The protein-only hypothesis predicts that infectious mammalian prions are composed solely of PrP Sc , a misfolded conformer of the normal prion protein, PrP C . However, protein-only PrP Sc preparations lack significant levels of prion infectivity, leading to the alternative hypothesis that cofactor molecules are required to form infectious prions. Here, we show that prions with parental strain properties and full specific infectivity can be restored from protein-only PrP Sc in vitro . The restoration reaction is rapid, potent, and requires bank vole PrP C substrate, post-translational modifications, and cofactor molecules. To our knowledge, this represents the first report in which the essential properties of an infectious mammalian prion have been restored from pure PrP without adaptation. These findings provide evidence for a unified hypothesis of prion infectivity in which the global structure of protein-only PrP Sc accurately stores latent infectious and strain information, but cofactor molecules control a reversible switch that unmasks biological infectivity.

Prions are unorthodox infectious agents that replicate by templating misfolded conformations of a host-encoded glycoprotein, collectively termed PrP Sc. Prion diseases are invariably fatal and currently incurable, but oral drugs that can prolong incubation times in prioninfected mice have been developed. Here, we tested the efficacy of combination therapy with two such drugs, IND24 and Anle138b, in scrapie-infected mice. The results indicate that combination therapy was no more effective than either IND24 or Anle138b monotherapy in prolonging scrapie incubation times. Moreover, combination therapy induced the formation of a new prion strain that is specifically resistant to the combination regimen but susceptible to Anle138b. To our knowledge, this is the first report of a pathogen with specific resistance to combination therapy despite being susceptible to monotherapy. Our findings also suggest that combination therapy may be a less effective strategy for treating prions than conventional pathogens.

Prion diseases are caused by the misfolding of a host-encoded glycoprotein, PrP C , into a pathogenic conformer, PrP Sc. Infectious prions can exist as different strains, composed of unique conformations of PrP Sc that generate strain-specific biological traits, including distinctive patterns of PrP Sc accumulation throughout the brain. Prion strains from different animal species display different cofactor and PrP C glycoform preferences to propagate efficiently in vitro, but it is unknown whether these molecular preferences are specified by the amino acid sequence of PrP C substrate or by the conformation of PrP Sc seed. To distinguish between these two possibilities, we used bank vole PrP C to propagate both hamster or mouse prions (which have distinct cofactor and glycosylation preferences) with a single, common substrate. We performed reconstituted sPMCA reactions using either (1) phospholipid or RNA cofactor molecules, or (2) di-or un-glycosylated bank vole PrP C substrate. We found that prion strains from either species are capable of propagating efficiently using bank vole PrP C substrates when reactions contained the same PrP C glycoform or cofactor molecule preferred by the PrP Sc seed in its host species. Thus, we conclude that it is the conformation of the input PrP Sc seed, not the amino acid sequence of the PrP C substrate, that primarily determines species-specific cofactor and glycosylation preferences. These results support the hypothesis that strain-specific patterns of prion neurotropism are generated by selection of differentially distributed cofactors molecules and/or PrP C glycoforms during prion replication.

Prions are unorthodox pathogens that cause fatal neurodegenerative diseases in humans and other mammals. Prion propagation occurs through the self-templating of the pathogenic conformer PrP Sc , onto the cell-expressed conformer, PrP C . Here we study the conversion of PrP C to PrP Sc using a recombinant mouse PrP Sc conformer (mouse protein-only recPrP Sc ) as a unique tool that can convert bank vole but not mouse PrP C substrates in vitro . Thus, its templating ability is not dependent on sequence homology with the substrate. In the present study, we used chimeric bank vole/mouse PrP C substrates to systematically determine the domain that allows for conversion by Mo protein-only recPrP Sc . Our results show that that either the presence of the bank vole amino acid residues E227 and S230 or the absence of the second N-linked glycan are sufficient to allow PrP C substrates to be converted by Mo protein-only recPrP Sc and several native infectious prion strains. We propose that residues 227 and 230 and the second glycan are part of a C-terminal domain that acts as a linchpin for bank vole and mouse prion conversion.

Epoxide hydrolases catalyze the conversion of epoxides to vicinal diols in a range of cellular processes such as signaling, detoxification, and virulence. These enzymes typically utilize a pair of tyrosine residues to orient the substrate epoxide ring in the active site and stabilize the hydrolysis intermediate. A new subclass of epoxide hydrolases that utilize a histidine in place of one of the tyrosines was established with the discovery of the CFTR Inhibitory Factor (Cif) from Pseudomonas aeruginosa . Although the presence of such Cif-like epoxide hydrolases was predicted in other opportunistic pathogens based on sequence analyses, only Cif and its homolog aCif from Acinetobacter nosocomialis have been characterized. Here we report the biochemical and structural characteristics of Cfl1 and Cfl2, two C i f - l ike epoxide hydrolases from Burkholderia cenocepacia . Cfl1 is able to hydrolyze xenobiotic as well as biological epoxides that might be encountered in the environment or during infection. In contrast, Cfl2 shows very low activity against a diverse set of epoxides. The crystal structures of the two proteins reveal quaternary structures that build on the well-known dimeric assembly of the α/β hydrolase domain, but broaden our understanding of the structural diversity encoded in novel oligomer interfaces. Analysis of the interfaces reveals both similarities and key differences in sequence conservation between the two assemblies, and between the canonical dimer and the novel oligomer interfaces of each assembly. Finally, we discuss the effects of these higher-order assemblies on the intra-monomer flexibility of Cfl1 and Cfl2 and their possible roles in regulating enzymatic activity.