Leu138 in bovine prion peptide fibrils is involved in seeding discrimination related to codon 129 M/V polymorphism in the prion peptide seeding experiment

The risk of acquiring variant Creutzfeldt-Jakob disease is closely related to polymorphism at codon 129 of the human prion gene, because almost all variant Creutzfeldt-Jakob disease patients are Met/Met homozygotes. Although animal transmission experiments corroborated this seeding discrimination, the origin of the differential seeding efficiency of the bovine prion seed for human codon 129 polymorphism remained elusive. Here, we used a short prion protein (PrP) peptide as a model system to test whether seeding discrimination can be found in this simple system. We used a previously developed 'seed-titration method' and time-resolved CD spectroscopy to compare sequence-dependent seeding efficiency regarding codon 129 polymorphism. Our results showed that the Met→Val substitution on the human PrP (huPrP) peptide decreased seeding efficiency by 10 times when fibrils formed from bovine PrP (bPrP) peptide were used as the seed. To explore whether the different seeding barrier is due to the chemical and structural properties of Met and Val or whether another residue is involved in this peptide model, we constructed three bPrP mutants, V112M, L138I and N143S, in each of which one residue was replaced by the corresponding human residue. Our data showed that Leu138 in the bPrP seed might be the key residue causing the different seeding efficiencies related to 129M/V polymorphism and the interference effect of huPrP129V in the huPrP129M/V mixture. We propose a 'surface competition hypothesis' to explain the big seeding barrier caused by 129V in the PrP peptide seeding experiment.     

Role of N-terminal familial mutations in prion protein fibrillization and prion amyloid propagation in vitro

A self-perpetuating conformational conversion of the prion protein (PrP) is believed to underlie pathology and transmission of prion diseases. Here we explore the effects of N-terminal pathogenic mutations (P102L, P105L, A117V) and the residue 129 polymorphism on amyloid fibril formation by the human PrP fragment 23-144, an in vitro conversion model that can reproduce certain characteristics of prion replication such as strains and species barriers. We find that these amino acid substitutions neither affect PrP23-144 amyloidogenicity nor introduce barriers to cross-seeding of soluble protein. However, the polymorphism strongly influences the conformation of the amyloid fibrils, as determined by infrared spectroscopy. Intriguingly, unlike conformational features governed by the critical amyloidogenic region of PrP23-144 (residues 138-139), the structural features distinguishing Met-129 and Val-129 PrP23-144 amyloid fibrils are not transmissible by cross-seeding. While based only on in vitro data, these findings provide fundamental insight into the mechanism of prion-based conformational transmission, indicating that only conformational features controlling seeding specificity (e.g. those in critical intermolecular contact sites of amyloid fibrils) are necessarily transmissible by cross-seeding; conformational traits in other parts of the PrP molecule may not be "heritable" from the amyloid template.     

Prion protein amino acid determinants of differential susceptibility and molecular feature of prion strains in mice and voles

The bank vole is a rodent susceptible to different prion strains from humans and various animal species. We analyzed the transmission features of different prions in a panel of seven rodent species which showed various degrees of phylogenetic affinity and specific prion protein (PrP) sequence divergences in order to investigate the basis of vole susceptibility in comparison to other rodent models. At first, we found a differential susceptibility of bank and field voles compared to C57Bl/6 and wood mice. Voles showed high susceptibility to sheep scrapie but were resistant to bovine spongiform encephalopathy, whereas C57Bl/6 and wood mice displayed opposite features. Infection with mouse-adapted scrapie 139A was faster in voles than in C57Bl/6 and wood mice. Moreover, a glycoprofile change was observed in voles, which was reverted upon back passage to mice. All strains replicated much faster in voles than in mice after adapting to the new species. PrP sequence comparison indicated a correlation between the transmission patterns and amino acids at positions 154 and 169 (Y and S in mice, N and N in voles). This correlation was confirmed when inoculating three additional rodent species: gerbils, spiny mice and oldfield mice with sheep scrapie and 139A. These rodents were chosen because oldfield mice do have the 154N and 169N substitutions, whereas gerbil and spiny mice do not have them. Our results suggest that PrP residues 154 and 169 drive the susceptibility, molecular phenotype and replication rate of prion strains in rodents. This might have implications for the assessment of host range and molecular traceability of prion strains, as well as for the development of improved animal models for prion diseases.     

