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Biochemical studies of prion composition and conversion.

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Call No.	QR502 .G46 2008
Author	Geoghegan, James C.
Title	Biochemical studies of prion composition and conversion.
Date	2008
Abstract	Prion diseases are fatal neurodegenerative disorders which affect a variety of different animal species including humans. The central pathogenic event underlying prion diseases is the conformational conversion of the cellular prion protein (PrP C ) into a disease-associated isoform (PrP Sc ). PrP C is characterized by extensive α-helical structure, solubility, and sensitivity to protease digestion. In contrast, PrP Sc contains primarily β-sheet structure, is insoluble, and is protease-resistant. PrP Sc is also self-propagating, but the precise molecular mechanism by which PrP C converts to form nascent PrP Sc is unknown. Several biochemical conversion assays have been developed to study the requirements for the conformational conversion of PrP C into PrP Sc. Utilizing an in vitro PrP Sc amplification assay, I demonstrated that partially purified PrP C , template PrP Sc , and synthetic polyanions can support the formation of new PrP Sc. This finding supported the hypothesis that these three molecules alone might be the minimal components required for the formation of PrP Sc. In subsequent studies using the protein-misfolding cyclic amplification (PMCA) assay, the generation of infectious hamster prions (HaPrP Sc ) in vitro required only purified hamster PrP C (HaPrP C ), co-purified lipids, and synthetic poly(A) RNA. Using PMCA, I showed that HaPrP C selectively incorporates poly(A) RNA molecules into a nuclease-resistant nucleo-protein complex during the formation of HaPrP Sc. The incorporation of poly(A) RNA molecules induces a conformational change in HaPrP C , resulting in the formation of an intermediate which resembles HaPrP Sc. These findings suggest that a polyanionic co-factor may act as a molecular chaperone to assist the generation of PrP Sc and may be a component of the infectious prion. Dominant negative PrP mutants are PrP molecules which can inhibit the process of prion replication. It was hypothesized that an unidentified co-factor, termed protein X, mediates the dominant negative property of these PrP mutants. Using recombinant hamster and mouse PrP (rPrP), and the PMCA assay, I investigated the mechanism of prion inhibition by dominant negative rPrP mutants in vitro. My findings indicate that both mouse and hamster rPrP retain dominant negative properties in vitro and that direct interaction between molecules of wild-type and mutant PrP, and not interaction with the putative protein X co-factor, underlies the mechanism of dominant negative inhibition.
Description	xi, 184 leaves : ill. (some col.) 28 cm.
Format	application/pdf
Advisor	Surachai Supattapone
Subject	Prions. Proteins -- Conformation. Prions -- physiology. Prions -- genetics.
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.195

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 Studies of Prion Composition and Conversion A Thesis Submitted to the Faculty in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry by James C. Geoghegan DARTMOUTH COLLEGE Hanover, New Hampshire May 13th, 2008 Examining Committee: _____________________________________ Dean Madden, Ph.D. (Chair) _____________________________________ Surachai Supattapone, M.D., Ph.D., D.Phil. _____________________________________ Victor Ambros, Ph.D. _____________________________________ Jiyan Ma, Ph.D. ________________________ Charles K. Barlowe, Ph.D. Dean of Graduate Studies

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Biochemical studies of prion composition and conversion.

Biochemical studies of prion composition and conversion.