NR AWOM
AU Stoehr,J.; Weinmann,N.; Wille,H.; Kaimann,T.; Nagel-Steger,L.; Prusiner,S.; Riesner,D.
TI Spontaneous, seeded and cyclic fibril formation of purified recombinant and posttranslationally modified PrP
QU International Conference - Prion 2006: Strategies, advances and trends towards protection of society - 3.10.-6.10.2006, Torino, Italy, Lingotto Conference Centre - Poster sessions S-25
PT Konferenz-Poster
AB
An in vitro conversion system has been described earlier (1) in which recombinant SHaPrP was shifted from alpha-helical, monomeric to alpha-helical dimeric, to beta-sheet rich oligomeric and to polymorphic insoluble aggregates by dilution of submicellar concentrations of SDS. The system was modified to produce regular fibrils (2). Here we show for the first time the conversion into fibrils with full length PrPc with glycosylations and the GPI-anchor obtained from transgenic CHO-cells (3, 4). These structures were characterized by electron microscopy (EM) including immunogold labelling. Similar fibrils were obtained from recombinant and native full length PrPc (2) The soluble initial state from which the fibrils were generated was characterized by Mr-determination, CD-spectroscopy and mass spectrometry. From these studies a system for seeded fibril formation was developed. Small amounts of purified full length PrPsc are able to convert recombinant SHaPrP into a fibrillar state as analyzed by Thioflavin T fluorescence, fluorescence correlation spectroscopy and EM. A cyclic amplification system could be developed which is similar to that of the Soto group (5) but clearly different in that only purified components were applied, i.e. no cellular extract.
(1) Leffers KW, Schell J, Jansen K, Lucassen R, Kaimann T, Nagel-Steger L, Tatzelt J, Riesner D. The structural transition of the prion protein into its pathogenic conformation is induced by unmasking hydrophobic sites. J Mol Biol. 2004 Nov 26;344(3):839-53. (2) Leffers KW, Wille H, Stohr J, Junger E, Prusiner SB, Riesner D. Assembly of natural and recombinant prion protein into fibrils. Biol. Chem. 2005 Jun;386(6):569-80. (3) Blochberger, T.C., Cooper, C., Peretz, D., Tatzelt, J., Griffith, O.H., Baldwin, M.A., Prusiner, S.B. (1997). Prion protein expression in Chinese hamster ovary cells using a glutamine synthetase selection and amplification system. Prot. Eng. 10, 1465-1473. (4) Elfrink, K. Riesner, D. (2004). Purification of PrPc. Techniques in Prion Research. ed. by S. Lehmann and J. Grassi (Birkhaeuser Verlag Basel), 4-15. (5) Saborio GP, Permanne B, Soto C. Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature. 2001 Jun 14;411(6839):810-3.
AD J. Stoehr, N. Weinmann, T. Kaimann, L. Nagel-Steger, D. Riesner: Institut für Physikalische Biologie, Heinrich-Heine-Universitaet Duesseldorf, 40225 Duesseldorf, Germany and Biologisch Medizinisches Forschungszentrum der Heinrich-Heine-Universitaet Duesseldorf,Germany; H. Wille; S. Prusiner: Institute for Neurodegenerative Diseases, UCSF, San Francisco, USA. E-mail: riesner@biophys.uniduesseldorf.de
SP englisch
PO Italien