Heterologous Stacking of Prion Protein Peptides Reveals Structural Details of Fibrils and Facilitates Complete Inhibition of Fibril Growth

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 ␤-helix. The ␤-helix is composed of spiraling rungs of parallel ␤-strands, and in the PrP model residues 105-143 of each PrP monomer can contribute two ␤-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 lefthanded ␤-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 lefthanded ␤-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 ␤-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.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. The deposition of aggregated prion protein fibrils on and in neurons is regarded to be the source of these neurodegenerative diseases and is frequently associated with occurrence of Congo red positivity (1-3). It is still unknown how much of the whole PrP Sc molecule is involved in the fibril growth. It is shown that the N-terminal part of PrP, specifically residues 112-141, can go through conformational changes involving ␤-strand formation, which subsequently triggers fibril growth (6 -8), and solid state NMR studies showed that residues 112-141 are part of the highly ordered core of huPrP-(23-144) (9). It was previously shown that peptides based on the 89 -143 region of the human PrP protein can form fibrils rich in ␤-sheet structure which are biologically active in transgenic mice (10). Within this region it is the huPrP-(106 -126) peptide that is the smallest known region of PrP that forms fibrils that are toxic and resemble the physiological properties of PrP . The formation of PrP Sc is considered to be a two-step event; first, there is the binding between Pr...

Detailed Biophysical Characterization of the Acid-Induced PrPc to PrPβ Conversion Process

“…All experiments were conducted using Varian BioPack pulse sequences (VNMRJ v2.1B). 2D 15 N-HSQC spectra were acquired with 2048 complex points in the 1 H dimension and 256 complex points in the 15 N dimension with a recycle delay of 1.5 s. A total of 160 transients were collected for each experiment. 3D 15 N-edited NOESY-HSQC spectra (32) were collected at 800 MHz for the pH 5.2 sample.…”

“…2D 15 N-HSQC spectra were acquired with 2048 complex points in the 1 H dimension and 256 complex points in the 15 N dimension with a recycle delay of 1.5 s. A total of 160 transients were collected for each experiment. 3D 15 N-edited NOESY-HSQC spectra (32) were collected at 800 MHz for the pH 5.2 sample. The spectral width was set to 8000 Hz for the directly and indirectly detected 1 H dimensions.…”

“…A total of 2048, 224, and 72 complex points were collected in the t 2 , t 1 , and t 3 dimensions, respectively. For pH values of 4.4, 3.5, 3.2, and 3.0, 3D 15 N-edited NOESY-HSQC data were obtained with spectral widths of 6000, 6000, and 1800 Hz for the direct and indirect proton dimensions and the indirectly detected 15 N dimension, respectively. A total of 1024, 96, and 64 complex points were obtained in the t 2 , t 1 and t 3 dimensions.…”

“…These methods have increased our insight into possible structures but have also resulted in multiple, sometimes competing, models of the protein structure. Electron crystallographic studies of 2D crystals obtained from truncated PrP sc fragments isolated from hamster brain tissue led to the development of an atomic resolution model characterized by a trimeric β-helix where the β-strand is largely localized to the N-terminus of the protein − . Molecular dynamic studies by Daggett and colleagues, coupled with data from epitope mapping and other biophysical studies, produced a model of PrP sc with similar N-terminal β-strand localization and trimeric stoichiometry but with a substantially different topology .…”

“…In particular, we have found that it is possible to convert a recombinant Syrian hamster prion fragment shPrP(90-232) from PrP c to PrP β using only a simple pH titration (from pH 7.0 to pH 1.0) at low salt (20 mM) concentrations. This allowed us to obtain comprehensive 1 H/ 15 N assignments as well as detailed NOE, T 2 , CD, and DLS data at multiple points throughout the conversion process. Our results implicate the flanking residues of the β-strand (B2) and the N-terminal residues of B1, H1, and H3 in the early stages of pHinduced conversion (as the pH drops from 5.2 to 2.8).…”