Structural changes in a hydrophobic domain of the prion protein induced by hydration and by ala-->Val and pro-->Leu substitutions

X-ray diffraction was used to study the structure of assemblies formed by synthetic peptide fragments of the prion protein (PrP) that include the hydrophobic domain implicated in the Gerstmann-Str?ussler-Scheinker (GSS) mutation (P102L). The effects of hydration on polypeptide assembly and of Ala-->Val substitutions in the hydrophobic domain were characterized. Synthetic peptides included: (i) Syrian hamster (SHa) hydrophobic core, SHa106-122 (KTNMKHMAGAAAAGAVV); (ii) SHa104-122(3A-V), with A-->V mutations at 113, 115 and 118 (KPKTNMKHMVGVAAVGAVV); (iii) mouse (Mo) wild-type sequence of the N-terminal hydrophobic domain, Mo89-143WT; and (iv) the same mouse sequence with leucine substitution for proline at residue number 101, Mo89-143(P101L). Samples of SHa106-122 that formed assemblies while drying under ambient conditions showed X-ray patterns indicative of 33 A thick slab-like structures having extensive H-bonding and intersheet stacking. By contrast, lyophilized peptide that was equilibrated against 100 % relative humidity showed assemblies with only a few layers of beta-sheets. The Ala-->Val substitutions in SHa104-122 and Mo89-143(P101L) resulted in the formation of 40 A wide, cross-beta fibrils. Observation of similar size beta-sheet fibrils formed by peptides SHa104-122(3A-V) and the longer Mo89-143(P101L) supports the notion that the hydrophobic sequence forms a template or core that promotes the beta-folding of the longer peptide. The substitution of amino acids in the mutants, e.g. 3A-->V and P101L, enhances the folding of the peptide into compact structural units, significantly enhancing the formation of the extensive beta-sheet fibrils.     

Sequence of Guinea Pig Myelin Basic Protein

This paper proposes a tentative amino acid sequence of guinea pig myelin basic protein obtained by comparison of peptide fragments of the guinea pig and bovine proteins. Analyses of the tryptic peptides confirmed the known sequence differences in the NH2-terminal half of the molecule and showed that in the COOH-terminal half of the guinea pig protein Ser131 was missing, Ala136 - His137 was deleted, Leu140 was replaced by Phe, and an extra Ala was inserted somewhere within sequence 142-151 (tryptic peptide T23 ). Sequence determination of guinea pig tryptic peptides corresponding to residues 130-134 ( T20 ), 135-138 ( T21 ), and 142-151 ( T23 ) of the bovine protein confirmed the above sequence changes and placed the extra Ala between Gly142 and His143 . The sequence of the region corresponding to bovine residues 130-143 is thus Ala-Asp-Tyr-Lys-Ser-Lys-Gly-Phe-Lys-Gly-Ala-His. No species differences were observed in the amino acid compositions of the remaining tryptic peptides obtained from the COOH-terminal half of the molecule. Based upon these results, the guinea pig basic protein contains 167 amino acid residues and has a molecular weight of 18,256.     

A Proposed Mechanism for the Promotion of Prion Conversion Involving a Strictly Conserved Tyrosine Residue in the β2-α2 Loop of PrPC

The transmission of infectious prions into different host species requires compatible prion protein (PrP) primary structures, and even one heterologous residue at a pivotal position can block prion infection. Mapping the key amino acid positions that govern cross-species prion conversion has not yet been possible, although certain residue positions have been identified as restrictive, including residues in the β₂-α₂ loop region of PrP. To further define how β₂-α₂ residues impact conversion, we investigated residue substitutions in PrP^C using an in vitro prion conversion assay. Within the β₂-α₂ loop, a tyrosine residue at position 169 is strictly conserved among mammals, and transgenic mice expressing mouse PrP having the Y169G, S170N, and N174T substitutions resist prion infection. To better understand the structural requirements of specific residues for conversion initiated by mouse prions, we substituted a diverse array of amino acids at position 169 of PrP. We found that the substitution of glycine, leucine, or glutamine at position 169 reduced conversion by ∼75%. In contrast, replacing tyrosine 169 with either of the bulky, aromatic residues, phenylalanine or tryptophan, supported efficient prion conversion. We propose a model based on a requirement for tightly interdigitating complementary amino acid side chains within specific domains of adjacent PrP molecules, known as “steric zippers,” to explain these results. Collectively, these studies suggest that an aromatic residue at position 169 supports efficient prion conversion.     

