Species and Strain Glycosylation Patterns of PrPSc

Background

A key event in transmissible spongiform encephalopathies (TSEs) is the conversion of the soluble, protease-sensitive glycosylated prion protein (PrP^C) to an abnormally structured, aggregated and partially protease-resistant isoform (PrP^Sc). Both PrP isoforms bear two potential glycosylation sites and thus in a typical western blot with an anti-PrP antibody three distinct bands appear, corresponding to the di-, mono- or unglycosylated forms of the protein. The relative intensity and electrophoretic mobility of the three bands are characteristic of each TSE strain and have been used to discriminate between them.

Methodology/Principal Findings

In the present study we used lectin-based western blotting to evaluate possible variations in composition within sugar chains carried by PrP^Sc purified from subjects affected with different TSEs. Our findings indicate that in addition to the already well-documented differences in electrophoretic mobility and amounts of the glycosylated PrP^Sc forms, TSE strains also vary in the abundance of specific N-linked sugars of the PrP^Sc protein.

Conclusions/Significance

These results imply that PrP glycosylation might fine-tune the conversion of PrP^C to PrP^Sc and could play an accessory role in the appearance of some of the characteristic features of TSE strains. The differences in sugar composition could also be used as an additional tool for discrimination between the various TSEs.     

The Octarepeat Region of the Prion Protein Is Conformationally Altered in PrPSc

Background

Prion diseases are fatal neurodegenerative disorders characterized by misfolding and aggregation of the normal prion protein PrP^C. Little is known about the details of the structural rearrangement of physiological PrP^C into a still-elusive disease-associated conformation termed PrP^Sc. Increasing evidence suggests that the amino-terminal octapeptide sequences of PrP (huPrP, residues 59–89), though not essential, play a role in modulating prion replication and disease presentation.

Methodology/Principal Findings

Here, we report that trypsin digestion of PrP^Sc from variant and sporadic human CJD results in a disease-specific trypsin-resistant PrP^Sc fragment including amino acids ∼49–231, thus preserving important epitopes such as the octapeptide domain for biochemical examination. Our immunodetection analyses reveal that several epitopes buried in this region of PrP^Sc are exposed in PrP^C.

Conclusions/Significance

We conclude that the octapeptide region undergoes a previously unrecognized conformational transition in the formation of PrP^Sc. This phenomenon may be relevant to the mechanism by which the amino terminus of PrP^C participates in PrP^Sc conversion, and may also be exploited for diagnostic purposes.     

Glimepiride Reduces the Expression of PrPC,Prevents PrPSc Formation and Protects against Prion Mediated Neurotoxicity

Background

A hallmark of the prion diseases is the conversion of the host-encoded cellular prion protein (PrP^C) into a disease related, alternatively folded isoform (PrP^Sc). The accumulation of PrP^Sc within the brain is associated with synapse loss and ultimately neuronal death. Novel therapeutics are desperately required to treat neurodegenerative diseases including the prion diseases.

Principal Findings

Treatment with glimepiride, a sulphonylurea approved for the treatment of diabetes mellitus, induced the release of PrP^C from the surface of prion-infected neuronal cells. The cell surface is a site where PrP^C molecules may be converted to PrP^Sc and glimepiride treatment reduced PrP^Sc formation in three prion infected neuronal cell lines (ScN2a, SMB and ScGT1 cells). Glimepiride also protected cortical and hippocampal neurones against the toxic effects of the prion-derived peptide PrP82–146. Glimepiride treatment significantly reduce both the amount of PrP82–146 that bound to neurones and PrP82–146 induced activation of cytoplasmic phospholipase A₂ (cPLA₂) and the production of prostaglandin E₂ that is associated with neuronal injury in prion diseases. Our results are consistent with reports that glimepiride activates an endogenous glycosylphosphatidylinositol (GPI)-phospholipase C which reduced PrP^C expression at the surface of neuronal cells. The effects of glimepiride were reproduced by treatment of cells with phosphatidylinositol-phospholipase C (PI-PLC) and were reversed by co-incubation with p-chloromercuriphenylsulphonate, an inhibitor of endogenous GPI-PLC.

Conclusions

Collectively, these results indicate that glimepiride may be a novel treatment to reduce PrP^Sc formation and neuronal damage in prion diseases.     

Molecular structure of amyloid fibrils controls the relationship between fibrillar size and toxicity

Background

According to the prevailing view, soluble oligomers or small fibrillar fragments are considered to be the most toxic species in prion diseases. To test this hypothesis, two conformationally different amyloid states were produced from the same highly pure recombinant full-length prion protein (rPrP). The cytotoxic potential of intact fibrils and fibrillar fragments generated by sonication from these two states was tested using cultured cells.

Methodology/Principal Findings

For one amyloid state, fibril fragmentation was found to enhance its cytotoxic potential, whereas for another amyloid state formed within the same amino acid sequence, the fragmented fibrils were found to be substantially less toxic than the intact fibrils. Consistent with the previous studies, the toxic effects were more pronounced for cell cultures expressing normal isoform of the prion protein (PrP^C) at high levels confirming that cytotoxicity was in part PrP^C-dependent. Silencing of PrP^C expression by small hairpin RNAs designed to silence expression of human PrP^C (shRNA-PrP^C) deminished the deleterious effects of the two amyloid states to a different extent, suggesting that the role of PrP^C-mediated and PrP^C-independent mechanisms depends on the structure of the aggregates.

Conclusions/Significance

This work provides a direct illustration that the relationship between an amyloid''s physical dimension and its toxic potential is not unidirectional but is controlled by the molecular structure of prion protein (PrP) molecules within aggregated states. Depending on the structure, a decrease in size of amyloid fibrils can either enhance or abolish their cytotoxic effect. Regardless of the molecular structure or size of PrP aggregates, silencing of PrP^C expression can be exploited to reduce their deleterious effects.     

Glycosaminoglycan Sulphation Affects the Seeded Misfolding of a Mutant Prion Protein

Background

The accumulation of protease resistant conformers of the prion protein (PrP^res) is a key pathological feature of prion diseases. Polyanions, including RNA and glycosaminoglycans have been identified as factors that contribute to the propagation, transmission and pathogenesis of prion disease. Recent studies have suggested that the contribution of these cofactors to prion propagation may be species specific.

Methodology/Principal Finding

In this study a cell-free assay was used to investigate the molecular basis of polyanion stimulated PrP^res formation using brain tissue or cell line derived murine PrP. Enzymatic depletion of endogenous nucleic acids or heparan sulphate (HS) from the PrP^C substrate was found to specifically prevent PrP^res formation seeded by mouse derived PrP^Sc. Modification of the negative charge afforded by the sulphation of glycosaminoglycans increased the ability of a familial PrP mutant to act as a substrate for PrP^res formation, while having no effect on PrP^res formed by wildtype PrP. This difference may be due to the observed differences in the binding of wild type and mutant PrP for glycosaminoglycans.

Conclusions/Significance

Cofactor requirements for PrP^res formation are host species and prion strain specific and affected by disease associated mutations of the prion protein. This may explain both species and strain dependent propagation characteristics and provide insights into the underlying mechanisms of familial prion disease. It further highlights the challenge of designing effective therapeutics against a disease which effects a range of mammalian species, caused by range of aetiologies and prion strains.     

PrP Expression,PrPSc Accumulation and Innervation of Splenic Compartments in Sheep Experimentally Infected with Scrapie

Background

In prion disease, the peripheral expression of PrP^C is necessary for the transfer of infectivity to the central nervous system. The spleen is involved in neuroinvasion and neural dissemination in prion diseases but the nature of this involvement is not known. The present study undertook the investigation of the spatial relationship between sites of PrP^Sc accumulation, localisation of nerve fibres and PrP^C expression in the tissue compartments of the spleen of scrapie-inoculated and control sheep.

Methodology/Principal Findings

Laser microdissection and quantitative PCR were used to determine PrP mRNA levels and results were compared with immunohistochemical protocols to distinguish PrP^C and PrP^Sc in tissue compartments of the spleen. In sheep experimentally infected with scrapie, the major sites of accumulation of PrP^Sc in the spleen, namely the lymphoid nodules and the marginal zone, expressed low levels of PrP mRNA. Double immunohistochemical labelling for PrP^Sc and the pan-nerve fibre marker, PGP, was used to evaluate the density of innervation of splenic tissue compartments and the intimacy of association between PrP^Sc and nerves. Some nerve fibres were observed to accompany blood vessels into the PrP^Sc-laden germinal centres. However, the close association between nerves and PrP^Sc was most apparent in the marginal zone. Other sites of close association were adjacent to the wall of the central artery of PALS and the outer rim of germinal centres.

Conclusions/Significance

The findings suggest that the degree of PrP^Sc accumulation does not depend on the expression level of PrP^C. Though several splenic compartments may contribute to neuroinvasion, the marginal zone may play a central role in being the compartment with most apparent association between nerves and PrP^Sc.     

Sulfated Dextrans Enhance In Vitro Amplification of Bovine Spongiform Encephalopathy PrPSc and Enable Ultrasensitive Detection of Bovine PrPSc

Background

Prions, infectious agents associated with prion diseases such as Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy (BSE) in cattle, and scrapie in sheep and goats, are primarily comprised of PrP^Sc, a protease-resistant misfolded isoform of the cellular prion protein PrP^C. Protein misfolding cyclic amplification (PMCA) is a highly sensitive technique used to detect minute amounts of scrapie PrP^Sc. However, the current PMCA technique has been unsuccessful in achieving good amplification in cattle. The detailed distribution of PrP^Sc in BSE-affected cattle therefore remains unknown.

Methodology/Principal Findings

We report here that PrP^Sc derived from BSE-affected cattle can be amplified ultra-efficiently by PMCA in the presence of sulfated dextran compounds. This method is capable of amplifying very small amounts of PrP^Sc from the saliva, palatine tonsils, lymph nodes, ileocecal region, and muscular tissues of BSE-affected cattle. Individual differences in the distribution of PrP^Sc in spleen and cerebrospinal fluid samples were observed in terminal-stage animals. However, the presence of PrP^Sc in blood was not substantiated in the BSE-affected cattle examined.

Conclusions/Significance

The distribution of PrP^Sc is not restricted to the nervous system and can spread to peripheral tissues in the terminal disease stage. The finding that PrP^Sc could be amplified in the saliva of an asymptomatic animal suggests a potential usefulness of this technique for BSE diagnosis. This highly sensitive method also has other practical applications, including safety evaluation or safety assurance of products and byproducts manufactured from bovine source materials.     

