DNA converts cellular prion protein into the beta-sheet conformation and inhibits prion peptide aggregation

The main hypothesis for prion diseases proposes that the cellular protein (PrP(C)) can be altered into a misfolded, beta-sheet-rich isoform (PrP(Sc)), which in most cases undergoes aggregation. In an organism infected with PrP(Sc), PrP(C) is converted into the beta-sheet form, generating more PrP(Sc). We find that sequence-specific DNA binding to recombinant murine prion protein (mPrP-(23-231)) converts it from an alpha-helical conformation (cellular isoform) into a soluble, beta-sheet isoform similar to that found in the fibrillar state. The recombinant murine prion protein and prion domains bind with high affinity to DNA sequences. Several double-stranded DNA sequences in molar excess above 2:1 (pH 4.0) or 0.5:1 (pH 5.0) completely inhibit aggregation of prion peptides, as measured by light scattering, fluorescence, and circular dichroism spectroscopy. However, at a high concentration, fibers (or peptide aggregates) can rescue the peptide bound to the DNA, converting it to the aggregating form. Our results indicate that a macromolecular complex of prion-DNA may act as an intermediate for the formation of the growing fiber. We propose that host nucleic acid may modulate the delicate balance between the cellular and the misfolded conformations by reducing the protein mobility and by making the protein-protein interactions more likely. In our model, the infectious material would act as a seed to rescue the protein bound to nucleic acid. Accordingly, DNA would act on the one hand as a guardian of the Sc conformation, preventing its propagation, but on the other hand may catalyze Sc conversion and aggregation if a threshold level is exceeded.     

Analysis of nucleic acid chaperoning by the prion protein and its inhibition by oligonucleotides

Prion diseases are unique neurodegenerative illnesses associated with the conversion of the cellular prion protein (PrP(C)) into the aggregated misfolded scrapie isoform, named PrP(Sc). Recent studies on the physiological role of PrP(C) revealed that this protein has probably multiple functions, notably in cell-cell adhesion and signal transduction, and in assisting nucleic acid folding. In fact, in vitro findings indicated that the human PrP (huPrP) possesses nucleic acid binding and annealing activities, similarly to nucleic acid chaperone proteins that play essential roles in cellular DNA and RNA metabolism. Here, we show that a peptide, representing the N-terminal domain of huPrP, facilitates nucleic acid annealing by two parallel pathways nucleated through the stem termini. We also show that PrP of human or ovine origin facilitates DNA strand exchange, ribozyme-directed cleavage of an RNA template and RNA trans-splicing in a manner similar to the nucleocapsid protein of HIV-1. In an attempt to characterize inhibitors of PrP-chaperoning in vitro we discovered that the thioaptamer 5'-GACACAAGCCGA-3' was extensively inhibiting the PrP chaperoning activities. At the same time a recently characterized methylated oligoribonucleotide inhibiting the chaperoning activity of the HIV-1 nucleocapsid protein was poorly impairing the PrP chaperoning activities.     

Experimental approaches to the interaction of the prion protein with nucleic acids and glycosaminoglycans: Modulators of the pathogenic conversion

The concept that transmissible spongiform encephalopathies (TSEs) are caused only by proteins has changed the traditional paradigm that disease transmission is due solely to an agent that carries genetic information. The central hypothesis for prion diseases proposes that the conversion of a cellular prion protein (PrP(C)) into a misfolded, β-sheet-rich isoform (PrP(Sc)) accounts for the development of (TSE). There is substantial evidence that the infectious material consists chiefly of a protein, PrP(Sc), with no genomic coding material, unlike a virus particle, which has both. However, prions seem to have other partners that chaperone their activities in converting the PrP(C) into the disease-causing isoform. Nucleic acids (NAs) and glycosaminoglycans (GAGs) are the most probable accomplices of prion conversion. Here, we review the recent experimental approaches that have been employed to characterize the interaction of prion proteins with nucleic acids and glycosaminoglycans. A PrP recognizes many nucleic acids and GAGs with high affinities, and this seems to be related to a pathophysiological role for this interaction. A PrP binds nucleic acids and GAGs with structural selectivity, and some PrP:NA complexes can become proteinase K-resistant, undergoing amyloid oligomerization and conversion to a β-sheet-rich structure. These results are consistent with the hypothesis that endogenous polyanions (such as NAs and GAGs) may accelerate the rate of prion disease progression by acting as scaffolds or lattices that mediate the interaction between PrP(C) and PrP(Sc) molecules. In addition to a still-possible hypothesis that nucleic acids and GAGs, especially those from the host, may modulate the conversion, the recent structural characterization of the complexes has raised the possibility of developing new diagnostic and therapeutic strategies.     

In vitro amplification of protease-resistant prion protein requires free sulfhydryl groups

Prions, the infectious agents of transmissible spongiform encephalopathies, are composed primarily of a misfolded protein designated PrP(Sc). Prion-infected neurons generate PrP(Sc) from a host glycoprotein designated PrP(C) through a process of induced conformational change, but the molecular mechanism by which PrP(C) undergoes conformational change into PrP(Sc) remains unknown. We employed an in vitro PrP(Sc) amplification technique adapted from protein misfolding cyclic amplification (PMCA) to investigate the mechanism of prion-induced protein conformational change. Using this technique, PrP(Sc) from diluted scrapie-infected brain homogenate can be amplified >10-fold without sonication when mixed with normal brain homogenate under nondenaturing conditions. PrP(Sc) amplification in vitro exhibits species and strain specificity, depends on both time and temperature, only requires membrane-bound components, and does not require divalent cations. In vitro amplification of Syrian hamster Sc237 PrP(Sc) displays an optimum pH of approximately 7, whereas amplification of CD-1 mouse RML PrP(Sc) is optimized at pH approximately 6. The thiolate-specific alkylating agent N-ethylmaleimide (NEM) as well as the reversible thiol-specific blockers p-hydroxymercuribenzoic acid (PHMB) and mersalyl acid inhibited PrP(Sc) amplification in vitro, indicating that the conformational change from PrP(C) to PrP(Sc) requires a thiol-containing factor. Our data provide the first evidence that a reactive chemical group plays an essential role in the conformational change from PrP(C) to PrP(Sc).     

Search for a prion-specific nucleic acid

Diversity of prion strains was attributed to an elusive nucleic acid, yet a search spanning nearly two decades has failed to identify a prion-specific polynucleotide. In our search for a prion-specific nucleic acid, we analyzed nucleic acids in purified fractions from the brains of Syrian hamsters infected with Sc237 prions. Purification of Sc237 prions removed nucleic acids larger than 50 nucleotides as measured by return refocusing electrophoresis (RRGE). To determine the size of the largest polynucleotide present in purified fractions at an abundance of one molecule per infectious (ID50) unit, we measured prions present after inoculation. In order to account for the rapid clearance of prions after intracerebral inoculation, we determined the number of PrP(Sc) molecules and ID50 units of prions that were retained in brain. Factoring in clearance after inoculation, we estimate that the largest polynucleotide present in our purified fractions at one molecule per ID50 unit is approximately 25 nucleotides in length. In the same fractions, there were approximately 3,000 protease-resistant PrP(Sc) molecules per ID50 unit after accounting for clearance of PrP(Sc) following inoculation. We compared the resistance of Sc237 and 139H prions to inactivation by UV irradiation at 254 nm. Irradiation of homogenates and microsomes diminished prion infectivity by a factor of approximately 1,000 but did not alter the strain-specified properties of the Sc237 and 139H prions. The data reported here combined with the production of synthetic prions argue that the 25-mer polynucleotides found in purified prion preparations are likely to be host encoded and of variable sequence; additionally, these 25-mers are unlikely to be prion specific.     

