Effect of enzymatic deimination on the conformation of recombinant prion protein.

Deimination is the post-translational conversion of arginine residues to citrulline. It has been implicated as a causative factor in autoimmune diseases such as multiple sclerosis and rheumatoid arthritis and more recently, as a marker of neurodegeneration. We have investigated the effect of the post-translational modification of arginine residues on the structure of recombinant ovine prion protein. Deiminated prion protein exhibited biophysical properties characteristic of the scrapie-associated conformer of prion protein viz. an increased beta-sheet secondary structure, congophilic structures indicative of amyloid and proteinase K resistance which could be templated onto normal unmodified prion protein. In the light of these findings, a potential role of post-translational modifications to prion protein in disease initiation or propagation is discussed.

Effects of post-translational modifications on prion protein aggregation and the propagation of scrapie-like characteristics in vitro.

Prion diseases, or transmissible spongiform encephalopathies (TSEs) are typically characterised by CNS accumulation of PrP(Sc), an aberrant conformer of a normal cellular protein PrP(C). It is thought PrP(Sc) is itself infectious and the causative agent of such diseases. To date, no chemical modifications of PrP(Sc), or a sub-population thereof, have been reported. In this study we have investigated whether chemical modification of amino acids within PrP might cause this protein to exhibit aberrant properties and whether these properties can be propagated onto unmodified prion protein. Of particular interest were post-translational modifications resulting from physiological conditions shown to be associated with TSE disease. Here we report that in vitro exposure of recombinant PrP to conditions that imitate the end effects of oxidative/nitrative stress in TSE-infected mouse brains cause the protein to adopt many of the physical characteristics of PrP(Sc). Most interestingly, these properties could be propagated onto unmodified PrP protein when the modified protein was used as a template. These data suggest that post-translational modifications of PrP might contribute to the initiation and/or propagation of prion protein-associated plaques in vivo during prion disease, thereby high-lighting novel biochemical pathways as possible therapeutic targets for these conditions.

The design, synthesis and evaluation of affinity ligands for prion proteins.

Bifunctional affinity ligands based on a triazine scaffold were rationally designed to target prion protein and shown to bind recombinant prion protein with high affinity and selectivity. The ligands were capable of discriminating between prion protein glycoforms and monomeric and dimeric forms of the prion protein. The ligands also discriminate between conformational differences in the prion protein, resulting from point mutations in the prion protein gene. These results suggest that derived compounds could be used selectively to detect the disease-associated form of the prion protein, and as such, could provide diagnostic or therapeutic tools for prion diseases.

Metabolite-sensitive holographic biosensors.

A new type of biosensor that combines the inexpensiveness and mass-produceability of reflection holograms with the selectivity and specificity of enzymes is described. pH-sensitive holographic sensors were fabricated from ionizable monomers incorporated into thin, polymeric, hydrogel films which were transformed into volume holograms using a diffusion method coupled with holographic recording, using a frequency-doubled Nd:YAG laser (532 nm). These holograms were used as transducer systems to monitor the pH changes associated with specific enzymatic reactions to construct prototype urea- and penicillin-sensitive biosensors. The diffraction wavelength (color) of the holographic biosensors was used to characterize their shrinkage and swelling behavior as a function of analyte concentration. The potential of these sensors for the measurement of the clinically and industrially important metabolites urea and penicillin G is demonstrated.