Cell-lysate conversion of prion protein into its protease-resistant isoform suggests the participation of a cellular chaperone.

A conformational transition between the normal cellular prion protein (PrPC) and the beta-sheet-rich pathological isoform (PrPSc) is a central event in the pathogenesis of spongiform encephalopathies. The prion infectious agent seems to contain mainly, if not exclusively, PrPSc, which has the ability to propagate its abnormal conformation by transforming the host PrPC into the pathological isoform. We have developed an in vitro system to induce the PrPC --> PrPSc conversion by incubating a cell-lysate containing mouse PrPC with partially purified mouse PrPSc. After 48 h of incubation with a 10-fold molar excess of PrPSc, the cellular protein acquired PK-resistance resembling a PrPSc-like state. Time course experiments suggest that the conversion follows a stepwise mechanism involving kinetic intermediates. The conversion was induced by PrPSc extracted from mice infected with two different prion strains, each propagating its characteristic Western blot profile. The latter results and the fact that all the cellular components are present in the conversion reaction suggest that PrPC-PrPSc interaction is highly specific and required for the conversion. No transformation was observed under the same conditions using purified proteins without cell-lysate. However, when PrPC-depleted cell-lysate was added to the purified proteins the conversion was recovered. These findings provide direct evidence for the participation of a chaperone-like activity involved in catalyzing the conversion of PrPC into PrPSc.     

Purification and properties of the cellular and scrapie hamster prion proteins

During scrapie infection an abnormal isoform of the prion protein (PrP), designated PrPSc, accumulates and is found to copurify with infectivity; to date, no nucleic acid has been found which is scrapie-specific. Both uninfected and scrapie-infected cells synthesize a PrP isoform, denoted PrPC, which exhibits physical properties that differentiate it from PrPSc. PrPC was purified by immunoaffinity chromatography using a PrP-specific monoclonal antibody cross-linked to protein-A--Avidgel. PrPSc was purified by detergent extraction, poly(ethylene glycol) precipitation and repeated differential centrifugation of PrPSc polymers. Both PrP isoforms were found to have the same N-terminal amino acid sequence which begins at a predicted signal peptide cleavage site. The first 8 residues of PrPC were found to be KKXPKPGG and the first 29 residues of PrPSc were found to be KKXPKPGGWNTGGSXYPGQGSPGGNRYPP. Arg residues 3 and 15 in PrPSc and 3 in PrPC appear to be modified since no detectable signals (denoted X) were found at these positions during gas-phase sequencing. Both PrP isoforms were found to contain an intramolecular disulfide bond, linking Cys 179 and 214, which creates a loop of 36 amino acids containing the two N-linked glycosylation sites. Development of a purification protocol for PrPC should facilitate comparisons of the two PrP isoforms and lead to an understanding of how PrPSc is synthesized either from PrPC or a precursor.     

Identification of cellular proteins binding to the scrapie prion protein

The scrapie prion protein (PrPSc) is an abnormal isoform of the cellular protein PrPc. PrPSc is found only in animals with scrapie or other prion diseases. The invariable association of PrPSc with infectivity suggests that PrPSc is a component of the infectious particle. In this study, we report the identification of two proteins from hamster brain of 45 and 110 kDa (denoted PrP ligands Pli 45 and Pli 110) which were able to bind to PrP 27-30, the protease-resistant core of PrPSc on ligand blots. Pli 45 and Pli 110 also bound PrPC. Both Pli's had isoelectric points of approximately 5. The dissociation rate constant of the Pli 45/PrP 27-30 complex was 3 x 10(-6) s-1. Amino acid and protein sequence analyses were performed on purified Pli 45. Both the composition and the sequence were almost identical with those predicted for mouse glial fibrillary acidic protein (GFAP). Furthermore, antibodies to Pli 45 reacted with recombinant GFAP. The identification of proteins which interact with the PrP isoforms in normal and diseased brain may provide new insights into the function of PrPC and into the molecular mechanisms underlying prion diseases.     

Characterization of prion proteins with monospecific antisera to synthetic peptides

The prion protein (PrP) 27-30 is the major macromolecular component in highly purified preparations of prions derived from scrapie-infected hamster brain. Immunoblotting studies demonstrated that this protein is generated by partial protease digestion of a larger precursor (PrPSc) with an apparent Mr of 33 to 35 kDa, and that a protease-sensitive cellular PrP isoform, designated PrPC, is present in normal hamster brain. To characterize the relationships among these proteins, ELISA and immunoblotting studies were undertaken with rabbit antisera raised against three synthetic PrP peptides. All three antisera were found to specifically react with the prion proteins, and failed to identify other lower or higher m.w. PrP proteins. Our results provide evidence that the primary structures of PrP 27-30, PrPSc, and PrPC are related; this conclusion supports molecular cloning studies indicating that these proteins are encoded by the same chromosomal gene.     

Integrity of H1 helix in prion protein revealed by molecular dynamic simulations to be especially vulnerable to changes in the relative orientation of H1 and its S1 flank

In the template-assistance model, normal prion protein (PrPC), the pathogenic cause of prion diseases such as Creutzfeldt-Jakob in human, bovine spongiform encephalopathy in cow, and scrapie in sheep, converts to infectious prion (PrPSc) through an autocatalytic process triggered by a transient interaction between PrPC and PrPSc. Conventional studies suggest the S1-H1-S2 region in PrPC to be the template of S1-S2 β-sheet in PrPSc, and the conformational conversion of PrPC into PrPSc may involve an unfolding of H1 in PrPC and its refolding into the β-sheet in PrPSc. Here we conduct a series of simulation experiments to test the idea of transient interaction of the template-assistance model. We find that the integrity of H1 in PrPC is vulnerable to a transient interaction that alters the native dihedral angles at residue Asn¹⁴³, which connects the S1 flank to H1, but not to interactions that alter the internal structure of the S1 flank, nor to those that alter the relative orientation between H1 and the S2 flank.     

Prions and prion proteins

Neurodegenerative diseases of animals and humans including scrapie, bovine spongiform encephalopathy, and Creutzfeldt-Jakob disease are caused by unusual infectious pathogens called prions. There is no evidence for a nucleic acid in the prion, but diverse experimental results indicate that a host-derived protein called PrPSc is a component of the infectious particle. Experiments with scrapie-infected cultured cells show that PrPSc is derived from a normal cellular protein called PrPC through an unknown posttranslational process. We have analyzed the amino acid sequence and posttranslational modifications of PrPSc and its proteolytically truncated core PrP 27-30 to identify potential candidate modifications that could distinguish PrPSc from PrPC. The amino acid sequence of PrP 27-30 corresponds to that predicted from the gene and cDNA. Mass spectrometry of peptides derived from PrPSc has revealed numerous modifications including two N-linked carbohydrate moieties, removal of an amino-terminal signal sequence, and alternative COOH termini. Most molecules contain a glycosylinositol phospholipid (GPI) attached at Ser-231 that results in removal of 23 amino acids from the COOH terminus, whereas 15% of the protein molecules are truncated to end at Gly-228. The structure of the GPI from PrPSc has been analyzed and found to be novel, including the presence of sialic acid. Other experiments suggest that the N-linked oligosaccharides are not necessary for PrPSc formation. Although detailed comparison of PrPSc with PrPC is required, there is no obvious way in which any of the modifications might confer upon PrPSc its unusual physical properties and allow it to act as a component of the prion. If no chemical difference is found between PrPC and PrPSc, then the two isoforms of the prion protein may differ only in their conformations or by the presence of bound cellular components.     

Insoluble aggregates and protease-resistant conformers of prion protein in uninfected human brains

Aggregated prion protein (PrPSc), which is detergent-insoluble and partially proteinase K (PK)-resistant, constitutes the major component of infectious prions that cause a group of transmissible spongiform encephalopathies in animals and humans. PrPSc derives from a detergent-soluble and PK-sensitive cellular prion protein (PrPC) through an alpha-helix to beta-sheet transition. This transition confers on the PrPSc molecule unique physicochemical and biological properties, including insolubility in nondenaturing detergents, an enhanced tendency to form aggregates, resistance to PK digestion, and infectivity, which together are regarded as the basis for distinguishing PrPSc from PrPC. Here we demonstrate, using sedimentation and size exclusion chromatography, that small amounts of detergent-insoluble PrP aggregates are present in uninfected human brains. Moreover, PK-resistant PrP core fragments are detectable following PK treatment. This is the first study that provides experimental evidence supporting the hypothesis that there might be silent prions lying dormant in normal human brains.     

