Formation of Soluble Oligomers and Amyloid Fibrils with Physical Properties of the Scrapie Isoform of the Prion Protein from the C-terminal Domain of Recombinant Murine Prion Protein mPrP-(121–231)

Samantha M Martins,Dóris J Frosoni,Ana M Blanco Martinez,Fernanda G De Felice,Sérgio T Ferreira

doi:10.1074/jbc.m605367200

Samantha M Martins, Dóris J Frosoni + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m605367200

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Abstract

Prion diseases are fatal neurodegenerative disorders associated with conformational conversion of the cellular prion protein, PrP(C), into a misfolded, protease-resistant form, PrP(Sc). Here we show, for the first time, the oligomerization and fibrillization of the C-terminal domain of murine PrP, mPrP-(121-231), which lacks the entire unstructured N-terminal domain of the protein. In particular, the construct we used lacks amino acid residues 106-120 from the so-called amyloidogenic core of PrP (residues 106-126). Amyloid formation was accompanied by acquisition of resistance to proteinase K digestion. Aggregation of mPrP-(121-231) was investigated using a combination of biophysical and biochemical techniques at pH 4.0, 5.5, and 7.0 and at 37 and 65 degrees C. Under partially denaturing conditions (65 degrees C), aggregates of different morphologies ranging from soluble oligomers to mature amyloid fibrils of mPrP-(121-231) were formed. Transmission electron microscopy analysis showed that roughly spherical aggregates were readily formed when the protein was incubated at pH 5.5 and 65 degrees C for 1 h, whereas prolonged incubation led to the formation of mature amyloid fibrils. Samples incubated at 65 degrees C at pH 4.0 or 7.0 presented an initial mixture of oligomers and protofibrils or fibrils. Electrophoretic analysis of samples incubated at 65 degrees C revealed formation of sodium dodecyl sulfate-resistant oligomers (dimers, trimers, and tetramers) and higher molecular weight aggregates of mPrP-(121-231). These results demonstrate that formation of an amyloid form with physical properties of PrP(Sc) can be achieved in the absence of the flexible N-terminal domain and, in particular, of residues 106-120 of PrP and does not require other cellular factors or a PrP(Sc) template.

Highlights

PrPSc is defined as an aggregated form of PrP that is largely resistant to proteinase K (PK) digestion under conditions in which PrPC and most other proteins are readily degraded [2]
Temperature and pH Effects on the Aggregation of mPrP[121–231]—To identify experimental conditions leading to the oligomerization or fibrillization of mPrP-(121–231), we initially monitored the aggregation of the protein at both pH 5.5 and 7.0 and at 37 and 65 °C
Incubation for 1 h at 65 °C and pH 4.0 in buffered medium hydrophobic region, we show that it has the ability to assemble produced relatively few oligomers and rare small protofibrillar into roughly spherical soluble aggregates, annular and linear structures (Fig. 3E), in few images acquired under this protofibrils, and amyloid fibrils under appropriate experimencondition it was possible to see occasional larger protofi- tal conditions in the absence of any other cellular factors

Summary

The abbreviations used are

PrPC, cellular isoform of the prion protein; PrPSc, pathogenic or scrapie isoform of the prion protein; PK, proteinase K; mPrP[121–231], recombinant murine PrP comprising residues 121–231; ThT, thioflavin T; Mes, 4-morpholineethanesulfonic acid. Several recent studies have shown that partially denaturing conditions are required for the in vitro formation of amyloid, which can be attributed to the necessity of destabilizing the native fold of a protein under conditions in which non-covalent interactions involving the polypeptide chain remain favorable (for reviews, see Ref. 12) In this context, use of partially denaturing in vitro conditions should be viewed as a tool to populate aggregation-prone conformations and to accelerate a process that might normally take much longer in vivo. We describe the oligomerization and fibrillization of the C-terminal domain of the murine prion protein, mPrP-(121–231) This construct lacks the entire N-terminal flexible region and most of the amino acid residues from the 106 –126 amyloidogenic core of PrP. Possible implications of the formation of PrP oligomers to the pathogenesis of prion diseases are discussed

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION