The murine B cell repertoire is severely selected against endogenous cellular prion protein

Abs to the prion protein (PrP) can protect against experimental prion infections, but efficient Ab responses are difficult to generate because PrP is expressed on many tissues and induces a strong tolerance. We previously showed that immunization of wild-type mice with PrP peptides and CpG oligodeoxynucleic acid overcomes tolerance and induces cellular and humoral responses to PrP. In this study, we compared Ab and T cell repertoires directed to PrP in wild-type and PrP knockout (Prnp o/o) C57BL/6 mice. Animals were immunized with mouse PrP-plasmid DNA or with 30-mer overlapping peptides either emulsified in CFA or CpG/IFA. In Prnp o/o mice, Abs raised by PrP-plasmid DNA immunization recognized only N-terminal PrP peptides; analyses of Ab responses after PrP peptide/CFA immunization allowed us to identify six distinct epitopes, five of which were also recognized by Abs raised by PrP peptides/CpG. By contrast, in wild-type mice, no Ab response was detected after PrP-plasmid DNA or peptide/CFA immunization. However, when using CpG, four C-terminal peptides induced Abs specific for distinct epitopes. Importantly, immune sera from Prnp o/o but not from wild-type mice bound cell surface PrP. Abs of IgG1 and IgG2b subclasses predominated in Prnp o/o mice while the strongest signals were for IgG2b in wild-type mice. Most anti-PrP Th cells were directed to a single epitope in both Prnp o/o and wild-type mice. We conclude that endogenous PrPC expression profoundly affects the Ab repertoire as B cells reactive for epitopes exposed on native PrPC are strongly tolerized. Implications for immunotherapy against prion diseases are discussed.     

Circumventing tolerance to the prion protein (PrP): vaccination with PrP-displaying retrovirus particles induces humoral immune responses against the native form of cellular PrP

Passive immunization with antibodies directed against the cellular form of the prion protein (PrPC) can protect against prion disease. However, active immunization with recombinant prion protein has so far failed to induce antibodies directed against native PrPC expressed on the cell surface. To develop an antiprion vaccine, a retroviral display system presenting either the full-length mouse PrP (PrP209) or the C-terminal 111 amino acids (PrP111) fused to the transmembrane domain of the platelet-derived growth factor receptor was established. Western blot analysis and immunogold electron microscopy of the retroviral display particles revealed successful incorporation of the fusion proteins into the particle membrane. Interestingly, retroviral particles displaying PrP111 (PrPD111 retroparticles) showed higher incorporation efficiencies than those displaying PrP209. Already 7 days after intravenous injection of PrPD111 retroparticles, PrPC-deficient mice (Prnp(o/o)) showed high immunoglobulin M (IgM) and IgG titers specifically binding the native PrPC molecule as expressed on the surface of T cells isolated from PrPC-overexpressing transgenic mice. More importantly, heterozygous Prnp(+/o) mice and also wild-type mice showed PrPC-specific IgM and IgG antibodies upon vaccination with PrPD111 retroparticles, albeit at considerably lower levels. Bacterially expressed recombinant PrP, in contrast, was unable to evoke IgG antibodies recognizing native PrPC in wild-type mice. Thus, our data show that PrP or parts thereof can be functionally displayed on retroviral particles and that immunization with PrP retroparticles may serve as a novel promising strategy for vaccination against transmissible spongiform encephalitis.     

Folding and intrinsic stability of deletion variants of PrP(121-231), the folded C-terminal domain of the prion protein

Transmissible spongiform encephalopathies in mammals are believed to be caused by PrPSc, the insoluble, oligomeric isoform of the cellular prion protein PrPC. PrPC and the subunits of PrPSc have identical covalent but different tertiary structure. To address the question of whether parts of the structure of PrPC are sufficiently stable to be retained in PrPSc, we have constructed two deletion variants of the C-terminal PrPC domain, PrP(121-231), which is the only part of recombinant PrP with defined tertiary structure. One of the variants, H2-H3, comprises the last two alpha-helices of PrP(121-231) that have been proposed to be preserved in models of PrP(Sc). In the other variant, PrP(121-231)-deltaH1, the first alpha-helix of PrP(121-231) was deleted and replaced by introduction of the beta-turn dipeptide Asn-Gly between the strands of the single beta-sheet of PrP(121-231). Although both deletion constructs still show alpha-helical CD-spectra, they are more disordered and thermodynamically strongly destabilized compared to PrP(121-231), with free energies of folding close to zero. These data demonstrate that the tertiary structure context is critical for the conformation of the segment comprising alpha-helix 2 and 3 in the solution structure of recombinant PrP.     

Disease-associated prion protein elicits immunoglobulin M responses in vivo

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

Influence of amino acid substitutions related to inherited human prion diseases on the thermodynamic stability of the cellular prion protein

Transmissible spongiform encephalopathies (TSEs) are caused by a unique infectious agent which appears to be identical with PrPSc, an oligomeric, misfolded isoform of the cellular prion protein, PrPC. All inherited forms of human TSEs, i.e., familial Creutzfeldt-Jakob disease, Gerstmann-Str?ussler-Scheinker syndrome, and fatal familial insomnia, segregate with specific point mutations or insertions in the gene coding for human PrP. Here we have tested the hypothesis that these mutations destabilize PrPC and thus facilitate its conversion into PrPSc. Eight of the disease-specific amino acid replacements are located in the C-terminal domain of PrPC, PrP(121-231), which constitutes the only part of PrPC with a defined tertiary structure. Introduction of all these replacements into PrP(121-231) yielded variants with the same spectroscopic characteristics as wild-type PrP(121-231) and similar to full-length PrP(23-231), which excludes the possibility that the exchanges a priori induce a PrPSc-like conformation. The thermodynamic stabilities of the variants do not correlate with specific disease phenotypes. Five of the amino acid replacements destabilize PrP(121-231), but the other variants have the same stability as the wild-type protein. These data suggest that destabilization of PrPC is neither a general mechanism underlying the formation of PrPSc nor the basis of disease phenotypes in inherited human TSEs.     

Clustered negative charges on the lipid membrane surface induce beta-sheet formation of prion protein fragment 106-126

The conformational conversion of prion protein (PrP) from an alpha-helix-rich normal cellular isoform (PrPC) to a beta-sheet-rich pathogenic isoform (PrPSc) is a key event in the development of prion diseases, and it takes place in caveolae, cavelike invaginations of the plasma membrane. A peptide homologous to residues 106-126 of human PrP (PrP106-126) is known to share several properties with PrPSc, e.g., the capability to form a beta-sheet and toxicity against PrPC-expressing cells. PrP106-126 is thus expected to represent a segment of PrP that is involved in the formation of PrPSc. We have examined the effect of lipid membranes containing negatively charged ganglioside, an important component of caveolae, on the secondary structure of PrP106-126 by circular dichroism. The peptide forms an alpha-helical or a beta-sheet structure on the ganglioside-containing membranes. The beta-sheet content increases with an increase of the peptide:lipid ratio, indicating that the beta-sheet formation is linked with self-association of the positively charged peptide on the negatively charged membrane surface. Analogous beta-sheet formation is also induced by membranes composed of negatively charged and neutral glycerophospholipids with high and low melting temperatures, respectively, in which lateral phase separation and clustering of negatively charged lipids occur as shown by Raman spectroscopy. Since ganglioside-containing membranes also exhibit lateral phase separation, clustered negative charges are concluded to be responsible for the beta-sheet formation of PrP106-126. In caveolae, clustered ganglioside molecules are likely to interact with the residue 106-126 region of PrPC to promote the PrPC-to-PrPSc conversion.     

Biological effects and use of PrPSc- and PrP-specific antibodies generated by immunization with purified full-length native mouse prions