Proteinase K-sensitive disease-associated ovine prion protein revealed by conformation-dependent immunoassay

PrPSc [abnormal disease-specific conformation of PrP (prion-related protein)] accumulates in prion-affected individuals in the form of amorphous aggregates. Limited proteolysis of PrPSc results in a protease-resistant core of PrPSc of molecular mass of 27-30 kDa (PrP27-30). Aggregated forms of PrP co-purify with prion infectivity, although infectivity does not always correlate with the presence of PrP27-30. This suggests that discrimination between PrPC (normal cellular PrP) and PrPSc by proteolysis may underestimate the repertoire and quantity of PrPSc subtypes. We have developed a CDI (conformation-dependent immunoassay) utilizing time-resolved fluorescence to study the conformers of disease-associated PrP in natural cases of sheep scrapie, without using PK (proteinase K) treatment to discriminate between PrPC and PrPSc. The capture-detector CDI utilizes N-terminal- and C-terminal-specific anti-PrP monoclonal antibodies that recognize regions of the prion protein differentially buried or exposed depending on the extent of denaturation of the molecule. PrPSc was precipitated from scrapie-infected brain stem and cerebellum tissue following sarkosyl extraction, with or without the use of sodium phosphotungstic acid, and native and denatured PrPSc detected by CDI. PrPSc was detectable in brain tissue from homozygous VRQ (V136 R154 Q171) and ARQ (A136 R154 Q171) scrapie-infected sheep brains. The highest levels of PrPSc were found in homozygous VRQ scrapie-infected brains. The quantity of PrPSc was significantly reduced, up to 90% in some cases, when samples were treated with PK prior to the CDI. Collectively, our results show that the level of PrPSc in brain samples from cases of natural scrapie display genotypic differences and that a significant amount of this material is PK-sensitive.     

Phosphorothioate oligonucleotides reduce PrP levels and prion infectivity in cultured cells

Prions are composed solely of the disease-causing prion protein (PrPSc) that is formed from the cellular isoform PrPC by a posttranslational process. Here we report that short phosphorothioate DNA (PS-DNA) oligonucleotides diminished the levels of both PrPC and PrPSc in prion-infected neuroblastoma (ScN2a) cells. The effect of PS-DNA on PrP levels was independent of the nucleotide sequence. The effective concentration (EC50) of PS-DNA required to achieve half-maximal diminution of PrPSc was approximately 70 nM, whereas the EC50 of PS-DNA for PrPC was more than 50-fold greater. This finding indicated that diminished levels of PrPSc after exposure to PS-DNA are unlikely to be due to decreased PrPC levels. Bioassays in transgenic mice demonstrated a substantial diminution in the prion infectivity after ScN2a cells were exposed to PS-DNAs. Whether PS-DNA will be useful in the treatment of prion disease in people or livestock remains to be established.     

Identification of novel proteinase K-resistant C-terminal fragments of PrP in Creutzfeldt-Jakob disease

The central event in the pathogenesis of prion diseases, a group of fatal, transmissible neurodegenerative disorders including Creutzfeldt-Jakob disease (CJD) in humans, is the conversion of the normal or cellular prion protein (PrPC) into the abnormal or scrapie isoform (PrPSc). The basis of the PrPC to PrPSc conversion is thought to involve the diminution of alpha-helical domains accompanied by the increase of beta structures within the PrP molecule. Consequently, treatment of PrPSc with proteinase K (PK) generates a large PK-resistant C-terminal core fragment termed PrP27-30 that in human prion diseases has a gel mobility of approximately 19-21 kDa for the unglycosylated form, and a ragged N terminus between residues 78 and 103. PrP27-30 is considered the pathogenic and infectious core of PrPSc. Here we report the identification of two novel PK-resistant, but much smaller C-terminal fragments of PrP (PrP-CTF 12/13) in brains of subjects with sporadic CJD. PrP-CTF 12/13, like PrP27-30, derive from both glycosylated as well as unglycosylated forms. The unglycosylated PrPCTF 12/13 migrate at 12 and 13 kDa and have the N terminus at residues 162/167 and 154/156, respectively. Therefore, PrP-CTF12/13 are 64-76 amino acids N-terminally shorter than PrP27-30 and are about half of the size of PrP27-30. PrP-CTF12/13 are likely to originate from a subpopulation of PrPSc distinct from that which generates PrP27-30. The finding of PrP-CTF12/13 in CJD brains widens the heterogeneity of the PK-resistant PrP fragments associated with prion diseases and may provide useful insights toward the understanding of the PrPSc structure and its formation.     

Differential release of cellular and scrapie prion proteins from cellular membranes by phosphatidylinositol-specific phospholipase C

The abnormal isoform of the scrapie prion protein PrPSc is both a host-derived protein and a component of the infectious agent causing scrapie. PrPSc and the normal cellular isoform PrPC have different physical properties that apparently arise from a posttranslational event. Both PrP isoforms are covalently modified at the carboxy terminus by a glycoinositol phospholipid. Using preparations of dissociated cells derived from normal and scrapie-infected hamster brain tissue, we find that the majority of PrPC is released from membranes by phosphatidylinositol-specific phospholipase C (PIPLC), while PrPSc is resistant to release. In contrast, purified denatured PrP 27-30 (which is formed from PrPSc during purification by proteolysis of the amino terminus) is completely cleaved by PIPLC. Incubation of the cell preparations with proteinase K cleaves PrPSc to form PrP 27-30, demonstrating that PrPSc is accessible to added enzymes. We have also developed a protocol involving biotinylation that gives a quantitative estimate of the fraction of a protein exposed to the cell exterior. Using this strategy, we find that a large portion of PrPSc in the cell preparations reacts with a membrane-impermeant biotinylation reagent. Whether alternative membrane anchoring of PrPSc, inaccessibility of the glycoinositol phospholipid anchor to PIPLC, or binding to another cellular component is responsible for the differential release of prion proteins from cells remains to be determined.     

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

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

Evidence for synthesis of scrapie prion proteins in the endocytic pathway.

Infectious scrapie prions are composed largely, if not entirely, of an abnormal isoform of the prion protein (PrP) which is designated PrPSc. A chromosomal gene encodes both the cellular prion protein (PrPC) as well as PrPSc. Pulse-chase experiments with scrapie-infected cultured cells indicate that PrPSc is formed by a post-translational process. PrP is translated in the endoplasmic reticulum, modified as it passes through the Golgi, and is transported to the cell surface. Release of nascent PrP from the cell surface by phosphatidylinositol-specific phospholipase C or hydrolysis with dispase prevented PrPSc synthesis. At 18 degrees C, the synthesis of PrPSc was inhibited under conditions that other investigators report a blockage of endosomal fusion with lysosomes. Our results suggest that PrPSc synthesis occurs after PrP transits from the cell surface. Whether all of the PrP molecules have an equal likelihood to be converted into PrPSc or only a distinct subset is eligible for conversion remains to be established. Identifying the subcellular compartment(s) of PrPSc synthesis should be of considerable importance in defining the molecular changes that distinguish PrPSc from PrPC.     

Cellular heparan sulfate participates in the metabolism of prions

During prion diseases, the host protein PrPC is refolded into an abnormal conformer "prion" PrPSc. Histological and pharmacological data have suggested that glycosaminoglycans may be involved in the development of prion diseases. Here we present the first direct evidence that cellular glycosaminoglycans play a role in the biogenesis of PrPSc in prion-infected ScN2a cells. When ScN2a cells were incubated with estradiol beta-d-xyloside to inhibit the glycosylation of proteoglycans, PrPSc was vastly reduced. Treating ScN2a-M cells with heparinase III, but not with heparinase I or chondroitinase ABC, caused a profound reduction of PrPSc. In contrast, neither the amount of PrPC nor its subcellular distribution were affected as assayed by immunofluorescence microscopy and flotation procedures. In vitro treatment of ScN2a membranes with heparinase III at either neutral or acidic pH did not reduce the level of protease-resistant PrPSc. The inhibitor of sulfation, sodium chlorate, vastly reduces PrPSc in ScN2a cells (Gabizon, R., Meiner, Z., Halimi, M., and Ben-Sasson, S. A. (1993) J. Cell. Physiol. 157, 319-325). Both soluble heparan sulfate and chondroitin sulfate partially restored the level of PrPSc in chlorate-treated cells. We conclude that heparinase III-sensitive, presumably undersulfated, cellular heparan sulfate plays a significant role in the biogenesis of PrPSc in ScN2a cells.     

