Biochemistry of Infectious Prions The """"""""protein-only"""""""" hypothesis proposes that prions are infectious proteins, which lack informational nucleic acids. Recent studies in filamentous fungi and yeast have proven this hypothesis for specific fungal proteins. Mammalian prions have also been generated in vitro. However, Koch's postulates remain unfulfilled for mammalian prions because in vitro prion propagation studies of wild type prions have used crude homogenates rather than purified proteins;and truncated, synthetic PrP molecules refolded into amyloid fibrils have thus far not caused disease when directly inoculated in non-transgenic animals. Our laboratory has recently developed an in vitro system that amplifies PrPres, the protease-resistant protein conformer associated with infectious mammalian prions, using only purified PrPC and synthetic nucleic acid molecules. Significantly, preliminary data indicate that this purified system can autocatalytically form PrPres molecules in vitro.
Our specific aims will focus upon rigorously testing the """"""""protein-only"""""""" hypothesis, and upon investigating the effects of post-translational modifications on PrPC-to-PrPres transformation in vitro.
Aim 1. To generate and propagate infectious mammalian prions in a purified system in vitro.
Aim 2. To determine whether strain properties of mammalian prions can be faithfully propagated by purified PrP molecules in vitro.
Aim 3. To investigate the effects of PrP post-translational modifications on the efficiency and specificity of prion propagation. In summary, we propose to test rigorously the unorthodox idea that the infectious agent of diseases such as Creutzfeldt-Jakob disease (CJD) can be composed of a single protein.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS055875-03
Application #
7579122
Study Section
Special Emphasis Panel (ZRG1-NDGB-E (09))
Program Officer
Wong, May
Project Start
2007-03-01
Project End
2012-02-28
Budget Start
2009-03-01
Budget End
2010-02-28
Support Year
3
Fiscal Year
2009
Total Cost
$279,825
Indirect Cost
Name
Dartmouth College
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041027822
City
Hanover
State
NH
Country
United States
Zip Code
03755
Turnbaugh, Jessie A; Unterberger, Ursula; SaĆ”, Paula et al. (2012) The N-terminal, polybasic region of PrP(C) dictates the efficiency of prion propagation by binding to PrP(Sc). J Neurosci 32:8817-30
Deleault, Nathan R; Piro, Justin R; Walsh, Daniel J et al. (2012) Isolation of phosphatidylethanolamine as a solitary cofactor for prion formation in the absence of nucleic acids. Proc Natl Acad Sci U S A 109:8546-51
Deleault, Nathan R; Walsh, Daniel J; Piro, Justin R et al. (2012) Cofactor molecules maintain infectious conformation and restrict strain properties in purified prions. Proc Natl Acad Sci U S A 109:E1938-46
Walsh, Daniel J; Noble, Geoffrey P; Piro, Justin R et al. (2012) Non-reducing alkaline solubilization and rapid on-column refolding of recombinant prion protein. Prep Biochem Biotechnol 42:77-86
Miller, Michael B; Supattapone, Surachai (2011) Superparamagnetic nanoparticle capture of prions for amplification. J Virol 85:2813-7
Piro, Justin R; Harris, Brent T; Supattapone, Surachai (2011) In situ photodegradation of incorporated polyanion does not alter prion infectivity. PLoS Pathog 7:e1002001
Piro, Justin R; Wang, Fei; Walsh, Daniel J et al. (2011) Seeding specificity and ultrastructural characteristics of infectious recombinant prions. Biochemistry 50:7111-6
Miller, Michael B; Geoghegan, James C; Supattapone, Surachai (2011) Dissociation of infectivity from seeding ability in prions with alternate docking mechanism. PLoS Pathog 7:e1002128
Deleault, Nathan R; Kascsak, Richard; Geoghegan, James C et al. (2010) Species-dependent differences in cofactor utilization for formation of the protease-resistant prion protein in vitro. Biochemistry 49:3928-34
Supattapone, Surachai (2010) Biochemistry. What makes a prion infectious? Science 327:1091-2

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