Abstract
Fibrils play an important role in the pathogenesis of amyloidosis; however, the underlying mechanisms of the growth process and the structural details of fibrils are poorly understood. Crucial in the fibril formation of prion proteins is the stacking of PrP monomers. We previously proposed that the structure of the prion protein fibril may be similar as a parallel left-handed beta-helix. The beta-helix is composed of spiraling rungs of parallel beta-strands, and in the PrP model residues 105-143 of each PrP monomer can contribute two beta-helical rungs to the growing fibril. Here we report data to support this model. We show that two cyclized human PrP peptides corresponding to residues 105-124 and 125-143, based on two single rungs of the left-handed beta-helical core of the human PrP(Sc) fibril, show spontaneous cooperative fibril growth in vitro by heterologous stacking. Because the structural model must have predictive value, peptides were designed based on the structure rules of the left-handed beta-helical fold that could stack with prion protein peptides to stimulate or to block fibril growth. The stimulator peptide was designed as an optimal left-handed beta-helical fold that can serve as a template for fibril growth initiation. The inhibiting peptide was designed to bind to the exposed rung but frustrate the propagation of the fibril growth. The single inhibitory peptide hardly shows inhibition, but the combination of the inhibitory with the stimulatory peptide showed complete inhibition of the fibril growth of peptide huPrP-(106-126). Moreover, the unique strategy based on stimulatory and inhibitory peptides seems a powerful new approach to study amyloidogenic fibril structures in general and could prove useful for the development of therapeutics.
Highlights
Transmissible spongiform encephalopathies are neurodegenerative disorders in a wide range of mammalian species, including Creutzfeldt-Jacob disease in man, scrapie in sheep, and bovine spongiform encephalopathy in cattle
We show that two cyclized human PrP peptides corresponding to residues 105–124 and 125–143, based on two single rungs of the lefthanded -helical core of the human PrPSc fibril, show spontaneous cooperative fibril growth in vitro by heterologous stacking
It is still unknown how much of the whole PrPSc molecule is involved in the fibril growth
Summary
PrPc, cellular isoform of PrP; PrPSc, scrapie isoform of PrP; huPrP, human prion protein; Fmoc, fluorenylmethoxycarbonyl; LpxA, UDP-N-acetylglucosamine acyltransferase; AU, arbitrary units; HPLC, high performance liquid chromatography; MS, mass spectroscopy; PBS, phosphate-buffered saline. We suggested that each PrPSc monomer contributes two left-handed -helical rungs to the fibril, comprising residues 105–124 and 125–143 (Fig. 1A). This two-rung structural model was recently confirmed for amyloid fibrils of the HET-s prion by NMR analysis [22]. To investigate whether the suggested rungs 105–123 and 125–143 from human PrP could be complementary [20], we studied the homologous stacking and the heterologous stacking of linear and cyclized prion protein peptides comprising the huPrP-(105–143) region (KTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGS). The findings in this study support models in which the sequential strands in a fibril must somehow spiral up- or downward along the fibril axis, e.g. like the hypothetical left-handed -helical structure of PrPSc fibrils [20]. It allows the development of well defined small protein modules which can be used for structure studies of the 82–143 domain of PrPSc and the development of therapeutics
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