Prion protein self-peptides modulate prion interactions and conversion
© Rigter et al; licensee BioMed Central Ltd. 2009
Received: 27 July 2009
Accepted: 30 November 2009
Published: 30 November 2009
Molecular mechanisms underlying prion agent replication, converting host-encoded cellular prion protein (PrPC) into the scrapie associated isoform (PrPSc), are poorly understood. Selective self-interaction between PrP molecules forms a basis underlying the observed differences of the PrPC into PrPSc conversion process (agent replication). The importance of previously peptide-scanning mapped ovine PrP self-interaction domains on this conversion was investigated by studying the ability of six of these ovine PrP based peptides to modulate two processes; PrP self-interaction and conversion.
Three peptides (octarepeat, binding domain 2 -and C-terminal) were capable of inhibiting self-interaction of PrP in a solid-phase PrP peptide array. Three peptides (N-terminal, binding domain 2, and amyloidogenic motif) modulated prion conversion when added before or after initiation of the prion protein misfolding cyclic amplification (PMCA) reaction using brain homogenates. The C-terminal peptides (core region and C-terminal) only affected conversion (increased PrPres formation) when added before mixing PrPC and PrPSc, whereas the octarepeat peptide only affected conversion when added after this mixing.
This study identified the putative PrP core binding domain that facilitates the PrPC-PrPSc interaction (not conversion), corroborating evidence that the region of PrP containing this domain is important in the species-barrier and/or scrapie susceptibility. The octarepeats can be involved in PrPC-PrPSc stabilization, whereas the N-terminal glycosaminoglycan binding motif and the amyloidogenic motif indirectly affected conversion. Binding domain 2 and the C-terminal domain are directly implicated in PrPC self-interaction during the conversion process and may prove to be prime targets in new therapeutic strategy development, potentially retaining PrPC function. These results emphasize the importance of probable PrPC-PrPC and required PrPC-PrPSc interactions during PrP conversion. All interactions are probably part of the complex process in which polymorphisms and species barriers affect TSE transmission and susceptibility.
Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative disorders characterized by accumulation of the pathological isoform of prion protein mainly in tissues of the central nervous system. Formation of this pathological isoform is a posttranslational process and involves refolding (conversion) of the host-encoded prion protein (PrPC) into a pathological isoform partially protease resistant PrPSc (derived from scrapie) or PrPres (PK-resistant PrP) . The molecular mechanisms involved in PrPC to PrPSc conversion are poorly understood, but polymorphisms in both PrP isoforms have been shown to be of importance in both interspecies and intraspecies transmissibilities . The formation of PrPSc aggregates probably requires self-interactions of PrPC molecules as well as with PrPSc [3, 4]. Thus binding and conformational changes are essential events in this conversion process. Cell-free conversion of PrPC provides a valuable in vitro model in which relative amounts of produced PrPres reflect important biological aspects of TSEs at the molecular level [5, 6]. A recent and very sensitive in vitro conversion system is the protein misfolding cyclic amplification (PMCA) assay [7–10], which has been shown to amplify minute amounts of PrPSc from a variety of sources including sheep scrapie . The effects of single polymorphisms and species-barriers in PrPC or PrPSc on PrP conversion can largely explain differences in susceptibility -and transmissibility in sheep scrapie [5, 11–13]. Even though these polymorphisms are involved in modulation of disease development they do not seem to affect the initial binding of PrPC to PrPSc  and do not seem to directly modulate PrPC-PrPSc binding. Furthermore, in a recent peptide-array mapping study of ovine PrPC we concluded that these polymorphisms are not part of the identified PrP binding domains likely to be involved in PrP self-interaction . However, this does not exclude these polymorphisms from posing indirect effects on binding behaviour of PrPC to PrPSc and other possible chaperoning molecules. In that peptide-array binding study we unequivocally demonstrated that ovine PrP binds with PrP derived (self) amino acid sequences (sequence specific) separate from the polymorphic scrapie susceptibility determinants . It remains to be elucidated whether the determined amino acid sequences play a role prior or during conversion in the self-interaction of PrPC molecules and/or in the interactions of PrPC with PrPSc. Simultaneously, whether these amino acid sequences play a role in the processes underlying PrP conversion needs to be elucidated. In the current study we selected several ovine PrP sequence derived synthetic peptides to study not only their capacity to affect PrP binding to a solid-phase (PrP) peptide-array but also their potential modulating effect on PrPC to PrPSc conversion.
Peptide information and peptide-array blocking results
amino acid sequence
PrP peptide inhibition of PrP self-binding to peptide-array
First these six peptides were tested for their capability to inhibit PrP binding to the PrP based peptide-array containing 242 peptides (15-mer) overlapping each other by increments of 1 AA, covering the complete ovine PrP amino acid sequence (results summarized in Table 1).
The capability of the N-terminal peptide NTG to moderately block binding of PrP to the peptide-array and also moderately modulated PrPres formation in the supplemented -and pre-incubated PMCA assay (described below) necessitates re-evaluation of the previously determined consensus domain [33-GWNTG-37] . Instead of this common consensus domain two binding domains seem present in the N-terminus. Binding motifs determined by motif-grafted antibodies  suggests that the domain of interest is [27-RPKPGGG-33], which encompasses most of the proposed glycosaminoglycan binding motif [25-KKRPK-29] .
