Generating Bona Fide Mammalian Prions with Internal Deletions

Abstract

Mammalian prions are PrP proteins with altered structures causing transmissible fatal neurodegenerative diseases. They are self-perpetuating through formation of beta-sheet-rich assemblies that seed conformational change of cellular PrP. Pathological PrP usually forms an insoluble protease-resistant core exhibiting beta-sheet structures but no more alpha-helical content, loosing the three alpha-helices contained in the correctly folded PrP. The lack of a high-resolution prion structure makes it difficult to understand the dynamics of conversion and to identify elements of the protein involved in this process. To determine whether completeness of residues within the protease-resistant domain is required for prions, we performed serial deletions in the helix H2 C terminus of ovine PrP, since this region has previously shown some tolerance to sequence changes without preventing prion replication. Deletions of either four or five residues essentially preserved the overall PrP structure and mutant PrP expressed in RK13 cells were efficiently converted into bona fide prions upon challenge by three different prion strains. Remarkably, deletions in PrP facilitated the replication of two strains that otherwise do not replicate in this cellular context. Prions with internal deletion were self-propagating and de novo infectious for naive homologous and wild-type PrP-expressing cells. Moreover, they caused transmissible spongiform encephalopathies in mice, with similar biochemical signatures and neuropathologies other than the original strains. Prion convertibility and transfer of strain-specific information are thus preserved despite shortening of an alpha-helix in PrP and removal of residues within prions. These findings provide new insights into sequence/structure/infectivity relationship for prions. IMPORTANCEPrions are misfolded PrP proteins that convert the normal protein into a replicate of their own abnormal form. They are responsible for invariably fatal neurodegenerative disorders. Other aggregation-prone proteins appear to have a prion-like mode of expansion in brains, such as in Alzheimer's or Parkinson's diseases. To date, the resolution of prion structure remains elusive. Thus, to genetically define the landscape of regions critical for prion conversion, we tested the effect of short deletions. We found that, surprisingly, removal of a portion of PrP, the C terminus of alpha-helix H2, did not hamper prion formation but generated infectious agents with an internal deletion that showed characteristics essentially similar to those of original infecting strains. Thus, we demonstrate that completeness of the residues inside prions is not necessary for maintaining infectivity and the main strain-specific information, while reporting one of the few if not the only bona fide prions with an internal deletion.

The role of the unusual threonine string in the conversion of prion protein

“…All single amino acid variants except for recPrP T191P had melting temperatures around 70 °C, and recPrP T191A had the highest T m among this group (70.3 °C), consistent with the idea that the α-helix prone alanine stabilizes the helical structure of recPrP. Interestingly, recPrP T191P had the highest melting temperature (74.3 °C) among all recPrP variants, suggesting that this recPrP, with a shortened α-helix 2, is quite stable; this result is consistent with a recent report34. Importantly, there was no correlation between thermal stability and PrP conversion, either by PMCA or by amyloid fibril growth assay.…”

“…1), and the role of this string in PrP conversion is controversial222425263334. In our PMCA study, all variants in this string had a reduced capability to form infectious prions, which could be due to the influence on three aspects of PrP conversion: (1) the seeding process, (2) the PrP conformational change, and (3) the final prion structure.…”

“…Further, a comprehensive mutagenesis study of mouse PrP showed that individually replacing the amino acids in the C-terminus of α-helix 2 with any other amino acid does not significantly influence the formation of infectious prions in scrapie-infected N2a cells33. Very recently, Munoz-Montestino reported that deleting 4 or 5 amino acids in this region (including all four threonines) does not affect the conversion of ovine PrP to infectious prions in RK13 cells34. These data apparently contradict the observations from prion transmission studies in Prnp b knock-in mice, which showed that the threonine 189 (mouse numbering) significantly affects disease incubation times20.…”

A stretch of residues within the protease-resistant core is not necessary for prion structure and infectivity

“…20 We found that PrPD193-196 and PrPD193-197 conferred to RK13 cells the same degree of susceptibility to 127S infection than the wild-type protein. 17 The levels of PrP res accumulated in cells also compared at least up to 12 passages of the cultures. The size distribution of cell-formed PrP res aggregates was assessed by sedimentation velocity and found to be the same for the wild-type and mutant proteins (Fig.…”

“…1) and found that removal of the last 5 residues of the helix H2 did not impair prion conversion. 17 This was the first clear-cut demonstration that a stretch of residues within the prion-associated domain of PrP is dispensable to generate bona fide prions.…”