Mutant prion protein acquires resistance to protease in mouse neuroblastoma cells

Abstract

Conversion of the cellular isoform of the prion protein (PrP C ) into the pathogenic isoform (PrP Sc ) is thought to be the causative event in prion diseases. Biochemically, PrP Sc differs from PrP C in its partial resistance to proteinase K (PK). The amino acid sequence AGAAAAGA, comprising residues 112-119 of the murine PrP C , has been shown to be amyloidogenic and evolutionarily conserved. To assess the effect of mutations at and around this hydrophobic sequence on protease resistance, the sequence was replaced either by alanines or by glycines and, in a third mutant, a large part surrounding this region was removed. The PrP mutant carrying substitutions of glycines for alanines showed PK resistance and aberrant proteolytic processing. Tetracycline-induced expression of this mutant indicated that resistance to protease is acquired concurrent with the synthesis of the protein. These findings indicate that mutations in the central hydrophobic region lead to immediate alterations in PrP structure and processing.

Unexpected Tolerance of α-Cleavage of the Prion Protein to Sequence Variations

“…In contrast, C39 displayed partial α-cleavage inhibition in both Hpl and HeLa cells, which is in agreement with the finding that a mutant bearing a deletion of the region 114–121 is inefficiently cleaved in the brain of transgenic mice [14]. We conclude that, in contrast to published claims [19], the substitution of the palindrome with a total or partial polyglycine chain does not have a measurable impact on α-cleavage of PrP C .…”

“…2A). A previous study reported that substitution of all palindromic glycines with alanines, as in construct BOM9, abrogated C1 generation in N2a cells [19]. We therefore assessed the cleavability of BOM9 and of BOM11, which bears only three palindromic alanine substitutions, in Prnp −/− Hpl cells [25] (Fig.…”

“…Other studies on PrP C processing in cultured cells suggest that the alanine and glycine residues in the PrP C palindromic region localized in the downstream flank of the α-cleavage site may be important for α-cleavage [19]. It was reported that in transfected N2a cells, PrP C bearing two point mutations that transformed the palindromic domain 112–119 into an alanine chain had no remarkable impairment in α-cleavage [19].…”

“…It was reported that in transfected N2a cells, PrP C bearing two point mutations that transformed the palindromic domain 112–119 into an alanine chain had no remarkable impairment in α-cleavage [19]. Conversely, the cleavage product C1 was not, or very weakly, detectable in constructs where this palindrome was converted into a glycine chain, or where region 105–125 was deleted [19]. Other studies also suggested that α-cleavage is not affected by the removal of the octapeptide region [15], [18], [20] or by the insertion of supernumerary octapeptide repeats [20].…”

Amyloid Formation by Recombinant Full-length Prion Proteins in Phospholipid Bicelle Solutions

“…36,37 Moreover, the peptide segment 112-119 has been reported to be essential for the replication of the TSE agent in tissue cultures. 38,39 Altogether, these results indicate that membrane-associated forms of PrP C may be involved in the amplification of the TSE agent or its pathology, and that the conformational transition of PrP C to PrP Sc might be a membrane-associated process. [40][41][42] …”