These results confirm the importance of the previously mapped domains  located within the first high binding area [PHGG] (octarepeat) and [102-WNK-104], as well as the importance of the C-terminal low binding domain [192-TTTTKGENFT-202] in PrP self-interaction. To a lesser extent the involvement of the N-terminal glycosaminoglycan binding motif contained within domain [27-RPKPGGG-33] and amyloidogenic motif [116-AGAAAAGA-123] in interaction is confirmed. Interestingly peptide CO, encompassing the previously mapped domain [140-PLIHFGNDYE-149] did not influence binding of PrP to the peptide-array.
PrP peptides modulation of PrPres formation in the PMCA-assay
The peptides analyzed in the prion protein peptide-array were also studied for their modulating capacity in the sheep PrP protein misfolding cyclic amplification (PMCA) assay [9, 10, 43] using sheep brain homogenates from confirmed scrapie-positive and scrapie-negative sheep in one round of sonication cycles. To test the influence of the peptides on conversion, peptides were either added after combining the scrapie positive -and negative brain homogenates (peptide supplemented PMCA) or alternatively peptide was added first to the scrapie-negative brain homogenate before addition of the scrapie-positive material (peptide pre-incubated PMCA). This allowed us to assess if the effect of the peptides on conversion was dependant on the first rapid interaction between PrPC and PrPSc  or not. Peptide was added in several molar ratios, relative to the calculated total amount of PrPC present in the reaction. PrPSc specific proteinase-K (PK) resistant fragments were quantified by Western blotting.
Peptide supplemented PMCA
Peptide pre-incubated PMCA
Peptide modulation of supplemented -and pre-incubated PMCA assay
PMCA assay negative controls
Even though each PMCA assay setup revealed at least one PrP specific peptide incapable of modulating PrPres formation, additional negative controls were performed. To rule out the possibility that factors other than PrP sequence specificity could be responsible for the observed results, the isoelectric point, net charge and average hydrophilicity were determined for each peptide (data not shown). Comparison revealed no clear correlation between these non-sequential features and the observed effects in the PMCA assay. Therefore the following additional negative controls were performed; In order to determine whether addition of just a random peptide is sufficient for modulating PrPres formation an unrelated peptide (canine parvo virus specific sequence peptide DGAVQPDGGQPAVRNER) was used in both testing setups described above. Addition of this peptide did not affect PrPres formation in either of the two PMCA assay setups (data not shown) at various concentrations, indicating that the observed increases in PrPres were a result of the specific PrP derived peptide amino acid sequences added to the reactions. Furthermore, PMCA assays were also performed for each peptide without scrapie positive homogenate, to determine whether de novo PrPres could be formed when the PrP derived peptide was combined with PrPC. Peptide was added at the optimum (in the supplemented PMCA) molar excess for peptide NTG (1.000), OR (500) and AM (5.000). While peptides TD2, CO and CT were added at the highest molar excess (25.000) used in the in the PMCA assays described above. No significant conversion induced by either of these peptides was detected after PK digestion (data not shown), showing that only addition of scrapie-positive brain homogenate resulted in initiation of the conversion reaction.
Ovine peptide-array analysis previously revealed two high binding areas within PrP  of which the first high binding area encompasses the octarepeats, more specifically the consensus domain P(H)GG. This study showed that the octarepeat peptide (OR) was capable of blocking the binding pattern of PrP to the peptide-array, probably as a result of peptide-induced changes in the tertiary structure of the N-terminal tail and thus affecting PrPC self-interaction. Only in the supplemented PMCA assay, a dose dependant and significant increase in PrPres is observed. The octarepeats can modulate [51–55] but are not a necessity for the molecular processes underlying conversion [21, 56]. Interaction between PrPC and PrPSc seems almost instantaneous , which would leave the peptide free to interact with the PrPC - PrPSc complex as a whole or with co-factors present in the homogenates. Because the octarepeat stabilizes the interaction of PrPC with the LRP-LR receptor , it seems that peptide OR indirectly affects the conversion process, either by affecting PrPi stability/formation (Figure 7B) or by stabilizing PrPi interaction with PrPSc (Figure 7C). Furthermore, a di-peptide containing the octarepeat self-aggregates into nanometric fibrils  and these may also be formed in the supplemented PMCA assay. Combined with our data, we propose that the flexible N-terminal tail containing the octarepeat region stabilizes PrPC-PrPSc interaction during conversion and that free peptide OR forms nanometric fibrils mimicking and increasing PrPC-PrPSc stabilization, thus aiding subsequent conversion. Also part of the peptide-array first high binding area is binding domain 2 ([102-WNK-104], Figure 1) and the data presented here shows that pre-incubation of the peptide TD2 (containing [102-WNK-104]) with PrP abolished binding of PrP to the peptide-array. This indicates the importance of this domain in PrPC self-interaction (Figure 7A and possibly 7B). Increased PrPresproduction was observed in both the supplemented -and the pre-incubated PMCA-assay. The mechanism by which the peptide stimulates PrPres formation may simply be due to peptide enhanced interaction between separate PrP molecules. Alternatively, peptide TD2 could aid unfolding and/or refolding of PrPC during conversion; binding domain 2 [102-WNK-104], together with the amyloidogenic motif, is part of the region of PrPC that is partially unfolded and refolded during oligomerization of PrPC into a β-sheet-rich soluble isoform of PrP .
The second high binding area in the peptide-array contains the domain [140-PLIHFGNDY-148] (domain 3, Figure 1). This study shows that the peptide CO containing the domain [140-PLIHFGNDY-148] does not affect binding of PrP to the peptide-array at all, thereby ruling out direct involvement in PrPC self-interaction. Several studies have established that polymorphisms at sheep PrP amino acid position 136, 154 and 171 surrounding [140-PLIHFGNDY-148] are most relevant in differential TSE susceptibility [5, 59–63] and that stability of this PrP region is a crucial determinant in whether PrPC is converted [40, 64] (affecting species-barrier and/or scrapie susceptibility). Therefore peptide CO induced PrPres formation in the pre-incubated PMCA assay is either due to this core region peptide facilitating binding of PrPC to PrPSc or the peptide affects the stability of this region of PrPC (interacting with the 'self-domain' or another domain of PrP) thereby facilitating refolding of PrP.
The N-terminal peptide NTG (containing the glycosaminoglycan binding motif) only moderately affects binding of PrPC throughout the peptide-array, suggesting that interaction of the peptide with PrPC results either in slight changes in the tertiary structuring affecting solubility of PrPC or in diminished availability of the previously determined domains  for interaction with the peptide-array. Intriguingly, peptide NTG induces PrPres formation in both the supplemented (dose dependant) and pre-incubated (dose optimum) PMCA assay. The glycosaminoglycan heparan sulphate proteoglycan (HSPG) and pentosan polysulphate (PPS) stimulate PrPres formation in vitro and suggests that free glycosaminoglycans acted as a contact-mediator allowing interaction of PrPC and PrPSc . The N-terminal peptide NTG likely indirectly affects in vitro conversion either by mimicking glycosaminoglycan binding to domain [27-RPKPGGG-33] or by recruiting glycosaminoglycans onto PrPC, facilitating conversion of PrPC into new PrPSc after seeding. These studies and our PMCA assay data strongly implicate glycosaminoglycans as an important cofactor in the conversion process.
The ability of peptide AM, which encompasses the amyloidogenic motif [116-AGAAAAGA-123], to moderately block binding of PrP to the peptide-array was somewhat surprising, since we previously showed that the amyloidogenic motif was not involved in PrP self-interaction . Peptide AM mainly inhibits binding of PrP to the peptides covering the N-terminal part of the mature PrP protein, suggesting that peptide AM interacts with one (or more) of the other previously determined binding domains. In contrast to earlier reports [41, 66, 67], we observed that peptide AM (containing the amyloidogenic motif) slightly but significantly increased PrPres formation in both the pre-incubated -and supplemented PMCA. However, all these inhibiting peptides contained two or more additional amino acids of the putative aggregation sites (flanking the amyloidogenic motif) implicated in aggregation/oligomerisation , suggesting inhibition by these peptides is due to interference with aggregation/oligomerization. Additionally, differences between the used conversion systems (i.e. availability of cofactors) are likely to play a role as well. The peptide AM used in this study specifically focuses only on the amyloidogenic motif. Our data suggests that peptide AM interacts with the N-terminal tail of PrPC (octarepeat motif or [102-WNK-104]), probably altering its tertiary structure and facilitating the proposed stabilizing effect of the N-terminal tail. Alternatively, peptides containing only the amyloidogenic motif are also capable of forming a β-sheet rich layer at the water-air interface when sonicated  and peptide AM may form a β-sheeted backbone that interacts with the PrPC-PrPSc complex, mimicking and/or complementing the proposed stabilizing effect of the N-terminal tail.
Peptide CT overlaps most of the third alpha helix of PrPC as well as the second glycosylation site and the second cysteine involved in the di-sulphide bridge formed in PrPC. The capacity to completely block PrP binding to the peptide-array suggests that the domain [225-SQAY-228] is of importance in PrPC self-interaction. This study shows a slight significant increase in PrPres formation when peptide CT is pre-incubated with scrapie negative brain homogenate. This contradicts results using a similar peptide capable of inhibiting cell free conversion . However, this inhibiting peptide is four amino acids larger than peptide CT, which may account for the difference in effects and/or it may just be due to the differences in experimental technique between the cell free conversion and the PMCA assay. This seems to be corroborated by the observation that in the supplemented PMCA (setup closest resembling conditions in cell free conversion ) peptide CT seems to slightly inhibit PrPres formation albeit not significantly. Fibrillization of a human PrP peptide fragment is hindered by disulfide bridge formation between two peptides  or when an additional disulfide bridge is introduced , which indicates that peptide CT (when pre-incubated with PrPC) likely compromises the disulfide bridge, destabilizing PrPC, which consequently promotes trimerisation or formation of a conversion intermediate (Figure 7B) and thus facilitating conversion.
In the PMCA-assay all peptides revealed an inducing effect on PrPres formation in the supplemented -and/or pre-incubated PMCA-assay. Above possible explanations for these effects have been discussed for each peptide. However it can not be ruled out that the peptides may have had an opposite effect; instead of interacting with PrP, peptide may have interacted with possible conversion inhibitory factors present in the homogenate, thus indirectly allowing conversion to take place more efficiently. Identifying these possible 'natural' inhibitory factors may prove an alternative line of investigation towards the underlying mechanisms involved in prion replication and may provide additional targets for future prion therapy.
The binding domains found for ovine PrPC using a prion protein peptide-array are primarily indicative of prion protein self-interaction. Apparently several specific self-interactions between individual PrP molecules occur, which include both PrPC-PrPC as well as PrPC-PrPSc interactions. The data presented here imply an influence of binding domain [140-PLIHFGNDY-148] on the stability of the region of PrP previously determined to be involved in the species-barrier and/or susceptibility to scrapie. Furthermore our data indicates a stabilizing function for the octarepeats region (N-terminal tail) in PrPC-PrPSc interaction and thus improving subsequent conversion. Our data further suggests that the N-terminal glycosaminoglycan binding motif [27-RPKPGGG-33] affects the conversion process indirectly, and implicates glycosaminoglycans as an important cofactor in prion disease pathogenicity. Peptide AM containing the amyloidogenic motif indirectly affects conversion either by aiding and/or complementing the proposed stabilizing function of the N-terminal tail of PrPC. Finally, the data implicates direct involvement of the two binding domains [102-WNK-104] and [225-SQAY-228] in self-interaction between PrPC molecules preceding binding to PrPSc and subsequent conversion. Therefore these two domains may prove prime targets for development of new therapeutic strategies. Our results emphasize the importance of the stability of the PrPC-PrPC and PrPC-PrPSc interactions in PrP conversion, which is an essential determinant in the effects of disease associated mutations, as well as the species-barrier. Focussing on the (stabilizing) self-interaction domains of PrP and the subsequent conversion processes may lead to further therapeutic strategies with the possibility to leave the physiological function of the prion protein unaffected.
MBP-PrP construction, expression and purification
The mature part of sheep PrP (ARQ) open reading frame (ORF) was cloned into the pMAL Protein Fusion and Purification System (New England Biolabs) as described before , resulting in the maltose binding protein (MBP) fusion to the N-terminus of PrP (MBP-PrP). MBP-PrP was expressed and purified by affinity chromatography as described in the manual of the pMAL Protein Fusion and Purifications System (method I; New England Biolabs) To improve binding of MBP-PrP and to prevent formation of interchain disulfide upon lysis (as suggested in the protocol), β-mercaptoethanol was added. Quantity and quality of the eluted MBP-PrP was determined before use in the peptide-array by SDS-PAGE (12% NuPAGE, Invitrogen). After separation the gel was either stained with Sypro Orange (total protein stain, Molecular Probes) or analyzed by Western blotting and immunodetection of MBP-PrP with polyclonal antiserum R521-7 specific for PrP .
Peptides were synthesized with an acetylated N-terminus and an amidated C-terminus as described before . The synthesized peptides were purified by high performance liquid chromatography using mass spectrometric analysis for identification. The resulting purified peptides were at least 90% pure. All peptides dissolved well in water and solutions were stored frozen. Sequential properties like iso-electric point were calculated with the Peptide Property Calculator made available online in the tools section of Innovagen http://www.innovagen.se.
Synthesis of complete sets of overlapping 15-mer peptides were carried out on grafted plastic surfaces, covering the ovine PrP amino acid sequence of mature PrP (residues 25-234 ). Coupling of the peptides to the plastic surface consisting of a 455-well credit-card size plastic (minicard) and subsequent ELISA analyses including subsequent background correction, relative density value calculation and binding pattern interpretation were performed as described before [15, 73]. This study also showed that linking of MBP to PrP did not have any disadvantageous effects and therefore its properties are indicative for PrP. Peptide blocking studies were performed by pre-incubating the MBP-PrP with molar excesses of prion peptides before incubating the PrP-peptide mixture as the antigen on the minicard. Binding to the peptide-array was considered relevant when at least at 3 consecutive peptides optical density values of at least 2.5 times the background were observed.
Protein Misfolding Cyclic Amplification assay
The protein misfolding cyclic amplification (PMCA) assay first described by Saborio et al.  and has been shown applicable to amplify PrPSc from different sources [10, 43]. In short; a 10% brain homogenate from a (confirmed) scrapie-positive sheep (SPH) was diluted with 50-100 times in 10% (confirmed) scrapie-negative sheep brain homogenate (SNH) after which the reaction was subjected to one round of sonication-incubation cycles (24 hours; 48 cycles). To test the influence of the peptides on conversion, the peptides were either added after combining the scrapie positive -and negative brain homogenates (supplemented PMCA) or peptide was pre-incubated with the scrapie-negative homogenate (pre-incubated PMCA) before addition of the scrapie-positive brain homogenate. The total amount of PrPC in the PMCA reaction was calculated based on the quantification of the amount of PrPC in brain tissue . The amount of peptide needed for a reaction was calculated based on the total amount of PrPC in the reaction, adjusted for the size difference between peptide and PrPC, so that the amount of peptide added represents various molar excesses of peptide molecules relative to the total number of PrPC molecules present in the reaction. The amount of formed PrPres was determined after proteinase K digestion (100 μg/ml) of the PMCA reaction and analysis by SDS-PAGE and subsequent Western blotting. Standard detection of PrPres was performed with monoclonal antibody 9A2 , except for PMCA reactions containing peptide SAU14, which contains the binding epitope of 9A2. In this case we used a proven combination of monoclonal antibodies L42  and Sha31 . To determine whether the determined amounts of PrPres were significantly different in comparison to its corresponding standard PMCA-assay reaction, an unpaired Student's t-test was performed and the p-value calculated.
transmissible spongiform encephalopathies
host encoded cellular prion protein
disease associated prion protein
proteinase K resistant prion protein
protein misfolding cyclic amplification
We thank Drs. Jaques Grassi (SPI, CEA) and Martin Groschup (FLI, Reims, GE) for generously supplying the monoclonal antibodies Sha31 and L42. This work was supported by grant 903-51-177 from the Dutch Organization for Scientific Research (NWO), by a grant from the Dutch Ministry of Agriculture, Nature Management and Fisheries (LNV) and by EU NeuroPrion project STOPPRIONs FOOD-CT-2004-506579.
- DeArmond SJ, Prusiner SB: Perspectives on prion biology, prion disease pathogenesis, and pharmacologic approaches to treatment. Clin Lab Med. 2003, 23 (1): 1-41.View ArticleGoogle Scholar
- Bossers A, Rigter A, de Vries R, Smits MA: In vitro conversion of normal prion protein into pathologic isoforms. Clin Lab Med. 2003, 23 (1): 227-247.View ArticleGoogle Scholar
- Govaerts C, Wille H, Prusiner SB, Cohen FE: Evidence for assembly of prions with left-handed beta-helices into trimers. Proc Natl Acad Sci USA. 2004, 101 (22): 8342-8347.PubMed CentralView ArticleGoogle Scholar
- Langedijk JP, Fuentes G, Boshuizen R, Bonvin AM: Two-rung Model of a Left-handed beta-Helix for Prions Explains Species Barrier and Strain Variation in Transmissible Spongiform Encephalopathies. J Mol Biol. 2006, 360 (4): 907-920.View ArticleGoogle Scholar
- Bossers A, de Vries R, Smits MA: Susceptibility of sheep for scrapie as assessed by in vitro conversion of nine naturally occurring variants of PrP. J Virol. 2000, 74 (3): 1407-1414.PubMed CentralView ArticleGoogle Scholar
- Kocisko DA, Priola SA, Raymond GJ, Chesebro B, Lansbury PT, Caughey B: Species specificity in the cell-free conversion of prion protein to protease-resistant forms: a model for the scrapie species barrier. Proc Natl Acad Sci USA. 1995, 92 (9): 3923-3927.PubMed CentralView ArticleGoogle Scholar
- Castilla J, Saa P, Morales R, Abid K, Maundrell K, Soto C: Protein misfolding cyclic amplification for diagnosis and prion propagation studies. Methods Enzymol. 2006, 412: 3-21.View ArticleGoogle Scholar
- Castilla J, Saa P, Soto C: Detection of prions in blood. Nat Med. 2005, 11 (9): 982-985.Google Scholar
- Saborio GP, Permanne B, Soto C: Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature. 2001, 411 (6839): 810-813.View ArticleGoogle Scholar
- Soto C, Anderes L, Suardi S, Cardone F, Castilla J, Frossard MJ, Peano S, Saa P, Limido L, Carbonatto M: Pre-symptomatic detection of prions by cyclic amplification of protein misfolding. FEBS Lett. 2005, 579 (3): 638-642.View ArticleGoogle Scholar
- Dubois MA, Sabatier P, Durand B, Calavas D, Ducrot C, Chalvet-Monfray K: Multiplicative genetic effects in scrapie disease susceptibility. C R Biol. 2002, 325 (5): 565-570.View ArticleGoogle Scholar
- Tranulis MA: Influence of the prion protein gene, Prnp, on scrapie susceptibility in sheep. Apmis. 2002, 110 (1): 33-43.View ArticleGoogle Scholar
- Sabuncu E, Petit S, Le Dur A, Lan Lai T, Vilotte JL, Laude H, Vilette D: PrP polymorphisms tightly control sheep prion replication in cultured cells. J Virol. 2003, 77 (4): 2696-2700.PubMed CentralView ArticleGoogle Scholar
- Rigter A, Bossers A: Sheep scrapie susceptibility-linked polymorphisms do not modulate the initial binding of cellular to disease-associated prion protein prior to conversion. J Gen Virol. 2005, 86 (Pt 9): 2627-2634.View ArticleGoogle Scholar
- Rigter A, Langeveld JP, Timmers-Parohi D, Jacobs JG, Moonen PL, Bossers A: Mapping of possible prion protein self interaction domains using peptide arrays. BMC Biochem. 2007, 8 (1): 6-PubMed CentralView ArticleGoogle Scholar
- Pan T, Wong BS, Liu T, Li R, Petersen RB, Sy MS: Cell-surface prion protein interacts with glycosaminoglycans. Biochem J. 2002, 368 (Pt 1): 81-90.PubMed CentralView ArticleGoogle Scholar
- Brown DR: Prion protein expression aids cellular uptake and veratridine-induced release of copper. J Neurosci Res. 1999, 58 (5): 717-725.View ArticleGoogle Scholar
- Brown DR: Role of the prion protein in copper turnover in astrocytes. Neurobiol Dis. 2004, 15 (3): 534-543.View ArticleGoogle Scholar
- Brown DR, Clive C, Haswell SJ: Antioxidant activity related to copper binding of native prion protein. J Neurochem. 2001, 76 (1): 69-76.View ArticleGoogle Scholar
- Dupuis L, Mbebi C, Gonzalez de Aguilar JL, Rene F, Muller A, de Tapia M, Loeffler JP: Loss of prion protein in a transgenic model of amyotrophic lateral sclerosis. Mol Cell Neurosci. 2002, 19 (2): 216-224.View ArticleGoogle Scholar
- Flechsig E, Shmerling D, Hegyi I, Raeber AJ, Fischer M, Cozzio A, von Mering C, Aguzzi A, Weissmann C: Prion protein devoid of the octapeptide repeat region restores susceptibility to scrapie in PrP knockout mice. Neuron. 2000, 27 (2): 399-408.View ArticleGoogle Scholar
- Frankenfield KN, Powers ET, Kelly JW: Influence of the N-terminal domain on the aggregation properties of the prion protein. Protein Sci. 2005, 14 (8): 2154-2166.PubMed CentralView ArticleGoogle Scholar
- Klamt F, Dal-Pizzol F, Conte da Frota MJ, Walz R, Andrades ME, da Silva EG, Brentani RR, Izquierdo I, Fonseca Moreira JC: Imbalance of antioxidant defense in mice lacking cellular prion protein. Free Radic Biol Med. 2001, 30 (10): 1137-1144.View ArticleGoogle Scholar
- Miele G, Jeffrey M, Turnbull D, Manson J, Clinton M: Ablation of cellular prion protein expression affects mitochondrial numbers and morphology. Biochem Biophys Res Commun. 2002, 291 (2): 372-377.View ArticleGoogle Scholar
- Pauly PC, Harris DA: Copper stimulates endocytosis of the prion protein. J Biol Chem. 1998, 273 (50): 33107-33110.View ArticleGoogle Scholar
- Rachidi W, Vilette D, Guiraud P, Arlotto M, Riondel J, Laude H, Lehmann S, Favier A: Expression of prion protein increases cellular copper binding and antioxidant enzyme activities but not copper delivery. J Biol Chem. 2003, 278 (11): 9064-9072.View ArticleGoogle Scholar
- Sakudo A, Lee DC, Saeki K, Nakamura Y, Inoue K, Matsumoto Y, Itohara S, Onodera T: Impairment of superoxide dismutase activation by N-terminally truncated prion protein (PrP) in PrP-deficient neuronal cell line. Biochem Biophys Res Commun. 2003, 308 (3): 660-667.View ArticleGoogle Scholar
- Walz R, Amaral OB, Rockenbach IC, Roesler R, Izquierdo I, Cavalheiro EA, Martins VR, Brentani RR: Increased sensitivity to seizures in mice lacking cellular prion protein. Epilepsia. 1999, 40 (12): 1679-1682.View ArticleGoogle Scholar
- Wong BS, Pan T, Liu T, Li R, Gambetti P, Sy MS: Differential contribution of superoxide dismutase activity by prion protein in vivo. Biochem Biophys Res Commun. 2000, 273 (1): 136-139.View ArticleGoogle Scholar
- Wong E, Thackray AM, Bujdoso R: Copper induces increased beta-sheet content in the scrapie-susceptible ovine prion protein PrPVRQ compared with the resistant allelic variant PrPARR. Biochem J. 2004, 380 (Pt 1): 273-282.PubMed CentralView ArticleGoogle Scholar
- Zeng F, Watt NT, Walmsley AR, Hooper NM: Tethering the N-terminus of the prion protein compromises the cellular response to oxidative stress. J Neurochem. 2003, 84 (3): 480-490.View ArticleGoogle Scholar
- Jacobs JG, Langeveld JP, Biacabe AG, Acutis PL, Polak MP, Gavier-Widen D, Buschmann A, Caramelli M, Casalone C, Mazza M: Molecular discrimination of atypical bovine spongiform encephalopathy strains from a geographical region spanning a wide area in Europe. J Clin Microbiol. 2007, 45 (6): 1821-1829.PubMed CentralView ArticleGoogle Scholar
- Thuring CM, Erkens JH, Jacobs JG, Bossers A, Van Keulen LJ, Garssen GJ, Van Zijderveld FG, Ryder SJ, Groschup MH, Sweeney T: Discrimination between scrapie and bovine spongiform encephalopathy in sheep by molecular size, immunoreactivity, and glycoprofile of prion protein. J Clin Microbiol. 2004, 42 (3): 972-980.PubMed CentralView ArticleGoogle Scholar
- Knowles TP, Zahn R: Enhanced stability of human prion proteins with two disulfide bridges. Biophys J. 2006, 91 (4): 1494-1500.PubMed CentralView ArticleGoogle Scholar
- Korth C, Stierli B, Streit P, Moser M, Schaller O, Fischer R, Schulz-Schaeffer W, Kretzschmar H, Raeber A, Braun U: Prion (PrPSc)-specific epitope defined by a monoclonal antibody. Nature. 1997, 390 (6655): 74-77.View ArticleGoogle Scholar
- Kuczius T, Grassi J, Karch H, Groschup MH: Binding of N- and C-terminal anti-prion protein antibodies generates distinct phenotypes of cellular prion proteins (PrPC) obtained from human, sheep, cattle and mouse. Febs J. 2007, 274 (6): 1492-1502.View ArticleGoogle Scholar
- Martucci F, Acutis P, Mazza M, Nodari S, Colussi S, Corona C, Barocci S, Gabrielli A, Caramelli M, Casalone C: Detection of typical and atypical BSE and scrapie prion strains by prion protein motif-grafted antibodies. J Gen Virol. 2009Google Scholar
- Moroncini G, Kanu N, Solforosi L, Abalos G, Telling GC, Head M, Ironside J, Brockes JP, Burton DR, Williamson RA: Motif-grafted antibodies containing the replicative interface of cellular PrP are specific for PrPSc. Proc Natl Acad Sci USA. 2004, 101 (28): 10404-10409.PubMed CentralView ArticleGoogle Scholar
- Moroncini G, Mangieri M, Morbin M, Mazzoleni G, Ghetti B, Gabrielli A, Williamson RA, Giaccone G, Tagliavini F: Pathologic prion protein is specifically recognized in situ by a novel PrP conformational antibody. Neurobiol Dis. 2006, 23 (3): 717-724.View ArticleGoogle Scholar
- Paramithiotis E, Pinard M, Lawton T, LaBoissiere S, Leathers VL, Zou WQ, Estey LA, Lamontagne J, Lehto MT, Kondejewski LH: A prion protein epitope selective for the pathologically misfolded conformation. Nat Med. 2003, 9 (7): 893-899.View ArticleGoogle Scholar
- Horiuchi M, Baron GS, Xiong LW, Caughey B: Inhibition of interactions and interconversions of prion protein isoforms by peptide fragments from the C-terminal folded domain. J Biol Chem. 2001, 276 (18): 15489-15497.View ArticleGoogle Scholar
- Solforosi L, Bellon A, Schaller M, Cruite JT, Abalos GC, Williamson RA: Toward Molecular Dissection of PrPC-PrPSc Interactions. J Biol Chem. 2007, 282 (10): 7465-7471.View ArticleGoogle Scholar
- Soto C, Saborio GP, Anderes L: Cyclic amplification of protein misfolding: application to prion-related disorders and beyond. Trends Neurosci. 2002, 25 (8): 390-394.View ArticleGoogle Scholar
- Wille H, Michelitsch MD, Guenebaut V, Supattapone S, Serban A, Cohen FE, Agard DA, Prusiner SB: Structural studies of the scrapie prion protein by electron crystallography. Proc Natl Acad Sci USA. 2002, 99 (6): 3563-3568.PubMed CentralView ArticleGoogle Scholar
- Apetri AC, Maki K, Roder H, Surewicz WK: Early intermediate in human prion protein folding as evidenced by ultrarapid mixing experiments. J Am Chem Soc. 2006, 128 (35): 11673-11678.PubMed CentralView ArticleGoogle Scholar
- Apetri AC, Surewicz K, Surewicz WK: The effect of disease-associated mutations on the folding pathway of human prion protein. J Biol Chem. 2004, 279 (17): 18008-18014.View ArticleGoogle Scholar
- Gerber R, Tahiri-Alaoui A, Hore PJ, James W: Oligomerization of the human prion protein proceeds via a molten globule intermediate. J Biol Chem. 2007, 282 (9): 6300-6307.View ArticleGoogle Scholar
- Hosszu LL, Trevitt CR, Jones S, Batchelor M, Scott DJ, Jackson GS, Collinge J, Waltho JP, Clarke AR: Conformational properties of beta -PrP. J Biol Chem. 2009Google Scholar
- Jenkins DC, Sylvester ID, Pinheiro TJ: The elusive intermediate on the folding pathway of the prion protein. Febs J. 2008, 275 (6): 1323-1335.View ArticleGoogle Scholar
- Stohr J, Weinmann N, Wille H, Kaimann T, Nagel-Steger L, Birkmann E, Panza G, Prusiner SB, Eigen M, Riesner D: Mechanisms of prion protein assembly into amyloid. Proc Natl Acad Sci USA. 2008, 105 (7): 2409-2414.PubMed CentralView ArticleGoogle Scholar
- Campbell TA, Palmer MS, Will RG, Gibb WR, Luthert PJ, Collinge J: A prion disease with a novel 96-base pair insertional mutation in the prion protein gene. Neurology. 1996, 46 (3): 761-766.View ArticleGoogle Scholar
- Cochran EJ, Bennett DA, Cervenakova L, Kenney K, Bernard B, Foster NL, Benson DF, Goldfarb LG, Brown P: Familial Creutzfeldt-Jakob disease with a five-repeat octapeptide insert mutation. Neurology. 1996, 47 (3): 727-733.View ArticleGoogle Scholar
- Goldfarb LG, Brown P, Little BW, Cervenakova L, Kenney K, Gibbs CJ, Gajdusek DC: A new (two-repeat) octapeptide coding insert mutation in Creutzfeldt-Jakob disease. Neurology. 1993, 43 (11): 2392-2394.View ArticleGoogle Scholar
- Goldfarb LG, Brown P, McCombie WR, Goldgaber D, Swergold GD, Wills PR, Cervenakova L, Baron H, Gibbs CJ, Gajdusek DC: Transmissible familial Creutzfeldt-Jakob disease associated with five, seven, and eight extra octapeptide coding repeats in the PRNP gene. Proc Natl Acad Sci USA. 1991, 88 (23): 10926-10930.PubMed CentralView ArticleGoogle Scholar
- Laplanche JL, Delasnerie-Laupretre N, Brandel JP, Dussaucy M, Chatelain J, Launay JM: Two novel insertions in the prion protein gene in patients with late-onset dementia. Hum Mol Genet. 1995, 4 (6): 1109-1111.View ArticleGoogle Scholar
- Lawson VA, Priola SA, Meade-White K, Lawson M, Chesebro B: 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. J Biol Chem. 2004, 279 (14): 13689-13695.View ArticleGoogle Scholar
- Hundt C, Peyrin JM, Haik S, Gauczynski S, Leucht C, Rieger R, Riley ML, Deslys JP, Dormont D, Lasmezas CI: Identification of interaction domains of the prion protein with its 37-kDa/67-kDa laminin receptor. Embo J. 2001, 20 (21): 5876-5886.PubMed CentralView ArticleGoogle Scholar
- Madhavaiah C, Verma S: Self-aggregation of reverse bis peptide conjugate derived from the unstructured region of the prion protein. Chem Commun (Camb). 2004, 638-639. 6
- Bossers A: Prion Disease: Susceptibility and Transmissibility; In vivo and in vitro studies with sheep scrapie. 1999, PhD. University of UtrechtGoogle Scholar
- Bossers A, Belt P, Raymond GJ, Caughey B, de Vries R, Smits MA: Scrapie susceptibility-linked polymorphisms modulate the in vitro conversion of sheep prion protein to protease-resistant forms. Proc Natl Acad Sci USA. 1997, 94 (10): 4931-4936.PubMed CentralView ArticleGoogle Scholar
- Bossers A, Schreuder BE, Muileman IH, Belt PB, Smits MA: PrP genotype contributes to determining survival times of sheep with natural scrapie. J Gen Virol. 1996, 77 (Pt 10): 2669-2673.View ArticleGoogle Scholar
- Goldmann W, Hunter N, Smith G, Foster J, Hope J: PrP genotypes and the Sip gene in Cheviot sheep form the basis for scrapie strain typing in sheep. Ann N Y Acad Sci. 1994, 724: 296-299.View ArticleGoogle Scholar
- Hunter N, Foster JD, Goldmann W, Stear MJ, Hope J, Bostock C: Natural scrapie in a closed flock of Cheviot sheep occurs only in specific PrP genotypes. Arch Virol. 1996, 141 (5): 809-824.View ArticleGoogle Scholar
- Surewicz WK, Jones EM, Apetri AC: The emerging principles of Mammalian prion propagation and transmissibility barriers: insight from studies in vitro. Acc Chem Res. 2006, 39 (9): 654-662.View ArticleGoogle Scholar
- Wong C, Xiong LW, Horiuchi M, Raymond L, Wehrly K, Chesebro B, Caughey B: Sulfated glycans and elevated temperature stimulate PrP(Sc)-dependent cell-free formation of protease-resistant prion protein. Embo J. 2001, 20 (3): 377-386.PubMed CentralView ArticleGoogle Scholar
- Chabry J, Caughey B, Chesebro B: Specific inhibition of in vitro formation of protease-resistant prion protein by synthetic peptides. J Biol Chem. 1998, 273 (21): 13203-13207.View ArticleGoogle Scholar
- Chabry J, Priola SA, Wehrly K, Nishio J, Hope J, Chesebro B: Species-independent inhibition of abnormal prion protein (PrP) formation by a peptide containing a conserved PrP sequence. J Virol. 1999, 73 (8): 6245-6250.PubMed CentralGoogle Scholar
- Ziegler J, Viehrig C, Geimer S, Rosch P, Schwarzinger S: Putative aggregation initiation sites in prion protein. FEBS Lett. 2006, 580 (8): 2033-2040.View ArticleGoogle Scholar
- Satheeshkumar KS, Jayakumar R: Sonication induced sheet formation at the air-water interface. Chem Commun (Camb). 2002, 2244-2245. 19
- Bosques CJ, Imperiali B: The interplay of glycosylation and disulfide formation influences fibrillization in a prion protein fragment. Proc Natl Acad Sci USA. 2003, 100 (13): 7593-7598.PubMed CentralView ArticleGoogle Scholar
- van Keulen LJ, Schreuder BE, Meloen RH, Poelen-van den Berg M, Mooij-Harkes G, Vromans ME, Langeveld JP: Immunohistochemical detection and localization of prion protein in brain tissue of sheep with natural scrapie. Vet Pathol. 1995, 32 (3): 299-308.View ArticleGoogle Scholar
- Goldmann W, Hunter N, Martin T, Dawson M, Hope J: Different forms of the bovine PrP gene have five or six copies of a short, G-C-rich element within the protein-coding exon. J Gen Virol. 1991, 72 (Pt 1): 201-204.View ArticleGoogle Scholar
- Rigter A, Priem J, Timmers-Parohi D, Langeveld JP, Bossers A: Mapping functional prion-prion protein interaction sites using prion protein based peptide-arrays. Methods Mol Biol. 2009, 570: 257-271.View ArticleGoogle Scholar
- Moudjou M, Frobert Y, Grassi J, La Bonnardiere C: Cellular prion protein status in sheep: tissue-specific biochemical signatures. J Gen Virol. 2001, 82 (Pt 8): 2017-2024.View ArticleGoogle Scholar
- Langeveld JP, Jacobs JG, Erkens JH, Bossers A, van Zijderveld FG, van Keulen LJ: Rapid and discriminatory diagnosis of scrapie and BSE in retro-pharyngeal lymph nodes of sheep. BMC Vet Res. 2006, 2: 19-PubMed CentralView ArticleGoogle Scholar
- Harmeyer S, Pfaff E, Groschup MH: Synthetic peptide vaccines yield monoclonal antibodies to cellular and pathological prion proteins of ruminants. J Gen Virol. 1998, 79 (Pt 4): 937-945.View ArticleGoogle Scholar
- Feraudet C, Morel N, Simon S, Volland H, Frobert Y, Creminon C, Vilette D, Lehmann S, Grassi J: Screening of 145 anti-PrP monoclonal antibodies for their capacity to inhibit PrPSc replication in infected cells. J Biol Chem. 2005, 280 (12): 11247-11258.View ArticleGoogle Scholar
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