Pathway Complexity of Prion Protein Assembly into Amyloid

Ilia V Baskakov,Giuseppe Legname,Michael A Baldwin,Stanley B Prusiner,Fred E Cohen

doi:10.1074/jbc.m111402200

Ilia V Baskakov, Giuseppe Legname + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m111402200

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Abstract

In vivo under pathological conditions, the normal cellular form of the prion protein, PrP(C) (residues 23-231), misfolds to the pathogenic isoform PrP(Sc), a beta-rich aggregated pathogenic multimer. Proteinase K digestion of PrP(Sc) leads to a proteolytically resistant core, PrP 27-30 (residues 90-231), that can form amyloid fibrils. To study the kinetic pathways of amyloid formation in vitro, we used unglycosylated recombinant PrP corresponding to the proteinase K-resistant core of PrP(Sc) and found that it can adopt two non-native abnormal isoforms, a beta-oligomer and an amyloid fibril. Several lines of kinetic data suggest that the beta-oligomer is not on the pathway to amyloid formation. The preferences for forming either a beta-oligomer or amyloid can be dictated by experimental conditions, with acidic pH similar to that seen in endocytic vesicles favoring the beta-oligomer and neutral pH favoring amyloid. Although both abnormal isoforms have high beta-sheet content and bind 1-anilinonaphthalene-8-sulfonate, they are dissimilar structurally. Multiple pathways of misfolding and the formation of distinct beta-sheet-rich abnormal isoforms may explain the difficulties in refolding PrP(Sc) in vitro, the need for a PrP(Sc) template, and the significant variation in disease presentation and neuropathology.

Highlights

In vivo under pathological conditions, the normal cellular form of the prion protein, PrPC, misfolds to the pathogenic isoform PrPSc, a ␤-rich aggregated pathogenic multimer
To study the kinetic pathways of amyloid formation in vitro, we used unglycosylated recombinant PrP corresponding to the proteinase K-resistant core of PrPSc and found that it can adopt two non-native abnormal isoforms, a ␤-oligomer and an amyloid fibril
By studying mouse and Syrian hamster PrP, we demonstrated that the protein can adopt the ␣-helical native isoform, two non-native ␤-sheet-rich isoforms, a ␤-oligomer, and an amyloid fibril

Summary

The abbreviations used are

PrP, prion protein; PK, proteinase K; ANS, 1-anilinonaphthalene-8-sulfonate; ESI, electrospray ionization; MS, mass spectrometry; Mo, mouse; rec, recombinant; SEC, size exclusion chromatography; SHa, Syrian hamster; ThT, thioflavin T; HPLC, high pressure liquid chromatography. By studying mouse and Syrian hamster PrP (designated Mo recPrP –231 and SHa recPrP –231, respectively), we demonstrated that the protein can adopt the ␣-helical native isoform, two non-native ␤-sheet-rich isoforms, a ␤-oligomer, and an amyloid fibril. These two abnormal isoforms may coexist under certain experimental conditions; the ␤-oligomer is not on the kinetic pathway to amyloid formation and is not a substructure in the assembling fibril. Our data demonstrate that PrP is capable of forming several abnormal isoforms and that the preference to fold into a particular abnormal isoform is influenced substantially by experimental conditions

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION