Recombinant PrP Research Articles

X-Ray Fiber Diffraction Reveals Major Structural Differences Between Brain-Derived Prions and Recombinant Prion Protein Amyloid

X-ray fiber diffraction was used to study the structure of brain-derived prions and recombinant protein amyloid. Partially oriented, dried fibers were prepared from brain-derived PrP 27-30 and recombinant PrP amyloid. Fiber diffraction patterns were analyzed and used to interrogate models for the structure of the infectious prion.Fiber diffraction patterns of recombinant PrP amyloid displayed characteristic, meridional reflections at ∼4.8 A and equatorial reflections at ∼10 A. These patterns were similar to those of other amyloids and are consistent with a basic cross-β architecture. In contrast, diffraction patterns from brain-derived PrP 27-30 displayed meridional reflections at ∼9.6, ∼6.4, and ∼4.8 A, which correspond to the 2nd, 3rd, and 4th order of a ∼19.2-A repeating unit, suggesting that PrP 27-30 contains four β-strands in a cross-β architecture. Furthermore, PrP 27-30 lacked the typical, equatorial reflection at ∼10 A, but instead produced equatorial reflections characterizing the diameter of the amyloid fiber and of individual protofilaments. Therefore, PrP 27-30 seems to have a structure consistent with a β-helix or β-solenoid, not unlike the model that was proposed earlier (Govaerts et al., 2004). This interpretation was also supported by extensive modeling, simulation of diffraction, electron microscopy, and FTIR.In a previous study, recombinant PrP amyloid induced a transmissible disease in transgenic mice overexpressing PrP, and was thus termed a prion (Legname et al., 2004). Serially transmitted, synthetic prions were purified from mouse brains and analyzed by fiber diffraction. These brain-derived, synthetic prions showed the same structural characteristics as natural isolates and not those of its recombinant protein precursor. The relationship between structural differences and infectivity can be explained by several hypotheses. It remains to be determined which one, if any, is correct.

Is RNA critical for the production of TSE infection?

Is RNA critical for the production of TSE infection?

Aggregation and Amyloid Fibril Formation Induced by Chemical Dimerization of Recombinant Prion Protein in Physiological-like Conditions

Prion diseases are caused by the conversion of a cellular protein (PrP(C)) into a misfolded, aggregated isoform (PrP(Res)). Misfolding of recombinant PrP(C) in the absence of PrP(Res) template, cellular factors, denaturing agents, or at neutral pH has not been achieved. A number of studies indicate that dimerization of PrP(C) may be a key step in the aggregation process. In an effort to understand the molecular event that may activate misfolding of PrP(C) in more relevant physiological conditions, we tested if enforced dimerization of PrP(C) may induce a conformational change reminiscent of the conversion of PrP(C) to PrP(Res). We used a well described inducible dimerization strategy whereby a chimeric PrP(C) composed of a modified FK506-binding protein (Fv) fused with PrP(C) and termed Fv-PrP is incubated in the presence of a monomeric FK506 or dimerizing AP20187 ligand. Addition of AP20187 but not FK506 to recombinant Fv-PrP (rFv-PrP) in physiological-like conditions resulted in a rapid conformational change characterized by an increase in beta-sheet structure and simultaneous aggregation of the protein. Aggregates were partially resistant to proteinase K and induced the conversion of soluble rFv-PrP in serial seeding experiments. As judged from thioflavin T binding and electron microscopy, aggregates converted to amyloid fibers. Aggregates were toxic to cultured cells, whereas soluble rFv-PrP and amyloid fibers were harmless. This study strongly supports the proposition that dimerization of PrP(C) is a key pathological primary event in the conversion of PrP(C) and may initiate the pathogenesis of prion diseases.