Liposome-siRNA-Peptide Complexes Cross the Blood-Brain Barrier and Significantly Decrease PrPC on Neuronal Cells and PrPRES in Infected Cell Cultures

Background

Recent advances toward an effective therapy for prion diseases employ RNA interference to suppress PrP^C expression and subsequent prion neuropathology, exploiting the phenomenon that disease severity and progression correlate with host PrP^C expression levels. However, delivery of lentivirus encoding PrP shRNA has demonstrated only modest efficacy in vivo.

Methodology/Principal Findings

Here we describe a new siRNA delivery system incorporating a small peptide that binds siRNA and acetylcholine receptors (AchRs), acting as a molecular messenger for delivery to neurons, and cationic liposomes that protect siRNA-peptide complexes from serum degradation.

Conclusions/Significance

Liposome-siRNA-peptide complexes (LSPCs) delivered PrP siRNA specifically to AchR-expressing cells, suppressed PrP^C expression and eliminated PrP^RES formation in vitro. LSPCs injected intravenously into mice resisted serum degradation and delivered PrP siRNA throughout the brain to AchR and PrP^C-expressing neurons. These data promote LSPCs as effective vehicles for delivery of PrP and other siRNAs specifically to neurons to treat prion and other neuropathological diseases.     

Protocol for aerosol-free recombinant production and NMR analysis of prion proteins

The central hallmark of prion diseases is the misfolding of cellular prion protein (PrP^C) into a disease-associated aggregated isoform known as scrapie prion protein (PrP^Sc). NMR spectroscopy has made many essential contributions to the characterization of recombinant PrP in its folded, unfolded and aggregated states. Recent studies reporting on de novo generation of prions from recombinant PrP and infection of animals using prion aerosols suggest that adjustment of current biosafety measures may be necessary, particularly given the relatively high protein concentrations required for NMR applications that favor aggregation. We here present a protocol for the production of recombinant PrP under biosafety level 2 conditions that avoids entirely exposure of the experimenter to aerosols that might contain harmful PrP aggregates. In addition, we introduce an NMR sample tube setup that allows for safe handling of PrP samples at the spectrometer that usually is not part of a dedicated biosafety level 2 laboratory.     

Methionine Sulfoxides on Prion Protein Helix-3 Switch on the α-Fold Destabilization Required for Conversion

Background

The conversion of the cellular prion protein (PrP^C) into the infectious form (PrP^Sc) is the key event in prion induced neurodegenerations. This process is believed to involve a multi-step conformational transition from an α-helical (PrP^C) form to a β-sheet-rich (PrP^Sc) state. In addition to the conformational difference, PrP^Sc exhibits as covalent signature the sulfoxidation of M213. To investigate whether such modification may play a role in the misfolding process we have studied the impact of methionine oxidation on the dynamics and energetics of the HuPrP(125–229) α-fold.

Methodology/Principal Findings

Using molecular dynamics simulation, essential dynamics, correlated motions and signal propagation analysis, we have found that substitution of the sulfur atom of M213 by a sulfoxide group impacts on the stability of the native state increasing the flexibility of regions preceding the site of the modification and perturbing the network of stabilizing interactions. Together, these changes favor the population of alternative states which maybe essential in the productive pathway of the pathogenic conversion. These changes are also observed when the sulfoxidation is placed at M206 and at both, M206 and M213.

Conclusions/Significance

Our results suggest that the sulfoxidation of Helix-3 methionines might be the switch for triggering the initial α-fold destabilization required for the productive pathogenic conversion.     

Prion Protein—Antibody Complexes Characterized by Chromatography-Coupled Small-Angle X-Ray Scattering

Aberrant self-assembly, induced by structural misfolding of the prion proteins, leads to a number of neurodegenerative disorders. In particular, misfolding of the mostly α-helical cellular prion protein (PrP^C) into a β-sheet-rich disease-causing isoform (PrP^Sc) is the key molecular event in the formation of PrP^Sc aggregates. The molecular mechanisms underlying the PrP^C-to-PrP^Sc conversion and subsequent aggregation remain to be elucidated. However, in persistently prion-infected cell-culture models, it was shown that treatment with monoclonal antibodies against defined regions of the prion protein (PrP) led to the clearing of PrP^Sc in cultured cells. To gain more insight into this process, we characterized PrP-antibody complexes in solution using a fast protein liquid chromatography coupled with small-angle x-ray scattering (FPLC-SAXS) procedure. High-quality SAXS data were collected for full-length recombinant mouse PrP [denoted recPrP(23–230)] and N-terminally truncated recPrP(89–230), as well as their complexes with each of two Fab fragments (HuM-P and HuM-R1), which recognize N- and C-terminal epitopes of PrP, respectively. In-line measurements by fast protein liquid chromatography coupled with SAXS minimized data artifacts caused by a non-monodispersed sample, allowing structural analysis of PrP alone and in complex with Fab antibodies. The resulting structural models suggest two mechanisms for how these Fabs may prevent the conversion of PrP^C into PrP^Sc.     

