Integrity of Helix 2-Helix 3 Domain of the PrP Protein Is Not Mandatory for Prion Replication

Hubert Laude,Human Rezaei,Jérôme Chapuis,Laetitia Herzog,Michel Dron,Mohammed Moudjou,Khalid Salamat,Vincent Béringue,Emilie Jaumain,Annalisa Pastore

doi:10.1074/jbc.m112.341677

Hubert Laude, Human Rezaei + Show 8 more

Open Access

https://doi.org/10.1074/jbc.m112.341677

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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.

Highlights

Prions are self-propagating ␤-sheet-rich conformers of PrP protein, domains involved in conformational modification remain undetermined
PrPC with Extended H2-H3 Loop Can Convert into Prion—In a previous study aimed at producing PrP glycosylation mutants [31], we identified a mutant that contained an insertion of 8 amino acids downstream from the second glycosylation site, within the unstructured loop joining helices H2 and H3 of sheep PrP (Fig. 1, A and B)
We introduced an 8-amino acid FLAG sequence into the original ins203 octapeptide, enabling us to recognize the presence of the insert using a relevant antibody (Fig. 5A)

Summary

Background

Prions are self-propagating ␤-sheet-rich conformers of PrP protein, domains involved in conformational modification remain undetermined. 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 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 PrPSc and elucidate the conformational changes underlying prions generation. Reverse genetic approaches to express modified PrP sequences have provided some clues but are constrained by the fact that mini-

The abbreviations used are

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION