RNA molecules stimulate prion protein conversion

Deleault, Nathan R.; Lucassen, Ralf; Supattapone, Surachai

doi:10.1038/nature01979

Cited by 464 publications

(452 citation statements)

References 13 publications

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“…1 Synthetic nucleic acids, in solution and in vitro, can catalyze conversion of recombinant and cellular PrP C to PrP Sc as evidenced from secondary structural studies of the protein and proteinase K (PK) resistance properties. [4][5][6][7] Our studies also indicated that bending and unwinding of nucleic acid occurred by prion protein and natural poly amines (spermine and spermidine) can inhibit this interaction. 8,9 Highly structured small RNA binds to PrP C at neutral pH and yields Proteinase K resistant component in the presence of cellular cofactors.…”

Section: Introductionsupporting

confidence: 52%

“…6 Studies using brain tissues have shown that an endogenous 300 nucleotide long RNA (100 kDa) can convert endogenous PrP C to Proteinase K resistant form in vitro. 7,10,11 These results indicate that nucleic acid can act as a cofactor for the conversion of PrP C to PrP Sc and can be the active transmissible spongiform encephalopathy (TSE) agent. In addition, the interaction between recombinant PrP C and nucleic acids (DNAs, tRNA, and PolyA) simultaneously produces a mixture of condensed and functionally active nucleoprotein complex, PrP Sc oligomers and linear and spherical amyloids.…”

Section: Introductionmentioning

confidence: 99%

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Nucleic acid induced unfolding of recombinant prion protein globular fragment is pH dependent

Bera

Nandi

2014

Protein Science

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Nucleic acid can catalyze the conversion of a-helical cellular prion protein to b-sheet rich Proteinase K resistant prion protein oligomers and amyloid polymers in vitro and in solution. Because unfolding of a protein molecule from its ordered a-helical structure is considered to be a necessary step for the structural conversion to its b-sheet rich isoform, we have studied the unfolding of the a-helical globular 121-231 fragment of mouse recombinant prion protein in the presence of different nucleic acids at neutral and acid pH. Nucleic acids, either single or double stranded, do not have any significant effect on the secondary structure of the protein fragment at neutral pH; however the protein secondary structure is modified by the nucleic acids at pH 5. Nucleic acids do not show any significant effect on the temperature induced unfolding of the globular prion protein domain at neutral pH which, however, undergoes a gross conformational change at pH 5 as evidenced from the lowering of the midpoint of thermal denaturation temperatures, Tm, of the protein. The extent of Tm decrease shows a dependence on the nature of nucleic acid. The interaction of nucleic acid with the nonpolar groups exposed from the protein interior at pH 5 probably contributes substantially to the unfolding process of the protein.

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Section: Introductionsupporting

confidence: 52%

Section: Introductionmentioning

confidence: 99%

Nucleic acid induced unfolding of recombinant prion protein globular fragment is pH dependent

Bera

Nandi

2014

Protein Science

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“…PMCA has been reported previously to increase the sensitivity of the detection of PrP Sc from brains of experimentally scrapie-infected rodents (Saborio et al, 2001;Bieschke et al, 2004;Deleault et al, 2003), cattle and sheep naturally infected with bovine spongiform encephalopathy and scrapie, respectively (Soto et al, 2005), and more recently from humans with Creutzfeldt-Jakob disease (Jones et al, 2007) and deer with chronic wasting disease (Kurt et al, 2007). Furthermore, the application of this technology for the detection of PrP Sc in blood, both at terminal stages of disease and in pre-symptomatic animals Fig.…”

Section: Discussionmentioning

confidence: 99%

In vitro amplification of PrPSc derived from the brain and blood of sheep infected with scrapie

Thorne

Terry

2008

Journal of General Virology

101

116

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Scrapie is a fatal, naturally transmissible, neurodegenerative prion disease that affects sheep and goats and is characterized by the accumulation of a misfolded protein, PrPSc, converted from host-encoded PrPc, in the central nervous system of affected animals. Highly efficient in vitro conversion of host PrPc to PrPSc has been achieved in models of scrapie and in natural prion diseases by protein misfolding cyclic amplification (PMCA). Here, we demonstrate amplification, by serial PMCA, of PrPSc from individual sources of scrapie-infected sheep. Efficiency of amplification was affected by the pairing of the source of PrPSc with the control brain substrate of different genotypes of PrP. In line with previous studies, efficiency of amplification was greatly enhanced with the addition of a synthetic polyanion, polyadenylic acid (PolyA), facilitating rapid detection of low levels of PrPSc from body fluids such as blood. To this end PrPSc was amplified, in a 3 day PMCA assay, from blood leukocyte preparations from VRQ/VRQ scrapie-affected sheep at clinical end point. While PolyA-assisted PMCA resulted in spontaneous conversion of PrPc, we were able to distinguish blood samples from unaffected and affected sheep under controlled conditions. This study demonstrates that highly efficient amplification of PrPSc can be achieved for ovine scrapie from both brain and blood from naturally infected sheep and shows potential applications for improvements in current diagnostics and pre-mortem testing.

