Program Director/Principal Investigator (Last, First, Middle): Prusiner, Stanley/Long, Jeffrey (Project 1) PROJECT SUMMARY (See instructions): The phosphotungstate anion (PTA) binds specifically to the infectious isoform of the prion protein (PrPSc), but not to its cellular precursor (PrPc). Although PTA is now widely used to precipitate and purify PrPSc, the molecular details and the mechanism of this interaction are not understood. We have observed that the interaction with PTA influences the quaternary structure of PrPSc and its N-terminally truncated form, PrP 27- 30. PTA is.a polyoxometalate (POM), an inorganic metal oxide cluster with a rigid polyhedral structure. POMs are a large family of compounds displaying substantial variations in chemical composition, size, shape, and charge density. We hypothesize that the binding between POMs and PrPSc can be manipulated by changing the chemical composition of the POM, thereby influencing its size, shape, and charge. In order to gain insights into the conformational selectivity of the POM-PrPSc interaction, we plan to investigate the structure, stoichiometry, and speciation of the bound POMs through NMR and infrared spectroscopy. In addition, we plan to use POMs as tools to modify the aggregation properties of PrP50 with the aim of producing higher-quality two-dimensional crystals and amyloidfibrils for structural analyses. The quaternary structure of the POM-prion aggregates will be analyzed by electron microscopy and scanning probe microscopy. In contrast, some larger POMs fail to precipitate PrPSc, and instead dissociate PrPSc aggregates. This unexpected property will be exploited to develop a solubilization protocol for infectious, native PrPSc. Different biophysical properties will be used as solubilization criteria. Bioassays will be utilized extensively to test whether solubilized forms of PrPSc remain infectious. A soluble and homogeneous preparation of PrPSc ^ c would enable numerous structural studies. The affinity of the various POMs for PrP will be analyzed by isothermal titration calorimetry (ITC). ITC allows determination of the stoichiometry, binding constant(Kg), reaction enthalpy (AH), and the standard free energy of binding (AG).

Public Health Relevance

Determining the reaction parameters for the PrPSc?POM complex formation should facilitate the structural understanding of the interaction between POMs and PrPSc, aid in the selection of new POMs with favorable properties, and enable more rational decisions about the synthesis of additional POMs. So far, relatively few POMs, based on tungsten and molybdenum with selected heteroatoms, have been explored. A wide variety of POM species remain to be tested, including water-soluble POMs based upon vanadium and niobium. PROJECT/

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Research Program Projects (P01)
Project #
5P01AG002132-33
Application #
8429415
Study Section
Special Emphasis Panel (ZAG1-ZIJ-2)
Project Start
Project End
2015-01-31
Budget Start
2013-02-01
Budget End
2014-01-31
Support Year
33
Fiscal Year
2013
Total Cost
$178,934
Indirect Cost
$58,870
Name
University of California San Francisco
Department
Type
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Wan, William; Stubbs, Gerald (2014) Heterogeneous seeding of HET-s(218-289) and the mutability of prion structures. Prion 8:
Silber, B Michael; Gever, Joel R; Rao, Satish et al. (2014) Novel compounds lowering the cellular isoform of the human prion protein in cultured human cells. Bioorg Med Chem 22:1960-72
Stöhr, Jan; Condello, Carlo; Watts, Joel C et al. (2014) Distinct synthetic A? prion strains producing different amyloid deposits in bigenic mice. Proc Natl Acad Sci U S A 111:10329-34
Watts, Joel C; Giles, Kurt; Patel, Smita et al. (2014) Evidence that bank vole PrP is a universal acceptor for prions. PLoS Pathog 10:e1003990
Watts, Joel C; Prusiner, Stanley B (2014) Mouse models for studying the formation and propagation of prions. J Biol Chem 289:19841-9
Watts, Joel C; Condello, Carlo; Stöhr, Jan et al. (2014) Serial propagation of distinct strains of A? prions from Alzheimer's disease patients. Proc Natl Acad Sci U S A 111:10323-8
Wan, William; Stubbs, Gerald (2014) Fiber diffraction of the prion-forming domain HET-s(218-289) shows dehydration-induced deformation of a complex amyloid structure. Biochemistry 53:2366-70
Wan, William; Stubbs, Gerald (2014) Fungal prion HET-s as a model for structural complexity and self-propagation in prions. Proc Natl Acad Sci U S A 111:5201-6
Kurouski, Dmitry; Lu, Xuefang; Popova, Ludmila et al. (2014) Is supramolecular filament chirality the underlying cause of major morphology differences in amyloid fibrils? J Am Chem Soc 136:2302-12
Li, Zhe; Gever, Joel; Rao, Satish et al. (2013) Discovery and Preliminary SAR of Arylpiperazines as Novel, Brainpenetrant Antiprion Compounds. ACS Med Chem Lett 4:397-401

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