Insoluble cellular prion protein and its association with prion and Alzheimer diseases

The soluble cellular prion protein (PrP^C) is best known for its association with prion disease (PrD) through its conversion to a pathogenic insoluble isoform (PrP^Sc). However, its deleterious effects independent of PrP^Sc have recently been observed not only in PrD but also in Alzheimer disease (AD), two diseases which mainly affect cognition. At the same time, PrP^C itself seems to have broad physiologic functions including involvement in cognitive processes. The PrP^C that is believed to be soluble and monomeric has so far been the only PrP conformer observed in the uninfected brain. In 2006, we identified an insoluble PrP^C conformer (termed iPrP^C) in uninfected human and animal brains. Remarkably, the PrP^Sc-like iPrP^C shares the immunoreactivity behavior and fragmentation with a newly-identified PrP^Sc species in a novel human PrD termed variably protease-sensitive prionopathy. Moreover, iPrP^C has been observed as the major PrP species that interacts with amyloid β (Aβ) in AD. This article highlights evidence of PrP involvement in two putatively beneficial and deleterious PrP-implicated pathways in cognition and hypothesizes first, that beneficial and deleterious effects of PrP^C are attributable to the chameleon-like conformation of the protein and second, that the iPrP^C conformer is associated with PrD and AD.Key words: prion protein, prion disease, cognition, cognitive deficit, insoluble prion protein, Alzheimer disease, variably protease-sensitive prionopathy, dementia, memory     

Experimental Verification of a Traceback Phenomenon in Prion Infection

The clinicopathological phenotypes of sporadic Creutzfeldt-Jakob disease (sCJD) correlate with the allelotypes (M or V) of the polymorphic codon 129 of the human prion protein (PrP) gene and the electrophoretic mobility patterns of abnormal prion protein (PrP^Sc). Transmission of sCJD prions to mice expressing human PrP with a heterologous genotype (referred to as cross-sequence transmission) results in prolonged incubation periods. We previously reported that cross-sequence transmission can generate a new prion strain with unique transmissibility, designated a traceback phenomenon. To verify experimentally the traceback of sCJD-VV2 prions, we inoculated sCJD-VV2 prions into mice expressing human PrP with the 129M/M genotype. These 129M/M mice showed altered neuropathology and a novel PrP^Sc type after a long incubation period. We then passaged the brain homogenate from the 129M/M mouse inoculated with sCJD-VV2 prions into other 129M/M or 129V/V mice. Despite cross-sequence transmission, 129V/V mice were highly susceptible to these prions compared to the 129M/M mice. The neuropathology and PrP^Sc type of the 129V/V mice inoculated with the 129M/M mouse-passaged sCJD-VV2 prions were identical to those of the 129V/V mice inoculated with sCJD-VV2 prions. Moreover, we generated for the first time a type 2 PrP^Sc-specific antibody in addition to type 1 PrP^Sc-specific antibody and discovered that drastic changes in the PrP^Sc subpopulation underlie the traceback phenomenon. Here, we report the first direct evidence of the traceback in prion infection.Creutzfeldt-Jakob disease (CJD) is a lethal transmissible neurodegenerative disease caused by an abnormal isoform of prion protein (PrP^Sc), which is converted from the normal cellular isoform (PrP^C) (1, 23). The genotype (M/M, M/V, or V/V, where M and V are allelotypes) at polymorphic codon 129 of the human prion protein (PrP) gene and the type (type 1 or type 2) of PrP^Sc in the brain are major determinants of the clinicopathological phenotypes of sporadic CJD (sCJD) (15-18). Type 1 and type 2 PrP^Sc are distinguishable according to the size of the proteinase K-resistant core of PrP^Sc (PrP^res) (21 and 19 kDa, respectively), reflecting differences in the proteinase K cleavage site (at residues 82 and 97, respectively) (15, 18). According to this molecular typing system, sCJD can be classified into six subgroups (MM1, MM2, MV1, MV2, VV1, or VV2).The homology of the PrP genes between inoculated animals and the inoculum determines the susceptibility to prion infection. Transmission of sCJD prions to mice expressing human PrP with a nonhomologous genotype (referred to as cross-sequence transmission) results in a relatively long incubation period (10, 12). Meanwhile, the cross-sequence transmission can generate a new prion strain. Transmission of sCJD-VV2 prions to mice expressing human PrP with the 129M/M genotype generates unusual PrP^res intermediate in size between type 1 and type 2 (10). We have designated this unusual PrP^res with an upward size shift (Sh+) from the inoculated type 2 template MM[VV2]2^Sh+ PrP^res, where the notation is of the following form: host genotype [type of inoculated prion] type of generated PrP^res.Similar to the MM[VV2]2^Sh+ PrP^res, the intermediate-sized PrP^res has been observed in the plaque-type of dura mater graft-associated CJD (p-dCJD) (10, 13). Furthermore, a transmission study using p-dCJD prions revealed that PrP-humanized mice with the 129V/V genotype were highly susceptible to p-dCJD prions despite cross-sequence transmission (10). In addition, these 129V/V mice inoculated with p-dCJD prions produced type 2 PrP^res (10). These findings suggest that p-dCJD could be caused by cross-sequence transmission of sCJD-VV2 prions to individuals with the 129M/M genotype. We have designated this phenomenon “traceback.” The traceback phenomenon was discovered for the first time by a transmission study using variant CJD (vCJD) prions (2). Mice expressing bovine PrP were highly susceptible to vCJD prions because vCJD was caused by cross-sequence transmission of bovine spongiform encephalopathy prions to human. These findings suggest that a traceback study can be a powerful tool to identify the origin of prions (2, 10, 11). However, the traceback phenomenon has not been verified experimentally despite the abundant circumstantial evidence described above.To verify the traceback of sCJD-VV2 prions, we inoculated sCJD-VV2 prions into PrP-humanized mice with the 129M/M genotype as an experimental model of p-dCJD. Thereafter, we inoculated these MM[VV2]2^Sh+ prions into PrP-humanized mice with the 129M/M or 129V/V genotype and compared the incubation period, neuropathology, and the type of PrP^res in the brain. Here, we report the first direct evidence of the traceback in prion infection.     