Copper and zinc binding modulates the aggregation and neurotoxic properties of the prion peptide PrP106-126.

The abnormal form of the prion protein (PrP) is believed to be responsible for the transmissible spongiform encephalopathies. A peptide encompassing residues 106-126 of human PrP (PrP106-126) is neurotoxic in vitro due its adoption of an amyloidogenic fibril structure. The Alzheimer's disease amyloid beta peptide (Abeta) also undergoes fibrillogenesis to become neurotoxic. Abeta aggregation and toxicity is highly sensitive to copper, zinc, or iron ions. We show that PrP106-126 aggregation, as assessed by turbidometry, is abolished in Chelex-100-treated buffer. ICP-MS analysis showed that the Chelex-100 treatment had reduced Cu(2+) and Zn(2+) levels approximately 3-fold. Restoring Cu(2+) and Zn(2+) to their original levels restored aggregation. Circular dichroism showed that the Chelex-100 treatment reduced the aggregated beta-sheet content of the peptide. Electron paramagnetic resonance spectroscopy identified a 2N1S1O coordination to the Cu(2+) atom, suggesting histidine 111 and methionine 109 or 112 are involved. Nuclear magnetic resonance confirmed Cu(2+) and Zn(2+) binding to His-111 and weaker binding to Met-112. An N-terminally acetylated PrP106-126 peptide did not bind Cu(2+), implicating the free amino group in metal binding. Mutagenesis of either His-111, Met-109, or Met-112 abolished PrP106-126 neurotoxicity and its ability to form fibrils. Therefore, Cu(2+) and/or Zn(2+) binding is critical for PrP106-126 aggregation and neurotoxicity.     

Competing intrachain interactions regulate the formation of beta-sheet fibrils in bovine PrP peptides

At the heart of the pathogenesis of transmissible spongiform encephalopathies (TSEs), such as BSE, scrapie, and Creutzfeldt-Jakob disease, lies a poorly understood structural rearrangement of PrP, an abundant glycoprotein of the nervous and lymphoid systems. The normal form (PrP(C)), rich in alpha-helix, converts into an aberrant beta-sheet-dominated form (PrP(Sc)), which seems to be at the center of the pathotoxic symptoms observed in TSEs. To understand this process better at a molecular level, we have studied the interactions between different peptides derived from bovine PrP and their structural significance. We show that two unstructured peptides derived from the central region of bovine PrP, residues 115-133 and 140-152, respectively, interact stoichiometrically under physiological conditions to generate beta-sheet-dominated fibrils. However, when both peptides are incubated in the presence of a third peptide derived from an adjoining alpha-helical region (residues 153-169), the formation of beta-sheet-rich fibrils is abolished. These data indicate that native PrP(C) helix 1 might inhibit the strong intrinsic beta-sheet-forming propensity of sequences immediately N-terminal to the globular core of PrP(C), by keeping in place intrachain interactions that would prevent these amyloidogenic regions from triggering aggregation. Moreover, these results indicate new ways in which PrP(Sc) formation could be prevented.     

A peptide from the eel pancreas with structural similarity to human pancreatic secretory trypsin inhibitor

The primary structure of a 61-amino-acid residue peptide from the pancreas of the European eel (Anguilla anguilla) has been established as E E K S G(5)L Y R K P(10)S C G E M(15)S A M H A(20)C P M N F(25)A P V C G(30)T D G N T(35)Y P N E C(40)S L C F Q(45)R Q N T K(50)T D I L I(55)T K D D R(60)C. There was no indication of microheterogeneity. This peptide shows structural similarity to pancreatic secretory trypsin inhibitors from several mammalian species and to a cholecystokinin-releasing peptide isolated from rat pancreatic juice. A comparison of the amino acid sequences of the peptides has identified a domain in the central region of the molecules that has been strongly conserved during evolution. In contrast, the amino acid sequence in the region corresponding to the reactive centre of the mammalian trypsin inhibitors is very poorly conserved in the eel peptide. The P1-P1' reactive site lysine-isoleucine (or arginine-isoleucine) bond in the mammalian trypsin inhibitors is replaced by a methionine-asparagine bond. This region does, however, show limited homology to the reactive centre of human alpha 1-protease inhibitor suggesting that the eel peptide may function as an inhibitor of other proteolytic enzymes in the pancreas.     

