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Biochemical properties of Ctm-PrP and Dpl 2

We expressed PrP construct which predispose the protein to form Ctm-PrP in neuronal cell lines (N2a, Hpl). Similarly, we also constructed and expressed constructs of Dpl in cell line, alone or along with PrP, in order to examine the role of Dpl and how it interacts with PrP, in particular whether Dpl has an antagonistic role to PrP in the cellular response to oxidative stress. We have generated polyclonal antibodies against Dpl used now by the other partners.

To study the role of Ctm-PrP, we constructed several plasmids to express the mutated protein MoPrP L9R/3AV (ORF generously provided by DA Harris, St Louis, USA). We stably expressed the protein after transfection in different cell lines including CHO and PrP-/- Hpl cells (generously provided by T Onodera, Tokyo Univ., Japan). We also attempted to generate antibodies against the amino terminal signal peptide which is preserved in Ctm-PrP but were so far unsuccessful, probably in relation with the high hydrophobicity of its sequence. Expression of Ctm-PrP was followed by western blot and immunofluorescence. Importantly, expression of Ctm-PrP had no major toxic effect on the cells.

A recent publication from the group of DA Harris (Mutational analysis of topological determinants in prion protein (PrP) and measurement of transmembrane and cytosolic PrP during prion infection. J Biol Chem. 278, 45960-8) suggested that Ctm PrP is not directly involved in Prion neurodegeneration, both in infectious and genetic TSEs. On the other hand, new studies from the groups of S. Lindquist, A. Taraboulos and C. Soto put forward the idea that proteasomal degradation, the endoplasmic reticulum and generation of a cytosoloic form of PrP could play an essential role in prion pathology. In a previous work, we suggested that PrP can be subjected to retrograde transport toward the endoplasmic reticulum and that this compartment may play a significant role in PrPSc conversion. We also recently observed that PrPSc can be readily detected in the nucleus of infected cells, associated to DNA. Taking in account all these data, we are now looking in parallel at the consequences of the expression of wild-type, cytosolic, transmembrane and nuclear PrPs in neuronal cells.

Peoc'h K, Serres C, Frobert Y, Martin C, Lehmann S, Chasseigneaux S, Sazdovitch V, Grassi J, Jouannet P, Launay JM, & Laplanche JL. (2002) The human "prion-like" protein Doppel is expressed in both Sertoli cells and spermatozoa. J Biol Chem. 277:43071-8.

Béranger F., Mangé M., Goud B.& Lehmann S. (2002) Stimulation of PrPC retrograde transport towards the Endoplasmic Reticulum increases accumulation of PrPSc in prion-infected cells J. Biol. Chem. 277 :38972-38977.

Mangé A., Crozet C., Lehmann S. & Béranger F. (2004) Scrapie-like prion protein is translocated to the nuclei of infected cells independently of proteasome inhibition and interacts with chromatin. Journal of Cell Science 117, 2411-6.

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