Comparison of trichloroacetic acid with other protein-precipitating agents in enriching abnormal prion protein for Western blot analysis

Detection of the abnormal or the pathogenic form of prion protein (PrP(Sc)) by Western blot (WB) is challenging, especially, for samples derived from cell cultures that contain low levels of PrP(Sc). A variety of PrP(Sc) concentration methods have been reported with various PrP(Sc) recovery efficiencies. Ultracentrifugation is one of the methods used frequently to enrich the pathogenic form of PrP(Sc) prior to WB analyses. The resulting PrP(Sc) pellet is extremely insoluble and often requires sonication to be dissolved, potentially generating aerosols. We modified the common protein-precipitating protocol using trichloroacetic acid to concentrate PrP(Sc) by slow-speed centrifugation, followed by solubilization of the pellets with 6 mol/L urea prior to sodium dodecyl sulphate -- polyacrylamide gel electrophoresis and WB analyses. Comparative studies suggest this simple trichloroacetic acid protocol was more effective in enriching PrP(Sc) presented in cell cultures and brain homogenates than other reported protein-precipitating methods. Furthermore, incorporation of the urea treatment step to dissolve the precipitated PrP(Sc) pellets helped to reduce the infectivity of PrP(Sc).     

Experimental approaches to TSE prevention via inhibition of prion formation

Transmissible spongiform encepahalopathies (TSEs) are fatal diseases that damage the central nervous system. TSEs are unique in that they may be inherited, infectious or spontaneous. The central pathogenic agent is thought to be a conformationally distinct form (PrP(Sc;)) of the endogenous prion protein(PrP(c)), which is high in beta-sheet content and is resistant to proteases; infectivity is thought to involve formation of PrP(Sc) via imprinting of abnormal conformation on the normal form of the protein (PrP(c)) by seeds of PrP(Sc). A number of compounds found to inhibit the conversion of PrP(c) to PrP(Sc) have been proposed as therapeutics to halt TSEs.     

A single prion protein peptide can elicit a panel of isoform specific monoclonal antibodies

The main step in the pathogenesis of transmissible spongiform encephalopathies (TSE) is the conformational change of the normal cellular prion protein (PrP(C)) into the abnormal isoform, named prion (PrP(Sc)). Since PrP is a highly conserved protein, the production of monoclonal antibodies (mAbs) of high specificity and affinity to PrP is a difficult task. In the present study we show that it is possible to overcome the unresponsiveness of the immune system by immunizing wild-type BALB/c mice with a 13 amino acid PrP peptide from the C-terminal part of PrP, bound to the keyhole limpet hemocyanin (KLH). Immunization induced predominantly anti-PrP(Sc) humoral immune response. Furthermore, we were able to obtain a panel of mAbs of IgG class specific for different non-self-conformations of PrP, with anti-PrP(Sc)-specific mAbs being the most abundant.     

CpG and LPS can interfere negatively with prion clearance in macrophage and microglial cells

Cells of the innate immune system play important roles in the progression of prion disease after peripheral infection. It has been found in vivo and in vitro that the expression of the cellular prion protein (PrP(c)) is up-regulated on stimulation of immune cells, also indicating the functional importance of PrP(c) in the immune system. The aim of our study was to investigate the impact of cytosine-phosphate-guanosine- and lipopolysaccharide-induced PrP(c) up-regulation on the uptake and processing of the pathological prion protein (PrP(Sc)) in phagocytic innate immune cells. For this purpose, we challenged the macrophage cell line J774, the microglial cell line BV-2 and primary bone marrow-derived macrophages in a resting or stimulated state with various prion strains, and monitored the uptake and clearance of PrP(Sc). Interestingly, stimulation led either to a transient increase in the level of PrP(Sc) relative to unstimulated cells or to a decelerated degradation of PrP(Sc). These features were dependent on cell type and prion strain. Our data indicate that the stimulation of innate immune cells may be able to support transient prion propagation, possibly explained by an increased PrP(c) cell surface expression in stimulated cells. We suggest that stimulation of innate immune cells can lead to an imbalance between the propagation and degradation of PrP(Sc).     

High-level expression and characterization of a glycosylated covalently linked dimer of the prion protein

There is evidence that prion protein dimers may be involved in the formation of the scrapie prion protein, PrP(Sc), from its normal (cellular) form, PrP(c). Recently, the crystal structure of the human prion protein in a dimeric form was reported. Here we report for the first time the overexpression of a human PrP dimer covalently linked by a FLAG peptide (PrP::FLAG::PrP) in the methylotrophic yeast Pichia pastoris. FLAG-tagged human PrP (aa1-aa253) (huPrP::FLAG) was also expressed in the same system. Treatment with tunicamycin and endoglycosidase H showed that both fusion proteins are expressed as various glycoforms. Both PrP proteins were completely digested by proteinase K (PK), suggesting that the proteins do not have a PrP(Sc) structure and are not infectious. Plasma membrane fractionation revealed that both proteins are transported to the plasma membrane of the cell. The glycosylated proteins might act as powerful tools for crystallization trials, PrP(c)/PrP(Sc) conversion studies and other applications in the life cycle of prions.     

Neurodegeneration induced by clustering of sialylated glycosylphosphatidylinositols of prion proteins

The transmissible spongiform encephalopathies, more commonly known as the prion diseases, are associated with the production and aggregation of disease-related isoforms of the prion protein (PrP(Sc)). The mechanisms by which PrP(Sc) accumulation causes neurodegeneration in these diseases are poorly understood. In cultured neurons, the addition of PrP(Sc) alters cell membranes, increasing cholesterol, activating cytoplasmic phospholipase A(2) (cPLA(2)), and triggering synapse damage. These effects of PrP(Sc) are dependent upon its glycosylphosphatidylinositol (GPI) anchor, suggesting that it is the increased density of GPIs that occurs following the aggregation of PrP(Sc) molecules that triggers neurodegeneration. This hypothesis was supported by observations that cross-linkage of the normal cellular prion protein (PrP(C)) also increased membrane cholesterol, activated cPLA(2), and triggered synapse damage. These effects were not seen after cross-linkage of Thy-1, another GPI-anchored protein, and were dependent on the GPI anchor attached to PrP(C) containing two acyl chains and sialic acid. We propose that the aggregation of PrP(Sc), or the cross-linkage of PrP(C), causes the clustering of sialic acid-containing GPI anchors at high densities, resulting in altered membrane composition, the pathological activation of cPLA(2), and synapse damage.     

The tyrosine kinase inhibitor STI571 induces cellular clearance of PrPSc in prion-infected cells

The conversion of the cellular prion protein (PrP(c)) into pathologic PrP(Sc) and the accumulation of aggregated PrP(Sc) are hallmarks of prion diseases. A variety of experimental approaches to interfere with prion conversion have been reported. Our interest was whether interference with intracellular signaling events has an impact on this conversion process. We screened approximately 50 prototype inhibitors of specific signaling pathways in prion-infected cells for their capacity to affect prion conversion. The tyrosine kinase inhibitor STI571 was highly effective against PrP(Sc) propagation, with an IC(50) of < or =1 microM. STI571 cleared prion-infected cells in a time- and dose-dependent manner from PrP(Sc) without influencing biogenesis, localization, or biochemical features of PrP(c). Interestingly, this compound did not interfere with the de novo formation of PrP(Sc) but activated the lysosomal degradation of pre-existing PrP(Sc), lowering the half-life of PrP(Sc) from > or =24 h to <9 h. Our data indicate that among the kinases known to be inhibited by STI571, c-Abl is likely responsible for the observed anti-prion effect. Taken together, we demonstrate that treatment with STI571 strongly activates the lysosomal degradation of PrP(Sc) and that substances specifically interfering with cellular signaling pathways might represent a novel class of anti-prion compounds.     

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.     

