In vitro amplification of protease-resistant prion protein requires free sulfhydryl groups.
Prions, the infectious agents of transmissible spongiform encephalopathies, are composed primarily of a misfolded protein designated PrP(Sc). Prion-infected neurons generate PrP(Sc) from a host glycoprotein designated PrP(C) through a process of induced conformational change, but the molecular mechanism by which PrP(C) undergoes conformational change into PrP(Sc) remains unknown. We employed an in vitro PrP(Sc) amplification technique adapted from protein misfolding cyclic amplification (PMCA) to investigate the mechanism of prion-induced protein conformational change. Using this technique, PrP(Sc) from diluted scrapie-infected brain homogenate can be amplified >10-fold without sonication when mixed with normal brain homogenate under nondenaturing conditions. PrP(Sc) amplification in vitro exhibits species and strain specificity, depends on both time and temperature, only requires membrane-bound components, and does not require divalent cations. In vitro amplification of Syrian hamster Sc237 PrP(Sc) displays an optimum pH of approximately 7, whereas amplification of CD-1 mouse RML PrP(Sc) is optimized at pH approximately 6. The thiolate-specific alkylating agent N-ethylmaleimide (NEM) as well as the reversible thiol-specific blockers p-hydroxymercuribenzoic acid (PHMB) and mersalyl acid inhibited PrP(Sc) amplification in vitro, indicating that the conformational change from PrP(C) to PrP(Sc) requires a thiol-containing factor. Our data provide the first evidence that a reactive chemical group plays an essential role in the conformational change from PrP(C) to PrP(Sc).
Lucassen
Ralf
R
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Nishina
Koren
K
Supattapone
Surachai
S
D000818:Animals; D006224:Cricetinae; D010450:Endopeptidases / Q000378:metabolism*; D008647:Mesocricetus; D051379:Mice; D018620:PrPSc Proteins / Q000737:chemistry / Q000378:metabolism*; D017510:Protein Folding; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*
D018620:PrPSc Proteins; D013438:Sulfhydryl Compounds; D010450:Endopeptidases