Paradoxical Role of Prion Protein Aggregates in Redox-Iron Induced Toxicity

Background

Imbalance of iron homeostasis has been reported in sporadic Creutzfeldt-Jakob-disease (sCJD) affected human and scrapie infected animal brains, but the contribution of this phenotype to disease associated neurotoxicity is unclear.

Methodology/Principal Findings

Using cell models of familial prion disorders, we demonstrate that exposure of cells expressing normal prion protein (PrP^C) or mutant PrP forms to a source of redox-iron induces aggregation of PrP^C and specific mutant PrP forms. Initially this response is cytoprotective, but becomes increasingly toxic with time due to accumulation of PrP-ferritin aggregates. Mutant PrP forms that do not aggregate are not cytoprotective, and cells show signs of acute toxicity. Intracellular PrP-ferritin aggregates induce the expression of LC3-II, indicating stimulation of autophagy in these cells. Similar observations are noted in sCJD and scrapie infected hamster brains, lending credence to these results. Furthermore, phagocytosis of PrP-ferritin aggregates by astrocytes is cytoprotective, while culture in astrocyte conditioned medium (CM) shows no measurable effect. Exposure to H₂O₂, on the other hand, does not cause aggregation of PrP, and cells show acute toxicity that is alleviated by CM.

Conclusions/Significance

These observations suggest that aggregation of PrP in response to redox-iron is cytoprotective. However, subsequent co-aggregation of PrP with ferritin induces intracellular toxicity unless the aggregates are degraded by autophagosomes or phagocytosed by adjacent scavenger cells. H₂O₂, on the other hand, does not cause aggregation of PrP, and induces toxicity through extra-cellular free radicals. Together with previous observations demonstrating imbalance of iron homeostasis in prion disease affected brains, these observations provide insight into the mechanism of neurotoxicity by redox-iron, and the role of PrP in this process.     

Familial CJD associated PrP mutants within transmembrane region induced Ctm-PrP retention in ER and triggered apoptosis by ER stress in SH-SY5Y cells

Background

Genetic prion diseases are linked to point and inserted mutations in the prion protein (PrP) gene that are presumed to favor conversion of the cellular isoform of PrP (PrP^C) to the pathogenic one (PrP^Sc). The pathogenic mechanisms and the subcellular sites of the conversion are not completely understood. Here we introduce several PRNP gene mutations (such as, PrP-KDEL, PrP-3AV, PrP-A117V, PrP-G114V, PrP-P102L and PrP-E200K) into the cultured cells in order to explore the pathogenic mechanism of familial prion disease.

Methodology/Principal Findings

To address the roles of aberrant retention of PrP in endoplasmic reticulum (ER), the recombinant plasmids expressing full-length human PrP tailed with an ER signal peptide at the COOH-terminal (PrP-KDEL) and PrP with three amino acids exchange in transmembrane region (PrP-3AV) were constructed. In the preparations of transient transfections, 18-kD COOH-terminal proteolytic resistant fragments (Ctm-PrP) were detected in the cells expressing PrP-KDEL and PrP-3AV. Analyses of the cell viabilities in the presences of tunicamycin and brefeldin A revealed that expressions of PrP-KDEL and PrP-3AV sensitized the transfected cells to ER stress stimuli. Western blots and RT-PCR identified the clear alternations of ER stress associated events in the cells expressing PrP-KDEL and PrP-3AV that induced ER mediated apoptosis by CHOP and capase-12 apoptosis pathway. Moreover, several familial CJD related PrP mutants were transiently introduced into the cultured cells. Only the mutants within the transmembrane region (G114V and A117V) induced the formation of Ctm-PrP and caused the ER stress, while the mutants outside the transmembrane region (P102L and E200K) failed.

Conclusions/Significance

The data indicate that the retention of PrP in ER through formation of Ctm-PrP results in ER stress and cell apoptosis. The cytopathic activities caused by different familial CJD associated PrP mutants may vary, among them the mutants within the transmembrane region undergo an ER-stress mediated cell apoptosis.     

Proteomic consequences of expression and pathological conversion of the prion protein in inducible neuroblastoma N2a cells

Neurodegenerative diseases are often associated with misfolding and deposition of specific proteins in the nervous system. The prion protein, which is associated with transmissible spongiform encephalopathies (TSEs), is one of them. The normal function of the cellular form of the prion protein (PrP^C) is mediated through specific signal transduction pathways and is linked to resistance to oxidative stress, neuronal outgrowth and cell survival. In TSEs, PrP^C is converted into an abnormally folded isoform, called PrP^Sc, that may impair the normal function of the protein and/or generate toxic aggregates. To investigate these molecular events we performed a two-dimensional gel electrophoresis comparison of neuroblastoma N2a cells expressing different amounts of PrP^C and eventually infected with the 22L prion strain. Mass spectrometry and peptide mass fingerprint analysis identified a series of proteins with modified expression. They included the chaperones Grp78/BiP, protein disulfide-isomerase A6, Grp75 and Hsp60 which had an opposite expression upon PrP^C expression and PrP^Sc production. The detection of these proteins was coherent with the idea that protein misfolding plays an important role in TSEs. Other proteins, such as calreticulin, tubulin, vimentin or the laminin receptor had their expression modified in infected cells, which was reminiscent of previous results. Altogether our data provide molecular information linking PrP expression and misfolding, which could be the basis of further therapeutic and pathophysiological research in this field.Key words: chaperones, neuroblastoma, prion, proteomics     

Lipid Rafts and Clathrin Cooperate in the Internalization of PrPC in Epithelial FRT Cells

Background

The cellular prion protein (PrP^C) plays a key role in the pathogenesis of Transmissible Spongiform Encephalopathies in which the protein undergoes post-translational conversion to the infectious form (PrP^Sc). Although endocytosis appears to be required for this conversion, the mechanism of PrP^C internalization is still debated, as caveolae/raft- and clathrin-dependent processes have all been reported to be involved.

Methodology/Principal Findings

We have investigated the mechanism of PrP^C endocytosis in Fischer Rat Thyroid (FRT) cells, which lack caveolin-1 (cav-1) and caveolae, and in FRT/cav-1 cells which form functional caveolae. We show that PrP^C internalization requires activated Cdc-42 and is sensitive to cholesterol depletion but not to cav-1 expression suggesting a role for rafts but not for caveolae in PrP^C endocytosis. PrP^C internalization is also affected by knock down of clathrin and by the expression of dominant negative Eps15 and Dynamin 2 mutants, indicating the involvement of a clathrin-dependent pathway. Notably, PrP^C co-immunoprecipitates with clathrin and remains associated with detergent-insoluble microdomains during internalization thus indicating that PrP^C can enter the cell via multiple pathways and that rafts and clathrin cooperate in its internalization.

Conclusions/Significance

These findings are of particular interest if we consider that the internalization route/s undertaken by PrP^C can be crucial for the ability of different prion strains to infect and to replicate in different cell lines.     

In Vivo Generation of Neurotoxic Prion Protein: Role for Hsp70 in Accumulation of Misfolded Isoforms

Prion diseases are incurable neurodegenerative disorders in which the normal cellular prion protein (PrP^C) converts into a misfolded isoform (PrP^Sc) with unique biochemical and structural properties that correlate with disease. In humans, prion disorders, such as Creutzfeldt-Jakob disease, present typically with a sporadic origin, where unknown mechanisms lead to the spontaneous misfolding and deposition of wild type PrP. To shed light on how wild-type PrP undergoes conformational changes and which are the cellular components involved in this process, we analyzed the dynamics of wild-type PrP from hamster in transgenic flies. In young flies, PrP demonstrates properties of the benign PrP^C; in older flies, PrP misfolds, acquires biochemical and structural properties of PrP^Sc, and induces spongiform degeneration of brain neurons. Aged flies accumulate insoluble PrP that resists high concentrations of denaturing agents and contains PrP^Sc-specific conformational epitopes. In contrast to PrP^Sc from mammals, PrP is proteinase-sensitive in flies. Thus, wild-type PrP rapidly converts in vivo into a neurotoxic, protease-sensitive isoform distinct from prototypical PrP^Sc. Next, we investigated the role of molecular chaperones in PrP misfolding in vivo. Remarkably, Hsp70 prevents the accumulation of PrP^Sc-like conformers and protects against PrP-dependent neurodegeneration. This protective activity involves the direct interaction between Hsp70 and PrP, which may occur in active membrane microdomains such as lipid rafts, where we detected Hsp70. These results highlight the ability of wild-type PrP to spontaneously convert in vivo into a protease-sensitive isoform that is neurotoxic, supporting the idea that protease-resistant PrP^Sc is not required for pathology. Moreover, we identify a new role for Hsp70 in the accumulation of misfolded PrP. Overall, we provide new insight into the mechanisms of spontaneous accumulation of neurotoxic PrP and uncover the potential therapeutic role of Hsp70 in treating these devastating disorders.     

Influence of the pathogenic mutations T188K/R/A on the structural stability and misfolding of human prion protein: Insight from molecular dynamics simulations

Background

Prion diseases are associated with a conformational switch for PrP from PrP^C to PrP^Sc. Many genetic mutations are linked with prion diseases, such as mutations T188K/R/A with fCJD.

Scope of review

MD simulations for the WT PrP and its mutants were performed to explore the underlying dynamic effects of T188 mutations on human PrP. Although the globular domains are fairly conserved, the three mutations have diverse effects on the dynamics properties of PrP, including the shift of H1, the elongation of native β-sheet and the conversion of S2-H2 loop to a 3₁₀ helix.

Major conclusions

Our present study indicates that the three mutants for PrP may undergo different pathogenic mechanisms and the realistic atomistic simulations can provide insights into the effects of disease-associated mutations on PrP dynamics and stability, which can enhance our understanding of how mutations induce the conversion from PrP^C to PrP^Sc.General significanceOur present study helps to understand the effects of T188K/R/A mutations on human PrP: despite the three pathogenic mutations almost do not alter the native structure of PrP, but perturb its stability. This instability may further modulate the oligomerization pathways and determine the features of the PrP^Sc assemblies.     

Solution structure and dynamics of the I214V mutant of the rabbit prion protein

Background

The conformational conversion of the host-derived cellular prion protein (PrP^C) into the disease-associated scrapie isoform (PrP^Sc) is responsible for the pathogenesis of transmissible spongiform encephalopathies (TSEs). Various single-point mutations in PrP^Cs could cause structural changes and thereby distinctly influence the conformational conversion. Elucidation of the differences between the wild-type rabbit PrP^C (RaPrP^C) and various mutants would be of great help to understand the ability of RaPrP^C to be resistant to TSE agents.

