In vitro cell-free conversion of bacterial recombinant PrP to PrPres as a model for conversion

Kirby, Louise; Birkett, Christopher R.; Rudyk, Hélène; Gilbert, Ian H.; Hope, James

doi:10.1099/vir.0.18903-0

Cited by 62 publications

(56 citation statements)

References 36 publications

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“…Conversion of recombinant PrP in the assay is also inhibited by known anti-prion drugs. 19 By use of the CFCA, we have recently published two studies 20,21 that appear unrelated, but which taken together suggest that partially stabilizing the structure of PrP C in a form that is amenable for conversion to PrP Sc is a potential role for prion cofactors. Our data agree with and extend previous observations on the balance between different misfolding pathways of PrP and the role of key areas of secondary structure of PrP C in directing conversion.…”

Section: Structural Requirements For Efficient Prion Protein Conversionmentioning

confidence: 99%

Structural requirements for efficient prion protein conversion

Gill¹,

Agarwal

Pinheiro

et al. 2010

Prion

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T o understand why cross-species infection of prion disease often results in inefficient transmission and reduced protein conversion, most research has focused on defining the effect of variations in PrP primary structures, including sequence compatibility of substrate and seed. By contrast, little research has been aimed at investigating structural differences between different variants of PrP C and secondary structural requirements for efficient conversion. This is despite a clear role for molecular chaperones in formation of prions in non-mammalian systems, indicating the importance of secondary/tertiary structure during the conversion process. Recent data from our laboratory on the cellular location of disease-specific prion cofactors supports the critical role of specific secondary structural motifs and the stability of these motifs in determining the efficiency of disease-specific prion protein conversion. In this paper we summarize our recent results and build on the hypothesis previously suggested by Wuthrich and colleagues, that stability of certain regions of the prion protein is crucial for protein conversion to abnormal isoforms in vivo. It is suggested that one role for molecular cofactors in the conversion process is to stabilize PrP C structure in a form that is amenable for conversion to PrP Sc .

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Section: Structural Requirements For Efficient Prion Protein Conversionmentioning

confidence: 99%

Structural requirements for efficient prion protein conversion

Gill¹,

Agarwal

Pinheiro

et al. 2010

Prion

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show abstract

“…10,11 Furthermore, recombinant PrP-sen derived from E. coli, which has no membrane anchor or glycosylation, can be induced to form proteaseresistant PrP in vitro when reacted with prion-infected brain homogenates. [12][13][14] Finally, in at least one instance, proteaseresistant recombinant PrP-res generated in ArticLe Addendum commentAry & View (Mo3F4 GPI -PrP-res) was detected within 96 hours indicating that acute PrP-res formation had occurred. 17 Thus, despite the fact that Mo3F4 GPI -PrP-sen is not expressed on the cell surface 16 ( Fig.…”

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confidence: 99%

The role of the prion protein membrane anchor in prion infection

Priola

McNally

2009

Prion

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“…By mixing together PrP Sc and recPrP expressed in mammalian cell lines to result in newly protease resistant PrP (PrP Res ) the group of Byron Caughey showed that PrP Sc can auto-catalytically seed the conformational conversion of recPrP (Kocisko et al, 1994). This technique was termed the cell free conversion assay (CFCA) and it was subsequently shown to mimic many aspects of disease seen in vivo, including species barriers (Kirby et al, 2003, Kocisko et al, 1995 and the inhibitory effects of specific chemicals , Demaimay et al, 2000, Demaimay et al, 1998. Quantifying conversion efficiency allows insights into mechanistic aspects of conversion: for example, there are two distinct phases of prion protein conversion -binding followed by conformational alteration (Horiuchi & Caughey, 1999) -and single amino acid substitutions were shown to dramatically affect the efficiency of conversion of the substrate (Bossers et al, 1997, Eiden et al, 2011, Kirby et al, 2006.…”

Section: Misfolding Of Prp Can Be Modelled In Vitromentioning

confidence: 99%