NR AWEQ
AU Falisse-Poirrier,N.; ElMoualij,B.; Pierrard,O.; Heinen,E.; Zorzi,W.
TI Unravelling the function of the prion protein: a proteomic approach using a 2D-LC tool
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 CE-13
PT Konferenz-Poster
AB
The loss of function of the host prion protein (PrPc), following its transconformation into an pathogenic isoform, is the causative factor of the fatal neurodegenerative transmissible spongiform encephalopathy. A role in circadian rhythm regulation, synaptic transmission, ion currents, nerve fibre organization, copper ion trafficking, nucleic acid chaperoning, anti-apoptotic processes and antioxidant has been suggested for PrPc(1). Interestingly, PrPc could play a role in cellular defence mechanisms against oxidative stress induced by Paraquat(2,3) but not against oxidative damage by redox active metals such as manganese(4). The mechanism by which PrPc protects against paraquat injury but not against manganese toxicity is unclear. This raises questions as to the cellular defence pathways in which the prion protein could be implied and opens a new area of investigation. In this work, we have used a proteomic approach based on a two dimensional liquid chromatography tool, the ProteomeLabTM PF 2D, in order to study the possible roles of the prion protein in cellular metabolism. Our proteomic technique separates proteins according to isoelectric point on an ionic exchanger in the first dimension and according to relative hydrophobicity on a non-porous reverse phase column in the second dimension. Proteins are detected by UV in the first and second dimensions. Data handling of whole proteomes, fractionated by this system, is realised using three software: 32karat, Proteovue and Deltavue(5). These software display fractionated proteins along their pI and relative hydrophobicity on an artificially generated 2D map and differentially expressed proteins are visualised along a third artificial map, facilitating proteome to proteome comparisons. Using this technique, we have fractionated and analysed the proteomes of three human neuroblastoma cell lines differing in their prion protein (PrP) levels of expression: SH-SY5Y, expressing basal levels of human PrP; wild type SH-SY5Y(wtPrP), expressing a murine PrP gene contained within a transfected plasmid(6); and a negative control SH-SY5Y, containing the plasmid control without the murine PrP gene. The identification of differentially expressed proteins following paraquat and manganese injury will contribute to clarify a fundamental metabolic state in correlation with PrP expression level. Indeed, it is likely that a loss of prion protein function is the causative factor for transmissible spongiform encephalopathies.
This work is supported by the Région Wallonne (R.Rwal. 14531 iPCRq and R.Rwal. 0845 "protéinopathies") and the Fonds social Européen (R.EURO.0557). Reference List: (1) Kovacs G.G et al., J Pathol 204, 241-247 (2004); (2) Dupiereux I et al., BBRC, submitted; (3) Senator A et al., Free Radic Biol Med, 37, 1224-30 (2004); (4) Falisse-Poirrier N et al., in preparation; (5) Yan F et al., Anal Chem 15, 2299-2308 (2003); (6) Walmsley A.R et al., Embo J 20, 703-712 (2001)
AD Institute of Pharmacy-CHU 1, Department of Human Histology, CRPP, University of Liège, 4000 Liège, Belgium. E-mail: N.Falisse@ulg.ac.be
SP englisch
PO Italien