Seeded conversion of recombinant prion protein to a disulfide-bonded oligomer by a reduction-oxidation process

The infectious form of prion protein, PrP(Sc), self-propagates by its conversion of the normal, cellular prion protein molecule PrP(C) to another PrP(Sc) molecule. It has not yet been demonstrated that recombinant prion protein can convert prion protein molecules from PrP(C) to PrP(Sc). Here we show that recombinant hamster prion protein is converted to a second form, PrP(RDX), by a redox process in vitro and that this PrP(RDX) form seeds the conversion of other PrP(C) molecules to the PrP(RDX) form. The converted form shows properties of oligomerization and seeded conversion that are characteristic of PrP(Sc). We also find that the oligomerization can be reversed in vitro. X-ray fiber diffraction suggests an amyloid-like structure for the oligomerized prion protein. A domain-swapping model involving intermolecular disulfide bonds can account for the stability and coexistence of two molecular forms of prion protein and the capacity of the second form for self-propagation.     

Concealment of epitope by reduction and alkylation in prion protein

Conversion of the cellular prion protein (PrP(C)) into its pathological isoform (PrP(Sc)), the key molecular event in the pathogenesis of prion diseases, is accompanied by a conformational transition of alpha-helix into beta-sheet structures involving alpha-helix 1 (alpha1) domain from residues 144 to 154 of the protein. Reduction and alkylation of PrP(C) have been found to inhibit the conversion of PrP(C) into PrP(Sc) in vitro. Here we report that while antibody affinity of epitopes in the N- and C-terminal domains remained unchanged, reduction and alkylation of the PrP molecule induced complete concealment of an epitope in alpha1 for anti-PrP antibody 6H4 that is able to cure prion infection in the cell model. Mass spectrometric analysis of recombinant PrP showed that the alkylation reaction takes place at reduced cysteines but no modification was observed in this cryptic epitope. Our study suggests that reduction and alkylation result in local or global rearrangement of PrP tertiary structure that is maintained in both liquid and solid phases. The implications in the conversion of PrP(C) into PrP(Sc) and the therapeutics of prion diseases are discussed.     

Preventing misfolding of the prion protein by trimethylamine N-oxide

Transmissible spongiform encephalopathies are a class of fatal neurodegenerative diseases linked to the prion protein. The prion protein normally exists in a soluble, globular state (PrP(C)) that appears to participate in copper metabolism in the central nervous system and/or signal transduction. Infection or disease occurs when an alternatively folded form of the prion protein (PrP(Sc)) converts soluble and predominantly alpha-helical PrP(C) into aggregates rich in beta-structure. The structurally disordered N-terminus adopts beta-structure upon conversion to PrP(Sc) at low pH. Chemical chaperones, such as trimethylamine N-oxide (TMAO), can prevent formation of PrP(Sc) in scrapie-infected mouse neuroblastoma cells [Tatzelt, J., et al. (1996) EMBO J. 15, 6363-6373]. To explore the mechanism of TMAO protection of PrP(C) at the atomic level, molecular dynamics simulations were performed under conditions normally leading to conversion (low pH) with and without 1 M TMAO. In PrP(C) simulations at low pH, the helix content drops and the N-terminus is brought into the small native beta-sheet, yielding a PrP(Sc)-like state. Addition of 1 M TMAO leads to a decreased radius of gyration, a greater number of protein-protein hydrogen bonds, and a greater number of tertiary contacts due to the N-terminus forming an Omega-loop and packing against the structured core of the protein, not due to an increase in the level of extended structure as with the PrP(C) to PrP(Sc) simulation. In simulations beginning with the "PrP(Sc)-like" structure (derived from PrP(C) simulated at low pH in pure water) in 1 M TMAO, similar structural reorganization at the N-terminus occurred, disrupting the extended sheet. The mechanism of protection by TMAO appears to be exclusionary in nature, consistent with previous theoretical and experimental studies. The TMAO-induced N-terminal conformational change prevents residues that are important in the conversion of PrP(C) to PrP(Sc) from assuming extended sheet structure at low pH.     

Locally disordered conformer of the hamster prion protein: a crucial intermediate to PrPSc?

A crucial step for transformation of the normal cellular isoform of the prion protein (PrP(C)) to the infectious prion protein (PrP(Sc)) is thought to entail a previously uncharacterized intermediate conformer, PrP*, which interacts with a template PrP(Sc) molecule in the conversion process. By carrying out (15)N-(1)H two-dimensional NMR measurements under variable pressure on Syrian hamster prion protein rPrP(90-231), we found a metastable conformer of PrP(C) coexisting at a population of approximately 1% at pH 5.2 and 30 degrees C, in which helices B and C are preferentially disordered. While the identity is still unproven, this observed metastable conformer is most logically PrP* or a closely related precursor. The structural characteristics of this metastable conformer are consistent with available immunological and pathological information about the prion protein.     

Toward molecular dissection of PrPC-PrPSc interactions

Direct interaction between endogenous cellular prion protein (PrP(C)) and misfolded, disease-associated (PrP(Sc)) conformers is a key event in prion propagation, which precedes templated conversion of PrP(C) into nascent PrP(Sc) and prion infectivity. Although almost none of the molecular details of this pivotal process are understood, the persistence of individual prion strains suggests that assembly of the prion replicative complex is mechanistically precise. To systematically map defined regions of PrP(C) sequence that bind tightly to PrP(Sc), we have generated a comprehensive panel of over 45 motif-grafted antibodies containing overlapping peptide grafts collectively spanning PrP residues 19-231. Grafted antibody binding experiments, performed under stringent conditions, clearly identified only three distinct and independent high affinity PrP(Sc) recognition motifs. The first of these binding motifs lies at the very N-terminal region of the mature PrP molecule within PrP-(23-33); the second motif lies within PrP-(98-110); and the third is contained within PrP-(136-158). Mutational analyses of these PrP(Sc)-binding regions revealed that reactivity of the 23-33 and 98-110 segments are largely dependent upon the presence of multiple positively charged amino acid residues. These studies yield new insight into critical peptidic components composing one side of the prion replicative interface.     

Novel heparan mimetics potently inhibit the scrapie prion protein and its endocytosis

During prion diseases the normal prion protein PrP(C) is refolded into an abnormal conformer PrP(Sc). We have studied the PrP(Sc) inhibiting activity of a library of synthetic heparan mimetic (HM) biopolymers. HMs are chemically derived dextrans obtained by successive substitutions with carboxymethyl, benzylamide, and sulfate groups on glucose residues. Some HMs eliminated PrP(Sc) from prion-infected cells after a 5 day course at 100 ng/ml and were 15 x potent than pentosan sulfate in this system. The anti-PrP(Sc) activity of HMs correlated with the degree of sulfation but was increased by benzylamidation. HMs did not reduce the synthesis of PrP(C) nor its attachment to lipid rafts, but instead blocked its conversion into PrP(Sc). The anti-PrP(Sc) HMs also prevented the uptake of prion rods by cultured cells. HMs may thus block the interaction of PrP(Sc) with a putative cellular receptor, possibly heparan sulfate. HMs provide an attractive chemical approach for the synthesis of TSE therapeutic and prophylactic reagents.     

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.     

The role of rafts in the fibrillization and aggregation of prions

A key molecular event in prion diseases is the conversion of the prion protein (PrP) from its normal cellular form (PrP(C)) to the disease-specific form (PrP(Sc)). The transition from PrP(C) to PrP(Sc) involves a major conformational change, resulting in amorphous aggregates and/or fibrillar amyloid deposits. Here several lines of evidence implicating membranes in the conversion of PrP are reviewed with a particular emphasis on the role of lipid rafts in the conformational transition of prion proteins. New correlations between in vitro biophysical studies and findings from cell biology work on the role of rafts in prion conversion are highlighted and a mechanism for the role of rafts in prion conversion is proposed.     

Strain-specific effects of reducing agents on the cell-free conversion of recombinant prion protein into a protease-resistant form

The pathogenic isoform (PrP(Sc) ) of the host-encoded normal cellular prion protein (PrP(C) ) is believed to be the infectious agent of transmissible spongiform encephalopathies. Spontaneous conversion of α-helix-rich recombinant PrP into the PrP(Sc) -like β-sheet-rich form or aggregation of cytosolic PrP has been found to be accelerated under reducing conditions. However, the effect of reducing conditions on PrP(Sc) -mediated conversion of PrP(C) into PrP(Sc) has remained unknown. In this study, the effect of reducing conditions on the binding of bacterial recombinant mouse PrP (MoPrP) with PrP(Sc) and the conversion of MoPrP into proteinase K-resistant PrP (PrP(res) ) using a cell-free conversion assay was investigated. High concentrations of dithiothreitol did not inhibit either the binding or conversion reactions of PrP(Sc) from five prion strains. Indeed, dithiothreitol significantly accelerated mouse-adapted BSE-seeded conversion. These data suggest that conversion of PrP(Sc) derived from a subset of prion strains is accelerated under reducing conditions, as has previously been shown for spontaneous conversion. Furthermore, the five prion strains used could be classified into three groups according to their efficiency at binding and conversion of MoPrP and cysteine-less mutants under both reducing and nonreducing conditions. The resulting classification is similar to that derived from biological and biochemical strain-specific features.     

