Introducing a Rigid Loop Structure from Deer into Mouse Prion Protein Increases Its Propensity for Misfolding In Vitro
Research Article
Introducing a Rigid Loop Structure from Deer into Mouse Prion Protein
Increases Its Propensity for Misfolding In Vitro
Leah M. Kyle, Affiliation: Department of Molecular Biology, University of
Wyoming, Laramie, Wyoming, United States of America
Theodore R. John, Affiliation: Department of Veterinary Sciences,
University of Wyoming, Laramie, Wyoming, United States of America
Hermann M. Schätzl mail, * E-mail: hschatzl@uwyo.edu
Affiliations: Department of Molecular Biology, University of Wyoming,
Laramie, Wyoming, United States of America, Department of Veterinary Sciences,
University of Wyoming, Laramie, Wyoming, United States of America
Randolph V. Lewis Affiliation: Department of Biology and Synthetic
Bioproducts Institute, Utah State University, Logan, Utah, United States of
America
Abstract
Prion diseases are fatal neurodegenerative disorders characterized by
misfolding of the cellular prion protein (PrPc) into the disease-associated
isoform (PrPSc) that has increased β-sheet content and partial resistance to
proteolytic digestion. Prion diseases from different mammalian species have
varying propensities for transmission upon exposure of an uninfected host to the
infectious agent. Chronic Wasting Disease (CWD) is a highly transmissible prion
disease that affects free ranging and farmed populations of cervids including
deer, elk and moose, as well as other mammals in experimental settings. The
molecular mechanisms allowing CWD to maintain comparatively high transmission
rates have not been determined. Previous work has identified a unique structural
feature in cervid PrP, a rigid loop between β-sheet 2 and α-helix 2 on the
surface of the protein. This study was designed to test the hypothesis that the
rigid loop has a direct influence on the misfolding process. The rigid loop was
introduced into murine PrP as the result of two amino acid substitutions: S170N
and N174T. Wild-type and rigid loop murine PrP were expressed in E. coli and
purified. Misfolding propensity was compared for the two proteins using
biochemical techniques and cell free misfolding and conversion systems. Murine
PrP with a rigid loop misfolded in cell free systems with greater propensity
than wild type murine PrP. In a lipid-based conversion assay, rigid loop PrP
converted to a PK resistant, aggregated isoform at lower concentrations than
wild-type PrP. Using both proteins as substrates in real time quaking-induced
conversion, rigid loop PrP adopted a misfolded isoform more readily than wild
type PrP. Taken together, these findings may help explain the high transmission
rates observed for CWD within cervids.
Citation: Kyle LM, John TR, Schätzl HM, Lewis RV (2013) Introducing a Rigid
Loop Structure from Deer into Mouse Prion Protein Increases Its Propensity for
Misfolding In Vitro. PLoS ONE 8(6): e66715.
doi:10.1371/journal.pone.0066715
Editor: Jiyan Ma, Ohio State University, United States of America
Received: March 25, 2013; Accepted: May 9, 2013; Published: June 25,
2013
Copyright: © 2013 Kyle et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Funding: This work was supported by grants from the Department of Defense
(DOD 030542, to RVL), the National Institute of Health (R01 NS076853-01A1, to
HMS), and was performed within the framework of the Wyoming Endowed Excellence
Chair. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests
exist.
snip...
In summary, introduction of the rigid loop, a structure inherent to cervid
PrP, increases the propensity for recombinant mouse PrP to misfold. This was
assessed in vitro using both an unseeded, lipid based conversion assay and
seeded conversion using RT-QuIC to detect differences in conversion rates
between two PrP variants. This finding may have relevance to the high levels of
shedding of CWD prions and transmission of CWD among cervids as compared to
prion diseases in other species.
disturbing...see this older study about this ;
Christina Sigurdson
Institute of Neuropathology, University Hospital of Zürich, Zurich
Switzerland
Award: DAMD17-03-1-0320
Spongiform Encephalopathy and Prion Plaque Generation by Mouse
Transgenesis
Chronic wasting disease (CWD) is a naturally-occurring, geographically
widespread prion disease in captive and free-ranging North American deer and
elk, with unknown potential as a human health threat. The elk PrPC structure
contains a well-defined loop connecting the second strand of the beta sheet with
alpha helix 2 (amino acids 165-175) which is rigidified by two local hydrogen
bond networks. These hydrogen bond networks are absent from mouse PrPC [1]. We
have expressed a PrPC mutant in transgenic mice that mimics the elk "rigid loop"
(RL). These transgenic mice develop a spontaneous neurologic disease with 100%
penetrance characterized by vacuolar change, gliosis, microglial activation, and
PrP plaques in the brain, similar to deer with CWD or patients with variant CJD
or Gerstmann-Straussler-Scheinker syndrome (GSS), and typical of a transmissible
spongiform encephalopathy.
1. Gossert, A.D., et al., Prion protein NMR structures of elk and of
mouse/elk hybrids. Proc Natl Acad Sci U S A, 2005. 102(3): p. 646-50 C.
Sigurdsona, M. Heikenwäldera, G. Mancoa, S. Hornemann b, P. Liberskic, J.
Pahnkea , F. Baumanna, S. Bonjourb, G. Mielea, L. Stitzd, P. Schwarza, F.
Heppnera, M. Glatzela, T. Rülickee, K. Wüthrichb, and A. Aguzzia
a. Institute of Neuropathology, University Hospital of Zürich,
Schmelzbergstrasse 12, CH- 8091 Zürich, Switzerland
b. Institute of Molecular Biology and Biophysics, Eidgenössischen
Technischen
you can see where the research money went here ;
Tuesday, June 25, 2013
Emerging Infectious Diseases (-$2.425 million) This request includes funds
to focus on necessary activities for prion disease
A FOOLISH MOVE BY THE GOVERNMENT...TSS
TSS
posted by Terry S. Singeltary Sr. at
1:17 PM
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