The prion agent is the infectious particle causing spongiform encephalopathies in animals and humans and is thought to consist of an altered conformation (PrP(Sc)) of the normal and ubiquitous prion protein PrP(C). The interaction of the prion agent with the immune system, particularly the humoral immune response, has remained unresolved. Here we investigated the immunogenicity of full-length native and infectious prions, as well as the specific biological effects of the resulting monoclonal antibodies (MAbs) on the binding and clearance of prions in cell culture and in in vivo therapy. Immunization of prion knockout (Prnp(0/0)) mice with phosphotungstic acid-purified mouse prions resulted in PrP-specific monoclonal antibodies with binding specificities selective for PrP(Sc) or for both PrP(C) and PrP(Sc). PrP(Sc)-specific MAb W261, of the IgG1 isotype, reacted with prions from mice, sheep with scrapie, deer with chronic wasting disease (CWD), and humans with sporadic and variant Creutzfeldt-Jakob disease (CJD) in assays including a capture enzyme-linked immunosorbent assay (ELISA) system. This PrP(Sc)-specific antibody was unable to clear prions from mouse neuroblastoma cells (ScN2a) permanently infected with scrapie, whereas the high-affinity MAb W226, recognizing both isoforms, PrP(Sc) and PrP(C), did clear prions from ScN2a cells, as determined by a bioassay. However, an attempt to treat intraperitoneally prion infected mice with full-length W226 or with a recombinant variable-chain fragment (scFv) from W226 could only slightly delay the incubation time. We conclude that (i) native, full-length PrP(Sc) elicits a prion-specific antibody response in PrP knockout mice, (ii) a PrP(Sc)-specific antibody had no prion-clearing effect, and (iii) even a high-affinity MAb that clears prions in vitro (W226) may not necessarily protect against prion infection, contrary to previous reports using different antibodies.     

Protease-resistant and detergent-insoluble prion protein is not necessarily associated with prion infectivity.

PrPSc, an abnormal isoform of PrPC, is the only known component of the prion, an agent causing fatal neurodegenerative disorders such as bovine spongiform encephalopathy (BSE) and Creutzfeldt-Jakob disease (CJD). It has been postulated that prion diseases propagate by the conversion of detergent-soluble and protease-sensitive PrPC molecules into protease-resistant and insoluble PrPSc molecules by a mechanism in which PrPSc serves as a template. We show here that the chemical chaperone dimethyl sulfoxide (Me2SO) can partially inhibit the aggregation of either PrPSc or that of its protease-resistant core PrP27-30. Following Me2SO removal by methanol precipitation, solubilized PrP27-30 molecules aggregated into small and amorphous structures that did not resemble the rod configuration observed when scrapie brain membranes were extracted with Sarkosyl and digested with proteinase K. Interestingly, aggregates derived from Me2SO-solubilized PrP27-30 presented less than 1% of the prion infectivity obtained when the same amount of PrP27-30 in rods was inoculated into hamsters. These results suggest that the conversion of PrPC into protease-resistant and detergent-insoluble PrP molecules is not the only crucial step in prion replication. Whether an additional requirement is the aggregation of newly formed proteinase K-resistant PrP molecules into uniquely structured aggregates remains to be established.     

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.     

Disease-associated F198S mutation increases the propensity of the recombinant prion protein for conformational conversion to scrapie-like form

The critical step in the pathogenesis of transmissible spongiform encephalopathies (prion diseases) is the conversion of a cellular prion protein (PrP(c)) into a protease-resistant, beta-sheet rich form (PrP(Sc)). Although the disease transmission normally requires direct interaction between exogenous PrP(Sc) and endogenous PrP(C), the pathogenic process in hereditary prion diseases appears to develop spontaneously (i.e. not requiring infection with exogenous PrP(Sc)). To gain insight into the molecular basis of hereditary spongiform encephalopathies, we have characterized the biophysical properties of the recombinant human prion protein variant containing the mutation (Phe(198) --> Ser) associated with familial Gerstmann-Straussler-Scheinker disease. Compared with the wild-type protein, the F198S variant shows a dramatically increased propensity to self-associate into beta-sheet-rich oligomers. In a guanidine HCl-containing buffer, the transition of the F198S variant from a normal alpha-helical conformation into an oligomeric beta-sheet structure is about 50 times faster than that of the wild-type protein. Importantly, in contrast to the wild-type PrP, the mutant protein undergoes a spontaneous conversion to oligomeric beta-sheet structure even in the absence of guanidine HCl or any other denaturants. In addition to beta-sheet structure, the oligomeric form of the protein is characterized by partial resistance to proteinase K digestion, affinity for amyloid-specific dye, thioflavine T, and fibrillar morphology. The increased propensity of the F198S variant to undergo a conversion to a PrP(Sc)-like form correlates with a markedly decreased thermodynamic stability of the native alpha-helical conformer of the mutant protein. This correlation supports the notion that partially unfolded intermediates may be involved in conformational conversion of the prion protein.     

