Protease-resistant prion protein amplification reconstituted with partially purified substrates and synthetic polyanions.
Little is currently known about the biochemical mechanism by which induced prion protein (PrP) conformational change occurs during mammalian prion propagation. In this study, we describe the reconstitution of PrPres amplification in vitro using partially purified and synthetic components. Overnight incubation of purified PrP27-30 and PrPC molecules at a molar ratio of 1:250 yielded approximately 2-fold baseline PrPres amplification. Addition of various polyanionic molecules increased the level of PrPres amplification to approximately 10-fold overall. Polyanionic compounds that stimulated purified PrPres amplification to varying degrees included synthetic, homopolymeric nucleic acids such as poly(A) and poly(dT), as well as non-nucleic acid polyanions, such as heparan sulfate proteoglycan. Size fractionation experiments showed that synthetic poly(A) polymers must be >0.2 kb in length to stimulate purified PrPres amplification. Thus, one possible set of minimal components for efficient conversion of PrP molecules in vitro may be surprisingly simple, consisting of PrP27-30, PrPC, and a stimulatory polyanionic compound.
Deleault
Nathan R
NR
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Geoghegan
James C
JC
Nishina
Koren
K
Kascsak
Richard
R
Williamson
R Anthony
RA
Supattapone
Surachai
S
D000818:Animals; D006224:Cricetinae; D006493:Heparin / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D010446:Peptide Fragments / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism*; D011061:Poly A / Q000494:pharmacology; D011071:Poly T / Q000494:pharmacology; D000071228:Polyelectrolytes; D011108:Polymers / Q000494:pharmacology*; D018621:PrPC Proteins / Q000378:metabolism*; D011487:Protein Conformation; D011509:Proteoglycans / Q000494:pharmacology
D006493:Heparin; D010446:Peptide Fragments; D010447:Peptide Hydrolases; D011061:Poly A; D011071:Poly T; D000071228:Polyelectrolytes; D011108:Polymers; D018621:PrPC Proteins; D011509:Proteoglycans; C036090:heparin proteoglycan