INTERACTIONS OF PRIONS WITH OTHER PROTEINS AND GLYCOSAMINOGLYCANS

Boston University Medical Campus, Boston MA

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. (Collaborator Pr. Harris group, Department of Biochemistry, Boston University). Prion diseases include rare neurological disorders such as Creutzfeldt-Jakob disease and Gerstmann-Str[unreadable]ussler syndrome. Prion diseases are caused by the conformational conversion of the normal, cellular prion protein (PrPC), a membrane glycoprotein of unknown function, into PrPSc, a misfolded isoform of PrPC. Despite compelling evidence about the conformational conversion of PrPC into PrPSc, our current understanding of the primary mechanisms of neurodegeneration in prion disease is still limited. Although it is commonly assumed that PrPSc is a hallmark of all prion diseases, its neurotoxicity most probably relies on the presence of functional PrPC molecules at the cell surface. A logic approach for gaining insights into the physiological function of PrPC is to identify its interaction partners. This strategy has been pursued by multiple laboratories identified a plethora of candidates. However, so far none of the proposed candidates has shown a definitive link with PrPC, or provided a clue for understanding its function. Moreover, important insights into the physiological activity of PrPC came also from the characterization of different mutations ([unreadable]CR PrP, PrPC deleted for residues 105-125). In order to elucidate the mechanism responsible for [unreadable]CR PrP toxicity, and for wild type PrPC rescuing activity, we are investigating the interaction partners (proteins and GAGs) of wild type PrPC, [unreadable]CR PrP, and PrP[unreadable]23-31. Towards this purpose, interaction partners are being identified by mass spectrometry. A bottom-up approach is performed from samples (immunoprecipitated using magnetic beads) containing the wild type or mutant PrPC. Digested peptides have been analyzed by MALDI-TOF MS and by nanoLC-MS/MS on the LTQ-Orbitrap (Thermo-Fisher). Because of the low abundance of the interaction partners, we have optimized the sample preparation to avoid contamination. The best conditions werre found to be: 1) ultrafiltration (10 kD) of the proteins attached to the magnetic beads, 2) digestion directly on the magnet beads, 3) purification of the peptides and 4) analysis of the samples by mass spectrometry. MALDI-TOF mass spectra showed slight differences between the wild type and the mutant PrPC. In future work, these samples will be analyzed by nanoLC-MS/MS on LTQ-Orbitrap (Thermo-Fisher) to identify the proteins.