NR ATQP
AU Wain,R.; Wille,H.; Prusiner,S.B.; Legname,G.
TI Biophysical characterization of the amyloid form of recombinant mouse prion protein
QU International Conference - Prion 2005: Between fundamentals and society's needs - 19.10.-21.10.2005, Congress Center Düsseldorf - Poster Session: Structure of PrP and molecular determinants of infectivity STRCT-05
PT Konferenz-Poster
AB
The discovery that recombinant (rec) mouse (Mo) prion protein (PrP) residues 89-230 can produce infectious prions when polymerized into amyloid fibrils in vitro opens many new avenues of research. Fibrils produced after seeding experiments were found to induce disease in transgenic (Tg) mice harboring the same sequence (Legname 2004; Legname 2005). Preliminary experiments have indicated that the conditions used for fibril formation can have a large effect on the kinetics and morphology of the amyloid fibrils formed. We have studied the factors affecting the stability of the kinetics of fibril formation. A 96-well plate assay has been developed in order to minimize handling during the fibril formation process.
Limited proteinase K digestion was performed on amyloid fibrils formed from recMoPrP(89-230) and full-length recMoPrP(23-230). Both proteins were partially resistant to digestion with high concentrations of proteinase K for 30 min at 37°. Electron microscopy of the undigested amyloid fibrils composed of recMoPrP(89-230) showed long and non-specifically aggregated mats while the digested fibrils were shorter and revealed a more exposed substructure with laterally aggregated polymers. Digested fibrils exhibited a morphology that is similar to that of prion rods composed of PrP 27-30, the protease-resistant core of disease-causing PrPsc. Denaturation studies show that these fibrils are highly resistant to disaggregation with some fibrils retaining their structure after exposure to 8 M guanidine hydrochloride. These results should provide the basis to investigate further the molecular mechanism of prion conversion and replication.
Moreover, immunofluorescence studies have shown that accessibility to the D13 epitope is lost after recMoPrP(89-230) assembles into amyloid fibrils as is observed after the conversion of PrPc to PrPsc (Peretz 1997; Leclerc 2003).
Leclerc, E., D. Peretz, et al. (2003). "Conformation of PrPc on the cell surface as probed by antibodies." J. Mol. Biol. 326: 475-483.
Legname, G., I. V. Baskakov, et al. (2004). "Synthetic mammalian prions." Science 305: 673-676.
Legname, G., H.-O. B. Nguyen, et al. (2005). "Strain-specified characteristics of mouse synthetic prions." Proc. Natl. Acad. Sci. USA 102: 2168-2173.
Peretz, D., R. A. Williamson, et al. (1997). "A conformational transition at the N-terminus of the prion protein features in formation of the scrapie isoform." J. Mol. Biol. 273: 614-622.
AD Rachel Wain, Holger Wille, Stanley B.Prusiner, Giuseppe Legname, Institute for Neurodegenerative Diseases, University of California, San Francisco, CA94143, USA
SP englisch
PO Deutschland