Limits...
Isolation of a Defective Prion Mutant from Natural Scrapie

View Article: PubMed Central - PubMed

ABSTRACT

It is widely known that prion strains can mutate in response to modification of the replication environment and we have recently reported that prion mutations can occur in vitro during amplification of vole-adapted prions by Protein Misfolding Cyclic Amplification on bank vole substrate (bvPMCA). Here we exploited the high efficiency of prion replication by bvPMCA to study the in vitro propagation of natural scrapie isolates. Although in vitro vole-adapted PrPSc conformers were usually similar to the sheep counterpart, we repeatedly isolated a PrPSc mutant exclusively when starting from extremely diluted seeds of a single sheep isolate. The mutant and faithful PrPSc conformers showed to be efficiently autocatalytic in vitro and were characterized by different PrP protease resistant cores, spanning aa ∼155–231 and ∼80–231 respectively, and by different conformational stabilities. The two conformers could thus be seen as different bona fide PrPSc types, putatively accounting for prion populations with different biological properties. Indeed, once inoculated in bank vole the faithful conformer was competent for in vivo replication while the mutant was unable to infect voles, de facto behaving like a defective prion mutant. Overall, our findings confirm that prions can adapt and evolve in the new replication environments and that the starting population size can affect their evolutionary landscape, at least in vitro. Furthermore, we report the first example of “authentic” defective prion mutant, composed of brain-derived PrPC and originating from a natural scrapie isolate. Our results clearly indicate that the defective mutant lacks of some structural characteristics, that presumably involve the central region ∼90–155, critical for infectivity but not for in vitro replication. Finally, we propose a molecular mechanism able to account for the discordant in vitro and in vivo behavior, suggesting possible new paths for investigating the molecular bases of prion infectivity.

No MeSH data available.


Related in: MedlinePlus

Hypothetical mechanisms underpinning the defective nature of 14K PrPSc.The cartoon depicts the hypothetical interaction between physiological proteases responsible for α-cleavage, here supposed to belong to the family of ADAM proteases [35] and 18K or 14K PrPSc (A), and how it changes in the in vitro (B) and the in vivo (C) replication environments. Distinct symbols indicate PrPC monomers, 18K or 14K PrPSc aggregates, as well as the α-cleavage site and the location of the polybasic domains in PrPSc, as shown in the graphical legend below the cartoon. (A) In 18K PrPSc aggregates the physiological α-cleavage site (residues 110–111, vole PrP numbering) and the central polybasic domain (in green, aa ~ 101–110), are tightly packed in the PK-resistant core of PrPSc. On the contrary, in mutant 14K PrPSc the α-cleavage site is available for hydrolysis. (B) During in vitro propagation by PMCA, the activity of ADAM proteases is purposely prevented by protease inhibitors, a factor which allows to keep full length PrP in solution. Under these conditions, both 18K and 14K PrPSc are full length and preserve intact polybasic domains, which would allow them to interact with PrPC and replicate. (C) In vivo, 18K PrPSc is still protected from ADAM proteolysis as the cleavage site is buried within the PK-resistant core, and thus it is still fully competent for replication as it retains the N-terminus. In contrast, 14K PrPSc would be cleaved at 110–111 and would lose the central polybasic domain, supposed to be a key mediator of the interaction with partners indispensable for prion replication (PrPC or cofactors).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5120856&req=5

ppat.1006016.g004: Hypothetical mechanisms underpinning the defective nature of 14K PrPSc.The cartoon depicts the hypothetical interaction between physiological proteases responsible for α-cleavage, here supposed to belong to the family of ADAM proteases [35] and 18K or 14K PrPSc (A), and how it changes in the in vitro (B) and the in vivo (C) replication environments. Distinct symbols indicate PrPC monomers, 18K or 14K PrPSc aggregates, as well as the α-cleavage site and the location of the polybasic domains in PrPSc, as shown in the graphical legend below the cartoon. (A) In 18K PrPSc aggregates the physiological α-cleavage site (residues 110–111, vole PrP numbering) and the central polybasic domain (in green, aa ~ 101–110), are tightly packed in the PK-resistant core of PrPSc. On the contrary, in mutant 14K PrPSc the α-cleavage site is available for hydrolysis. (B) During in vitro propagation by PMCA, the activity of ADAM proteases is purposely prevented by protease inhibitors, a factor which allows to keep full length PrP in solution. Under these conditions, both 18K and 14K PrPSc are full length and preserve intact polybasic domains, which would allow them to interact with PrPC and replicate. (C) In vivo, 18K PrPSc is still protected from ADAM proteolysis as the cleavage site is buried within the PK-resistant core, and thus it is still fully competent for replication as it retains the N-terminus. In contrast, 14K PrPSc would be cleaved at 110–111 and would lose the central polybasic domain, supposed to be a key mediator of the interaction with partners indispensable for prion replication (PrPC or cofactors).

Mentions: The dominant PrPC processing event, α-cleavage, occurs at the start of the hydrophobic core region, at approximately K110↓H111 (vole PrP numbering) [34, 35], producing the membrane anchored C-terminal C1 fragment and releasing the corresponding N-terminal N1 fragment, which contains the polybasic domain. This cleavage event is prevented in infectious PrPSc, as the α-cleavage site is in the tightly packed PK-resistant core and is solvent excluded, but it is allowed in PrPSc with less extended ~ 155–231 PK-resistant core, in which residues 110–111 are solvent exposed and available to the endoproteolytic processing. Interestingly, noninfectious PrPSc with ~ 155–231 PK-resistant core have only been observed in vitro under conditions in which α-cleavage do not occur, i.e. in experiments involving purified preparations of recombinant PrP or in brain homogenates supplemented with protease inhibitors, that are routinely added in PMCA reactions to prevent PrPC proteolysis. This observation prompted us to hypothesize that preserving PrPSc from α-cleavage could be indispensable for prion replication. In Fig 4 we describe a molecular mechanism based on this hypothesis, which is able to reconcile the contrasting in vitro and in vivo behavior of 14K PrPres. The model proposes that the short 100–110 polybasic peptide containing the lysine cluster plays a crucial role in prion replication and that the infectivity of 14K PrPSc was prevented by the removal of the polybasic domain through in vivo α-endoproteolysis, which is structurally inhibited in infectious 18K PrPSc. Overall, taking into account the hypothesis of the quasispecies nature of prion strains, we might suppose that even defective PrPSc conformations such as 14K could be continuously generated and eliminated by the host endoproteolitic cleavage, i.e. the in vivo negative selection. Once the negative selection is abrogated, as occurs in PMCA experiments in which brain homogenates are supplemented with protease inhibitors, such innate continuous generation of PrPSc conformational variants might eventually result in the selective emergence of defective mutants.


Isolation of a Defective Prion Mutant from Natural Scrapie
Hypothetical mechanisms underpinning the defective nature of 14K PrPSc.The cartoon depicts the hypothetical interaction between physiological proteases responsible for α-cleavage, here supposed to belong to the family of ADAM proteases [35] and 18K or 14K PrPSc (A), and how it changes in the in vitro (B) and the in vivo (C) replication environments. Distinct symbols indicate PrPC monomers, 18K or 14K PrPSc aggregates, as well as the α-cleavage site and the location of the polybasic domains in PrPSc, as shown in the graphical legend below the cartoon. (A) In 18K PrPSc aggregates the physiological α-cleavage site (residues 110–111, vole PrP numbering) and the central polybasic domain (in green, aa ~ 101–110), are tightly packed in the PK-resistant core of PrPSc. On the contrary, in mutant 14K PrPSc the α-cleavage site is available for hydrolysis. (B) During in vitro propagation by PMCA, the activity of ADAM proteases is purposely prevented by protease inhibitors, a factor which allows to keep full length PrP in solution. Under these conditions, both 18K and 14K PrPSc are full length and preserve intact polybasic domains, which would allow them to interact with PrPC and replicate. (C) In vivo, 18K PrPSc is still protected from ADAM proteolysis as the cleavage site is buried within the PK-resistant core, and thus it is still fully competent for replication as it retains the N-terminus. In contrast, 14K PrPSc would be cleaved at 110–111 and would lose the central polybasic domain, supposed to be a key mediator of the interaction with partners indispensable for prion replication (PrPC or cofactors).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120856&req=5