Differences between the prion protein and its homolog Doppel: a partially structured state with implications for scrapie formation

The key event in the pathogenesis of prion diseases is a conformational change in the prion protein (PrP). Models for conversion of PrP(C) into PrP(Sc) typically implicate an, as yet, unidentified intermediate. In an attempt to identify such an intermediate, we used native-state hydrogen exchange monitored with NMR. Although we were unable to detect an intermediate directly, we observed substantial protection above that expected based upon measurements of the global stability of PrP (>2 kcal mol(-1) super protection). This super protection implicates either structure in the denatured state or the presence of an intermediate. Similar experiments with Doppel, a homolog of PrP that does not form infectious prions, failed to demonstrate such super protection. This suggests that the partially structured state of PrP encompassing portions of the B and C helices, may be a significant factor in the ability of PrP to convert from PrP(C) to PrP(Sc).     

Dissociation of infectivity from seeding ability in prions with alternate docking mechanism

Previous studies identified two mammalian prion protein (PrP) polybasic domains that bind the disease-associated conformer PrP(Sc), suggesting that these domains of cellular prion protein (PrP(C)) serve as docking sites for PrP(Sc) during prion propagation. To examine the role of polybasic domains in the context of full-length PrP(C), we used prion proteins lacking one or both polybasic domains expressed from Chinese hamster ovary (CHO) cells as substrates in serial protein misfolding cyclic amplification (sPMCA) reactions. After ～5 rounds of sPMCA, PrP(Sc) molecules lacking the central polybasic domain (ΔC) were formed. Surprisingly, in contrast to wild-type prions, ΔC-PrP(Sc) prions could bind to and induce quantitative conversion of all the polybasic domain mutant substrates into PrP(Sc) molecules. Remarkably, ΔC-PrP(Sc) and other polybasic domain PrP(Sc) molecules displayed diminished or absent biological infectivity relative to wild-type PrP(Sc), despite their ability to seed sPMCA reactions of normal mouse brain homogenate. Thus, ΔC-PrP(Sc) prions interact with PrP(C) molecules through a novel interaction mechanism, yielding an expanded substrate range and highly efficient PrP(Sc) propagation. Furthermore, polybasic domain deficient PrP(Sc) molecules provide the first example of dissociation between normal brain homogenate sPMCA seeding ability from biological prion infectivity. These results suggest that the propagation of PrP(Sc) molecules may not depend on a single stereotypic mechanism, but that normal PrP(C)/PrP(Sc) interaction through polybasic domains may be required to generate prion infectivity.     

Integrity of helix 2-helix 3 domain of the PrP protein is not mandatory for prion replication

The process of prion conversion is not yet well understood at the molecular level. The regions critical for the conformational change of PrP remain mostly debated and the extent of sequence change acceptable for prion conversion is poorly documented. To achieve progress on these issues, we applied a reverse genetic approach using the Rov cell system. This allowed us to test the susceptibility of a number of insertion mutants to conversion into prion in the absence of wild-type PrP molecules. We were able to propagate several prions with 8 to 16 extra amino acids, including a polyglycine stretch and His or FLAG tags, inserted in the middle of the protease-resistant fragment. These results demonstrate the possibility to increase the length of the loop between helices H2 and H3 up to 4-fold, without preventing prion replication. They also indicate that this loop probably remains unstructured in PrP(Sc). We also showed that bona fide prions can be produced following insertion of octapeptides in the two C-terminal turns of H2. These insertions do not interfere with the overall fold of the H2-H3 domain indicating that the highly conserved sequence of the terminal part of H2 is not critical for the conversion. Altogether these data showed that the amplitude of modifications acceptable for prion conversion in the core of the globular domain of PrP is much greater than one might have assumed. These observations should help to refine structural models of PrP(Sc) and elucidate the conformational changes underlying prions generation.     

