Selective oxidation of methionine residues in prion proteins.

Prion proteins are central to the pathogenesis of several neurodegenerative diseases through the postulated conversion of the endogenous cellular isoform (PrPc) into a pathogenic isoform (PrPSc). Although the cellular function of normal prion protein remains unresolved a number of studies have shown that prion proteins may be involved in the cellular response to oxidative stress. Here, using purified recombinant sources of mouse and chicken PrP refolded in the presence of copper (II) we show that the methionine residues of the protein are uniquely susceptible to oxidation. We suggest that Met residues may form an essential part of the mechanism of the antioxidant activity exhibited by normal prion protein.     

Expansion of the octarepeat domain alters the misfolding pathway but not the folding pathway of the prion protein

A misfolded conformation of the prion protein (PrP), PrP (Sc), is the essential component of prions, the infectious agents that cause transmissible neurodegenerative diseases. Insertional mutations that lead to an increase in the number of octarepeats (ORs) in PrP are linked to familial human prion disease. In this study, we investigated how expansion of the OR domain causes PrP to favor a prion-like conformation. Therefore, we compared the conformational and aggregation modulating properties of wild-type versus expanded OR domains, either as a fusion construct with the protein G B1 domain (GB1-OR) or as an integral part of full-length mouse PrP (MoPrP). Using circular dichroism spectroscopy, we first demonstrated that ORs are not unfolded but exist as an ensemble of three distinct conformers: polyproline helix-like, beta-turn, and "Trp-related". Domain expansion had little effect on the conformation of GB1-OR fusion proteins. When part of MoPrP however, OR domain expansion changed PrP's folding landscape, not by hampering the production of native alpha-helical monomers but by greatly reducing the propensity to form amyloid and by altering the assembly of misfolded, beta-rich aggregates. These features may relate to subtle pH-dependent conformational differences between wild-type and mutant monomers. In conclusion, we propose that PrP insertional mutations are pathogenic because they enhance specific misfolding pathways of PrP rather than by undermining native folding. This idea was supported by a trial bioassay in transgenic mice overexpressing wild-type MoPrP, where intracerebral injection of recombinant MoPrP with an expanded OR domain but not wild-type MoPrP caused prion disease.     

Folding of prion protein to its native alpha-helical conformation is under kinetic control

The recombinant mouse prion protein (MoPrP) can be folded either to a monomeric alpha-helical or oligomeric beta-sheet-rich isoform. By using circular dichroism spectroscopy and size-exclusion chromatography, we show that the beta-rich isoform of MoPrP is thermodynamically more stable than the native alpha-helical isoform. The conformational transition from the alpha-helical to beta-rich isoform is separated by a large energetic barrier that is associated with unfolding and with a higher order kinetic process related to oligomerization. Under partially denaturing acidic conditions, MoPrP avoids the kinetic trap posed by the alpha-helical isoform and folds directly to the thermodynamically more stable beta-rich isoform. Our data demonstrate that the folding of the prion protein to its native alpha-helical monomeric conformation is under kinetic control.     

Epitope mapping of the Syrian hamster prion protein utilizing chimeric and mutant genes in a vaccinia virus expression system.

The cellular prion protein (PrPc) is a host-encoded sialoglycoprotein bound to the external surface of the cell membrane by a glycosyl phosphatidylinositol anchor. A posttranslationally modified PrP isoform (PrPSc) is a component of the infectious particle causing scrapie and the other prion diseases. mAb have been raised against the protease-resistant core of Syrian hamster (SHa) PrPSc designated PrP 27-30. To map the epitopes within PrP reacting to these antibodies, we have expressed wild-type, chimeric mouse (Mo)/SHa and mutant MoPrP genes using recombinant vaccinia virus systems. The fidelity of the expression of recombinant PrPC was examined using vaccinia viruses expressing SHa-PrPC. It is full length, possesses Asn-linked carbohydrates and is attached to the external surface of the cell membrane by a glycosyl phosphatidylinositol anchor that is sensitive to cleavage by phosphatidylinositol-specific phospholipase C. We have tested 18 mAb for their ability to bind to chimeric prion proteins on immunoblots. Three distinct epitopes were identified that mapped to amino acid differences between SHa and MoPrP sequences. The first epitope, recognized by three of the antibodies tested, was defined by methionines at amino acids 108 and 111 in the mouse protein. The second epitope was dependent upon the presence of asparagines at positions 154 and 174 in MoPrP and was recognized by four of the antibodies tested. The third epitope mapped to a single amino acid substitution at residue 138 in MoPrP. mAb raised against SHaPrP 27-30 specific for this epitope are able to bind MoPrPC which has a single amino acid change (Ile to Met) at position 138. Eleven of the 18 antibodies tested mapped to this immunodominant epitope. It is located within a postulated amphipathic helix, a structure associated with immunodominant Ag. Inasmuch as PrPC, in its native form on the cell surface, is detected by the mAb 13A5 (a prototypic antibody of the immunodominant third epitope class), it is likely that this epitope is accessible in the native conformation of this protein.     

Substitution of serine for non-disulphide-bond-forming cysteine in grass carp (Ctenopharygodon idellus) growth hormone improves in vitro oxidative renaturation

Native grass carp (Ctenopharygodon idellus) growth hormone, has 5 cysteine amino acid residues, forms two disulphide bridges in its mature form. Recombinant grass carp growth hormone, when over-expressed in E. coli, forms inclusion bodies. In vitro oxidative renaturation of guanidine-hydrochloride dissolved recombinant grass carp growth hormone was achieved by sequential dilution and stepwise dialysis at pH 8.5. The redox potential of the refolding cocktail was maintained by glutathione disulphide/glutathione couple. The oxidative refolded protein is heterogeneous, and contains multimers, oligomers and monomers. The presence of non-disulphide-bond-forming cysteine in recombinant grass carp growth hormone enhances intermolecular disulphide bond formation and also nonnative intramolecular disulphide bond formation during protein folding. The non-disulphide-bond-forming cysteine was converted to serine by PCR-mediated site-directed mutagenesis. The resulting 4-cysteine grass carp growth hormone has improved in vitro oxidative refolding properties when studied by gel filtration and reverse phase chromatography. The refolded 4-cysteine form has less hydrophobic aggregate and has only one monomeric isoform. Both refolded 4-cysteine and 5-cysteine forms are active in radioreceptor binding assay.     

Inhibition of proteinase K activity by copper(II) ions

Disease-related prion protein, PrPSc, can be distinguished from its normal cellular precursor, PrPC, by its detergent insolubility and partial resistance to proteolysis. Several studies have suggested that copper(II) ions can convert PrPC to a proteinase K-resistant conformation; however, interpretation of these studies is complicated by potential inhibition of proteinase K (PK) by copper(II) ions. Here we have examined directly the kinetic and equilibrium effects of copper(II) ions on PK activity using a simple synthetic substrate, p-nitrophenyl acetate. We show that at equilibrium two to three copper(II) ions bind stoichiometrically to PK and destroy its activity (Kd < 1 microM). This inhibition has two components, an initial reversible and weak binding phase and a slower, irreversible abolition of activity with a half-time of 6 min at saturating copper(II) ion concentrations. Copper(II) ions produce a similar biphasic inhibition of PK activity in the presence of brain homogenate but only when the copper(II) ion concentration exceeds that of the chelating components present in brain tissue. Under these conditions, the apparent resistance of PrPC to proteolysis by PK appears to be directly attributable to the inhibition of PK activity by copper(II) ions.     

Copper has differential effect on prion protein with polymorphism of position 129

The pathology of human prion diseases is affected by polymorphism at amino acid residue 129 of the prion protein gene. Recombinant mouse prion proteins mimicking either form of the polymorphism were prepared to examine their effect on the conformation and the level of superoxide dismutase (SOD) activity of the prion protein. Following the binding of copper atoms to prion protein, antibody mapping and CD analysis detected conformational differences between the two forms of protein. However, neither the level of copper binding nor the level of SOD activity associated with this form of prion protein altered with the identity of codon 129. These results suggest that in the holo-metal binding form of the protein, prion structure but not its SOD activity is affected by polymorphism at codon 129.     

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.     

Absence of superoxide dismutase activity in a soluble cellular isoform of prion protein produced by baculovirus expression system

