Transmissible spongiform encephalopathies (TSEs) are fatal, neurodegenerative diseases in human and animals, caused by prion protein (PrP). The functions of PrP and the basis of these diseases are still under debate. In the present study characteristics of possible prion protein interactor PINT, have been studied.
Four ovine PINT constructs were used for expression of protein in prokaryotic (E.coli) and eukaryotic cell lines (murine neuroblastoma cells (N2a) and human neuroblastoma cells (SH-SY5Y)). Overexpression of PINT-His6 protein in E.coli led to the formation of insoluble inclusion bodies, which did not depend on growth temperature (16- 37ºC was used). Transient and stable transfections, and fluorescent protease protection (FPP) assay allowed a detailed study of the cellular localization of PINT protein. PINT was shown to be localized both in cytosol and in the cell nucleus; some of the cytosolic PINT-DsRed-Ex appeared also to be bound to membranes. It was shown that PINT-DsRed-Ex is resistant to trypsin digestion, but not to proteinase K. Application of not boiled lysates on the gel showed that PINT-DsRed-Ex tends to form oligomers in transiently transfected cells. To study the possible interaction between PINT and PrP, immunoprecipitation assay was performed. Lysates of transfected N2a-PrP-EGFP cells were added anti-RFP (directed against PINT-DsRed-Ex) or P4 (directed against PrP) antibodies and antibody binding proteins such as Protein A or Protein G. Signals were detected with antibodies directed against both proteins. Immunoprecipitation assay confirmed weak interaction between PrP and PINT protein.