Chemical chaperones interfere with the formation of scrapie prion protein.

The fundamental event in prion diseases involves a conformational change in one or more of the alpha-helices of the cellular prion protein (PrP(C)) as they are converted into beta-sheets during the formation of the pathogenic isoform (PrP(Sc)). Here, we show that exposure of scrapie-infected mouse neuroblastoma (ScN2a) cells to reagents known to stabilize proteins in their native conformation reduced the rate and extent of PrP(Sc) formation. Such reagents include the cellular osmolytes glycerol and trimethylamine N-oxide (TMAO) and the organic solvent dimethylsulfoxide (DMSO), which we refer to as 'chemical chaperones' because of their influence on protein folding. Although the chemical chaperones did not appear to affect the existing population of PrP(Sc) molecules in ScN2a cells, they did interfere with the formation of PrP(Sc) from newly synthesized PrP(C). We suggest that the chemical chaperones act to stabilize the alpha-helical conformation of PrP(C) and thereby prevent the protein from undergoing a conformational change to produce PrP(Sc). These observations provide further support for the idea that prions arise due to a change in protein conformation and reveal potential strategies for preventing PrP(Sc) formation.     

Selective re-routing of prion protein to proteasomes and alteration of its vesicular secretion prevent PrP(Sc) formation

Conversion of the cellular prion protein (PrP(C)) into the abnormal scrapie isoform (PrP(Sc)) is the hallmark of prion diseases, which are fatal and transmissible neurodegenerative disorders. ER-retained anti-prion recombinant single-chain Fv fragments have been proved to be an effective tool for inhibition of PrP(C) trafficking to the cell surface and antagonize PrP(Sc) formation and infectivity. In the present study, we have generated the secreted version of 8H4 intrabody (Sec-8H4) in order to compel PrP(C) outside the cells. The stable expression of the Sec-8H4 intrabodies induces proteasome degradation of endogenous prion protein but does not influence its glycosylation profile and maturation. Moreover, we found a dramatic diverting of PrP(C) traffic from its vesicular secretion and, most importantly, a total inhibition of PrP(Sc) accumulation in NGF-differentiated Sec-8H4 PC12 cells. These results confirm that perturbing the intracellular traffic of endogenous PrP(C) is an effective strategy to inhibit PrP(Sc) accumulation and provide convincing evidences for application of intracellular antibodies in prion diseases.     

The effect of disease-associated mutations on the folding pathway of human prion protein

Propagation of transmissible spongiform encephalopathies is believed to involve the conversion of cellular prion protein, PrP(C), into a misfolded oligomeric form, PrP(Sc). An important step toward understanding the mechanism of this conversion is to elucidate the folding pathway(s) of the prion protein. We reported recently (Apetri, A. C., and Surewicz, W. K. (2002) J. Biol. Chem. 277, 44589-44592) that the folding of wild-type prion protein can best be described by a three-state sequential model involving a partially folded intermediate. Here we have performed kinetic stopped-flow studies for a number of recombinant prion protein variants carrying mutations associated with familial forms of prion disease. Analysis of kinetic data clearly demonstrates the presence of partially structured intermediates on the refolding pathway of each PrP variant studied. In each case, the partially folded state is at least one order of magnitude more populated than the fully unfolded state. The present study also reveals that, for the majority of PrP variants tested, mutations linked to familial prion diseases result in a pronounced increase in the thermodynamic stability, and thus the population, of the folding intermediate. These data strongly suggest that partially structured intermediates of PrP may play a crucial role in prion protein conversion, serving as direct precursors of the pathogenic PrP(Sc) isoform.     

Molecular dynamics simulations of human prion protein: importance of correct treatment of electrostatic interactions.

