PrP Sc Formation Research Articles

NMDA Receptor and L-Type Calcium Channel Modulate Prion Formation.

Transmissible neurodegenerative prion diseases are characterized by the conversion of the cellular prion protein (PrPC) to misfolded isoforms denoted as prions or PrPSc. Although the conversion can occur in the test tube containing recombinant prion protein or cell lysates, efficient prion formation depends on the integrity of intact cell functions. Since neurons are main targets for prion replication, we asked whether their most specialized function, i.e. synaptic plasticity, could be a factor by which PrPSc formation can be modulated.Immortalized gonadotropin-releasing hormone cells infected with the Rocky Mountain Laboratory prion strain were treated with L-type calcium channels (LTCCs) and NMDA receptors (NMDARs) stimulators or inhibitors. Western blotting was used to monitor the effects on PrPSc formation in relation to ERK signalling.Infected cells showed enhanced levels of phosphorylated ERK (pERK) compared with uninfected cells. Exposure of infected cells to the LTCC agonist Bay K8644 enhanced pERK and PrPSc levels. Although treatment with an LTCC blocker (nimodipine) or an NMDAR competitive antagonist (D-AP5) had no effects, their combination reduced both pERK and PrPSc levels. Treatment with the non-competitive NMDAR channel blocker MK-801 markedly reduced pERK and PrPSc levels.Our study shows that changes in LTCCs and NMDARs activities can modulate PrPSc formation through ERK signalling. During synaptic plasticity, while ERK signalling promotes long-term potentiation accompanied by expansion of post-synaptic lipid rafts, other NMDA receptor-depending signalling pathways, p38-JNK, have opposing effects. Our findings indicate that contrasting intracellular signals of synaptic plasticity can influence time-dependent prion conversion.

Development and structural determination of an anti-PrPC aptamer that blocks pathological conformational conversion of prion protein

Prion diseases comprise a fatal neuropathy caused by the conversion of prion protein from a cellular (PrPC) to a pathological (PrPSc) isoform. Previously, we obtained an RNA aptamer, r(GGAGGAGGAGGA) (R12), that folds into a unique G-quadruplex. The R12 homodimer binds to a PrPC molecule, inhibiting PrPC-to-PrPSc conversion. Here, we developed a new RNA aptamer, r(GGAGGAGGAGGAGGAGGAGGAGGA) (R24), where two R12s are tandemly connected. The 50% inhibitory concentration for the formation of PrPSc (IC50) of R24 in scrapie-infected cell lines was ca. 100 nM, i.e., much lower than that of R12 by two orders. Except for some antibodies, R24 exhibited the lowest recorded IC50 and the highest anti-prion activity. We also developed a related aptamer, r(GGAGGAGGAGGA-A-GGAGGAGGAGGA) (R12-A-R12), IC50 being ca. 500 nM. The structure of a single R12-A-R12 molecule determined by NMR resembled that of the R12 homodimer. The quadruplex structure of either R24 or R12-A-R12 is unimolecular, and therefore the structure could be stably formed when they are administered to a prion-infected cell culture. This may be the reason they can exert high anti-prion activity.

Direct Observation of Prion Protein Fibril Elongation Kinetics

It is widely accepted that prion diseases are caused by the conversion of cellular prion protein (PrPC) to the misfolded form PrPSc, which is self-propagating by seeded aggregation. The molecular mechanism and propagation kinetics are not well understood. Here, we use Total Internal Reflection Fluorescence and Transient Amyloid Binding super-resolution microscopy to investigate the elongation kinetics of mouse prion protein at single fibril level. Recombinant mouse prion protein (recMoPrP 91-231) fibrils formed under partially denaturing conditions were deposited on a glass slide, incubated with recMoPrP 91-231 monomer solution and imaged for up to 70h. We found that PrP fibrils elongated by an intermittent ‘stop-and-go’ mechanism. The elongation rates of growing phase are obtained under different temperature, various concentrations of monomer, denaturant, and sodium chloride, which are then used to calculate the kinetic parameters. This study sheds light on the mechanism of prion protein seeded propagation in vitro and provides a useful system to study the influence of PrP mutants and screening of inhibitors.

Structural evidence for the critical role of the prion protein hydrophobic region in forming an infectious prion.

Prion or PrPSc is the proteinaceous infectious agent causing prion diseases in various mammalian species. Despite decades of research, the structural basis for PrPSc formation and prion infectivity remains elusive. To understand the role of the hydrophobic region in forming infectious prion at the molecular level, we report X-ray crystal structures of mouse (Mo) prion protein (PrP) (residues 89–230) in complex with a nanobody (Nb484). Using the recombinant prion propagation system, we show that the binding of Nb484 to the hydrophobic region of MoPrP efficiently inhibits the propagation of proteinase K resistant PrPSc and prion infectivity. In addition, when added to cultured mouse brain slices in high concentrations, Nb484 exhibits no neurotoxicity, which is drastically different from other neurotoxic anti-PrP antibodies, suggesting that the Nb484 can be a potential therapeutic agent against prion disease. In summary, our data provides the first structure-function evidence supporting a crucial role of the hydrophobic region of PrP in forming an infectious prion.

Prion protein-Semisynthetic prion protein (PrP) variants with posttranslational modifications.