Analysis of the interactions between HIV-1 and the cellular prion protein in a human cell line

The cellular prion protein (PrP(c)) is highly conserved in mammals and expressed widely in different tissues but its physiological role remains elusive. Recently, the human PrP(c) was shown to possess nucleic acid binding and chaperoning properties similar to human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein, a key viral factor in virus structure and replication. These findings prompted us to determine if PrP(c) could influence HIV-1 replication. We used the human 293T cell line as a model system, since only a very low level of PrP(c) accumulates in these cells. Expression of PrP at a high level resulted in a specific decrease of HIV-1 Env and Vpr expression. Despite similar levels of intracellular Gag, virus production was reduced by eightfold and infectivity by three- to fourfold in the presence of PrP(c). A PrP(c) mutant lacking the glycosylphosphatidylinositol (GPI) anchor peptide did not impair HIV-1 production, suggesting that PrP(c) trafficking is critical for this inhibitory effect. Coexpressing HIV-1 and PrP(c) in these cells also caused a fraction of PrP(c) to become partially proteinase K-resistant (PrP(res)), further illustrating the interactions between HIV-1 and PrP(c).     

The cellular prion protein (PrP(C)): its physiological function and role in disease

Prion diseases are caused by conversion of a normal cell-surface glycoprotein (PrP(C)) into a conformationally altered isoform (PrP(Sc)) that is infectious in the absence of nucleic acid. Although a great deal has been learned about PrP(Sc) and its role in prion propagation, much less is known about the physiological function of PrP(C). In this review, we will summarize some of the major proposed functions for PrP(C), including protection against apoptotic and oxidative stress, cellular uptake or binding of copper ions, transmembrane signaling, formation and maintenance of synapses, and adhesion to the extracellular matrix. We will also outline how loss or subversion of the cytoprotective or neuronal survival activities of PrP(C) might contribute to the pathogenesis of prion diseases, and how similar mechanisms are probably operative in other neurodegenerative disorders.     

Protease-sensitive scrapie prion protein in aggregates of heterogeneous sizes

The pathological prion protein PrP(Sc) is the only known component of the infectious prion. In cells infected with prions, PrP(Sc) is formed posttranslationally by the refolding of the benign cell surface glycoprotein PrP(C) into an aberrant conformation. The two PrP isoforms possess very different properties, as PrP(Sc) has a protease-resistant core, forms very large amyloidic aggregates in detergents, and is only weakly immunoreactive in its native form. We now show that prion-infected rodent brains and cultured cells contain previously unrecognized protease-sensitive PrP(Sc) varieties. In both ionic (Sarkosyl) and nonionic (n-octyl beta-D-glucopyranoside) detergents, the novel protease-sensitive PrP(Sc) species formed aggregates as small as 600 kDa, as measured by gel filtration. The denaturation dependence of PrP(Sc) immunoreactivity correlated with the size of the aggregate. The small PrP(Sc) aggregates described here are consistent with the previous demonstration of scrapie infectivity in brain fractions with a sedimentation coefficient as small as 40 S [Prusiner et al. (1980) J. Neurochem. 35, 574-582]. Our results demonstrate for the first time that prion-infected tissues contain protease-sensitive PrP(Sc) molecules that form low MW aggregates. Whether these new PrP(Sc) species play a role in the biogenesis or the pathogenesis of prions remains to be established.     

PrP(c) mRNA, but not PrP(Sc) is found in the salivary glands of scrapie-infected sheep

Transmission studies in transmissible spongiform encephalopathies (TSEs) have become increasingly important due to the possible transmission of bovine spongiform encephalopathy to humans resulting in new variant Creutzfeldt-Jacob disease. The horizontal transmission of scrapie, a TSE of sheep, is poorly understood. Possible sources of horizontal transmission are the submandibular and parotid salivary glands. TSEs like natural sheep scrapie are characterized by the conversion of a normal protease sensitive prion protein, PrP(c), to an abnormal protease resistant prion protein, PrP(Sc). Since the presence of PrP(Sc) is an indicator of disease, the salivary glands of scrapie-infected sheep were examined for the presence of PrP(Sc). Although PrP(c) mRNA was detected in the salivary glands, PrP(Sc) was not found in the salivary glands of scrapie-infected sheep. These data suggest that the salivary glands are unlikely sources of horizontal transmission of natural sheep scrapie.     

