The long-term objective of our research is to understand the molecular mechanisms of the pathogenic process in transmissible spongiform encephalopathies (TSE), also known as prion diseases. Although the precise nature of an infectious agent in TSEs is controversial, a prevailing model is based on the 'protein-only' hypothesis. According to this hypothesis, the key event in the pathogenic process is the conversion of the prion protein from its normal, ct-helical form, PrPC, to a conformationally altered, protease-resistant, beta-sheet rich form, PrPSc The major goal of this project is to elucidate the mechanism of the conformational conversion(s) of the prion protein and gain insight into the structure of the abnormal, beta-sheet-rich conformer. The proposed study involves experiments with the recombinant human prion protein as well as with the abnormal PrPSc isoform(s) isolated from diseased brain. Recently, we have shown that, under appropriate experimental conditions, the recombinant human prion protein huPrP9O-23 1 can be converted to an oligomeric beta-sheet-rich form with physicochemical properties similar to those of brain PrPS. The first Specific Aim is to characterize the conformational structure of this recombinant PrPSc-like model. The structure of the protein will be probed by fluorescence spectroscopic methods (fluorescence quenching, resonance energy transfer) using a series of protein variants with genetically engineered single tryptophan residues and extrinsic fluorescent probes. The second Specific Aim is to determine the mechanism of amyloid formation by the disease-associated, C-truncated Y145Stop variant (residues 23-144) of the human prion protein. The recombinant protein corresponding to this variant spontaneously undergoes a self-propagating transition to amyloid fibrils, providing an attractive and experimentally accessible model for studying mechanistic aspects of the conformational conversion(s) in the prion protein. The final Specific Aim is to determine the effect of mutations associated with inherited prion diseases on conformational properties of authentic PrPSC from diseased human brain. The main technique to be used for this purpose is Fourier-transform infrared spectroscopy.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS044158-02
Application #
6629481
Study Section
Special Emphasis Panel (ZRG1-BDCN-3 (01))
Program Officer
Nunn, Michael
Project Start
2002-06-15
Project End
2007-05-31
Budget Start
2003-06-01
Budget End
2004-05-31
Support Year
2
Fiscal Year
2003
Total Cost
$363,375
Indirect Cost
Name
Case Western Reserve University
Department
Physiology
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Nieznanski, Krzysztof; Surewicz, Krystyna; Chen, Shugui et al. (2014) Interaction between prion protein and A? amyloid fibrils revisited. ACS Chem Neurosci 5:340-5
Cobb, Nathan J; Apostol, Marcin I; Chen, Shugui et al. (2014) Conformational stability of mammalian prion protein amyloid fibrils is dictated by a packing polymorphism within the core region. J Biol Chem 289:2643-50
Apostol, Marcin I; Perry, Kay; Surewicz, Witold K (2013) Crystal structure of a human prion protein fragment reveals a motif for oligomer formation. J Am Chem Soc 135:10202-5
Dutta, Arpana; Chen, Shugui; Surewicz, Witold K (2013) The effect of ?2-?2 loop mutation on amyloidogenic properties of the prion protein. FEBS Lett 587:2918-23
Kong, Qingzhong; Mills, Jeffrey L; Kundu, Bishwajit et al. (2013) Thermodynamic stabilization of the folded domain of prion protein inhibits prion infection in vivo. Cell Rep 4:248-54
Nieznanski, Krzysztof; Choi, Jin-Kyu; Chen, Shugui et al. (2012) Soluble prion protein inhibits amyloid-? (A?) fibrillization and toxicity. J Biol Chem 287:33104-8
Helmus, Jonathan J; Surewicz, Krystyna; Apostol, Marcin I et al. (2011) Intermolecular alignment in Y145Stop human prion protein amyloid fibrils probed by solid-state NMR spectroscopy. J Am Chem Soc 133:13934-7
Surewicz, Witold K; Apostol, Marcin I (2011) Prion protein and its conformational conversion: a structural perspective. Top Curr Chem 305:135-67
Smirnovas, Vytautas; Baron, Gerald S; Offerdahl, Danielle K et al. (2011) Structural organization of brain-derived mammalian prions examined by hydrogen-deuterium exchange. Nat Struct Mol Biol 18:504-6
Jones, Eric M; Wu, Bo; Surewicz, Krystyna et al. (2011) Structural polymorphism in amyloids: new insights from studies with Y145Stop prion protein fibrils. J Biol Chem 286:42777-84

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