Truncated forms of the prion protein PrP demonstrate the need for complexity in prion structure

Self-propagation of aberrant protein folds is the defining characteristic of prions. Knowing the structural basis of self-propagation is essential to understanding prions and their related diseases. Prion rods are amyloid fibrils, but not all amyloids are prions. Prions have been remarkably intractable to structural studies, so many investigators have preferred to work with peptide fragments, particularly in the case of the mammalian prion protein PrP. We compared the structures of a number of fragments of PrP by X-ray fiber diffraction, and found that although all of the peptides adopted amyloid conformations, only the larger fragments adopted conformations that modeled the complexity of self-propagating prions, and even these fragments did not always adopt the PrP structure. It appears that the relatively complex structure of the prion form of PrP is not accessible to short model peptides, and that self-propagation may be tied to a level of structural complexity unobtainable in simple model systems. The larger fragments of PrP, however, are useful to illustrate the phenomenon of deformed templating (heterogeneous seeding), which has important biological consequences.     

Dichotomous versus palm-type mechanisms of lateral assembly of amyloid fibrils

Despite possessing a common cross-beta core, amyloid fibrils are known to exhibit great variations in their morphologies. To date, the mechanism responsible for the polymorphism in amyloid fibrils is poorly understood. Here we report that two variants of mammalian full-length prion protein (PrP), hamster (Ha) and mouse (Mo) PrPs, produced morphologically distinguishable subsets of mature fibrils under identical solvent conditions. To gain insight into the origin of this morphological diversity we analyzed the early stages of polymerization. Unexpectedly, we found that despite a highly conserved amyloidogenic region (94% identity within the residues 90-230), Ha and Mo PrPs followed two distinct pathways for lateral assembly of protofibrils into mature, higher order fibrils. The protofibrils of Ha PrP first formed irregular bundles characterized by a peculiar palm-type shape, which ultimately condensed into mature fibrils. The protofibrils of Mo PrP, on the other hand, associated in pairs in a pattern resembling dichotomous coalescence. These pathways are referred to here as the palm-type and dichotomous mechanisms. Two distinct mechanisms for lateral assembly explain striking differences in morphology of mature fibrils produced from closely related Mo and Ha PrPs. Remarkable similarities between subtypes of amyloid fibrils generated from different proteins and peptides suggest that the two mechanisms of lateral assembly may not be limited to prion proteins but may be a common characteristic of polymerization of amyloidogenic proteins and peptides in general.     

The prion peptide forms ion channels in planar lipid bilayers

One of the hypotheses concerning the pathogenic properties of the prion protein considers its influence on cellular ion homeostasis. Using the lipid bilayer technique, the influence of prion-derived peptides on the lipid bilayer conductance was characterized. To evaluate the physiological significance and possible pathological functions of the peptides, their effect on the membrane potential and respiration rate of hippocampal mitochondria was also studied. We used a peptide bearing the human prion protein sequence YSNQNNF (PrP [169-175]), and peptide SSQNNF (PrP [170-175]) bearing a naturally-occurring mutation in position 171 [N(r)S] linked to schizoaffective diseases in humans (Samaia, H.B., Mari, J.J., Vallada, H.P., Moura R.P., Simpson A.J.G., Brentani R.R. A prion-linked psychiatric disorder. Nature 390 (1997) 241). In this report, we show that PrP [170-175] N171S increases the conductance of planar lipid bilayers. Based on the conductance of single channel currents recorded in 500/500 mM KCl (cis/trans), we found a single channel conductance of 8 to 26 pS. The native prion peptide PrP [169-175] does not form ion channels in the lipid bilayer. Neither of the peptides significantly changed the membrane potential or respiration rate of isolated rat hippocampal mitochondria. We propose a possible mechanism for channel formation by aggregation of the prion-derived peptide.     