Antimicrobial Activity of Human Prion Protein Is Mediated by Its N-Terminal Region

BackgroundCellular prion-related protein (PrPc) is a cell-surface protein that is ubiquitously expressed in the human body. The multifunctionality of PrPc, and presence of an exposed cationic and heparin-binding N-terminus, a feature characterizing many antimicrobial peptides, made us hypotesize that PrPc could exert antimicrobial activity.Methodology and Principal FindingsIntact recombinant PrP exerted antibacterial and antifungal effects at normal and low pH. Studies employing recombinant PrP and N- and C-terminally truncated variants, as well as overlapping peptide 20mers, demonstrated that the antimicrobial activity is mediated by the unstructured N-terminal part of the protein. Synthetic peptides of the N-terminus of PrP killed the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, and the Gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungus Candida parapsilosis. Fluorescence studies of peptide-treated bacteria, paired with analysis of peptide effects on liposomes, showed that the peptides exerted membrane-breaking effects similar to those seen after treatment with the “classical” human antimicrobial peptide LL-37. In contrast to LL-37, however, no marked helix induction was detected for the PrP-derived peptides in presence of negatively charged (bacteria-mimicking) liposomes. PrP furthermore showed an inducible expression during wounding of human skin ex vivo and in vivo, as well as stimulation of keratinocytes with TGF-α in vitro.ConclusionsThe demonstration of an antimicrobial activity of PrP, localisation of its activity to the N-terminal and heparin-binding region, combined with results showing an increased expression of PrP during wounding, indicate that PrPs could have a previously undisclosed role in host defense.

Natural and synthetic prion structure from X-ray fiber diffraction

A conformational isoform of the mammalian prion protein (PrP(Sc)) is the sole component of the infectious pathogen that causes the prion diseases. We have obtained X-ray fiber diffraction patterns from infectious prions that show cross-beta diffraction: meridional intensity at 4.8 A resolution, indicating the presence of beta strands running approximately at right angles to the filament axis and characteristic of amyloid structure. Some of the patterns also indicated the presence of a repeating unit along the fiber axis, corresponding to four beta-strands. We found that recombinant (rec) PrP amyloid differs substantially from highly infectious brain-derived prions, both in structure as demonstrated by the diffraction data, and in heterogeneity as shown by electron microscopy. In addition to the strong 4.8 A meridional reflection, the recPrP amyloid diffraction is characterized by strong equatorial intensity at approximately 10.5 A, absent from brain-derived prions, and indicating the presence of stacked beta-sheets. Synthetic prions recovered from transgenic mice inoculated with recPrP amyloid displayed structural characteristics and homogeneity similar to those of naturally occurring prions. The relationship between the structural differences and prion infectivity is uncertain, but might be explained by any of several hypotheses: only a minority of recPrP amyloid possesses a replication-competent conformation, the majority of recPrP amyloid has to undergo a conformational maturation to acquire replication competency, or inhibitory forms of recPrP amyloid interfere with replication during the initial transmission.

Differential stability of the bovine prion protein upon urea unfolding

Prion diseases, or transmissible spongiform encephalopathies, are a group of infectious neurological diseases associated with the structural conversion of an endogenous protein (PrP) in the central nervous system. There are two major forms of this protein: the native and noninfectious cellular form, PrP(C); and the misfolded, infectious, and proteinase K-resistant form, PrP(Sc). The C-terminal domain of PrP(C) is mainly alpha-helical in structure, whereas PrP(Sc) in known to aggregate into an assembly of beta-sheets, forming amyloid fibrils. To identify the regions of PrP(C) potentially involved in the initial steps of the conversion to the infectious conformation, we have used high-resolution NMR spectroscopy to characterize the stability and structure of bovine recombinant PrP(C) (residues 121 to 230) during unfolding with the denaturant urea. Analysis of the 800 MHz (1)H NMR spectra reveals region-specific information about the structural changes occurring upon unfolding. Our data suggest that the dissociation of the native beta-sheet of PrP(C) is a primary step in the urea-induced unfolding process, while strong hydrophobic interactions between helices alpha1 and alpha3, and between alpha2 and alpha3, stabilize these regions even at very high concentrations of urea.

Prion Protein-Detergent Micelle Interactions Studied by NMR in Solution

Cellular prion proteins, PrP(C), carrying the amino acid substitutions P102L, P105L, or A117V, which confer increased susceptibility to human transmissible spongiform encephalopathies, are known to form structures that include transmembrane polypeptide segments. Herein, we investigated the interactions between dodecylphosphocholine micelles and the polypeptide fragments 90-231 of the recombinant mouse PrP variants carrying the amino acid replacements P102L, P105L, A117V, A113V/A115V/A118V, K110I/H111I, M129V, P105L/M129V, and A117V/M129V. Wild-type mPrP-(90-231) and mPrP[M129V]-(91-231) showed only weak interactions with dodecylphosphocholine micelles in aqueous solution at pH 7.0, whereas discrete interaction sites within the polypeptide segment 102-127 were identified for all other aforementioned mPrP variants by NMR chemical shift mapping. These model studies thus provide evidence that amino acid substitutions within the polypeptide segment 102-127 affect the interactions of PrP(C) with membranous structures, which might in turn modulate the physiological function of the protein in health and disease.

