NR AORT
AU Deleault,N.R.; Lucassen,R.W.; Nishina,K.A.; Supattapone,S.
TI Host Factors Required for Amplification of Protease-Resistant PrP Molecules In Vitro
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Oral sessions OS-14
PT Konferenz-Vortrag
AB
Prions, the infectious agents of transmissible spongiform encephalopathies, are composed primarily of a misfolded protein designated PrPsc. Prion-infected neurons generate PrPsc from a host glycoprotein designated PrPc through a process of induced conformational change, but the molecular mechanism by which PrPc undergoes conformational change into PrPsc remains unknown. We employed an in vitro amplification technique adapted from Protein Misfolding Cyclic Ampification (PMCA) method of Saborio and Soto to investigate the mechanism of prion-induced protein conformational change biochemically.
Using this modified technique, we amplified protease-resistant PrPsc-like molecules (PrPres) >10-fold by mixing diluted scrapie-infected brain homogenate and normal brain homogenate without ultrasonication. PrPres amplification in vitro exhibits species and strain specificity, depends upon both time and temperature, is optimized at neutral pH, and does not require divalent cations.
Treatment of normal brain homogenate with the sulfhydryl inhibitors N-ethylmaleimide (NEM), p-hydroxymercuribenzoic acid (PHMB), or mersalyl acid inhibited PrPsc amplification in vitro, indicating that the conformational change from PrPc to PrPsc requires a thiol-containing factor. These data suggest that a reactive chemical group plays an essential role in the conformational change from PrPc to PrPres.
Using this system, host requirements for PrPres amplification can be studied biochemically. The results of ongoing investigations using this approach will be discussed.
AD Nathan R. Deleault, Ralf W. Lucassen, Koren Nishina, Surachai Supattapone, Dartmouth Medical School, USA
SP englisch
PO Deutschland