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“…4a,b). These results indicate that the presence of endogenous bovine PrP C is essential for PrP BSE propagation in vitro and that any other host-derived cellular cofactors included in the brain homogenates, such as RNA 19 and sulfated glycosaminoglycan 20 , cannot support the in vitro PrP BSE propagation in the absence of endogenous bovine PrP C . We also performed a similar PMCA assay using a brain homogenate from cattle infected with transmissible mink encephalopathy (TME), another prion strain infectious to cattle, as inoculum and detected no propagation of the PrP TME (Fig.…”

mentioning

confidence: 91%

Production of cattle lacking prion protein

et al. 2006

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Prion diseases are caused by propagation of misfolded forms of the normal cellular prion protein PrP C , such as PrP BSE in bovine spongiform encephalopathy (BSE) in cattle and PrP CJD in Creutzfeldt-Jakob disease (CJD) in humans 1 . Disruption of PrP C expression in mice, a species that does not naturally contract prion diseases, results in no apparent developmental abnormalities [2][3][4][5] . However, the impact of ablating PrP C function in natural host species of prion diseases is unknown. Here we report the generation and characterization of PrP C -deficient cattle produced by a sequential gene-targeting system 6 . At over 20 months of age, the cattle are clinically, physiologically, histopathologically, immunologically and reproductively normal. Brain tissue homogenates are resistant to prion propagation in vitro as assessed by protein misfolding cyclic amplification 7 . PrP C -deficient cattle may be a useful model for prion research and could provide industrial bovine products free of prion proteins.To generate PrP C -deficient (PRNP −/− ) cattle, we transfected a male Holstein primary fetal fibroblast line 6594 with first and second knockout (KO) vectors (pBPrP(H)KOneo and pBPrP (H)KOpuro vectors) 6 to sequentially disrupt the two alleles of PRNP. PRNP −/− fetal cell lines were established at 40-60 d of gestation and three of the PRNP −/− fetal cell lines (5211, 5232 and 4296) were recloned to produce calves (Table 1 and Fig. 1a). To verify that the calves possess the PRNP −/− genotype, we collected ear biopsies and established fibroblast cell lines for genotyping. Genotyping was done by genomic PCR specific to each gene targeting event 6 (primer pairs: neoF7 × neoR7 and puroF14 × puroR14, Fig. 1b COMPETING INTERESTS STATEMENTThe authors declare competing financial interests (see the Nature Biotechnology website for details).Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/ NIH Public Access Fig. 1c) and confirmed the disruption of PRNP-specific mRNA expression in PRNP −/− calves. For protein expression analysis, we performed PrP-specific western blot analyses on fibroblasts (Fig. 1d), peripheral blood lymphocytes (Fig. 1e) and brain stem (Fig. 1f) from wild-type and PRNP −/− calves using the mouse anti-bovine PrP monoclonal antibody F89. We detected PrP-specific bands in the wild-type calves, whereas no reaction was observed in PRNP −/− calves and negative control mouse fibroblasts. These data clearly demonstrate that the PRNP gene is functionally inactivated in the PRNP −/− calves.PRNP −/− cattle were monitored for growth and general health status from birth to 20 months of age. Mean birth weight was 46 kg and average daily gain was 0.91 kg/d to 10 months. Both values were in the normal range for Holstein bulls. Serum chemistry was evaluated at 6 months of age and compared with published reference ranges. All the values for PRNP −/− calves (n = 12) were well within the reference range (Supplementary Table 1) and obvious abnormalities w...

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RNA molecules stimulate prion protein conversion

Cited by 464 publications

References 13 publications

Nucleic acid induced unfolding of recombinant prion protein globular fragment is pH dependent

Nucleic acid induced unfolding of recombinant prion protein globular fragment is pH dependent

In vitro amplification of PrPSc derived from the brain and blood of sheep infected with scrapie

Production of cattle lacking prion protein

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