Conformational Properties of β-PrP

Prion propagation involves a conformational transition of the cellular form of prion protein (PrPC) to a disease-specific isomer (PrPSc), shifting from a predominantly alpha-helical conformation to one dominated by beta-sheet structure. This conformational transition is of critical importance in understanding the molecular basis for prion disease. Here, we elucidate the conformational properties of a disulfide-reduced fragment of human PrP spanning residues 91-231 under acidic conditions, using a combination of heteronuclear NMR, analytical ultracentrifugation, and circular dichroism. We find that this form of the protein, which similarly to PrPSc, is a potent inhibitor of the 26 S proteasome, assembles into soluble oligomers that have significant beta-sheet content. The monomeric precursor to these oligomers exhibits many of the characteristics of a molten globule intermediate with some helical character in regions that form helices I and III in the PrPC conformation, whereas helix II exhibits little evidence for adopting a helical conformation, suggesting that this region is a likely source of interaction within the initial phases of the transformation to a beta-rich conformation. This precursor state is almost as compact as the folded PrPC structure and, as it assembles, only residues 126-227 are immobilized within the oligomeric structure, leaving the remainder in a mobile, random-coil state.

Design of anti- and pro-aggregation variants to assess the effects of methionine oxidation in human prion protein.

Prion disease is characterized by the alpha-->beta structural conversion of the cellular prion protein (PrP(C)) into the misfolded and aggregated "scrapie" (PrP(Sc)) isoform. It has been speculated that methionine (Met) oxidation in PrP(C) may have a special role in this process, but has not been detailed and assigned individually to the 9 Met residues of full-length, recombinant human PrP(C) [rhPrP(C)(23-231)]. To better understand this oxidative event in PrP aggregation, the extent of periodate-induced Met oxidation was monitored by electrospray ionization-MS and correlated with aggregation propensity. Also, the Met residues were replaced with isosteric and chemically stable, nonoxidizable analogs, i.e., with the more hydrophobic norleucine (Nle) and the highly hydrophilic methoxinine (Mox). The Nle-rhPrP(C) variant is an alpha-helix rich protein (like Met-rhPrP(C)) resistant to oxidation that lacks the in vitro aggregation properties of the parent protein. Conversely, the Mox-rhPrP(C) variant is a beta-sheet rich protein that features strong proaggregation behavior. In contrast to the parent Met-rhPrP(C), the Nle/Mox-containing variants are not sensitive to periodate-induced in vitro aggregation. The experimental results fully support a direct correlation of the alpha-->beta secondary structure conversion in rhPrP(C) with the conformational preferences of Met/Nle/Mox residues. Accordingly, sporadic prion and other neurodegenerative diseases, as well as various aging processes, might also be caused by oxidative stress leading to Met oxidation.

Hydrogen/deuterium exchange mass spectrometry identifies two highly protected regions in recombinant full‐length prion protein amyloid fibrils

Understanding the structural basis that distinguishes the amyloid form of the prion protein from its monomeric homologue is of crucial importance to elucidate the mechanism of the lethal diseases related to this protein. Recently, an in vitro conversion system was established which reproduces the transition of recombinant prion protein PrP(23-230) from its native alpha-helical rich form into an aggregated amyloid beta-sheet rich form with physicochemical properties reminiscent to those of the disease-related isoform of the prion protein, PrPSc. To study the tertiary and quaternary structural organization within recombinant amyloid fibrils from mouse, mPrP(23-231)betaf; bovine, bPrP(23-230)betaf; and elk, ePrP(23-230)betaf; we utilized hydrogen/deuterium (H/D) exchange analyzed by matrix-assisted laser desorption/ionization (MALDI) and nano-electrospray (nano-ESI) mass spectrometry. No significant differences were found by measuring the deuterium exchange kinetics of the aggregated fibrillar forms for mPrP(23-231)betaf, bPrP(23-230)betaf and ePrP(23-230)betaf, indicating a similar overall structural organization of the fibrils from all three species. Next, we characterized the solvent accessibility for the soluble and fibrillar forms of the mouse prion protein by hydrogen exchange, pepsin proteolysis and nano-ESI ion trap mass spectrometry analysis. In its amyloid form, two highly protected regions of mPrP(23-231) comprising residues [24-98] and [182-212] were identified. The residues between the two highly protected stretches were found to be more solvent exposed, but less than in the soluble protein, and might therefore rather form part of a fibrillar interface.

Surround optical fiber immunoassay (SOFIA): An ultra-sensitive assay for prion protein detection

We describe the development of a new technology (SOFIA) and demonstrate its utility by establishing a sensitive and specific assay for PrP Sc. SOFIA is a surround optical fiber immunoassay which is comprised of a set of specific monoclonal antibodies and comprehensive capture of high energy fluorescence emission. In its current format, this system is capable of detecting less than 10 attogram (ag) of hamster, sheep and deer recombinant PrP. Approximately 10 ag of PrP Sc from 263 K-infected hamster brains can be detected with similar lower limits of PrP Sc detection from the brains of scrapie-infected sheep and deer infected with chronic wasting disease. These detection limits allow protease treated and untreated material to be diluted beyond the point where PrP C, non-specific proteins or other extraneous material may interfere with PrP Sc signal detection and/or specificity. This not only eliminates the issue of specificity of PrP Sc detection but also increases sensitivity since the possibility of partial PrP Sc proteolysis is no longer a concern. SOFIA will likely lead to early antemortem detection of transmissible encephalopathies and is also amenable for use with additional target amplification protocols. SOFIA represents a sensitive means for detecting specific proteins involved in disease pathogenesis and/or diagnosis that extends beyond the scope of the transmissible spongiform encephalopathies.

The propagation of hamster‐adapted scrapie PrPSc can be enhanced by reduced pyridine nucleotide in vitro

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are fatal neurodegenerative disorders caused by an infectious agent termed a prion, which can convert normal cellular prion protein (PrP(C)) into a pathologically misfolded isoform (PrP(Sc)). Taking advantage of protein misfolding cyclic amplification (PMCA), a series of experiments was conducted to investigate the possible influences of pyridine nucleotides on the propagation activities of hamster-adapted scrapie agents 263K and 139A in vitro using normal hamster brain homogenates and recombinant hamster PrP as the substrates. The results showed that PrP(Sc) from both scrapie agent 263K- and 139A-infected brains propagated more efficiently in PMCA with the addition of reduced NADPH, showing an obvious dose-dependent enhancement. Reduced NADH also prompted PrP(Sc) propagation, whereas NADP, NAD and vitamin C failed. Moreover, following incubation with NADPH, recombinant hamster PrP could be efficiently converted into the proteinase K-resistant form when exposed to the trace of PrP(Sc) from infected hamsters. Our data provide evidence that the reduced pyridine nucleotide plays an important role in the propagation of prion and this process seems to target PrP(C) molecules.

Conformational detection of prion protein with biarsenical labeling and FlAsH fluorescence

Prion diseases are associated with the misfolding of the host-encoded cellular prion protein (PrP C) into a disease associated form (PrP Sc). Recombinant PrP can be refolded into either an α-helical rich conformation (α-PrP) resembling PrP C or a β-sheet rich, protease resistant form similar to PrP Sc. Here, we generated tetracysteine tagged recombinant PrP, folded this into α- or β-PrP and determined the levels of FlAsH fluorescence. Insertion of the tetracysteine tag at three different sites within the 91–111 epitope readily distinguished β-PrP from α-PrP upon FlAsH labeling. Labelling of tetracysteine tagged PrP in the α-helical form showed minimal fluorescence, whereas labeling of tagged PrP in the β-sheet form showed high fluorescence indicating that this region is exposed upon conversion. This highlights a region of PrP that can be implicated in the development of diagnostics and is a novel, protease free mechanism for distinguishing PrP Sc from PrP C. This technique may also be applied to any protein that undergoes conformational change and/or misfolding such as those involved in other neurodegenerative disorders including Alzheimer’s, Huntington’s and Parkinson’s diseases.

Proteinase K-Resistant Aggregates of Recombinant Prion Protein PrP-(23-98) Are Toxic to Cultured Cells

Here, we show for the first time that non-fibrillar and spherical aggregates produced from PrP-(23-98) in the presence of NADPH plus copper ions are toxic to cultured cells and induce apoptotic signals. It is also confirmed that endogenous cellular PrP isoform is not required for toxicity to occur.

Biosynthesis of Prion Protein Nucleocytoplasmic Isoforms by Alternative Initiation of Translation

The cellular prion protein PrP(C) is synthesized as a family of four distinct forms. Of these, (Cyt)PrP is a minor member that segregates outside of the secretory route and can generate cytotoxic forms. Using signal sequence mutants, we found that (Cyt)PrP is translated from a downstream AUG (coding for Met-8 in human PrP or Met-15 in Syrian hamster PrP). Shortening of the signal sequence dictated the spillage of this isoform into the cytosol, from where it accessed the nucleus or formed insoluble cytosolic aggregates if the proteasome is inhibited. The PrP isoform isolated from the nuclear fractions of cell and brain homogenates was partially SUMO-1-conjugated. Expression of HaPrP(M15) in cells caused an antiproliferative phenotype due to a cell cycle arrest at the G(0)/G(1) phase. The identification of this PrP isoform and its properties provides novel insight into PrP(C) physiological and pathological functions.

Prion Protein Amyloid Formation under Native-like Conditions Involves Refolding of the C-terminal α-Helical Domain

Transmissible spongiform encephalopathies are associated with conformational conversion of the cellular prion protein, PrP(C), into a proteinase K-resistant, amyloid-like aggregate, PrP(Sc). Although the structure of PrP(Sc) remains enigmatic, recent studies have afforded increasingly detailed characterization of recombinant PrP amyloid. However, all previous studies were performed using amyloid fibrils formed in the presence of denaturing agents that significantly alter the folding state(s) of the precursor monomer. Here we report that PrP amyloid can also be generated under physiologically relevant conditions, where the monomeric protein is natively folded. Remarkably, site-directed spin labeling studies reveal that these fibrils possess a beta-core structure nearly indistinguishable from that of amyloid grown under denaturing conditions, where the C-terminal alpha-helical domain of the PrP monomer undergoes major refolding to a parallel and in-register beta-structure upon conversion. The structural similarity of fibrils formed under drastically different conditions strongly suggests that the common beta-sheet architecture within the approximately 160-220 core region represents a distinct global minimum in the PrP conversion free energy landscape. We also show that the N-terminal region of fibrillar PrP displays conformational plasticity, undergoing a reversible structural transition with an apparent pK(a) of approximately 5.3. The C-terminal region, on the other hand, retains its beta-structure over the pH range 1-11, whereas more alkaline buffer conditions denature the fibrils into constituent PrP monomers. This profile of pH-dependent stability is reminiscent of the behavior of brain-derived PrP(Sc), suggesting a substantial degree of structural similarity within the beta-core region of these PrP aggregates.

Complementarity determining regions of an anti-prion protein scFv fragment orchestrate conformation specificity and antiprion activity

The prion protein, PrP, exists in several stable conformations, with the presence of one conformation, PrPSc, associated with transmissible neurodegenerative diseases. Targeting PrP by high-affinity ligands has been proven to be an effective way of preventing peripheral prion infections. Here, we have generated bacterially expressed single chain fragments of the variable domains (scFv) of a monoclonal antibody in Escherichia coli, originally raised against purified PrPSc that recognizes both PrPC and PrPSc. This scFv fragment had a dissociation constant (KD) with recombinant PrP of 2nM and cleared prions in ScN2a cells at 4nM, as demonstrated by a mouse prion bioassay. A peptide corresponding to the complementarity determining region 3 of the heavy chain (CDR3H) selectively bound PrPSc but had lost antiprion activity. However, synthesis and application of an improved peptide mimicking side chain topology of CDR3H while exhibiting increased protease resistance, a retro-inverso d-peptide of CDR3H, still bound PrPSc and reinstated antiprion activity. We conclude that (1) scFvW226 is so far the smallest polypeptide with bioassay confirmed antiprion activity, and (2) differential conformation specificity and bioactivity can be regulated by orchestrating the participation of different CDRs.

