TY - JOUR
T1 - Polyanion induced fibril growth enables the development of a reproducible assay in solution for the screening of fibril interfering compounds, and the investigation of the prion nucleation site
AU - Boshuizen, Ronald S.
AU - Morbin, Michela
AU - Mazzoleni, Giulia
AU - Tagliavini, Fabrizio
AU - Meloen, Rob H.
AU - Langedijk, J. P M
PY - 2007
Y1 - 2007
N2 - 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.
AB - 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.
KW - Amyloid
KW - BSE
KW - CJD
KW - GAG
KW - Prion
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U2 - 10.1080/13506120701464628
DO - 10.1080/13506120701464628
M3 - Article
C2 - 17701468
AN - SCOPUS:34547855876
VL - 14
SP - 205
EP - 219
JO - Amyloid : the international journal of experimental and clinical investigation : the official journal of the International Society of Amyloidosis
JF - Amyloid : the international journal of experimental and clinical investigation : the official journal of the International Society of Amyloidosis
SN - 1350-6129
IS - 3
ER -