Oxidation reduces the fibrillation but not the neurotoxicity of the prion peptide PrP106-126

There is increasing evidence that soluble oligomers of misfolded protein may play a role in the pathogenesis of protein misfolding diseases including the transmissible spongiform encephalopathies (TSE) where the protein involved is the prion protein, PrP. The effect of oxidation on fibrillation tendency and neurotoxicity of different molecular variants of the prion peptide PrP106-126 was investigated. It was found that methionine oxidation significantly reduced amyloid fibril formation and proteinase K resistance, but it did not reduce (but rather increase slightly) the neurotoxicity of the peptides in vivo (electroretinography after intraocular injections in mice) and in vitro (in primary neuronal cultures). We furthermore found that the bovine variant of PrP106-126, containing only one methionine residue, showed both reduced fibril forming capacity and in vivo and in vitro neurotoxicity. The findings imply (I) that there is not a simple relation between the formation of amyloid fibrils and neurotoxicity of PrP106-126 derived peptides, (II) that putative, soluble, non-amyloid protofibrils, presumed to be present in increased proportions in oxidized PrP106-126, could play a role in the pathogenesis of TSE and III) that the number of methionine residues in the PrP106-126 peptide seems to have a pivotal role in determining the physical and biological properties of PrP106-126.     

Relative and regional stabilities of the hamster, mouse, rabbit, and bovine prion proteins toward urea unfolding assessed by nuclear magnetic resonance and circular dichroism spectroscopies

The residue-specific urea-induced unfolding patterns of recombinant prion proteins from different species (bovine, rabbit, mouse, and Syrian hamster) were monitored using high-resolution (1)H nuclear magnetic resonance (NMR) spectroscopy. Protein constructs of different lengths, and with and without a His tag attached at the N-terminus, were studied. The various species showed different overall sensitivities toward urea denaturation with stabilities in the following order: hamster ≤ mouse < rabbit < bovine protein. This order is in agreement with recent circular dichroism (CD) spectroscopic measurements for several species [Khan, M. Q. (2010) Proc. Natl. Acad. Sci. U.S.A.107, 19808-19813] and for the bovine protein presented herein. The [urea](1/2) values determined by CD spectroscopy parallel those of the most stable residues observed by NMR spectroscopy. Neither the longer constructs containing an additional hydrophobic region nor the His tag influenced the stability of the structured domain of the constructs studied. The effect of the S174N mutation in rabbit PrP(C) was also investigated. The rank order of the regional stabilities within each protein remained the same for all species. In particular, the residues in the β-sheet region in all four species were more sensitive to urea-induced unfolding than residues in the α2 and α3 helical regions. These observations indicate that the regional specific unfolding pattern is the same for the four mammalian prion proteins studied but militate against the idea that PrP(Sc) formation is linked with the global stability of PrP(C).     

Amino acid sequence and prion strain specific effects on the in vitro and in vivo convertibility of ovine/murine and bovine/murine prion protein chimeras

Prion diseases are characterised by the conversion of a cellular prion protein (PrP(C)) by its misfolded, hence pathogenic, isoform (PrP(Sc)). The efficiency of this transition depends on the molecular similarities between both interaction partners and on the intrinsic convertibility of PrP(C). Transgenic mice expressing chimeric murine/ovine PrP(C) (Tgmushp mice) are susceptible to BSE and/or scrapie prions of bovine or ovine origin while transgenic mice expressing similar murine/bovine PrP(C) chimera (Tgmubo mice) are essentially resistant. We have studied this phenomenon by cell-free conversion on procaryotically expressed chimeric PrP(C). Mouse passaged scrapie or BSE PrP(Sc) was used as a seed and the conversion reaction was carried out under semi-native conditions. The results obtained in this assay were similar to those of our in vivo experiments. Since mubo- and mushp-PrP(C) differ only at four amino acid positions (S96G, N142S, Y154H and Q185E), single or double point mutations of mushp-PrP(C) were examined in the cell-free conversion assay. While the scrapie Me7 prion induced conversion was largely reduced by the N142S and Q185E but not by the S96G and Y154H mutation, the BSE induced conversion was retained in all mutants. Newly formed PrP(res) exhibited strain specific characteristics, such as the localisation of the proteinase K cleavage site, even in the chimeric PrP(C) mutants. We therefore postulate that the efficiency of the conversion of chimeric PrP(C) depends on the amino acid sequence as well as on prion strain specific effects.     

