Flexible N-terminal Region of Prion Protein Influences Conformation of Protease-resistant Prion Protein Isoforms Associated with Cross-species Scrapie Infection in Vivo and in Vitro

Lawson, Victoria; Priola, Suzette A.; Meade-White, Kimberly; Lawson, McKinley; Chesebro, Bruce

doi:10.1074/jbc.m303697200

Cited by 60 publications

(65 citation statements)

References 47 publications

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“…Additional studies have also suggested that the N terminus is important in PrP C function and pathogenesis. The N terminus modifies disease phenotype (43) and influences the conformation and generation of PK-resistant PrP Sc (44,45) as well as rPrP aggregation (37).…”

Section: Discussionmentioning

confidence: 99%

Human prion proteins with pathogenic mutations share common conformational changes resulting in enhanced binding to glycosaminoglycans

Yin

Pham

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Mutation in the prion gene PRNP accounts for 10 -15% of human prion diseases. However, little is known about the mechanisms by which mutant prion proteins (PrPs) cause disease. Here we investigated the effects of 10 different pathogenic mutations on the conformation and ligand-binding activity of recombinant human PrP (rPrP). We found that mutant rPrPs react more strongly with N terminus-specific antibodies, indicative of a more exposed N terminus. The N terminus of PrP contains a glycosaminoglycan (GAG)-binding motif. Binding of GAG is important in prion disease. Accordingly, all mutant rPrPs bind more GAG, and GAG promotes the aggregation of mutant rPrPs more efficiently than wild-type recombinant normal cellular PrP (rPrP C ). Furthermore, point mutations in PRNP also cause conformational changes in the region between residues 109 and 136, resulting in the exposure of a second, normally buried, GAG-binding motif. Importantly, brainderived PrP from transgenic mice, which express a pathogenic mutant with nine extra octapeptide repeats, also binds more strongly to GAG than wild-type PrP C . Thus, several rPrPs with distinct pathogenic mutations have common conformational changes, which enhance binding to GAG. These changes may contribute to the pathogenesis of inherited prion diseases.genetics ͉ structure

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Section: Discussionmentioning

confidence: 99%

Human prion proteins with pathogenic mutations share common conformational changes resulting in enhanced binding to glycosaminoglycans

Yin

Pham

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…The construct used in the present work corresponds to the structured 111-residue C-terminal domain of the murine cellular prion protein and lacks the entire flexible N-terminal region, which has been implicated in transmissible spongiform encephalopathy transmission and in the formation of protease-resistant aggregates (21). Moreover, the segment comprising amino acid residues 106 -126 in full-length PrP represents the most conserved and hydrophobic region, is known to form neurotoxic aggregates in vitro, and is generally considered to form the amyloidogenic core of the protein (33).…”

Section: Discussionmentioning

confidence: 99%

“…Ref. 21). The N-terminal domain also contains amino acid sequences that are generally considered important in the control of aggregation (e.g.…”

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confidence: 99%

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Formation of Soluble Oligomers and Amyloid Fibrils with Physical Properties of the Scrapie Isoform of the Prion Protein from the C-terminal Domain of Recombinant Murine Prion Protein mPrP-(121–231)

Martins

Frosoni

Martínez

et al. 2006

Journal of Biological Chemistry

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Prion diseases are fatal neurodegenerative disorders associated with conformational conversion of the cellular prion protein, PrP C , into a misfolded, protease-resistant form, PrP Sc . Here we show, for the first time, the oligomerization and fibrillization of the C-terminal domain of murine PrP, mPrP-(121-231), which lacks the entire unstructured N-terminal domain of the protein. In particular, the construct we used lacks amino acid residues 106 -120 from the so-called amyloidogenic core of PrP (residues 106 -126). Amyloid formation was accompanied by acquisition of resistance to proteinase K digestion. Aggregation of mPrP-(121-231) was investigated using a combination of biophysical and biochemical techniques at pH 4.0, 5.5, and 7.0 and at 37 and 65°C. Under partially denaturing conditions (65°C), aggregates of different morphologies ranging from soluble oligomers to mature amyloid fibrils of mPrP-(121-231) were formed. Transmission electron microscopy analysis showed that roughly spherical aggregates were readily formed when the protein was incubated at pH 5.5 and 65°C for 1 h, whereas prolonged incubation led to the formation of mature amyloid fibrils. Samples incubated at 65°C at pH 4.0 or 7.0 presented an initial mixture of oligomers and protofibrils or fibrils. Electrophoretic analysis of samples incubated at 65°C revealed formation of sodium dodecyl sulfate-resistant oligomers (dimers, trimers, and tetramers) and higher molecular weight aggregates of mPrP-(121-231). These results demonstrate that formation of an amyloid form with physical properties of PrP Sc can be achieved in the absence of the flexible N-terminal domain and, in particular, of residues 106 -120 of PrP and does not require other cellular factors or a PrP Sc template.The conformational conversion of the normal cellular isoform of the prion protein, PrP C , 4 into an abnormal pathological isoform, PrP Sc , underlies a group of fatal neurodegenerative disorders known as transmissible spongiform encephalopathies or prion diseases, which includes Creutzfeldt-Jakob disease in humans, scrapie in sheep and goats, and bovine spongiform encephalopathy in cattle (1). Neuropathologically, prion diseases are characterized by neuronal loss and astrogliosis and often by spongiform degeneration of the brain and deposition of amyloid plaques (1). The unique feature of these diseases is that, in addition to sporadic and inherited forms, they may be acquired by transmission of an infectious agent. The "proteinonly" hypothesis of prion propagation postulates that the abnormal isoform, PrP Sc , acts as a transmissible agent of the disease and self-propagates its pathological conformation using PrP C as a substrate (1).PrP Sc is defined as an aggregated form of PrP that is largely resistant to proteinase K (PK) digestion under conditions in which PrP C and most other proteins are readily degraded (2). In addition to their different stabilities to proteolytic degradation, the secondary, tertiary, and quaternary structures of PrP C and PrP Sc also differ (3-7...

