Low density subcellular fractions enhance disease-specific prion protein misfolding.
The production of prion particles in vitro by amplification with or without exogenous seed typically results in infectivity titers less than those associated with PrP(Sc) isolated ex vivo and highlights the potential role of co-factors that can catalyze disease-specific prion protein misfolding in vivo. We used a cell-free conversion assay previously shown to replicate many aspects of transmissible spongiform encephalopathy disease to investigate the cellular location of disease-specific co-factors using fractions derived from gradient centrifugation of a scrapie-susceptible cell line. Fractions from the low density region of the gradient doubled the efficiency of conversion of recombinant PrP. These fractions contain plasma membrane and cytoplasmic proteins, and conversion enhancement can be achieved using PrP(Sc) derived from two different strains of mouse-passaged scrapie as seed. Equivalent fractions from a second scrapie-susceptible cell line also stimulate conversion. We also show that subcellular fractions enhancing disease-specific prion protein conversion prevent in vitro fibrillization of recombinant prion protein, suggesting the existence of separate, competing mechanisms of disease-specific and nonspecific misfolding in vivo.
Graham
James F
JF
Neuropathogenesis Division, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Alexander Robertson Building, Easter Bush Veterinary Centre, Roslin, Midlothian EH25 9RG.
Agarwal
Sonya
S
Neuropathogenesis Division, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Alexander Robertson Building, Easter Bush Veterinary Centre, Roslin, Midlothian EH25 9RG.
Kurian
Dominic
D
Institute for Animal Health, Compton, Newbury, Berkshire RG20 7NN.
Kirby
Louise
L
Neuropathogenesis Division, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Alexander Robertson Building, Easter Bush Veterinary Centre, Roslin, Midlothian EH25 9RG.
Pinheiro
Teresa J T
TJT
School of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, United Kingdom.
Gill
Andrew C
AC
Neuropathogenesis Division, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Alexander Robertson Building, Easter Bush Veterinary Centre, Roslin, Midlothian EH25 9RG. Electronic address: andrew.gill@roslin.ed.ac.uk.
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D003593:Cytoplasm / Q000378:metabolism; D051379:Mice; D011328:Prions / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000737:chemistry; D012608:Scrapie / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism*
D011328:Prions; D020033:Protein Isoforms; D011994:Recombinant Proteins