP fibrils.

Rotation project
The initiation of PrP amyloid formation from recombinant PrP can occur spontaneously or be seeded by the addition of prion-infected material in ‘amyloid-seeding-assays’ (ASA)1, although this does not result in the formation of infectious prions. The limited information currently available clearly indicates a different fibril conformation is propagated following the seeding of recombinant PrP with infectious material as opposed to that which is generated spontaneously. This project will involve biochemical and biophysical characterisation of the fibrils formed from recombinant PrP both by spontaneous fibrillisation and by seeding with different prion strains. The ability to easily distinguish between fibrils formed spontaneously or those resulting from the presence of seed material will be of considerable benefit in enhancing the specificity of amyloid seeding assays in diagnostic applications, and will facilitate the study of prion strains at the molecular level.

PhD project
Prions, the causative agents of diseases such as Creutzfeldt-Jakob disease can also be generated in vitro using the protein misfolding cyclic amplification (PMCA) reaction1, which exploits the ability of prions to template the conversion of normal PrP (PrPC), present in brain and other tissue homogenates leading to amplification of the seed prion. To date, there remains controversy surrounding the additional requirements for the generation of authentic prions by PMCA; rather than non-infectious amyloid by ASA and the most reliable substrate for PMCA remains whole brain homogenate. The three month project will be broadened to include redaction of PMCA. The aim will be to investigate by systematic fractionation, component depletion and supplementation, the minimal requirements for generation of infectious prions in vitro. The major techniques employed will be biochemical fractionation, enzymatic treatment of substrates, PMCA and analysis of products by western blotting and enzyme-linked immunosorbent assay (ELISA) and an automated cell assay for prion infectivity (ASCA). In conjunction with ASA, the simplest system that can encode strain specific properties will be used to generate material (potentially isotopically labelled) for detailed structural studies by collaborators using; electron microscopy (EM), atomic force microscopy (AFM), NMR and optical spectroscopy in addition to the methods already learnt and applied in the 3 month project.
The demonstration that strain properties may be imprinted onto recombinant PrP and detected has the potential for the differentiation of sporadic, iatrogenic and variant CJD in future assays utilising seeded fibrillisation and the study of prion strains at the molecular level.

1. Saborio GP, Permanne B, Soto C. Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature 2001; 411:810-813.