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, 109 (22), 8546-51

Isolation of Phosphatidylethanolamine as a Solitary Cofactor for Prion Formation in the Absence of Nucleic Acids

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Isolation of Phosphatidylethanolamine as a Solitary Cofactor for Prion Formation in the Absence of Nucleic Acids

Nathan R Deleault et al. Proc Natl Acad Sci U S A.

Abstract

Infectious prions containing the pathogenic conformer of the mammalian prion protein (PrP(Sc)) can be produced de novo from a mixture of the normal conformer (PrP(C)) with RNA and lipid molecules. Recent reconstitution studies indicate that nucleic acids are not required for the propagation of mouse prions in vitro, suggesting the existence of an alternative prion propagation cofactor in brain tissue. However, the identity and functional properties of this unique cofactor are unknown. Here, we show by purification and reconstitution that the molecule responsible for the nuclease-resistant cofactor activity in brain is endogenous phosphatidylethanolamine (PE). Synthetic PE alone facilitates conversion of purified recombinant (rec)PrP substrate into infectious recPrP(Sc) molecules. Other phospholipids, including phosphatidylcholine, phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol, were unable to facilitate recPrP(Sc) formation in the absence of RNA. PE facilitated the propagation of PrP(Sc) molecules derived from all four different animal species tested including mouse, suggesting that unlike RNA, PE is a promiscuous cofactor for PrP(Sc) formation in vitro. Phospholipase treatment abolished the ability of brain homogenate to reconstitute the propagation of both mouse and hamster PrP(Sc) molecules. Our results identify a single endogenous cofactor able to facilitate the formation of prions from multiple species in the absence of nucleic acids or other polyanions.

Conflict of interest statement

Conflict of interest statement: S.S. and N.D. are inventors of a patent application that covers the use of phosphatidylethanolamine as a prion cofactor.

Figures

Fig. 1.
Fig. 1.
Characterization of cofactor preparation. Western blots of reconstituted sPMCA reactions probed with 6D11 anti-PrP mAb. (A) Species specificity. Immunopurified native mouse PrPC substrate initially seeded with Me7 prions (Upper) or hamster PrPC substrate seeded Sc237 prions (Lower) were supplemented with either 50 μg/mL total rat brain RNA (Left) or a purified cofactor (Right). (B and C) Saponification and PLC treatment. Saponified and phospholipase-treated cofactor samples were used to reconstitute sPMCA reactions with recPrP substrate seeded with recPrPSc template, as indicated. (D) Silica chromatography fractionation. recPrP substrate was supplemented with normal-phase chromatographic fractions and subjected to sPMCA reactions seeded with recPrPSc template, as indicated. -PK, samples not subjected to proteinase K digestion; all other samples were proteolyzed.
Fig. 2.
Fig. 2.
Effect of various phospholipids on prion formation. Western blots showing three-round sPMCA reactions using recPrP substrate and seeded with recPrPSc template, supplemented with various commercial preparations of purified and synthetic phospholipids at 2.5 mM final concentration, as indicated.
Fig. 3.
Fig. 3.
Generation of infectious prions with synthetic plasmalogen PE. (A) Western blot showing 18-round sPMCA reactions containing only recPrP and 1 mM synthetic plasmalogen PE seeded with recPrPSc in the first round. (B) Hematoxylin and eosin stained microscopic section of hypothalamus from mice inoculated with final sPMCA product of experiment shown in A, unseeded substrate mixture not subjected to sPMCA, or seeded mock sPMCA reactions in control buffer, as indicated. (C) Western blot to detect proteinase K-resistant PrPSc in brain homogenates of inoculated mice. Samples from three different animals inoculated with the same preparation of recPrPSc (seeded + PE) are shown.
Fig. 4.
Fig. 4.
Effect of PLC treatment on PrPSc formation in brain homogenates. PLC-treated and control crude Prnp0/0 brain homogenates were treated as described in Materials and Methods and used to reconstitute purified native hamster or mouse PrPC substrate in duplicate three-round sPMCA reactions seeded with either hamster Sc237 or mouse Me7 prions, as indicated. The control samples were not exposed to PLC, but otherwise mock processed together with the experimental samples (i.e., incubated with ZnCl2, dialyzyed, and exposed to EDTA).

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