Methionine oxidation interferes with conversion of the prion protein into the fibrillar proteinase K-resistant conformation.
In recent studies, we developed a protocol for in vitro conversion of full-length mouse recombinant PrP (Mo rPrP23-230) into amyloid fibrils [Bocharova et al. (2005) J. Mol. Biol. 346, 645-659]. Because amyloid fibrils produced from recombinant Mo PrP89-230 display infectivity [Legname et al. (2004) Science 305, 673-676], polymerizatiom of rPrPs in vitro represents a valuable model for elucidating the mechanism of prion conversion. Unexpectedly, when the same conversion protocol was used for hamster (Ha) rPrP23-231, we experienced substantial difficulties in forming fibrils. While searching for potential reasons of our failure to produce fibrils, we probed the effect of methionine oxidation in rPrP. We found that oxidation of methionines interferes with the formation of rPrP fibrils and that this effect is more profound for Ha than for Mo rPrP. To minimize the level of spontaneous oxidation, we developed a new protocol for rPrP purification, in which highly amyloidogenic Ha rPrP with minimal levels of oxidized residues was produced. Furthermore, our studies revealed that oxidation of methionines in preformed fibrils inhibited subsequent maturation of fibrils into proteinase K-resistant PrP(Sc)-like conformation (PrP-res). Our data are consistent with the proposition that conformational changes within the central region of the protein (residues 90-140) are essential for adopting PrP-res conformation and demonstrate that methionine oxidation interferes with this process. These studies provide new insight into the mechanism of prion polymerization, solve a long-standing practical problem in producing PrP-res fibrils from full-length PrP, and may help in identifying new genetic and environmental factors that modulate prion disease.
Breydo
Leonid
L
Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, Maryland 21201, USA.
Bocharova
Olga V
OV
Makarava
Natallia
N
Salnikov
Vadim V
VV
Anderson
Maighdlin
M
Baskakov
Ilia V
IV
D000682:Amyloid / Q000096:biosynthesis; D000818:Animals; D001704:Biopolymers / Q000096:biosynthesis; D006224:Cricetinae; D019286:Endopeptidase K / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism*; D010084:Oxidation-Reduction; D011328:Prions / Q000737:chemistry*; D011487:Protein Conformation
D000682:Amyloid; D001704:Biopolymers; D011328:Prions; D008715:Methionine; D019286:Endopeptidase K