Regulating Factors of PrPres Glycosylation in Creutzfeldt-Jakob Disease - Implications for the Dissemination and the Diagnosis of Human Prion Strains

Objective

The glycoprofile of pathological prion protein (PrP^res) is widely used as a diagnosis marker in Creutzfeldt-Jakob disease (CJD) and is thought to vary in a strain-specific manner. However, that the same glycoprofile of PrP^res always accumulates in the whole brain of one individual has been questioned. We aimed to determine whether and how PrP^res glycosylation is regulated in the brain of patients with sporadic and variant Creutzfeldt-Jakob disease.

Methods

PrP^res glycoprofiles in four brain regions from 134 patients with sporadic or variant CJD were analyzed as a function of the genotype at codon 129 of PRNP and the Western blot type of PrP^res.

Results

The regional distribution of PrP^res glycoforms within one individual was heterogeneous in sporadic but not in variant CJD. PrP^res glycoforms ratio significantly correlated with the genotype at codon 129 of the prion protein gene and the Western blot type of PrP^res in a region-specific manner. In some cases of sCJD, the glycoprofile of thalamic PrP^res was undistinguishable from that observed in variant CJD.

Interpretation

Regulations leading to variations of PrP^res pattern between brain regions in sCJD patients, involving host genotype and Western blot type of PrP^res may contribute to the specific brain targeting of prion strains and have direct implications for the diagnosis of the different forms of CJD.     

Small Ruminant Nor98 Prions Share Biochemical Features with Human Gerstmann-Str?ussler-Scheinker Disease and Variably Protease-Sensitive Prionopathy

Prion diseases are classically characterized by the accumulation of pathological prion protein (PrP^Sc) with the protease resistant C-terminal fragment (PrP^res) of 27–30 kDa. However, in both humans and animals, prion diseases with atypical biochemical features, characterized by PK-resistant PrP internal fragments (PrP^res) cleaved at both the N and C termini, have been described. In this study we performed a detailed comparison of the biochemical features of PrP^Sc from atypical prion diseases including human Gerstmann-Sträussler-Scheinker disease (GSS) and variably protease-sensitive prionopathy (VPSPr) and in small ruminant Nor98 or atypical scrapie. The kinetics of PrP^res production and its cleavage sites after PK digestion were analyzed, along with the PrP^Sc conformational stability, using a new method able to characterize both protease-resistant and protease-sensitive PrP^Sc components. All these PrP^Sc types shared common and distinctive biochemical features compared to PrP^Sc from classical prion diseases such as sporadic Creutzfeldt-Jakob disease and scrapie. Notwithstanding, distinct biochemical signatures based on PrP^res cleavage sites and PrP^Sc conformational stability were identified in GSS A117V, GSS F198S, GSS P102L and VPSPr, which allowed their specific identification. Importantly, the biochemical properties of PrP^Sc from Nor98 and GSS P102L largely overlapped, but were distinct from the other human prions investigated. Finally, our study paves the way towards more refined comparative approaches to the characterization of prions at the animal–human interface.     

