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Abstract
The misfolded conformer of the prion protein (PrP) that aggregates into fibrils is believed to be the pathogenic agent in transmissible spongiform encephalopathies. In order to find fibril interfering compounds a screening assay in solution would be the preferred format to approximate more closely to physical conditions and enable the performance of kinetic studies. However, such an assay is hampered by the high irreproducibility because of the stochastic nature of the fibril formation process. According to published fibril models, the fibrillar core may be composed of stacked parallel β-strands. In these models positive charge repulsion may reduce the chance of favorable stacking and cause the irreproducibility in the fibril formation. This study shows that the charge compensation by polyanions induced a very strong fibril growth which made it possible to develop a highly reproducible fibril interference assay. The stimulating effect of the polyanions depended on the presence of the basic residues Lys106, Lys110 and His111. The assay was validated by comparison of the 50% fibril inhibition levels of peptide huPrP106-126 by six tetracyclic compounds. With this new assay, the fibrillogenic core (GAAAAGAVVG) of peptide huPrP106-126 was determined and for the first time it was possible to test the inhibition potentials of peptide analogues. Also it was found that variants of peptide huPrP106-126 with proline substitutions at positions Ala115, Ala120, or Val122 inhibited the fibril formation of huPrP106-126.
| Abbreviations | ||
| Aβ | = | amyloid β protein |
| BSE | = | bovine spongiform encephalopathy |
| CJD | = | Creutzfeldt-Jacob disease |
| Fmoc | = | fluorenylmethoxycarbonyl |
| GAG | = | glycosaminoglycan |
| huPrP | = | human prion protein |
| moPrP | = | mouse prion protein |
| PrPc | = | cellular isoform of PrP |
| PrPSc | = | Scrapie isoform of PrP |
| TFA | = | trifluoroacetic acid |
| ThS | = | thioflavin S |
| TSE | = | transmissible spongiform encephalopathy |
| Abbreviations | ||
| Aβ | = | amyloid β protein |
| BSE | = | bovine spongiform encephalopathy |
| CJD | = | Creutzfeldt-Jacob disease |
| Fmoc | = | fluorenylmethoxycarbonyl |
| GAG | = | glycosaminoglycan |
| huPrP | = | human prion protein |
| moPrP | = | mouse prion protein |
| PrPc | = | cellular isoform of PrP |
| PrPSc | = | Scrapie isoform of PrP |
| TFA | = | trifluoroacetic acid |
| ThS | = | thioflavin S |
| TSE | = | transmissible spongiform encephalopathy |