ppat.1006016.g004: Hypothetical mechanisms underpinning the defective nature of 14K PrPSc.The cartoon depicts the hypothetical interaction between physiological proteases responsible for α-cleavage, here supposed to belong to the family of ADAM proteases [35] and 18K or 14K PrPSc (A), and how it changes in the in vitro (B) and the in vivo (C) replication environments. Distinct symbols indicate PrPC monomers, 18K or 14K PrPSc aggregates, as well as the α-cleavage site and the location of the polybasic domains in PrPSc, as shown in the graphical legend below the cartoon. (A) In 18K PrPSc aggregates the physiological α-cleavage site (residues 110–111, vole PrP numbering) and the central polybasic domain (in green, aa ~ 101–110), are tightly packed in the PK-resistant core of PrPSc. On the contrary, in mutant 14K PrPSc the α-cleavage site is available for hydrolysis. (B) During in vitro propagation by PMCA, the activity of ADAM proteases is purposely prevented by protease inhibitors, a factor which allows to keep full length PrP in solution. Under these conditions, both 18K and 14K PrPSc are full length and preserve intact polybasic domains, which would allow them to interact with PrPC and replicate. (C) In vivo, 18K PrPSc is still protected from ADAM proteolysis as the cleavage site is buried within the PK-resistant core, and thus it is still fully competent for replication as it retains the N-terminus. In contrast, 14K PrPSc would be cleaved at 110–111 and would lose the central polybasic domain, supposed to be a key mediator of the interaction with partners indispensable for prion replication (PrPC or cofactors).
Mentions: The dominant PrPC processing event, α-cleavage, occurs at the start of the hydrophobic core region, at approximately K110↓H111 (vole PrP numbering) [34, 35], producing the membrane anchored C-terminal C1 fragment and releasing the corresponding N-terminal N1 fragment, which contains the polybasic domain. This cleavage event is prevented in infectious PrPSc, as the α-cleavage site is in the tightly packed PK-resistant core and is solvent excluded, but it is allowed in PrPSc with less extended ~ 155–231 PK-resistant core, in which residues 110–111 are solvent exposed and available to the endoproteolytic processing. Interestingly, noninfectious PrPSc with ~ 155–231 PK-resistant core have only been observed in vitro under conditions in which α-cleavage do not occur, i.e. in experiments involving purified preparations of recombinant PrP or in brain homogenates supplemented with protease inhibitors, that are routinely added in PMCA reactions to prevent PrPC proteolysis. This observation prompted us to hypothesize that preserving PrPSc from α-cleavage could be indispensable for prion replication. In Fig 4 we describe a molecular mechanism based on this hypothesis, which is able to reconcile the contrasting in vitro and in vivo behavior of 14K PrPres. The model proposes that the short 100–110 polybasic peptide containing the lysine cluster plays a crucial role in prion replication and that the infectivity of 14K PrPSc was prevented by the removal of the polybasic domain through in vivo α-endoproteolysis, which is structurally inhibited in infectious 18K PrPSc. Overall, taking into account the hypothesis of the quasispecies nature of prion strains, we might suppose that even defective PrPSc conformations such as 14K could be continuously generated and eliminated by the host endoproteolitic cleavage, i.e. the in vivo negative selection. Once the negative selection is abrogated, as occurs in PMCA experiments in which brain homogenates are supplemented with protease inhibitors, such innate continuous generation of PrPSc conformational variants might eventually result in the selective emergence of defective mutants.

View Article: PubMed Central - PubMed

ABSTRACT

It is widely known that prion strains can mutate in response to modification of the replication environment and we have recently reported that prion mutations can occur in vitro during amplification of vole-adapted prions by Protein Misfolding Cyclic Amplification on bank vole substrate (bvPMCA). Here we exploited the high efficiency of prion replication by bvPMCA to study the in vitro propagation of natural scrapie isolates. Although in vitro vole-adapted PrPSc conformers were usually similar to the sheep counterpart, we repeatedly isolated a PrPSc mutant exclusively when starting from extremely diluted seeds of a single sheep isolate. The mutant and faithful PrPSc conformers showed to be efficiently autocatalytic in vitro and were characterized by different PrP protease resistant cores, spanning aa ∼155–231 and ∼80–231 respectively, and by different conformational stabilities. The two conformers could thus be seen as different bona fide PrPSc types, putatively accounting for prion populations with different biological properties. Indeed, once inoculated in bank vole the faithful conformer was competent for in vivo replication while the mutant was unable to infect voles, de facto behaving like a defective prion mutant. Overall, our findings confirm that prions can adapt and evolve in the new replication environments and that the starting population size can affect their evolutionary landscape, at least in vitro. Furthermore, we report the first example of “authentic” defective prion mutant, composed of brain-derived PrPC and originating from a natural scrapie isolate. Our results clearly indicate that the defective mutant lacks of some structural characteristics, that presumably involve the central region ∼90–155, critical for infectivity but not for in vitro replication. Finally, we propose a molecular mechanism able to account for the discordant in vitro and in vivo behavior, suggesting possible new paths for investigating the molecular bases of prion infectivity.

No MeSH data available.


Related in: MedlinePlus