Identifying key components of the PrPC-PrPSc replicative interface

In prion disease, direct interaction between the cellular prion protein (PrP(C)) and its misfolded disease-associated conformer PrP(Sc) is a crucial, although poorly understood step promoting the formation of nascent PrP(Sc) and prion infectivity. Recently, we hypothesized that three regions of PrP (corresponding to amino acid residues 23-33, 98-110, and 136-158) interacting specifically and robustly with PrP(Sc), likely represent peptidic components of one flank of the prion replicative interface. In this study, we created epitope-tagged mouse PrP(C) molecules in which the PrP sequences 23-33, 98-110, and 136-158 were modified. These novel PrP molecules were individually expressed in the prion-infected neuroblastoma cell line (ScN2a) and the conversion of each mutated mouse PrP(C) substrate to PrP(Sc) compared with that of the epitope-tagged wild-type mouse PrP(C). Mutations within PrP 98-110, substituting all 4 wild-type lysine residues with alanine residues, prevented conversion to PrP(Sc). Furthermore, when residues within PrP 136-140 were collectively scrambled, changed to alanines, or amino acids at positions 136, 137, and 139 individually replaced by alanine, conversion to PrP(Sc) was similarly halted. However, other PrP molecules containing mutations within regions 23-33 and 101-104 were able to readily convert to PrP(Sc). These results suggest that PrP sequence comprising residues 98-110 and 136-140 not only participates in the specific binding interaction between PrP(C) and PrP(Sc), but also in the process leading to conversion of PrP(Sc)-sequestered PrP(C) into its disease-associated form.     

Truncated forms of the prion protein PrP demonstrate the need for complexity in prion structure

Abstract

Self-propagation of aberrant protein folds is the defining characteristic of prions. Knowing the structural basis of self-propagation is essential to understanding prions and their related diseases. Prion rods are amyloid fibrils, but not all amyloids are prions. Prions have been remarkably intractable to structural studies, so many investigators have preferred to work with peptide fragments, particularly in the case of the mammalian prion protein PrP. We compared the structures of a number of fragments of PrP by X-ray fiber diffraction, and found that although all of the peptides adopted amyloid conformations, only the larger fragments adopted conformations that modeled the complexity of self-propagating prions, and even these fragments did not always adopt the PrP structure. It appears that the relatively complex structure of the prion form of PrP is not accessible to short model peptides, and that self-propagation may be tied to a level of structural complexity unobtainable in simple model systems. The larger fragments of PrP, however, are useful to illustrate the phenomenon of deformed templating (heterogeneous seeding), which has important biological consequences.     

Styryl-based and tricyclic compounds as potential anti-prion agents

Prion diseases currently have no effective therapy. These illnesses affect both animal and human populations, and are characterized by the conformational change of a normal self protein PrP(C) (C for cellular) to a pathological and infectious conformer, PrP(Sc) (Sc for scrapie). We used a well characterized tissue culture model of prion infection, where mouse neuroblastoma cells (N2a) were infected with 22L PrP(Sc), to screen compounds for anti-prion activity. In a prior study we designed a library of styryl based, potential imaging compounds which were selected for high affinity binding to Alzheimer's disease β-amyloid plaques and good blood-brain barrier permeability. In the current study we screened this library for activity in the N2a/22L tissue culture system. We also tested the anti-prion activity of two clinically used drugs, trimipramine and fluphenazine, in the N2a/22L system. These were selected based on their structural similarity to quinacrine, which was previously reported to have anti-prion activity. All the compounds were also screened for toxicity in tissue culture and their ability to disaggregate amyloid fibrils composed of PrP and β-amyloid synthetic peptides in vitro. Two of the imaging agents, 23I and 59, were found to be both effective at inhibiting prion infection in N2a/22L tissue culture and to be non-toxic. These two compounds, as well as trimipramine and fluphenazine were evaluated in vivo using wild-type CD-1 mice infected peripherally with 139A PrP(Sc). All four agents significantly prolonged the asymptomatic incubation period of prion infection (p<0.0001 log-rank test), as well as significantly reducing the degree of spongiform change, astrocytosis and PrP(Sc) levels in the brains of treated mice. These four compounds can be considered, with further development, as candidates for prion therapy.     