A method for expression and purification of a soluble form of histidine (HIS)-tagged murine prion protein (bacMuPrP), which lacks the entire C-terminal cleavage and glycosyl phosphatidyl inositol (GPI) addition site, has been developed using a recombinant baculovirus expression system and purification with Ni-NTA agarose affinity chromatography. In mammalian sources, PrP(C) is attached to the cell membrane by a GPI anchor. However, in our system, bacMuPrP was secreted into the media, enabling its easy purification in abundance. Indirect immunofluorescence studies and immunoblot analysis localized not in cell membrane but in the perinuclear endoplasmic reticulum region in cells and is secreted into the media. Tunicamycin treatment revealed non-glycosylated proteins were secreted into the media, suggesting that glycosylation is not necessary for bacMuPrP secretion. Density-gradient sedimentation analysis demonstrated a sedimentation coefficient of secretory bacMuPrP as 2.3 S, indicating a monomeric form. Although affinity-purified PrP from mouse brain or recombinant prion protein (PrP) produced by Escherichia coli and refolded in the presence of copper has been reported to display superoxide dismutase (SOD) activity, bacMuPrP did not show SOD activity. These results suggest that bacMuPrP has a different biochemical and biophysical characterization from mammalian and bacterial-derived PrP. Furthermore, this simple expression system may provide an adequate source for structural, functional, and biochemical analyses of PrP.     

An unusual soluble beta-turn-rich conformation of prion is involved in fibril formation and toxic to neuronal cells

A key molecular event in prion diseases is the conversion of the prion protein (PrP) from its normal cellular form (PrPC) to the disease-specific form (PrPSc). The transition from PrPC to PrPSc involves a major conformational change, resulting in amorphous protein aggregates and fibrillar amyloid deposits with increased beta-sheet structure. Using recombinant PrP refolded into a beta-sheet-rich form (beta-PrP) we have studied the fibrillization of beta-PrP both in solution and in association with raft membranes. In low ionic strength thick dense fibrils form large networks, which coexist with amorphous aggregates. High ionic strength results in less compact fibrils, that assemble in large sheets packed with globular PrP particles, resembling diffuse aggregates found in ex vivo preparations of PrPSc. Here we report on the finding of a beta-turn-rich conformation involved in prion fibrillization that is toxic to neuronal cells in culture. This is the first account of an intermediate in prion fibril formation that is toxic to neuronal cells. We propose that this unusual beta-turn-rich form of PrP may be a precursor of PrPSc and a candidate for the neurotoxic molecule in prion pathogenesis.     

In vitro conversion of full-length mammalian prion protein produces amyloid form with physical properties of PrP(Sc)

The "protein only" hypothesis postulates that the infectious agent of prion diseases, PrP(Sc), is composed of the prion protein (PrP) converted into an amyloid-specific conformation. However, cell-free conversion of the full-length PrP into the amyloid conformation has not been achieved. In an effort to understand the mechanism of PrP(Sc) formation, we developed a cell-free conversion system using recombinant mouse full-length PrP with an intact disulfide bond (rPrP). We demonstrate that rPrP will convert into the beta-sheet-rich oligomeric form at highly acidic pH (<5.5) and at high concentrations, while at slightly acidic or neutral pH (>5.5) it assembles into the amyloid form. As judged from electron microscopy, the amyloid form had a ribbon-like assembly composed of two non-twisted filaments. In contrast to the formation of the beta-oligomer, the conversion to the amyloid occurred at concentrations close to physiological and displayed key features of an autocatalytic process. Moreover, using a shortened rPrP consisting of 106 residues (rPrP 106, deletions: Delta23-88 and Delta141-176), we showed that the in vitro conversion mimicked a transmission barrier observed in vivo. Furthermore, the amyloid form displayed a remarkable resistance to proteinase K (PK) and produced a PK-resistant core identical with that of PrP(Sc). Fourier transform infrared spectroscopy analyses showed that the beta-sheet-rich core of the amyloid form remained intact upon PK-digestion and accounted for the extremely high thermal stability. Electron and real-time fluorescent microscopy revealed that proteolytic digestion induces either aggregation of the amyloid ribbons into large clumps or further assembly into fibrils composed of several ribbons. Fibrils composed of ribbons were very fragile and had a tendency to fragment into short pieces. Remarkably, the amyloid form treated with PK preserved high seeding activity. Our work supports the protein only hypothesis of prion propagation and demonstrates that formation of the amyloid form that recapitulates key physical properties of PrP(Sc) can be achieved in vitro in the absence of cellular factors or a PrP(Sc) template.     

Production of a recombinant full-length prion protein in a soluble form without refolding or detergents

Recombinant prion protein has been produced in insoluble form and refolded following solubilization with denaturants. It is, however, preferable to use a soluble recombinant protein prepared without artificial solubilization. In this study, a soluble recombinant prion protein was produced in Escherichia coli cells by coexpression of neuregulin I-β1 and purified to high purity.     