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“…High pressure liquid chromatography-purified synthetic single-stranded RNA fragment 43-59 of SAF93 aptamer (SAF93 [43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59] ) (22) and opRNA, a random 18-mer sequence, derived from a translational operator of MS2 bacteriophage (37) were obtained from Integrated DNA Technologies, Inc. (Coralville, IA). Nucleotide sequences were: SAF93 [43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59] , 5Ј-GGA UGC AAU CUC CAU CCC-3Ј; and opRNA, 5Ј-AAA CAU GGG UUC CCA UGU-3Ј. Synthetic RNA samples were maintained lyophilized at Ϫ20°C and used in RNase-free water.…”

Section: Methodsmentioning

confidence: 99%

Prion Protein Complexed to N2a Cellular RNAs through Its N-terminal Domain Forms Aggregates and Is Toxic to Murine Neuroblastoma Cells

Gomes¹,

Millen²,

Ferreira³

et al. 2008

Journal of Biological Chemistry

123

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Conversion of the cellular prion protein (PrP C ) into its altered conformation, PrPSc , is believed to be the major cause of prion diseases. Although PrP is the only identified agent for these diseases, there is increasing evidence that other molecules can modulate the conversion. We have found that interaction of PrP with double-stranded DNA leads to a protein with higher ␤-sheet content and characteristics similar to those of PrP Sc . RNA molecules can also interact with PrP and potentially modulate PrP C to PrP Sc conversion or even bind differentially to both PrP isoforms. Here, we investigated the interaction of recombinant murine PrP with synthetic RNA sequences and with total RNA extracted from cultured neuroblastoma cells (N2aRNA). We found that PrP interacts with N2aRNA with nanomolar affinity, aggregates upon this interaction, and forms species partially resistant to proteolysis. RNA does not bind to N-terminal deletion mutants of PrP, indicating that the N-terminal region is important for this process. Cell viability assays showed that only the N2aRNA extract induces PrP-RNA aggregates that can alter the homeostasis of cultured cells. Small RNAs bound to PrP give rise to nontoxic small oligomers. Nuclear magnetic resonance measurements of the PrP-RNA complex revealed structural changes in PrP, but most of its native fold is maintained. These results indicate that there is selectivity in the species generated by interaction with different molecules of RNA. The catalytic effect of RNA on the PrP C 3 PrP Sc conversion depends on the RNA sequence, and small RNA molecules may exert a protective effect.Prion diseases can be infectious, sporadic, or inherited (1). Regardless of their origin, they are related to modifications of a ubiquitous membrane-anchored protein, the prion protein (PrP).3 Through a poorly understood process, the cellular PrP isoform (PrP C ), an ␣-helix-rich protein, undergoes a profound conformational change, acquiring higher ␤-sheet content; the latter isoform is known as PrP Sc (Sc from scrapie) and is the only known component of the infectious prion particle (1-4).The protein-only hypothesis postulates that PrP Sc "multiplies" by catalyzing the conversion of PrP C into a likeness of itself, thus becoming responsible for its own propagation (5). This hypothesis is based strongly on the fact that PrP knock-out mice are resistant to prion infection, suggesting that endogenous PrP is necessary for prion propagation and infection (6). It was also suggested, however, that an additional unknown factor could influence the PrP C to PrP Sc conversion (7-10). This molecule would act by lowering the free energy barrier between PrP C and PrP Sc and triggering conversion (11,12). In this field, a great number of biological macromolecules have emerged as candidates for conversion catalysts. Cellular adhesion molecules, nucleic acids (NAs), basal membrane molecules, and sulfated glycans, among other biological macromolecules, have been reported to interact with PrP C and to induce its conversion in...

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Flexible N-terminal Region of Prion Protein Influences Conformation of Protease-resistant Prion Protein Isoforms Associated with Cross-species Scrapie Infection in Vivo and in Vitro

Cited by 60 publications

References 47 publications

Human prion proteins with pathogenic mutations share common conformational changes resulting in enhanced binding to glycosaminoglycans

Human prion proteins with pathogenic mutations share common conformational changes resulting in enhanced binding to glycosaminoglycans

Formation of Soluble Oligomers and Amyloid Fibrils with Physical Properties of the Scrapie Isoform of the Prion Protein from the C-terminal Domain of Recombinant Murine Prion Protein mPrP-(121–231)

Prion Protein Complexed to N2a Cellular RNAs through Its N-terminal Domain Forms Aggregates and Is Toxic to Murine Neuroblastoma Cells

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