Disease-associated prion protein elicits immunoglobulin M responses in vivo

Prion diseases such as Creutzfeldt-Jakob disease are believed to result from the misfolding of a widely expressed normal cellular prion protein, PrPc. The resulting disease-associated isoforms, PrP(Sc), have much higher beta-sheet content, are insoluble in detergents, and acquire relative resistance to proteases. Although known to be highly aggregated and to form amyloid fibrils, the molecular architecture of PrP9Sc) is poorly understood. To date, it has been impossible to elicit antibodies to native PrP(Sc) that are capable of recognizing PrP(Sc) without denaturation, even in Pm-P(o/o) mice that are intolerant of it. Here we demonstrate that antibodies for native PrPc and PrP(Sc) can be produced by immunization of Pm-P(o/o) mice with partially purified PrPc and PrP(Sc) adsorbed to immunomagnetic particles using high-affinity anti-PrP monoclonal antibodies (mAbs). Interestingly, the polyclonal response to PrP(Sc) was predominantly of the immunoglobulin M (IgM) isotype, unlike the immunoglobulin G (IgG) responses elicited by PrP(c) or by recombinant PrP adsorbed or not to immunomagnetic particles, presumably reflecting the polymeric structure of disease-associated prion protein. Although heat-denatured PrP(Sc) elicited more diverse antibodies with the revelation of C-terminal epitopes, remarkably, these were also predominantly IgM suggesting that the increasing immunogenicity, acquisition of protease sensitivity, and reduction in infectivity induced by heat are not associated with dissociation of the PrP molecules in the diseased-associated protein. Adsorbing native proteins to immunomagnetic particles may have general applicability for raising polyclonal or monoclonal antibodies to any native protein, without attempting laborious purification steps that might affect protein conformation.     

Influence of Mabs on PrP(Sc) Formation Using In Vitro and Cell-Free Systems

PrP(Sc) is believed to serve as a template for the conversion of PrP(C) to the abnormal isoform. This process requires contact between the two proteins and implies that there may be critical contact sites that are important for conversion. We hypothesized that antibodies binding to either PrP(c)or PrP(Sc) would hinder or prevent the formation of the PrP(C)-PrP(Sc) complex and thus slow down or prevent the conversion process. Two systems were used to analyze the effect of different antibodies on PrP(Sc) formation: (i) neuroblastoma cells persistently infected with the 22L mouse-adapted scrapie stain, and (ii) protein misfolding cyclic amplification (PMCA), which uses PrP(Sc) as a template or seed, and a series of incubations and sonications, to convert PrP(C) to PrP(Sc). The two systems yielded similar results, in most cases, and demonstrate that PrP-specific monoclonal antibodies (Mabs) vary in their ability to inhibit the PrP(C)-PrP(Sc) conversion process. Based on the numerous and varied Mabs analyzed, the inhibitory effect does not appear to be epitope specific, related to PrP(C) conformation, or to cell membrane localization, but is influenced by the targeted PrP region (amino vs carboxy).     

Intriguing nucleic-acid-binding features of mammalian prion protein

In transmissible spongiform encephalopathies, the infectious material consists chiefly of a protein, the scrapie prion protein PrP(Sc), that carries no genetic coding material; however, prions are likely to have accomplices that chaperone their activity and promote the conversion of the cellular prion protein PrP(C) into the disease-causing isoform (PrP(Sc)). Recent studies from several laboratories indicate that PrP(C) recognizes many nucleic acids (NAs) with high affinities, and we correlate these findings with a possible pathophysiological role for this interaction. Thus, of the chaperones, NA is the most likely candidate for prions. The participation of NAs in prion propagation opens new avenues for developing new diagnostic tools and therapeutics to target prion diseases, as well as for understanding the function of PrP(C), probably as a NA chaperone.