Prions and the proteasome

Prion diseases are fatal neurodegenerative disorders that include Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy in animals. They are unique in terms of their biology because they are caused by the conformational re-arrangement of a normal host-encoded prion protein, PrPC, to an abnormal infectious isoform, PrPSc. Currently the precise mechanism behind prion-mediated neurodegeneration remains unclear. It is hypothesised than an unknown toxic gain of function of PrPSc, or an intermediate oligomeric form, underlies neuronal death. Increasing evidence suggests a role for the ubiquitin proteasome system (UPS) in prion disease. Both wild-type PrPC and disease-associated PrP isoforms accumulate in cells after proteasome inhibition leading to increased cell death, and abnormal beta-sheet-rich PrP isoforms have been shown to inhibit the catalytic activity of the proteasome. Here we review potential interactions between prions and the proteasome outlining how the UPS may be implicated in prion-mediated neurodegeneration.     

Functional implication of cellular prion protein in antigen-driven interactions between T cells and dendritic cells

The cellular prion protein (PrPC) is a host-encoded, GPI-anchored cell surface protein, expressed on a wide range of tissues including neuronal and lymphoreticular cells. PrPC may undergo posttranslational conversion, giving rise to scrapie PrP, the pathogenic conformer considered as responsible for prion diseases. Despite intensive studies, the normal function of PrPC is still enigmatic. Starting from microscope observations showing an accumulation of PrPC at the sites of contact between T cells and Ag-loaded dendritic cells (DC), we have studied the contribution of PrPC in alloantigen and peptide-MHC-driven T/DC interactions. Whereas the absence of PrPC on the DC results in a reduced allogeneic T cell response, its absence on the T cell partner has no apparent effect upon this response. Therefore, PrPC seems to fulfill different functions on the two cell partners forming the synapse. In contrast, PrPC mobilization by Ab reduces the stimulatory properties of DC and the proliferative potential of responding T cells. The contrasted consequences, regarding T cell function, between PrPC deletion and PrPC coating by Abs, suggests that the prion protein acts as a signaling molecule on T cells. Furthermore, our results show that the absence of PrPC has consequences in vivo also, upon the ability of APCs to stimulate proliferative T cell responses. Thus, independent of neurological considerations, some of the evolutionary constraints that may have contributed to the conservation of the Prnp gene in mammalians, could be of immunological origin.     

The deletion of amino acids 114-121 in the TM1 domain of mouse prion protein stabilizes its conformation but does not affect the overall structure

A mutant of mouse prion protein (PrPC) carrying a deletion of residues 114-121 (PrPDelta114-121) has previously been described to lack convertibility into the scrapie-associated isoform of PrP (PrPSc) and to exhibit a dominant-negative effect on the conversion of wild-type PrPC into PrPSc in living cells. Here we report the characterization of recombinantly expressed PrPDelta114-121 by Fourier-transformation infrared spectroscopy (FTIR) and circular dichroism (CD) spectroscopy. The analysis of spectra revealed an increased antiparallel beta-sheet content in the deletion mutant compared to wild-type PrPC. This additional short beta-sheet stabilized the fold of the mutant protein by DeltaDeltaG(0)'=3.4+/-0.3 kJ mol(-1) as shown by chemical unfolding experiments using guanidine hydrochloride. Secondary structure predictions suggest that the additional beta-sheet in PrPDelta114-121 is close to the antiparallel beta-sheet in PrPC. The high-affinity Cu2+-binding site outside the octarepeats, which is located close to the deletion and involves His110 as a ligand, was not affected, as detected by electron paramagnetic resonance (EPR) spectroscopy, suggesting that Cu2+ binding does not contribute to the protection of PrPDelta114-121 from conversion into PrPSc. We propose that the deletion of residues 114-121 stabilizes the mutant protein. This stabilization most likely does not obstruct the interaction of PrPDelta114-121 with PrPSc but represents an energy barrier that blocks the conversion of PrPDelta114-121 into PrPSc.     

