The dominant‐negative effect of the Q218K variant of the prion protein does not require protein X

Abstract

Previous studies identified several single-point mutants of the prion protein that displayed dominantnegative effects on prion replication. The dominant-negative effect was assumed to be mediated by protein X, an as-yet-unknown cellular cofactor that is believed to be essential for prion replication. To gain insight into the mechanism that underlies the dominant-negative phenomena, we evaluated the effect of the Q218K variant of full-length recombinant prion protein (Q218K rPrP), one of the dominant-negative mutants, on cell-free polymerization of wild-type rPrP into amyloid fibrils. We found that both Q218K and wild-type (WT) rPrPs were incorporated into fibrils when incubated as a mixture; however, the yield of polymerization was substantially decreased in the presence of Q218K rPrP. Furthermore, in contrast to fibrils produced from WT rPrP, the fibrils generated in the mixture of WT and Q218K rPrPs did not acquire the proteinase K-resistant core of 16 kDa that was shown previously to encompass residues 97-230 and was similar to that of PrPSc . Our studies demonstrate that the Q218K variant exhibits the dominant-negative effect in cell-free conversion in the absence of protein X, and that this effect is, presumably, mediated by physical interaction between Q218K and WT rPrP during the polymerization process.Keywords: prion protein; dominant-negative effect; prion diseases; amyloid fibrils; cell-free conversion Prion diseases are a group of fatal neurodegenerative maladies that can arise spontaneously or be inherited, and that can also be infectious (Prusiner 1998). All three forms of the prion diseases, sporadic, familial, and infectious, are induced by misfolding and aggregation of the prion protein (PrP). Single-point mutations at several positions within the PrP open reading frame were linked to familial prion diseases. Remarkably, the polymorphisms at other codons of PrP gene were found to protect against scrapie infection or development of sporadic forms of prion diseases. For example, sheep with Q/R polymorphisms at the codon 171 (equivalent to the codon 167 in mouse PrP) were resistant to scrapie, while sheep carrying the Q/Q alleles developed prion disease (Ikeda et al. 1995;Hunter et al. 1997). Human genetic studies revealed that 12% of the Japanese population carries the polymorphism E/K at the codon 219 (equivalent to the codon 218 of mouse PrP) (Shibuya et al. 1998b Abbreviations: PrP, prion protein; PrP C , normal cellular isoform of the prion protein; PrP Sc , abnormal pathological isoform of the prion protein; rPrP, full-length recombinant prion protein; WT, wild type; FTIR, Fourier transform infrared spectroscopy; ThT, Thioflavin T; GdnHCl, guanidine hydrochloride; GdnSCN, guanidine thiocyanate.Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi

Role of the Goat K 222 -PrP C Polymorphic Variant in Prion Infection Resistance

“…The human K 219 -PrP C allele (homologous to the codon 222 allele in goat PrP C ) was reported to protect humans against Creutzfeldt-Jakob disease (42), while the equivalent K 218 variant of mouse PrP C significantly reduced the fibril aggregation kinetics and generated non-proteinase K-resistant PrP (43). A plausible explanation for this inhibitory effect could be the insertion of an additional positive charge at codon 222, provided by the lysine amino acid (K), thus interfering with the PrP C -PrP Sc interaction and resulting in abolished or low conversion rates of PrP Sc (44).…”

A Naturally Occurring C-terminal Fragment of the Prion Protein (PrP) Delays Disease and Acts as a Dominant-negative Inhibitor of PrPSc Formation

“…In addition to these point mutations, short deletions within the central region of the protein (⌬112-119, ⌬114 -121, and ⌬105-125) led to a dominant-negative effect in infected cells and mice, respectively (42,54). In vitro conversion reactions showed that the dominant-negative effect of point mutations at residues 218 or 171 does not require the presence of auxiliary molecules (55,56), implying a direct interaction between the C terminus of PrP and the PrP Sc seed. Collectively, these results demonstrate that modifications in the C-terminal half of the protein (after 111) can significantly impact the generation of PK-resistant PrP.…”

Insights into prion biology

“…Thus, in either case, expression of dominant-negative mutants skews the competition between aggregate assembly and disassembly pathways toward the latter, allowing the effective clearance of an established prion conformer in vivo proposed for a Q172R variant, conversion of wild-type PrP, depending on their affinities for one another. 125,126 Potentially supporting this affinity-based model, PrP dominant-negative mutants differ in their effective inhibitory ratios relative to wild-type protein, 111,[125][126][127][128][129] but the same observation is also consistent with a model in which these mutants target different events in prion propagation in vivo. According to this latter idea, mutants that act at the templating surface would be effective at lower doses than those affecting fragmentation, which occurs along the length of the aggregate.…”

“…28,117,125,135 In contrast, PrP Q219K, like, Sup35 G58D, converts to the prion conformer efficiently, destabilizes aggregates and is incompatible only with prion variants of reduced thermodynamic stability. 28,105,117,125,126 Thus, despite the distinct cellular environments in which mammalian and yeast prions propagate, the pathways of dominant inhibition that we have uncovered for Sup35 mutants may be applicable to PrP mutants and may provide mechanistic insight into the phenotypic outcomes of prion infections in that system.…”

“…[114][115][116] Early models suggested that PrP dominant-negative mutants acted by titrating away a host-encoded cofactor (protein X) required for the conversion reaction; 122 however, these sequence variants also inhibit prion propagation in cell-free systems, suggesting that their effects are mediated directly through prion-prion interactions. [125][126][127] In this scenario, PrP sequence variants would interact with the templating surface on an aggregate and either slow, as has been proposed for a Q219K variant, or block, as has been ©2 0 1 1 L a n d e s B i o s c i e n c e .…”