High hydrophobic amino acid exposure is responsible of the neurotoxic effects induced by E200K or D202N disease-related mutations of the human prion protein

Mutations in prion protein are thought to be causative of inherited prion diseases favoring the spontaneous conversion of the normal prion protein into the scrapie-like pathological prion protein. We previously reported that, by controlled thermal denaturation, human prion protein fragment 90-231 acquires neurotoxic properties when transformed in a β-rich conformation, resembling the scrapie-like conformation. In this study we generated prion protein fragment 90-231 bearing mutations identified in familial prion diseases (D202N and E200K), to analyze their role in the induction of a neurotoxic conformation. Prion protein fragment 90-231(wild type) and the D202N mutant were not toxic in native conformation but induced cell death only after thermal denaturation. Conversely, prion protein fragment 90-231(E200K) was highly toxic in its native structure, suggesting that E200K mutation per se favors the acquisition of a peptide neurotoxic conformation. To identify the structural determinants of prion protein fragment 90-231 toxicity, we show that while the wild type peptide is structured in α-helix, hPrP90-231 E200K is spontaneously refolded in a β-structured conformer characterized by increased proteinase K resistance and propensity to generate fibrils. However, the most significant difference induced by E200K mutation in prion protein fragment 90-231 structure in native conformation we observed, was an increase in the exposure of hydrophobic amino-acids on protein surface that was detected in wild type and D202N proteins only after thermal denaturation. In conclusion, we propose that increased hydrophobicity is one of the main determinants of toxicity induced by different mutations in prion protein-derived peptides.     

Computational studies on prion proteins: effect of Ala(117)-->Val mutation

Molecular dynamics calculations demonstrated the conformational change in the prion protein due to Ala(117)-->Val mutation, which is related to Gerstmann-Str?ussler-Sheinker disease, one of the familial prion diseases. Three kinds of model structures of human and mouse prion proteins were examined: (model 1) nuclear magnetic resonance structures of human prion protein HuPrP (125-228) and mouse prion protein MoPrP (124-224), each having a globular domain consisting of three alpha-helices and an antiparallel beta-sheet; (model 2) extra peptides including Ala(117) (109-124 in HuPrP and 109-123 in MoPrP) plus the nuclear magnetic resonance structures of model 1; and (model 3) extra peptides including Val(117) (109-124 in HuPrP and 109-123 in MoPrP) plus the nuclear magnetic resonance structures of model 1. The results of molecular dynamics calculations indicated that the globular domains of models 1 and 2 were stable and that the extra peptide in model 2 tended to form a new alpha-helix. On the other hand, the globular domain of model 3 was unstable, and the beta-sheet region increased especially in HuPrP.     

The role of disulfide bridge in the folding and stability of the recombinant human prion protein

It is believed that the critical step in the pathogenesis of transmissible spongiform encephalopathies is a transition of prion protein (PrP) from an alpha-helical conformation, PrP(C), to a beta-sheet-rich form, PrP(Sc). Native prion protein contains a single disulfide bond linking Cys residues at positions 179 and 214. To elucidate the role of this bridge in the stability and folding of the protein, we studied the reduced form of the recombinant human PrP as well as the variant of PrP in which cysteines were replaced with alanine residues. At neutral pH, the reduced prion protein and the Cys-free mutant were insoluble and formed amorphous aggregates. However, the proteins could be refolded in a monomeric form under the conditions of mildly acidic pH. Spectroscopic experiments indicate that the monomeric Cys-free and reduced PrP have molten globule-like properties, i.e. they are characterized by compromised tertiary interactions, an increased exposure of hydrophobic surfaces, lack of cooperative unfolding transition in urea, and partial loss of native (alpha-helical) secondary structure. In the presence of sodium chloride, these partially unfolded proteins undergo a transition to a beta-sheet-rich structure. However, this transition is invariably associated with protein oligomerization. The present data argue against the notion that reduced prion protein can exist in a stable monomeric form that is rich in beta-sheet structure.     

Cytoplasmic expression of mouse prion protein causes severe toxicity in Caenorhabditis elegans

To test if Caenorhabditis elegans could be established as a model organism for prion study, we created transgenic C. elegans expressing the cytosolic form of the mouse prion protein, MoPrP(23-231), which lacks the N-terminal signal sequence and the C-terminal glycosylphosphatidylinisotol (GPI) anchor site. We report here that transgenic worms expressing MoPrP(23-231)-CFP exhibited a wide range of distinct phenotypes: from normal growth and development, reduced mobility and development delay, complete paralysis and development arrest, to embryonic lethality. Similar levels of MoPrP(23-231)-CFP were produced in animals exhibiting these distinct phenotypes, suggesting that MoPrP(23-231)-CFP might have misfolded into distinct toxic species. In combining with the observation that mutations in PrP that affect prion pathogenesis also affect the toxic phenotypes in C. elegans, we conclude that the prion protein-folding mechanism is similar in mammals and C. elegans. Thus, C. elegans can be a useful model organism for prion research.     