Ligand binding promotes prion protein aggregation – role of the octapeptide repeats

Aggregation of the normal cellular prion protein, PrP, is important in the pathogenesis of prion disease. PrP binds glycosaminoglycan (GAG) and divalent cations, such as Cu(2+) and Zn(2+). Here, we report our findings that GAG and Cu(2+) promote the aggregation of recombinant human PrP (rPrP). The normal cellular prion protein has five octapeptide repeats. In the presence of either GAG or Cu(2+), mutant rPrPs with eight or ten octapeptide repeats are more aggregation prone, exhibit faster kinetics and form larger aggregates than wild-type PrP. When the GAG-binding motif, KKRPK, is deleted the effect of GAG but not that of Cu(2+) is abolished. By contrast, when the Cu(2+)-binding motif, the octapeptide-repeat region, is deleted, neither GAG nor Cu(2+) is able to promote aggregation. Therefore, the octapeptide-repeat region is critical in the aggregation of rPrP, irrespective of the promoting ligand. Furthermore, aggregation of rPrP in the presence of GAG is blocked with anti-PrP mAbs, whereas none of the tested anti-PrP mAbs block Cu(2+)-promoted aggregation. However, a mAb that is specific for an epitope at the N-terminus enhances aggregation in the presence of either GAG or Cu(2+). Therefore, although binding of either GAG or Cu(2+) promotes the aggregation of rPrP, their aggregation processes are different, suggesting multiple pathways of rPrP aggregation.

Pathologic Prion Protein Infects Cells by Lipid-Raft Dependent Macropinocytosis

Transmissible spongiform encephalopathies, including variant-Creutzfeldt-Jakob disease (vCJD) in humans and bovine spongiform encephalopathies in cattle, are fatal neurodegenerative disorders characterized by protein misfolding of the host cellular prion protein (PrPC) to the infectious scrapie form (PrPSc). However, the mechanism that exogenous PrPSc infects cells and where pathologic conversion of PrPC to the PrPSc form occurs remains uncertain. Here we report that similar to the mechanism of HIV-1 TAT-mediated peptide transduction, processed mature, full length PrP contains a conserved N-terminal cationic domain that stimulates cellular uptake by lipid raft-dependent, macropinocytosis. Inhibition of macropinocytosis by three independent means prevented cellular uptake of recombinant PrP; however, it did not affect recombinant PrP cell surface association. In addition, fusion of the cationic N-terminal PrP domain to a Cre recombinase reporter protein was sufficient to promote both cellular uptake and escape from the macropinosomes into the cytoplasm. Inhibition of macropinocytosis was sufficient to prevent conversion of PrPC to the pathologic PrPSc form in N2a cells exposed to strain RML PrPSc infected brain homogenates, suggesting that a critical determinant of PrPC conversion occurs following macropinocytotic internalization and not through mere membrane association. Taken together, these observations provide a cellular mechanism that exogenous pathological PrPSc infects cells by lipid raft dependent, macropinocytosis.

The Polybasic N-Terminal Region of the Prion Protein Controls the Physical Properties of Both the Cellular and Fibrillar Forms of PrP

Individual variations in structure and morphology of amyloid fibrils produced from a single polypeptide are likely to underlie the molecular origin of prion strains and control the efficiency of the species barrier in the transmission of prions. Previously, we observed that the shape of amyloid fibrils produced from full-length prion protein (PrP 23–231) varied substantially for different batches of purified recombinant PrP. Variations in fibril morphology were also observed for different fractions that corresponded to the highly pure PrP peak collected at the last step of purification. A series of biochemical experiments revealed that the variation in fibril morphology was attributable to the presence of miniscule amounts of N-terminally truncated PrPs, where a PrP encompassing residue 31–231 was the most abundant of the truncated polypeptides. Subsequent experiments showed that the presence of small amounts of recombinant PrP 31–231 (0.1–1%) in mixtures with full-length PrP 23–231 had a dramatic impact on fibril morphology and conformation. Furthermore, the deletion of the short polybasic N-terminal region 23–30 was found to reduce the folding efficiency to the native α-helical forms and the conformational stability of α-PrP. These findings are very surprising considering that residues 23–30 are very distant from the C-terminal globular folded domain in α-PrP and from the prion folding domain in the fibrillar form. However, our studies suggest that the N-terminal polybasic region 23–30 is essential for effective folding of PrP to its native cellular conformation. This work also suggests that this region could regulate diversity of prion strains or subtypes despite its remote location from the prion folding domain.