Mutational analysis of topological determinants in prion protein (PrP) and measurement of transmembrane and cytosolic PrP during prion infection

The prion protein (PrP) can adopt multiple membrane topologies, including a fully translocated form (SecPrP), two transmembrane forms (NtmPrP and CtmPrP), and a cytosolic form. It is important to understand the factors that influence production of these species, because two of them, CtmPrP and cytosolic PrP, have been proposed to be key neurotoxic intermediates in certain prion diseases. In this paper, we perform a mutational analysis of PrP synthesized using an in vitro translation system in order to further define sequence elements that influence the formation of CtmPrP. We find that substitution of charged residues in the hydrophobic core of the signal peptide increases synthesis of CtmPrP and also reduces the efficiency of translocation into microsomes. Combining these mutations with substitutions in the transmembrane domain causes the protein to be synthesized exclusively with the CtmPrP topology. Reducing the spacing between the signal peptide and the transmembrane domain also increases CtmPrP. In contrast, topology is not altered by mutations that prevent signal peptide cleavage or by deletion of the C-terminal signal for glycosylphosphatidylinositol anchor addition. Removal of the signal peptide completely blocks translocation. Taken together, our results are consistent with a model in which the signal peptide and transmembrane domain function in distinct ways as determinants of PrP topology. We also present characterization of an antibody that selectively recognizes CtmPrP and cytosolic PrP by virtue of their uncleaved signal peptides. By using this antibody, as well as the distinctive gel mobility of CtmPrP and cytosolic PrP, we show that the amounts of these two forms in cultured cells and rodent brain are not altered by infection with scrapie prions. We conclude that CtmPrP and cytosolic PrP are unlikely to be obligate neurotoxic intermediates in familial or infectiously acquired prion diseases.     

The charge structure of helix 1 in the prion protein regulates conversion to pathogenic PrPSc

The prion diseases are transmissible neurodegenerative disorders linked to a pathogenic conformer (PrP(Sc)) of the normal prion protein (PrP(C)). Accumulation of PrP(Sc) occurs via a poorly defined process in which PrP(Sc) complexes with and converts endogenous PrP(C) to nascent PrP(Sc). Recent experiments have focused on the highly charged first alpha helix (H1) of PrP. It has been proposed that two putative asparagine-to-arginine intrahelical salt bridges stabilize H1 in PrP(C) yet form intermolecular ionic bonds with adjacent PrP molecules during conversion of PrP(C) to PrP(Sc) (M. P. Morrissey and E. I. Shakhnovich, Proc. Natl. Acad. Sci. USA 96:11293-11298, 1999). Subsequent work (J. O. Speare et al., J. Biol. Chem. 278:12522-12529, 2003 using a cell-free assay of PrP(Sc) conversion suggested that rather than promoting conversion, the salt bridges stabilize PrP(C) against it. However, the role of individual H1 charges in PrP(Sc) generation has not yet been investigated. To approach this question, we systematically reversed or neutralized each charged residue in H1 and tested the effect on conversion to PrP(Sc) in scrapie-infected murine neuroblastoma (ScN2a) cells. We find that replacements of charged H1 residues with like charges permit conversion, while charge reversals hinder it. Neutralization of charges in the N-terminal (amino acids 143 to 146) but not the C-terminal (amino acids 147 to 151) half of H1 permits conversion, while complete reversal of charge orientation of the putative salt bridges produces a nonconvertible PrP. Circular dichroism spectroscopy studies and confocal microscopy immunofluorescence localization studies indicated that charge substitutions did not alter the secondary structure or cell surface expression of PrP(C). These data support the necessity of specific charge orientations in H1 for a productive PrP(Sc)-PrP(C) complex.