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Biochemical characterization of prion conversion.

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Call No.	QR201.P737 N57 2006
Author	Nishina, Koren A.
Title	Biochemical characterization of prion conversion.
Date	2006
Abstract	Prion diseases are infectious neurodegenerative disorders characterized by a conformational change of the host-encoded prion protein. The normal nonpathogenic isoform of the prion protein (PrPc) is primarily composed of α-helices and very low β-sheet content. Conversely, the infectious conformer (PrP Sc ) has high β-sheet content and lowered levels of α-helices. In cases of disease, PrP C is converted to PrP Sc through a conformational change. The mechanism of this structural conversion, however, remains unknown. The development of the in vitro protein misfolding cyclic amplification (PMCA) assay allows for analysis of PrP C to PrP Sc conversion. While biochemical purification is the most direct strategy to identify the components needed for conversion, it first requires generating a soluble preparation capable of supporting prion conversion. By screening detergents for efficient solubilization of membrane-bound PrP C and for compatability in the PMCA reaction, I was able to demonstrate that contiguous membranes are not required for the amplification reaction. This finding indicated that PrP C could be purified and utilized to further characterize prion conversion. Differences in protease sensitivity biochemically distinguish the two conformers of prion protein: PrP C is protease sensitive while PrP Sc is protease resistant. Typically, protease resistance of PrP Sc is associated with infection; this trait is consequently exploited by current disease detection techniques. However, I have discovered that the protease resistance of PrP Sc is dependant on the presence of physiological levels of divalent cations (copper and zinc) or high ionic strength buffers. Without the divalent cations or in low ionic strength conditions, PrP Sc becomes protease sensitive. This demonstrates that cations can affect protease sensitivity of PrP Sc , possibly by altering structure. Finally, different strains of prions have distinct pathological and biochemical characteristics that are maintained through subsequent passages in animals. One of the distinctive characteristics of strains is differential ratios of PrP Sc glycoforms. Yet, it is not known how differential glycosylation of PrP C affects prion conversion. Using PMCA and a purification technique that isolates natively processed, wild type PrP C glycoforms, I provide the first direct evidence that distinct glycoform ratios of PrP C influences strain specificity in prion conversion.
Description	x, 173 leaves : ill. (some col.) 28 cm.
Format	application/pdf
Advisor	William T. Wickner.
Subject	Prion diseases. Prions. Proteolytic enzymes -- Pathophysiology. Proteins -- Conformation.
College	Dartmouth Medical School -- Department of Biochemistry.
Issuing Body	Dartmouth Dissertations are made available through the Dartmouth Digital Library.
Degree	Thesis (Ph.D.)--Dartmouth College.
Copyright	All rights reserved. No part of this book may be reproduced in any form by any electronic or mechanical means without permission in writing from the author.
DOI	http://dx.doi.org/10.1349/ddlp.107

Title	Page 1
Format	application/pdf
Issuing Body	Dartmouth Dissertations are made available through the Dartmouth Digital Library.
Degree	Thesis (Ph.D.)--Dartmouth College.
Full Text	Biochemical Characterization of Prion Conversion A Thesis submitted to the Faculty in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry by Koren A. Nishina DARTMOUTH COLLEGE Hanover, New Hampshire April 20, 2006 Examining committee Charles K. Barlowe, Ph.D. Dean of Graduate Studies William T. Wickner, M.D. (Chair) Surachai Supattapone, M.D., Ph.D., D.Phil. John Hwa, M.D., Ph.D. Marc Diamond, M.D.

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Biochemical characterization of prion conversion.

Biochemical characterization of prion conversion.