Prion protein (PrP) underlies a spectrum of diseases with no established treatment and devastating human and economic consequences. The """"""""protein-only"""""""" hypothesis postulates that an abnormal prion protein conformation (PrPSc) propagates itself in an autocatalytic manner by recruiting normal isoform of the same protein (PrPC) and, therefore, acts as a transmissible agent of disease. The reconstitution of PrPSc in vitro from synthetic components has been difficult to achieve despite many years of effort. These difficulties are attributed to the lack of reliable biochemical markers of prion infectivity and to our poor understanding of the physical properties that are essential for infectivity. During the previous funding period, we developed the first experimental procedure for cell-free conversion of full-length PrP into self-propagating amyloid fibrils;we described several pathways of PrP polymerization;we introduced the most comprehensive mechanism of PrP conversion;we also established several novel assays including an immunoconformational assay for probing conformation within a single PrP fibril or particle. In the present application, we propose to elucidate PrPSc ultrastructure and to establish a link between infectivity and physical property of prion fibrils. The first specific aim will elucidate the substructure of PrPSc using novel immunoconformational assay developed in our laboratory combined with high resolution Atomic Force Microscopy. The second specific aim will elucidate the ultrastructure of prion fibrils generated in vitro, and the third specific aim is designed to test a relationship between conformational properties of the in vitro generated fibrils and their intrinsic infectivity. Such knowledge should lay the foundation for development of sensitive antemortem diagnostics and efficient therapeutics for treating prion diseases.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-MDCN-E (02))
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Wong, May
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University of Maryland Baltimore
Schools of Medicine
United States
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Makarava, Natallia; Savtchenko, Regina; Lasch, Peter et al. (2018) Preserving prion strain identity upon replication of prions in vitro using recombinant prion protein. Acta Neuropathol Commun 6:92
Srivastava, Saurabh; Katorcha, Elizaveta; Makarava, Natallia et al. (2018) Inflammatory response of microglia to prions is controlled by sialylation of PrPSc. Sci Rep 8:11326
Katorcha, Elizaveta; Gonzalez-Montalban, Nuria; Makarava, Natallia et al. (2018) Prion replication environment defines the fate of prion strain adaptation. PLoS Pathog 14:e1007093
Katorcha, Elizaveta; Baskakov, Ilia V (2018) Analysis of Covalent Modifications of Amyloidogenic Proteins Using Two-Dimensional Electrophoresis: Prion Protein and Its Sialylation. Methods Mol Biol 1779:241-255
Katorcha, Elizaveta; Baskakov, Ilia V (2017) Analyses of N-linked glycans of PrPSc revealed predominantly 2,6-linked sialic acid residues. FEBS J 284:3727-3738
Makarava, Natallia; Savtchenko, Regina; Baskakov, Ilia V (2017) Purification and Fibrillation of Full-Length Recombinant PrP. Methods Mol Biol 1658:3-22
Baskakov, Ilia V (2017) Limited understanding of the functional diversity of N-linked glycans as a major gap of prion biology. Prion 11:82-88
Srivastava, Saurabh; Katorcha, Elizaveta; Daus, Martin L et al. (2017) Sialylation Controls Prion Fate in Vivo. J Biol Chem 292:2359-2368
Makarava, Natallia; Savtchenko, Regina; Baskakov, Ilia V (2017) Methods of Protein Misfolding Cyclic Amplification. Methods Mol Biol 1658:169-183
Katorcha, Elizaveta; Daus, Martin L; Gonzalez-Montalban, Nuria et al. (2016) Reversible off and on switching of prion infectivity via removing and reinstalling prion sialylation. Sci Rep 6:33119

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