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.     

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.     

Small Protease Sensitive Oligomers of PrPSc in Distinct Human Prions Determine Conversion Rate of PrPC

The mammalian prions replicate by converting cellular prion protein (PrP^C) into pathogenic conformational isoform (PrP^Sc). Variations in prions, which cause different disease phenotypes, are referred to as strains. The mechanism of high-fidelity replication of prion strains in the absence of nucleic acid remains unsolved. We investigated the impact of different conformational characteristics of PrP^Sc on conversion of PrP^C in vitro using PrP^Sc seeds from the most frequent human prion disease worldwide, the Creutzfeldt-Jakob disease (sCJD). The conversion potency of a broad spectrum of distinct sCJD prions was governed by the level, conformation, and stability of small oligomers of the protease-sensitive (s) PrP^Sc. The smallest most potent prions present in sCJD brains were composed only of∼20 monomers of PrP^Sc. The tight correlation between conversion potency of small oligomers of human sPrP^Sc observed in vitro and duration of the disease suggests that sPrP^Sc conformers are an important determinant of prion strain characteristics that control the progression rate of the disease.     

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.     

Ultra-sensitive detection of prion protein fibrils by flow cytometry in blood from cattle affected with bovine spongiform encephalopathy

Background  

The definite diagnosis of prion diseases such as Creutzfeldt-Jakob disease (CJD) in humans or bovine spongiform encephalopathy (BSE) in cattle currently relies on the post mortem detection of the pathological form of the prion protein (PrP^Sc) in brain tissue. Infectivity studies indicate that PrP^Sc may also be present in body fluids, even at presymptomatic stages of the disease, albeit at concentrations well below the detection limits of currently available analytical methods.     

The Distribution of Prion Protein Allotypes Differs Between Sporadic and Iatrogenic Creutzfeldt-Jakob Disease Patients

Sporadic Creutzfeldt-Jakob disease (sCJD) is the most prevalent of the human prion diseases, which are fatal and transmissible neurodegenerative diseases caused by the infectious prion protein (PrP^Sc). The origin of sCJD is unknown, although the initiating event is thought to be the stochastic misfolding of endogenous prion protein (PrP^C) into infectious PrP^Sc. By contrast, human growth hormone-associated cases of iatrogenic CJD (iCJD) in the United Kingdom (UK) are associated with exposure to an exogenous source of PrP^Sc. In both forms of CJD, heterozygosity at residue 129 for methionine (M) or valine (V) in the prion protein gene may affect disease phenotype, onset and progression. However, the relative contribution of each PrP^C allotype to PrP^Sc in heterozygous cases of CJD is unknown. Using mass spectrometry, we determined that the relative abundance of PrP^Sc with M or V at residue 129 in brain specimens from MV cases of sCJD was highly variable. This result is consistent with PrP^C containing an M or V at residue 129 having a similar propensity to misfold into PrP^Sc thus causing sCJD. By contrast, PrP^Sc with V at residue 129 predominated in the majority of the UK human growth hormone associated iCJD cases, consistent with exposure to infectious PrP^Sc containing V at residue 129. In both types of CJD, the PrP^Sc allotype ratio had no correlation with CJD type, age at clinical onset, or disease duration. Therefore, factors other than PrP^Sc allotype abundance must influence the clinical progression and phenotype of heterozygous cases of CJD.     

Rapid, high-throughput detection of PrPSc by 96-well immunoassay

Prion diseases are emerging infectious disorders that affect several mammalian species including humans. The transmissible agent is comprised of PrP^Sc, a misfolded isoform of the normal host-encoded prion protein PrP^C. Immunodetection of PrP^Sc is often utilized for prion disease diagnosis and tracking spread of the infectious agent through the host. We have developed a rapid, high-throughput 96-well immunoassay, which is specific for the detection of PrP^Sc. This assay has PrP^Sc detection limits similar to western blot and is advantageous because of its comparatively shorter running time, smaller start-up and operation costs and large sample capacity.Key words: prion disease, immunodetection, PrP^Sc     

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.     