Methodology/Principal Findings

We determined the solution structure of the I214V mutant of RaPrP^C(91–228) and detected the backbone dynamics of its structured C-terminal domain (121–228). The I214V mutant displays a visible shift of surface charge distribution that may have a potential effect on the binding specificity and affinity with other chaperones. The number of hydrogen bonds declines dramatically. Urea-induced transition experiments reveal an obvious decrease in the conformational stability. Furthermore, the NMR dynamics analysis discloses a significant increase in the backbone flexibility on the pico- to nanosecond time scale, indicative of lower energy barrier for structural rearrangement.

Conclusions/Significance

Our results suggest that both the surface charge distribution and the intrinsic backbone flexibility greatly contribute to species barriers for the transmission of TSEs, and thereby provide valuable hints for understanding the inability of the conformational conversion for RaPrP^C.     

Plasminogen: A cellular protein cofactor for PrPSc propagation

The biochemical essence of prion replication is the molecular multiplication of the disease-associated misfolded isoform of prion protein (PrP), termed PrP^Sc, in a nucleic acid-free manner. PrP^Sc is generated by the protein misfolding process facilitated by conformational conversion of the host-encoded cellular PrP to PrP^Sc. Evidence suggests that an auxiliary factor may play a role in PrP^Sc propagation. We and others previously discovered that plasminogen interacts with PrP, while its functional role for PrP^Sc propagation remained undetermined. In our recent in vitro PrP conversion study, we showed that plasminogen substantially stimulates PrP^Sc propagation in a concentration-dependent manner by accelerating the rate of PrP^Sc generation while depletion of plasminogen, destabilization of its structure and interference with the PrP-plasminogen interaction hinder PrP^Sc propagation. Further investigation in cell culture models confirmed an increase of PrP^Sc formation by plasminogen. Although molecular basis of the observed activity for plasminogen remain to be addressed, our results demonstrate that plasminogen is the first cellular protein auxiliary factor proven to stimulate PrP^Sc propagation.Key words: prion, PrP^Sc, protein misfolding, auxiliary factor, plasminogen, PMCA, cell culture modelPrions are unique infectious particles that replicate in the absence of nucleic acids¹ and cause fatal neurologic disorders in mammals.² In fact, the prion particle is composed of an alternatively folded form of the cellular prion protein (PrP^C) encoded by the Prnp gene. The misfolded form of PrP^C, referred to as PrP^Sc, shares the same primary structure with PrP^C,³ but exhibits distinctively different biochemical and biophysical properties.^4,5 Moreover, animals lacking expression of the Prnp gene are resistant to prion infection,⁶ suggesting that PrP^C serves as a precursor for PrP^Sc.The essence of prion biosynthesis is based on the protein-only hypothesis that postulates self-perpetuating replication of the prion protein.¹ Although the exact replication process remains elusive, the template-assisted conversion model proposes an idea that PrP^Sc serves as a template to convert α-helices of PrP^C into the β-sheets of PrP^Sc during prion replication.⁷ According to many lines of evidence, there exists an unidentified auxiliary factor, designated protein X, which favorably interacts with PrP^C to produce a thermodynamically stable intermediate conformation called PrP*.⁸ By introducing PrP^Sc to a host, dimers will readily form between PrP* and PrP^Sc. This interaction induces PrP* to take on the conformation of PrP^Sc and results in a complex consisting of the template and the newly formed PrP^Sc molecules. Once the dimer disassociates protein X and the two PrP^Sc molecules are released and allowed to continue replicating in an exponential fashion. Recent observations that infectious material can be generated in vitro using recombinant PrP have authenticated the protein-only hypothesis.^9,10 However, the infectivity of these in vitro generated PrP^Sc products is lower than that of brain-derived PrP^Sc, leading to the possibility that insufficient levels of the unidentified auxiliary factor are the limiting factor for these in vitro assays.To date, non-mammalian chaperone proteins, sulfated glycans and certain polyanionic macromolecules, such as RNA, have shown to increase the level of PrP^Sc in several different in vitro assays.^11–14 However, defining these molecules as cellular auxiliary factors that promote the conversion of PrP^C to PrP^Sc has been prevented for several reasons. First, overexpression of yeast heat shock protein Hsp 104 in transgenic mice does not modulate the incubation time of disease and PrP^Sc accumulation upon prion inoculation.¹⁵ Although mammalian Hsp 70 is upregulated in humans and animals with prion diseases,^16,17 it participates in downregulation, but not upregulation, of PrP^Sc accumulation.¹⁸ Second, the effect of sulfated glycans on PrP^Sc formation has been inconsistent^11–13 and certain sulfated glycans inhibit PrP^Sc propagation in animals and cultured cells.^19–21 Lastly, although RNA is able to increase PrP^Sc propagation and induce the formation of PrP^Sc de novo from purified PrP^C in the absence of a PrP^Sc seed,²² its specificity is in question. Thus, an auxiliary factor that positively assists PrP^Sc replication and is composed of a mammalian cellular protein remains to be identified.Our approach to identify an auxiliary factor relies on the idea that the auxiliary factor interacts with PrP^C as proposed in the protein X hypothesis.