The role of the prion protein membrane anchor in prion infection

In transmissible spongiform encephalopathies (TSE or prion diseases) such as sheep scrapie, bovine spongiform encephalopathy and human Creutzfeldt-Jakob disease, normally soluble and protease-sensitive prion protein (PrP-sen or PrPC) is converted to an abnormal, insoluble and protease-resistant form termed PrP-res or PrPSc. PrP-res/PrPSc is believed to be the main component of the prion, the infectious agent of the TSE/prion diseases. Its precursor, PrP-sen, is anchored to the cell surface at the C-terminus by a co-translationally added glycophosphatidyl-inositol (GPI) membrane anchor which can be cleaved by the enzyme phosphatidyl-inositol specific phospholipase (PIPLC). The GPI anchor is also present in PrP-res, but is inaccessible to PIPLC digestion suggesting that conformational changes in PrP associated with PrP-res formation have blocked the PIPLC cleavage site. Although the GPI anchor is present in both PrP-sen and PrP-res, its precise role in TSE diseases remains unclear primarily because there are data to suggest that it both is and is not necessary for PrP-res formation and prion infection.     

Mutant prion protein-mediated aggregation of normal prion protein in the endoplasmic reticulum: implications for prion propagation and neurotoxicity

Familial prion disorders are believed to result from spontaneous conversion of mutant prion protein (PrPM) to the pathogenic isoform (PrPSc). While most familial cases are heterozygous and thus express the normal (PrPC) and mutant alleles of PrP, the role of PrPC in the pathogenic process is unclear. Plaques from affected cases reveal a heterogeneous picture; in some cases only PrPM is detected, whereas in others both PrPC and PrPM are transformed to PrPSc. To understand if the coaggregation of PrPC is governed by PrP mutations or is a consequence of the cellular compartment of PrPM aggregation, we coexpressed PrPM and PrPC in neuroblastoma cells, the latter tagged with green fluorescent protein (PrPC-GFP) for differentiation. Two PrPM forms (PrP231T, PrP217R/231T) that aggregate spontaneously in the endoplasmic reticulum (ER) were generated for this analysis. We report that PrPC-GFP aggregates when coexpressed with PrP231T or PrP217R/231T, regardless of sequence homology between the interacting forms. Furthermore, intracellular aggregates of PrP231T induce the accumulation of a C-terminal fragment of PrP, most likely derived from a potentially neurotoxic transmembrane form of PrP (CtmPrP) in the ER. These findings have implications for prion pathogenesis in familial prion disorders, especially in cases where transport of PrPM from the ER is blocked by the cellular quality control.     

Scrapie prion protein contains a phosphatidylinositol glycolipid

The scrapie (PrPSc) and cellular (PrPC) prion proteins are encoded by the same gene, and their different properties are thought to arise from posttranslational modifications. We have found a phosphatidylinositol glycolipid on both PrPC and PrP 27-30 (derived from PrPSc by limited proteolysis at the amino terminus). Ethanolamine, myo-inositol, phosphate, and stearic acid were identified as glycolipid components of gel-purified PrP 27-30. PrP 27-30 contains 2.8 moles of ethanolamine per mole. Incubation of PrP 27-30 with a bacterial phosphatidylinositol-specific phospholipase C (PIPLC) releases covalently bound stearic acid, and allows PrP 27-30 to react with antiserum specific for the PIPLC-digested glycolipid linked to the carboxyl terminus of the trypanosomal variant surface glycoprotein. PIPLC catalyzes the release of PrPC from cultured mammalian cells into the medium. These observations indicate that PrPC is anchored to the cell surface by the glycolipid.     

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.     

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.     

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.     

朊粒蛋白PrP~(Sc)寡聚体的形成与跨膜毒性

朊粒蛋白(prionprotein,PrP)传染致病机制一直是朊粒(prion)研究领域的焦点.由正常型朊粒蛋白(PrPC)向致病型朊粒蛋白(PrPSc)的转变是致病的关键步骤.本文综述了近年来PrPC向PrPSc转变的结构变化特征、PrPSc由单体形成寡聚体的组装机制、以及PrPSc寡聚体的跨膜机制与细胞毒性间的关系等方面的研究进展.