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Tài liệu Báo cáo khoa học: The elusive intermediate on the folding pathway of the prion protein pptx
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Tài liệu Báo cáo khoa học: The elusive intermediate on the folding pathway of the prion protein pptx

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The elusive intermediate on the folding pathway

of the prion protein

David C. Jenkins, Ian D. Sylvester and Teresa J. T. Pinheiro

Department of Biological Sciences, University of Warwick, UK

Prion diseases, which include Creutzfeldt–Jakob dis￾ease in humans, bovine spongiform encephalopathy in

cattle and scrapie in sheep, are associated with conver￾sion of the normal cellular form of the prion protein

(PrPC) to an altered pathological form, generally desig￾nated as the scrapie isoform (PrPSc). Such diseases can

be sporadic, inherited or acquired by transmission.

Sporadic Creutzfeldt–Jakob disease accounts for 85%

of all cases of the disease; around 10–15% are associ￾ated with the familial cases and fewer than 5% are

transmitted [1].

Although coded for by the same gene [2,3] and

covalently identical [4], the structure and properties of

PrPC and PrPSc contrast greatly. Whereas PrPC is a

globular protein, composed primarily of an unstruc￾tured N-terminal region and a C-terminal domain

comprising three a-helices and two short b-strands

(Fig. 1) [5–8], PrPSc has a much higher proportion of

b-sheet structure [9]. Physicochemical studies have

shown that PrPC is monomeric, soluble in aqueous

buffer and sensitive to protease digestion, while, in

comparison, PrPSc has a high propensity to aggregate,

is water- and detergent-insoluble, and partially resis￾tant to proteinases [2,10,11].

The details of prion conversion are not fully under￾stood, but it is generally accepted that the key mole￾cular event in sporadic and familial cases of prion

diseases involves a conformational transition of the

prion protein from its cellular state to the altered dis￾ease-associated form [12]. Thus, an understanding of

the folding and refolding mechanisms of the prion pro￾tein should provide insight into the process of prion

conversion, and represent a major step forward in our

understanding of the misfolding and aggregation of

PrP during disease.

Studies of the folding kinetics of PrP have indicated

that it may fold via an intermediate state [13–15], and

that this intermediate may, under as yet uncharacter￾ized conditions, be recruited to form PrPSc. Results

from equilibrium folding studies have been ambiguous

on defining the presence of an intermediate in the fold￾ing of PrP. Initial studies apparently revealed a folding

intermediate rich in b-structure [16], later shown to be

off-pathway aggregated species in the folding of PrP

Keywords

denaturant unfolding; molten globule; phase￾diagram; prion conversion; prion diseases

Correspondence

T. J. T. Pinheiro, Department of Biological

Sciences, Gibbet Hill Road, University of

Warwick, Coventry CV4 7AL, UK

Fax: +44 2476 523 701

Tel: +44 2476 528 364

E-mail: t.pinheiro@warwick.ac.uk

(Received 14 August 2007, revised 20

December 2007, accepted 15 January 2008)

doi:10.1111/j.1742-4658.2008.06293.x

A key molecular event in prion diseases is the conversion of the cellular

conformation of the prion protein (PrPC) to an altered disease-associated

form, generally denoted as scrapie isoform (PrPSc). The molecular details

of this conformational transition are not fully understood, but it has been

suggested that an intermediate on the folding pathway of PrPC may be

recruited to form PrPSc. In order to investigate the folding pathway of PrP

we designed and expressed two mutants, each possessing a single strategi￾cally located tryptophan residue. The secondary structure and folding

properties of the mutants were examined. Using conventional analyses

of folding transition data determined by fluorescence and CD, and novel

phase-diagram analyses, we present compelling evidence for the presence of

an intermediate species on the folding pathway of PrP. The potential role

of this intermediate in prion conversion is discussed.

Abbreviations

PrP, prion protein; SHaPrP, Syrian hamster prion protein.

FEBS Journal 275 (2008) 1323–1335 ª 2008 The Authors Journal compilation ª 2008 FEBS 1323

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