Identification of proteins with high affinity for refolded and native PrPC

Abstract

PrPC, the cellular prion protein, is widely expressed in most tissues, including brain, muscle and the gastrointestinal tract, but its physiological role remains unclear. During propagation of transmissible spongiform encephalopathies (TSEs), prion protein is converted to the pathological isoform, PrPSc, in a process believed to be mediated by as-yet-unknown host factors. The identification of proteins associated with PrP may provide information about the biology of prions and the pathogenesis of TSEs. In the present work, we report proteins identified from brain tissue based on their ability to bind to recombinant PrP (recPrP) or form multimolecular complexes with native PrPC in the presence of cross-linkers. Immobilized his-tagged recPrP was used as an affinity matrix to isolate PrP-interacting proteins from brain homogenates of normal individuals. In parallel, PrPC-associated proteins were characterized by cross-linking and co-immunoprecipitation assays. The unknown molecules were identified by MS and the results of LC-MS/MS analysis were subsequently verified by Western blot. Both techniques resulted in identification of proteins participating in the formation of cytoskeleton and signal transduction, further supporting the hypothesis that PrP is involved in the organization and function of receptors throughout the nervous system.

Prion Diseases

“…87,88 PrP C also appears to play a role in neuronal development, differentiation, and neurite outgrowth, a feature that may be related to its interaction with adhesion molecules such as laminin and N-CAM. [89][90][91] In addition to these molecules, several other binding partners of PrP C encompassing a range of functions from endocytosis to apoptotic signaling have been detected (see 92,93 for details), although based on the use of in vitro systems and the propensity of PrP to aggregate, it is not clear whether many of these binding partners are physiologically relevant.…”

Physiological role of the cellular prion protein

“…PrP C localization in lipid rafts, membrane microdomains enriched in sphingolipids and cholesterol, and the association with signal transduction mechanisms, indicate that PrP may participate in cell signaling pathways [82]. Many signal transduction patterns have been uncovered during PrP C function analysis.…”

“…Many others ligands have been identified, using various approaches, as possibly interacting with PrP C , among them : αβ-crystalline, Na + /K + -ATPase α3 subunit, CNPase, β-actin, α-spectrin and creatine kinase-β, synapsin Ib, the adaptator protein Grb2, and the prion interactor Pint1 [82]. It is, however, difficult to propose a specific role for such a number of possibilities.…”

Anti-LRP/LR Antibody W3 Hampers Peripheral PrPSc Propagation in Scrapie Infected Mice

“…12 Besides monoBesides monoclonal antibodies targeting the prion protein, 13,14 also single chain anti-PrP antibodies are currently investigated for a TSE therapy. 15 �mong many interaction partners identified for PrP c , 10,[16][17][18] the non-integrin 37/67 kDa laminin receptor (LRP/LR) has been discovered as a receptor for both the cellular PrP c 19,20 and the disease associated PrP Sc . 21,22 Downregulation of LRP/LR by antisense Downregulation of LRP/LR by antisense LRP RN� or siRN�s directed against LRP mRN� abrogates PrP Sc propagation in ScN2a cells.…”