Immunopurification of Pathological Prion Protein Aggregates

Background

Prion diseases are fatal neurodegenerative disorders that can arise sporadically, be genetically inherited or acquired through infection. The key event in these diseases is misfolding of the cellular prion protein (PrP^C) into a pathogenic isoform that is rich in β-sheet structure. This conformational change may result in the formation of PrP^Sc, the prion isoform of PrP, which propagates itself by imprinting its aberrant conformation onto PrP^C molecules. A great deal of effort has been devoted to developing protocols for purifying PrP^Sc for structural studies, and testing its biological properties. Most procedures rely on protease digestion, allowing efficient purification of PrP27-30, the protease-resistant core of PrP^Sc. However, protease treatment cannot be used to isolate abnormal forms of PrP lacking conventional protease resistance, such as those found in several genetic and atypical sporadic cases.

Principal Findings

We developed a method for purifying pathological PrP molecules based on sequential centrifugation and immunoprecipitation with a monoclonal antibody selective for aggregated PrP. With this procedure we purified full-length PrP^Sc and mutant PrP aggregates at electrophoretic homogeneity. PrP^Sc purified from prion-infected mice was able to seed misfolding of PrP^C in a protein misfolding cyclic amplification reaction, and mutant PrP aggregates from transgenic mice were toxic to cultured neurons.

Significance

The immunopurification protocol described here isolates biologically active forms of aggregated PrP. These preparations may be useful for investigating the structural and chemico-physical properties of infectious and neurotoxic PrP aggregates.     

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.     

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.     

The Prion Protein Preference of Sporadic Creutzfeldt-Jakob Disease Subtypes

Sporadic Creutzfeldt-Jakob disease (CJD) is the most prevalent manifestation of the transmissible spongiform encephalopathies or prion diseases affecting humans. The disease encompasses a spectrum of clinical phenotypes that have been correlated with molecular subtypes that are characterized by the molecular mass of the protease-resistant fragment of the disease-related conformation of the prion protein and a polymorphism at codon 129 of the gene encoding the prion protein. A cell-free assay of prion protein misfolding was used to investigate the ability of these sporadic CJD molecular subtypes to propagate using brain-derived sources of the cellular prion protein (PrP^C). This study confirmed the presence of three distinct sporadic CJD molecular subtypes with PrP^C substrate requirements that reflected their codon 129 associations in vivo. However, the ability of a sporadic CJD molecular subtype to use a specific PrP^C substrate was not determined solely by codon 129 as the efficiency of prion propagation was also influenced by the composition of the brain tissue from which the PrP^C substrate was sourced, thus indicating that nuances in PrP^C or additional factors may determine sporadic CJD subtype. The results of this study will aid in the design of diagnostic assays that can detect prion disease across the diversity of sporadic CJD subtypes.     

Distinct Structures of Scrapie Prion Protein (PrPSc)-seeded Versus Spontaneous Recombinant Prion Protein Fibrils Revealed by Hydrogen/Deuterium Exchange

