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Integrity of helix 2-helix 3 domain of the PrP protein is not mandatory for prion replication.

ABSTRACT: The process of prion conversion is not yet well understood at the molecular level. The regions critical for the conformational change of PrP remain mostly debated and the extent of sequence change acceptable for prion conversion is poorly documented. To achieve progress on these issues, we applied a reverse genetic approach using the Rov cell system. This allowed us to test the susceptibility of a number of insertion mutants to conversion into prion in the absence of wild-type PrP molecules. We were able to propagate several prions with 8 to 16 extra amino acids, including a polyglycine stretch and His or FLAG tags, inserted in the middle of the protease-resistant fragment. These results demonstrate the possibility to increase the length of the loop between helices H2 and H3 up to 4-fold, without preventing prion replication. They also indicate that this loop probably remains unstructured in PrP(Sc). We also showed that bona fide prions can be produced following insertion of octapeptides in the two C-terminal turns of H2. These insertions do not interfere with the overall fold of the H2-H3 domain indicating that the highly conserved sequence of the terminal part of H2 is not critical for the conversion. Altogether these data showed that the amplitude of modifications acceptable for prion conversion in the core of the globular domain of PrP is much greater than one might have assumed. These observations should help to refine structural models of PrP(Sc) and elucidate the conformational changes underlying prions generation.

SUBMITTER: Salamat K

PROVIDER: S-EPMC3365929 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Charge neutralization of the central lysine cluster in prion protein (PrP) promotes PrP(Sc)-like folding of recombinant PrP amyloids.

Project description:The structure of the infectious form of prion protein, PrP(Sc), remains unclear. Most pure recombinant prion protein (PrP) amyloids generated in vitro are not infectious and lack the extent of the protease-resistant core and solvent exclusion of infectious PrP(Sc), especially within residues ∼90-160. Polyanionic cofactors can enhance infectivity and PrP(Sc)-like characteristics of such fibrils, but the mechanism of this enhancement is unknown. In considering structural models of PrP(Sc) multimers, we identified an obstacle to tight packing that might be overcome with polyanionic cofactors, namely, electrostatic repulsion between four closely spaced cationic lysines within a central lysine cluster of residues 101-110. For example, in our parallel in-register intermolecular β-sheet model of PrP(Sc), not only would these lysines be clustered within the 101-110 region of the primary sequence, but they would have intermolecular spacings of only ∼4.8 Å between stacked β-strands. We have now performed molecular dynamics simulations predicting that neutralization of the charges on these lysine residues would allow more stable parallel in-register packing in this region. We also show empirically that substitution of these clustered lysine residues with alanines or asparagines results in recombinant PrP amyloid fibrils with extended proteinase-K resistant β-sheet cores and infrared spectra that are more reminiscent of bona fide PrP(Sc). These findings indicate that charge neutralization at the central lysine cluster is critical for the folding and tight packing of N-proximal residues within PrP amyloid fibrils. This charge neutralization may be a key aspect of the mechanism by which anionic cofactors promote PrP(Sc) formation.

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The amino-terminal PrP domain is crucial to modulate prion misfolding and aggregation.

Project description:The main hypothesis for prion diseases is that the cellular protein (PrP(C)) can be altered into a misfolded, beta-sheet-rich isoform (PrP(Sc)), which undergoes aggregation and triggers the onset of transmissible spongiform encephalopathies. Here, we investigate the effects of amino-terminal deletion mutations, rPrP(Delta51-90) and rPrP(Delta32-121), on the stability and the packing properties of recombinant murine PrP. The region lacking in rPrP(Delta51-90) is involved physiologically in copper binding and the other construct lacks more amino-terminal residues (from 32 to 121). The pressure stability is dramatically reduced with decreasing N-domain length and the process is not reversible for rPrP(Delta51-90) and rPrP(Delta32-121), whereas it is completely reversible for the wild-type form. Decompression to atmospheric pressure triggers immediate aggregation for the mutants in contrast to a slow aggregation process for the wild-type, as observed by Fourier-transform infrared spectroscopy. The temperature-induced transition leads to aggregation of all rPrPs, but the unfolding temperature is lower for the rPrP amino-terminal deletion mutants. The higher susceptibility to pressure of the amino-terminal deletion mutants can be explained by a change in hydration and cavity distribution. Taken together, our results show that the amino-terminal region has a pivotal role on the development of prion misfolding and aggregation.

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