⁷ Therefore, we considered PrP ligands as the best candidates for this unidentified factor. By screening a phage display cDNA expression library from ScN2a cells, we identified kringle domains of plasminogen that interact with recombinant PrP folded in an α-helical conformation (α-PrP).²³ In vitro binding assays showed that interaction between plasminogen and α-PrP that represents the conformational state of PrP^C was enhanced by the introduction of a dominant negative mutation and the presence of the basic N-terminal sequences in PrP.²³ Interaction of PrP^C and plasminogen was further confirmed by the ability of plasminogen and its kringle domains to readily interact with α-PrP.^24–29 However, despite greater interaction with α-PrP, plasminogen also interacted with PrP in β-sheet conformations.²³ Prior to our study, it was shown that PrP^Sc was immunoprecipitated with beads linked to plasminogen, its first three kringle domains [K(1+2+3)], and more recently the repeating YRG motif found within the plasminogen kringle domains.^30–33 However, the ability of plasminogen to bind to PrP^Sc was dependent on conditions of the lipid rafts and plasminogen was actually associated with PrP^C in the intact lipid rafts.³⁴To determine the functional relevance of this interaction for PrP^Sc replication, we explored whether plasminogen enhances PrP^Sc propagation using cell culture models and the in vitro PrP conversion assay, termed protein misfolding cyclic amplification (PMCA).³⁵ The addition of plasminogen in PMCA resulted in the generation of significantly more PrP^Sc in a concentration-dependent manner (Fig. 1A), suggesting that plasminogen stimulates the conversion of PrP^C to PrP^Sc. Indeed, our kinetic studies showed that plasminogen accelerates the rate of PrP^Sc generation during the early stages of the in vitro PrP conversion reaction (reviewed in ref. ³⁵). In contrast, the addition of plasminogen in PMCA lacking either PrP^C or PrP^Sc failed to generate PrP^Sc (Fig. 1B and C). These results suggest that both PrP^C and PrP^Sc are required for PrP^Sc replication stimulated by plasminogen and that plasminogen facilitates neither spontaneous PrP conversion nor PrP^Sc augmentation through aggregating pre-existing PrP^Sc. Incubation of plasminogen with pre-formed PrP^Sc followed by treatments with proteinase K failed to either increase or decrease the PrP^Sc level compared to controls (Fig. 1D and E), suggesting that plasminogen is not involved in stabilization of PrP^Sc, enhancement of PrP^Sc resistance to protease or promotion of PrP^Sc binding to the membrane for western blotting. The activity to stimulate PrP conversion was a specific property of plasminogen and was not shared with other proteins such as a known PrP ligand and proteins abundantly found in the serum or at the extracellular matrices where plasminogen is present (reviewed in ref. ³⁵). In addition, we found that the ability of plasminogen to assist in PrP^Sc propagation is preserved in its kringle domains (reviewed in ref. ³⁵). Furthermore, the activity associated with plasminogen under cell-free conditions was reproduced in cell culture models. Plasminogen and its kringle domains increased PrP^Sc propagation in cultured cells chronically infected with mouse-adapted scrapie or chronic wasting disease prions (Fig. 2A–D). This suggests that the activity of plasminogen in PrP^Sc replication has biological relevance.Open in a separate windowFigure 1The role of plasminogen in PrP^Sc propagation. The effect of plasminogen (Plg) was assessed by PMCA using normal brain material supplemented with or without 0.5 µM human Glu-Plg (A–D) or using Plg-deficient (Plg^-/-) brain material (F and G). Pre- (−) and post- (+) PMCA samples were treated with proteinase K (PK) and analyzed by western blotting. Seeds for PMCA were diluted either as indicated or 1:900 (G) −8,100 (F). (A) Stimulation of PrP^Sc propagation by Plg. (B) Plg-supplemented PMCA in the absence of SBH seeds. (C) Plg-supplemented PMCA in the absence of NBH. (D) Comparison of PrP^Sc levels in Plg-supplemented PMCA samples (during) vs. PMCA samples only incubated with Plg prior to PK digestion (after). (E) Comparison of PrP^Sc levels of ScN2a cell lysate after incubation with or without Plg prior to PK digestion. (F) PMCA with brain material of Plg^-/- mice and genetically unaltered littermate controls (C). (G) Restoration of PMCA using Plg^-/- brain material with Plg-supplementation. NBH, normal brain homogenate; SBH, sick brain homogenate; PrPKOBH, brain homogenate of PrP^C-deficient mice; CL, cell lysate. Reproduced with permission from The FASEB Journal, Mays and Ryou 2010.³⁵Open in a separate windowFigure 2PrP^Sc propagation increased by plasminogen in prion-infected cells. (A) The levels of PrP in ScN2a cells incubated with 0–0.5 µM human Glu-plasminogen (Plg) for two days. (B) The levels of PrP in ScN2a cells incubated with 0, 0.1 and 1.0 µM Plg or the first three kringle domains of Plg [K(1+2+3)] for six days. (C) The levels of 3F4-tagged PrP^C and nascent PrP^Sc formation in ScN2a cells transiently transfected (Tfx) with plasmids encoding the 3F4-tagged PrP gene (PrP-3F4) and with an empty vector (mock). The transfected cells were treated with 0 or 1 µM K (1+2+3) for three days. (D) The levels of PrP in Elk21⁺ cells incubated with 0 and 0.5 µM Plg for two days. PrP was detected by anti-PrP antibody D13 (A and B), 3F4 (C) or 6H4 (D) before (−) and after (+) PK treatment. Reproduced by permission of the The FASEB Journal, Mays and Ryou 2010.