Abstract
Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal diseases associated with the conversion of the cellular prion protein (PrPC) to the abnormal prion protein (PrPSc). Since the molecular mechanisms in pathogenesis are widely unclear, we analyzed the global phospho-proteome and detected a differential pattern of tyrosine- and threonine phosphorylated proteins in PrPSc-replicating and pentosan polysulfate (PPS)-rescued N2a cells in two-dimensional gel electrophoresis. To quantify phosphorylated proteins, we performed a SILAC (stable isotope labeling by amino acids in cell culture) analysis and identified 105 proteins, which showed a regulated phosphorylation upon PrPSc infection. Among those proteins, we validated the dephosphorylation of stathmin and Cdc2 and the induced phosphorylation of cofilin in PrPSc-infected N2a cells in Western blot analyses. Our analysis showed for the first time a differentially regulated phospho-proteome in PrPSc infection, which could contribute to the establishment of novel protein markers and to the development of novel therapeutic intervention strategies in targeting prion-associated disease.
Highlights
Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal diseases associated with the conversion of the cellular prion protein (PrPC) to the abnormal prion protein (PrPSc)
Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases occurring in many different host species including humans, which develop e.g. Creutzfeld Jacob disease [1]
The development of TSEs is associated with the self-propagating conversion of the normal host cellular prion protein (PrPC) into the abnormal protease-resistant isoform (PrPSc or PrPres) in an autocatalytic manner [2]
Summary
Cell culture N2a58/22L cells have been described previously [11] and were kindly provided by Prof. Cells were cultured in DMEM containing 10% FCS and 4 mM L-glutamine at 37°C. Cells were treated with 5 μg/ml pentosan polysulfate Cell lysates were prepared by scraping cells in lysis buffer containing 150 mM NaCl, 0.5% Triton X-100, 0.5% DOC, 50 mM Tris pH 7.5, 1 mM Na-vanadate, 1 mM Na-molybdate, 20 mM NaF, 10 mM NaPP, 20 mM β-glycerophosphat, 1× protease inhibitor cocktail (Roche, Mannheim, Germany). For digestion with proteinase K (PK) 80 μg protein were treated with 20 μg/ml PK for 30 min at 37°C. PK digestion was stopped by addition of laemmli sample buffer and protein denaturation at 95°C for 7 min. The cell layer was soaked in lysis buffer
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