The detailed structures of prion disease-associated, partially protease-resistant forms of prion protein (e.g. PrP^Sc) are largely unknown. PrP^Sc appears to propagate itself by autocatalyzing the conformational conversion and oligomerization of normal prion protein (PrP^C). One manifestation of PrP^Sc templating activity is its ability, in protein misfolding cyclic amplification reactions, to seed the conversion of recombinant prion protein (rPrP) into aggregates that more closely resemble PrP^Sc than spontaneously nucleated rPrP amyloids in terms of proteolytic fragmentation and infrared spectra. The absence of posttranslational modifications makes these rPrP aggregates more amenable to detailed structural analyses than bona fide PrP^Sc. Here, we compare the structures of PrP^Sc-seeded and spontaneously nucleated aggregates of hamster rPrP by using H/D exchange coupled with mass spectrometry. In spontaneously formed fibrils, very slow H/D exchange in region ∼163–223 represents a systematically H-bonded cross-β amyloid core structure. PrP^Sc-seeded aggregates have a subpopulation of molecules in which this core region extends N-terminally as far as to residue ∼145, and there is a significant degree of order within residues ∼117–133. The formation of tightly H-bonded structures by these more N-terminal residues may account partially for the generation of longer protease-resistant regions in the PrP^Sc-seeded rPrP aggregates; however, part of the added protease resistance is dependent on the presence of SDS during proteolysis, emphasizing the multifactorial influences on proteolytic fragmentation patterns. These results demonstrate that PrP^Sc has a distinct templating activity that induces ordered, systematically H-bonded structure in regions that are dynamic and poorly defined in spontaneously formed aggregates of rPrP.Transmissible spongiform encephalopathies (TSEs),² or prion diseases, are a group of infectious neurodegenerative disorders that affect many mammalian species and include Creutzfeldt-Jakob disease in humans, scrapie in sheep, chronic wasting disease in cervids, and bovine spongiform encephalopathy (“mad cow” disease) (1–7). All of these diseases appear to be intimately associated with conformational conversion of the normal host-encoded prion protein, termed PrP^C, to a pathological isoform, PrP^Sc (1–5). According to the “protein-only” model, PrP^Sc itself represents the infectious prion agent (1, 8); it is believed to self-propagate by an autocatalytic mechanism involving binding to PrP^C and templating the conversion of the latter protein to the PrP^Sc state (9, 10). Although molecular details of such a mechanism of disease propagation remain largely unknown, the general principle of protein-based infectivity is supported by a wealth of experimental data (1–7).PrP^C is a monomeric glycophosphatidylinositol-linked glycoprotein that is highly protease-sensitive and soluble in nonionic detergents. High resolution NMR data show that the recombinant PrP (rPrP), a nonglycosylated model of PrP^C, consists of a flexible N-terminal region and a folded C-terminal domain encompassing three α-helices and two short β-strands (11–13). Conversely, the PrP^Sc isoform is aggregate in nature, rich in β-sheet structure, insoluble in nonionic detergents, and partially resistant to proteinase K (PK) digestion, with a PK-resistant core encompassing the C-terminal ∼140 residues (1–5, 14, 15). Little specific structural information is available, however, for this isoform beyond low resolution biochemical and spectroscopic characterization. Thus, the structure of PrP^Sc conformer(s) associated with prion infectivity remains one of the best guarded mysteries, hindering efforts to understand the molecular basis of TSE diseases.Many efforts have been made over the years to recapitulate PrP^Sc formation and prion propagation in vitro. Early studies have shown that PrP^C can be converted with remarkable species and strain specificities to a PrP^Sc-like conformation (as judged by PK resistance) simply by incubation with PrP^Sc from prion-infected animals (16, 17). The yields of these original cell-free conversion experiments were low, and no new infectivity could be attributed to the newly converted material (18). An important more recent study showed that both PrP^Sc and TSE infectivity can be amplified indefinitely in crude brain homogenates using successive rounds of sonication and incubation (19), a procedure called protein misfolding cyclic amplification (PMCA) (20). Similar amplification of the TSE infectivity was also accomplished by PMCA employing purified PrP^C as a substrate, although only in the presence of polyanions such as RNA and copurified lipids (21). Unfortunately, the quantities of infectious PrP^Sc generated by PMCA using purified brain-derived PrP^C are very small, precluding most structural studies.In contrast to brain-derived PrP^C, large scale purification can be readily accomplished for bacterially expressed rPrP, a form of PrP lacking glycosylation and the glycophosphatidylinositol anchor. The latter protein can spontaneously polymerize into amyloid fibrils, and much insight has been gained into mechanistic and structural aspects of this reaction (22–28). However, although rPrP fibrils were shown to cause or accelerate a transmissible neurodegenerative disorder in transgenic mice overexpressing a PrP^C variant encompassing residues 89–231, the infectivity titer of these “synthetic prions” was extremely low (29) or absent altogether (4). This low infectivity coincides with much shorter PK-resistant core of rPrP amyloid fibrils compared with brain-derived PrP^Sc (26, 30), raising questions regarding the relationship between these fibrils and the authentic TSE agent. In this context, an important recent development was the finding that the PrP^Sc-seeded PMCA method can be extended to rPrP, yielding protease-resistant recombinant PrP aggregates (rPrP^PMCA or rPrP-res^(Sc)) (31). These aggregates display a PK digestion pattern that is much more closely related to PrP^Sc than that of previously studied spontaneously formed rPrP fibrils, offering a potentially more relevant model for biochemical and biophysical studies. Here, we provide, for the first time, a direct insight into the structure of rPrP^PMCA. H/D exchange data coupled with MS analysis (HXMS) allowed us to identify systematically H-bonded core region(s) of these aggregates, shedding a new light on the mechanisms underlying formation of PK-resistant structures.     

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