³⁵Corresponding to the results that plasminogen positively assists in PrP^Sc replication by stimulating conversion of PrP^C to PrP^Sc, depletion of plasminogen from the PMCA reaction by using brain material derived from plasminogen-deficient mice restricted PrP^Sc replication to the basal level (Fig. 1F). Supplementation with plasminogen for PMCA using plasminogen-deficient brain homogenate restored PrP^Sc propagation to levels equivalent to that of control PMCA in which only brain material of their non-genetically altered littermates was used (Fig. 1G). Furthermore, structural destabilization of plasminogen affected the activity of plasminogen that enhances PrP^Sc propagation. Because intact disulfide bonds are critical in maintaining structural integrity and the binding activity of plasminogen, we conducted PMCA supplemented with either structurally intact or modified plasminogen to investigate the functionality of plasminogen. The result showed that plasminogen pre-treated sequentially with chemical agents that disrupt disulfide bonds and modify free sulfhydryl groups failed to stimulate PrP^Sc propagation (reviewed in ref. ³⁵). In addition, we showed that interference with the plasminogen-PrP interaction using L-lysine abolished the plasminogen-mediated stimulation of PrP^Sc propagation in PMCA (reviewed in ref. ³⁵). Because previous observations in immunoprecipitation³⁰ and ELISA binding assays²³ described that L-lysine specifically inhibited formation of the PrP-plasminogen complex, the presence of L-lysine in PMCA is considered to saturate the lysine binding motifs of kringle domains, which competitively prevents the kringle domains of plasminogen from interacting with PrP and inhibited PrP conversion. These various inhibition studies of PrP^Sc propagation provides confirmatory evidence that plasminogen plays an important role in PrP^Sc replication.Despite our progress in understanding the role of plasminogen in PrP^Sc propagation, we are still unable to address mechanistic details by which plasminogen exerts its function. In fact, plasminogen shares a number of the expected characteristics of the previously proposed auxiliary factor, although there are minor but distinctive discrepancies in their properties (summarized in Fig. 3). First, plasminogen may control conformational rearrangement of PrP^C to PrP*, resembling a molecular chaperone. This scenario is identical to the protein X hypothesis, in which plasminogen replaces protein X to assist the conversion process. Second, plasminogen may promote aggregation of PrP^Sc already converted from PrP^C. This will result in efficient formation of PrP^Sc multimers. Third, plasminogen may stabilize the pre-existing PrP^Sc aggregates so that stabilized aggregates are better protected from degradation or processing by the intrinsic clearance mechanism. This will result in increased accumulation and stability of PrP^Sc. In the second and third scenarios, the function of plasminogen is not involved in PrP^C or its conversion process, but limited to the interaction with pre-formed PrP^Sc. Fourth, plasminogen may play a role as a scaffolding molecule that simply brings both PrP^C and PrP^Sc together within a proximity. This will increase the frequency of interaction between PrP^C and PrP^Sc for conversion. This scenario is distinguished from the other three by postulating plasminogen interaction with both isoforms of PrP. In contrast, plasminogen is assumed to interact with only PrP^C or PrP^Sc in other cases. Although accumulating data including our recent studies provide critical pieces of evidence to envision described mechanistic insights, it is still premature to conclude the mechanism involved in plasminogen-mediated stimulation of PrP^Sc propagation.Open in a separate windowFigure 3Plausible mechanisms for plasminogen to enhance PrP^Sc propagation. Plasminogen may stimulate PrP^Sc propagation via conformational alteration of PrP^C to PrP* (i), enhancement of PrP^Sc aggregation (ii), stabilization of pre-exisiting PrP^Sc aggregates (iii) or scaffolding to gather PrP^C and PrP^Sc together (iv).

Table 1

Properties of plasminogen as an auxiliary factor for PrP^Sc propagation

Peroxiredoxin 6 promotes upregulation of the prion protein (PrP) in neuronal cells of prion-infected mice

Background

It has been widely established that the conversion of the cellular prion protein (PrP^C) into its abnormal isoform (PrP^Sc) is responsible for the development of transmissible spongiform encephalopathies (TSEs). However, the knowledge of the detailed molecular mechanisms and direct functional consequences within the cell is rare. In this study, we aimed at the identification of deregulated proteins which might be involved in prion pathogenesis.

Findings

Apolipoprotein E and peroxiredoxin 6 (PRDX6) were identified as upregulated proteins in brains of scrapie-infected mice and cultured neuronal cell lines. Downregulation of PrP gene expression using specific siRNA did not result in a decrease of PRDX6 amounts. Interestingly, selective siRNA targeting PRDX6 or overexpression of PRDX6 controlled PrP^C and PrP^Sc protein amounts in neuronal cells.

Conclusions

Besides its possible function as a novel marker protein in the diagnosis of TSEs, PDRX6 represents an attractive target molecule in putative pharmacological intervention strategies in the future.