A pitfall in diagnosis of human prion diseases using detection of protease-resistant prion protein in urine. Contamination with bacterial outer membrane proteins

Because a definite diagnosis of prion diseases relies on the detection of the abnormal isoform of prion protein (PrPSc), it has been urgently necessary to establish a non-invasive diagnostic test to detect PrPSc in human prion diseases. To evaluate diagnostic usefulness and reliability of the detection of protease-resistant prion protein in urine, we extensively analyzed proteinase K (PK)-resistant proteins in patients affected with prion diseases and control subjects by Western blot, a coupled liquid chromatography and mass spectrometry analysis, and N-terminal sequence analysis. The PK-resistant signal migrating around 32 kDa previously reported by Shaked et al. (Shaked, G. M., Shaked, Y., Kariv-Inbal, Z., Halimi, M., Avraham, I., and Gabizon, R. (2001) J. Biol. Chem. 276, 31479-31482) was not observed in this study. Instead, discrete protein bands with an apparent molecular mass of approximately 37 kDa were detected in the urine of many patients affected with prion diseases and two diseased controls. Although these proteins also gave strong signals in the Western blot using a variety of anti-PrP antibodies as a primary antibody, we found that the signals were still detectable by incubation of secondary antibodies alone, i.e. in the absence of the primary anti-PrP antibodies. Mass spectrometry and N-terminal protein sequencing analysis revealed that the majority of the PK-resistant 37-kDa proteins in the urine of patients were outer membrane proteins (OMPs) of the Enterobacterial species. OMPs isolated from these bacteria were resistant to PK and the PK-resistant OMPs from the Enterobacterial species migrated around 37 kDa on SDS-PAGE. Furthermore, nonspecific binding of OMPs to antibodies could be mistaken for PrPSc. These findings caution that bacterial contamination can affect the immunological detection of prion protein. Therefore, the presence of Enterobacterial species should be excluded in the immunological tests for PrPSc in clinical samples, in particular, urine.     

朊蛋白PrP~C与神经元的凋亡和存活

朊病毒是不含核酸的一种蛋白质感染颗粒,感染宿主后可诱导细胞固有的同类朊蛋白(PrPC)构象改变、转化成具有蛋白酶抗性的致病性朊粒蛋白(PrPSc),导致可传播性海绵状脑病的发生.PrPC既作为朊病毒复制和疾病发生的分子基础,又是正常的细胞膜蛋白,作为细胞信号转导的参与者调控多条信号通路.因此,揭示PrPC在各条信号途径中发挥的作用将有助于深入了解PrPC的生理功能,进一步理解疾病发生发展过程,为今后的诊断治疗奠定基础.     

Cholesterol depletion and modification of COOH-terminal targeting sequence of the prion protein inhibit formation of the scrapie isoform [published erratum appears in J Cell Biol 1995 Jul;130(2):501]

After the cellular prion protein (PrPC) transits to the cell surface where it is bound by a glycophosphatidyl inositol (GPI) anchor, PrPC is either metabolized or converted into the scrapie isoform (PrPSc). Because most GPI-anchored proteins are associated with cholesterol-rich membranous microdomains, we asked whether such structures participate in the metabolism of PrPC or the formation of PrPSc. The initial degradation of PrPC involves removal of the NH2 terminus of PrPC to produce a 17-kD polypeptide which was found in a Triton X-100 insoluble fraction. Both the formation of PrPSc and the initial degradation of PrPC were diminished by lovastatin-mediated depletion of cellular cholesterol but were insensitive to NH4Cl. Further degradation of the 17-kD polypeptide did occur within an NH4Cl-sensitive, acidic compartment. Replacing the GPI addition signal with the transmembrane and cytoplasmic domains of mouse CD4 rendered chimeric CD4PrPC soluble in cold Triton X-100. Both CD4PrPC and truncated PrPC without the GPI addition signal (Rogers, M., F. Yehieley, M. Scott, and S. B. Prusiner. 1993. Proc. Natl. Acad. Sci. USA. 90:3182-3186) were poor substrates for PrPSc formation. Thus, it seems likely that both the initial degradation of PrPC to the 17-kD polypeptide and the formation of PrPSc occur within a non-acidic compartment bound by cholesterol-rich membranes, possibly glycolipid-rich microdomains, where the metabolic fate of PrPC is determined. The pathway remains to be identified by which the 17-kD polypeptide and PrPSc are transported to an acidic compartment, presumably endosomes, where the 17-kD polypeptide is hydrolyzed and limited proteolysis of PrPSc produces PrP 27-30.     