High pressure induces scrapie-like prion protein misfolding and amyloid fibril formation

Our understanding of conformational conversion of proteins in diseases is essential for any diagnostic and therapeutic approach. Although not fully understood, misfolding of the prion protein (PrP) is implicated in the pathogenesis of prion diseases. Despite several efforts to produce the pathologically misfolded conformation in vitro from a recombinant PrP, no positive result has yet been obtained. Within the "protein-only hypothesis", the reason for this hindrance may be that the experimental conditions used did not allow selection of the pathway adopted in vivo resulting in conversion into the infectious form. Here, using a pressure perturbation approach, we show that recombinant PrP is converted to a novel misfolded conformer, which is prone to aggregate and ultimately form amyloid fibrils. A short incubation at high pressure (600 MPa) of the truncated form of hamster prion protein (SHaPrP(90-231)) resulted in the formation of pre-amyloid structures. The mostly globular aggregates were characterized by ThT and ANS binding, and by a beta-sheet-rich secondary structure. After overnight incubation at 600 MPa, amyloid fibrils were formed. In contrast to pre-amyloid structures, they showed birefringency of polarized light after Congo red staining and a strongly decreased ANS binding capacity, but enhanced ThT binding. Both aggregate types were resistant to digestion by PK, and can be considered as potential scrapie-like forms or precursors. These results may be useful for the search for compounds preventing pathogenic PrP misfolding and aggregation.     

Binding of prion proteins to lipid membranes

A key molecular event in prion diseases is the conversion of the normal cellular form of the prion protein (PrPC) to an aberrant form known as the scrapie isoform, PrPSc. Under normal physiological conditions PrPC is attached to the outer leaflet of the plasma membrane via a GPI-anchor. It has been proposed that a direct interaction between PrP and lipid membranes could be involved in the conversion of PrPC to its disease-associated corrupted conformation, PrPSc. Recombinant PrP can be refolded into an alpha-helical structure, designated alpha-PrP isoform, or into beta-sheet-rich states, designated beta-PrP isoform. The current study investigates the binding of recombinant PrP isoforms to model lipid membranes using surface plasmon resonance spectroscopy. The binding of alpha- and beta-PrP to negatively charged lipid membranes of POPG, zwitterionic membranes of DPPC, and model raft membranes composed of DPPC, cholesterol, and sphingomyelin is compared at pH 7 and 5, to simulate the environment at the plasma membrane and within endosomes, respectively. It is found that PrP binds strongly to lipid membranes. The strength of the association of PrP with lipid membranes depends on the protein conformation and pH, and involves both hydrophobic and electrostatic lipid-protein interactions. Competition binding measurements established that the binding of alpha-PrP to lipid membranes follows a decreasing order of affinity to POPG>DPPC>rafts.     

Globular and pre-fibrillar prion aggregates are toxic to neuronal cells and perturb their electrophysiology

Prion diseases are characterised at autopsy by neuronal loss and accumulation of amorphous protein aggregates and/or amyloid fibrils in the brains of humans and animals. These protein deposits result from the conversion of the cellular, mainly alpha-helical prion protein (PrP(C)) to the beta-sheet-rich isoform (PrP(Sc)). Although the pathogenic mechanism of prion diseases is not fully understood, it appears that protein aggregation is itself neurotoxic and not the product of cell death. The precise nature of the neurotoxic species and mechanism of cell death are yet to be determined, although recent studies with other amyloidogenic proteins suggest that ordered pre-fibrillar or oligomeric forms may be responsible for cellular dysfunction. In this study we have refolded recombinant prion protein (rPrP) to two distinct forms rich in beta-sheet structure with an intact disulphide bond. Here we report on the structural properties of globular aggregates and pre-fibrils of rPrP and show that both states are toxic to neuronal cells in culture. We show that exogenous rPrP aggregates are internalised by neuronal cells and found in the cytoplasm. We also measured the changes in electrophysiological properties of cultured neuronal cells on exposure to exogenous prion aggregates and discuss the implications of these findings.