Molecular dynamics simulations have been used to investigate the dynamical and structural behavior of a homology model of human prion protein HuPrP(90-230) generated from the NMR structure of the Syrian hamster prion protein ShPrP(90-231) and of ShPrP(<90-231) itself. These PrPs have a large number of charged residues on the protein surface. At the simulation pH 7, HuPrP(90-230) has a net charge of -1 eu from 15 positively and 14 negatively charged residues. Simulations for both PrPs, using the AMBER94 force field in a periodic box model with explicit water molecules, showed high sensitivity to the correct treatment of the electrostatic interactions. Highly unstable behavior of the structured region of the PrPs (127-230) was found using the truncation method, and stable trajectories could be achieved only by including all the long-range electrostatic interactions using the particle mesh Ewald (PME) method. The instability using the truncation method could not be reduced by adding sodium and chloride ions nor by replacing some of the sodium ions with calcium ions. The PME simulations showed, in accordance with NMR experiments with ShPrP and mouse PrP, a flexibly disordered N-terminal part, PrP(90-126), and a structured C-terminal part, PrP(127-230), which includes three alpha-helices and a short antiparallel beta-strand. The simulations showed some tendency for the highly conserved hydrophobic segment PrP(112-131) to adopt an alpha-helical conformation and for helix C to split at residues 212-213, a known disease-associated mutation site (Q212P). Three highly occupied salt bridges could be identified (E146/D144<-->R208, R164<-->D178, and R156<-->E196) which appear to be important for the stability of PrP by linking the stable main structured core (helices B and C) with the more flexible structured part (helix A and strands A and B). Two of these salt bridges involve disease-associated mutations (R208H and D178N). Decreased PrP stability shown by protein unfolding experiments on mutants of these residues and guanidinium chloride or temperature-induced unfolding studies indicating reduced stability at low pH are consistent with stabilization by salt bridges. The fact that electrostatic interactions, in general, and salt bridges, in particular, appear to play an important role in PrP stability has implications for PrP structure and stability at different pHs it may encounter physiologically during normal or abnormal recycling from the pH neutral membrane surface into endosomes or lysomes (acidic pHs) or in NMR experiments (5.2 for ShPrP and 4.5 for mouse PrP).     

Conversion efficiency of bank vole prion protein in vitro is determined by residues 155 and 170, but does not correlate with the high susceptibility of bank voles to sheep scrapie in vivo

The misfolded infectious isoform of the prion protein (PrP(Sc)) is thought to replicate in an autocatalytic manner by converting the cellular form (PrP(C)) into its pathogenic folding variant. The similarity in the amino acid sequence of PrP(C) and PrP(Sc) influences the conversion efficiency and is considered as the major determinant for the species barrier. We performed in vitro conversion reactions on wild-type and mutated PrP(C) to determine the role of the primary sequence for the high susceptibility of bank voles to scrapie. Different conversion efficiencies obtained with bank vole and mouse PrP(C) in reactions with several prion strains were due to differences at amino acid residues 155 and 170. However, the conversion efficiencies obtained with mouse and vole PrP(C) in reactions with sheep scrapie did not correlate with the susceptibility of the respective species to this prion strain. This discrepancy between in vitro and in vivo data may indicate that at least in the case of scrapie transmission to bank voles additional host factors can strongly modulate the species barrier. Furthermore, in vitro conversion reactions with different prion strains revealed that the degree of alteration of the conversion efficiency induced by amino acid exchanges was varying according to the prion strain. These results support the assumption that the repertoire of conformations adopted by a certain PrP(C) primary sequence is decisive for its convertibility to the strain-specific PrP(Sc) conformation.     

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.     

Reconstitution of prion infectivity from solubilized protease-resistant PrP and nonprotein components of prion rods

The scrapie isoform of the prion protein, PrP(Sc), is the only identified component of the infectious prion, an agent causing neurodegenerative diseases such as Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. Following proteolysis, PrP(Sc) is trimmed to a fragment designated PrP 27-30. Both PrP(Sc) and PrP 27-30 molecules tend to aggregate and precipitate as amyloid rods when membranes from prion-infected brain are extracted with detergents. Although prion rods were also shown to contain lipids and sugar polymers, no physiological role has yet been attributed to these molecules. In this work, we show that prion infectivity can be reconstituted by combining Me(2)SO-solubilized PrP 27-30, which at best contained low prion infectivity, with nonprotein components of prion rods (heavy fraction after deproteination, originating from a scrapie-infected hamster brain), which did not present any infectivity. Whereas heparanase digestion of the heavy fraction after deproteination (originating from a scrapie-infected hamster brain), before its combination with solubilized PrP 27-30, considerably reduced the reconstitution of infectivity, preliminary results suggest that infectivity can be greatly increased by combining nonaggregated protease-resistant PrP with heparan sulfate, a known component of amyloid plaques in the brain. We submit that whereas PrP 27-30 is probably the obligatory template for the conversion of PrP(C) to PrP(Sc), sulfated sugar polymers may play an important role in the pathogenesis of prion diseases.