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Introducing a Rigid Loop Structure from Deer into Mouse Prion Protein Increases Its Propensity for Misfolding In Vitro

Posted Jun 27 2013 2:17pm
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
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