N-terminally tagged prion protein supports prion propagation in transgenic mice.

The eight amino acid sequence, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, representing the FLAG peptide, was inserted after codons 22 or 88 of the mouse (Mo) prion protein (PrP) gene. Inclusion of the FLAG sequence at these locations interfered neither with the cellular processing of PrPC nor its conversion into PrPSc. Inclusion of the FLAG epitope at residue 22 but not at residue 88 facilitated immunodetection of tagged PrP by anti-FLAG monoclonal antibodies (mAbs). Inoculation of transgenic (Tg) mice expressing N-terminally tagged MoPrP with Mo prions resulted in abbreviated incubation times, indicating that the FLAG sequence was not deleterious to prion propagation. Immunopurification of FLAG-tagged MoPrPC in the brains of Tg mice was achieved using the calcium-dependent anti-FLAG M1 mAb and non-denaturing procedures. Although the function of PrPC remains unknown, our studies demonstrate that some modifications of PrPC do not inhibit the one biological activity that can be measured, i.e., conversion into PrPSc. Tagged PrP molecules may prove useful in the development of improved assays for prions as well as structural studies of the PrP isoforms.     

The AGAAAAGA palindrome in PrP is required to generate a productive PrPSc-PrPC complex that leads to prion propagation

The molecular hallmark of prion disease is the conversion of normal prion protein (PrPC) to an insoluble, proteinase K-resistant, pathogenic isoform (PrPSc). Once generated, PrPSc propagates by complexing with, and transferring its pathogenic conformation onto, PrPC. Defining the specific nature of this PrPSc-PrPC interaction is critical to understanding prion genesis. To begin to approach this question, we employed a prion-infected neuroblastoma cell line (ScN2a) combined with a heterologous yeast expression system to independently model PrPSc generation and propagation. We additionally applied fluorescence resonance energy transfer analysis to the latter to specifically study PrP-PrP interactions. In this report we focus on an N-terminal hydrophobic palindrome of PrP (112-AGAAAAGA-119) thought to feature intimately in prion generation via an unclear mechanism. We found that, in contrast to wild type (wt) PrP, PrP lacking the palindrome (PrPDelta112-119) neither converted to PrPSc when expressed in ScN2a cells nor generated proteinase K-resistant PrP when expressed in yeast. Furthermore, PrPDelta112-119 was a dominant-negative inhibitor of wtPrP in ScN2a cells. Both wtPrP and PrPDelta112-119 were highly insoluble when expressed in yeast and produced distinct cytosolic aggregates when expressed as fluorescent fusion proteins (PrP::YFP). Although self-aggregation was evident, fluorescence resonance energy transfer studies in live yeast co-expressing PrPSc-like protein and PrPDelta112-119 indicated altered interaction properties. These results suggest that the palindrome is required, not only for the attainment of the PrPSc conformation but also to facilitate the proper association of PrPSc with PrPC to effect prion propagation.     

Immunolocalisation of PrPSc in scrapie-infected N2a mouse neuroblastoma cells by light and electron microscopy

The causative agent of transmissible spongiform encephalopathies (TSE) is PrPSc, an infectious, misfolded isoform of the cellular prion protein (PrPC). The localisation and trafficking of PrPSc and sites of conversion from PrPC to PrPSc are under debate, particularly since most published work did not discriminate between PrPC and PrPSc. Here we describe the localisation of PrPC and PrPSc in a scrapie-infected neuroblastoma cell line, ScN2a, by light and electron microscopic immunolocalisation. After eliminating PrPC with proteinase K, PrPSc was detected at the plasma membrane, endocytosed via clathrin-coated pits and delivered to early endosomes. Finally, PrPSc was detected in late endosomes/lysosomes. As we detected PrPSc at the cell surface, in early endosomes and in late endosomes/lysosomes, i.e. locations where PrPC is also present, our data imply that the conversion process could take place at the plasma membrane and/or along the endocytic pathway. Finally, we observed the release of PrPC/PrPSc via exocytotic pathways, i.e. via exosomes and as an opaque electron-dense mass which may represent a mechanism of intercellular spreading of infectious prions.