Deciphering the pathophysiologic events in prion diseases is challenging, and the role of posttranslational modifications (PTMs) such as glypidation and glycosylation remains elusive due to the lack of homogeneous protein preparations. So far, experimental studies have been limited in directly analyzing the earliest events of the conformational change of cellular prion protein (PrPC) into scrapie prion protein (PrPSc) that further propagates PrPC misfolding and aggregation at the cellular membrane, the initial site of prion infection, and PrP misfolding, by a lack of suitably modified PrP variants. PTMs of PrP, especially attachment of the glycosylphosphatidylinositol (GPI) anchor, have been shown to be crucially involved in the PrPSc formation. To this end, semisynthesis offers a unique possibility to understand PrP behavior in vitro and in vivo as it provides access to defined site‐selectively modified PrP variants. This approach relies on the production and chemoselective linkage of peptide segments, amenable to chemical modifications, with recombinantly produced protein segments. In this article, advances in understanding PrP conversion using semisynthesis as a tool to obtain homogeneous posttranslationally modified PrP will be discussed.

Different effects of lipid on conformational conversion of chicken and murine prion proteins

Prion diseases are characterized by the conformational conversion of the cellular prion protein (PrPC) to the pathogenic isoform (PrPSc). Lipids have been found to interact with PrPC and contribute to the efficient formation of PrPSc. Non-mammalian PrPs are not readily to undergo the conversion process into an infectious isoform, yet the effect of lipid on the conformational conversion of non-mammalian PrPC remains to be explored. Herein, the effects of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) on full-length recombinant chicken PrP (ChPrP) 24–249 and murine PrP (MoPrP) 23–230 were investigated. Firstly, it was found that in the presence of chemical denaturant, POPG remarkably inhibited MoPrP amyloid fibril growth, while had slight effect on that of ChPrP as estimated by amyloid fibril growth and transmissible electronic microscope assays. Secondly, under physiological condition, POPG induced conformation changes in both MoPrP and ChPrP using Thioflavin T (ThT) fluorescence, circular dichroism, proteinase K digestion and transmission electron microscopy assays. With a POPG:PrP molar ratio of 30:1, the ThT fluorescence of MoPrP was found to be lower than that of ChPrP, however, the POPG-induced MoPrP had higher β-sheet content and was more proteinase K-resistant than POPG-induced ChPrP. In summary, the present results suggested that the effects of POPG on conformational conversion of MoPrP and ChPrP were different under both denaturation and physiological conditions.

Anti-prion Protein Antibody 6D11 Restores Cellular Proteostasis of Prion Protein Through Disrupting Recycling Propagation of PrPSc and Targeting PrPSc for Lysosomal Degradation.

PrPSc is an infectious and disease-specific conformer of the prion protein, which accumulation in the CNS underlies the pathology of prion diseases. PrPSc replicates by binding to the cellular conformer of the prion protein (PrPC) expressed by host cells and rendering its secondary structure a likeness of itself. PrPC is a plasma membrane anchored protein, which constitutively recirculates between the cell surface and the endocytic compartment. Since PrPSc engages PrPC along this trafficking pathway, its replication process is often referred to as "recycling propagation." Certain monoclonal antibodies (mAbs) directed against prion protein can abrogate the presence of PrPSc from prion-infected cells. However, the precise mechanism(s) underlying their therapeutic propensities remains obscure. Using N2A murine neuroblastoma cell line stably infected with 22L mouse-adapted scrapie strain (N2A/22L), we investigated here the modus operandi of the 6D11 clone, which was raised against the PrPSc conformer and has been shown to permanently clear prion-infected cells from PrPSc presence. We determined that 6D11 mAb engages and sequesters PrPC and PrPSc at the cell surface. PrPC/6D11 and PrPSc/6D11 complexes are then endocytosed from the plasma membrane and are directed to lysosomes, therefore precluding recirculation of nascent PrPSc back to the cell surface. Targeting PrPSc by 6D11 mAb to the lysosomal compartment facilitates its proteolysis and eventually shifts the balance between PrPSc formation and degradation. Ongoing translation of PrPC allows maintaining the steady-state level of prion protein within the cells, which was not depleted under 6D11 mAb treatment. Our findings demonstrate that through disrupting recycling propagation of PrPSc and promoting its degradation, 6D11 mAb restores cellular proteostasis of prion protein.

Residue-specific mobility changes in soluble oligomers of the prion protein define regions involved in aggregation

Prion (PrP) diseases are neurodegenerative diseases characterized by the formation of β-sheet rich, insoluble and protease resistant protein deposits (called PrPSc) that occur throughout the brain. Formation of synthetic or in vitro PrPSc can occur through on-pathway toxic oligomers. Similarly, toxic and infectious oligomers identified in cell and animal models of prion disease indicate that soluble oligomers are likely intermediates in the formation of insoluble PrPSc. Despite the critical role of prion oligomers in disease progression, little is known about their structure. In order, to obtain structural insight into prion oligomers, we generated oligomers by shaking-induced conversion of recombinant, monomeric prion protein PrPc (spanning residues 90–231). We then obtained two-dimensional solution NMR spectra of the PrPc monomer, a 40% converted oligomer, and a 94% converted oligomer. Heteronuclear single-quantum correlation (1H–15N) studies revealed that, in comparison to monomeric PrPc, the oligomer has intense amide peak signals in the N-terminal (residues 90–114) and C-terminal regions (residues 226–231). Furthermore, a core region with decreased mobility is revealed from residues ~127 to 225. Within this core oligomer region with decreased mobility, there is a pocket of increased amide peak signal corresponding to the middle of α-helix 2 and the loop between α-helices 2 and 3 in the PrPc monomer structure. Using high-resolution solution-state NMR, this work reveals detailed and divergent residue-specific changes in soluble oligomeric models of PrP.

Mutations Alter RNA-Mediated Conversion of Human Prions.

Prion diseases are connected with self-replication and self-propagation of misfolded proteins. The rate-limiting factor is the formation of the initial seed. We have recently studied the early stages in the conversion between functional PrPC and the infectious scrapie PrPSC form, triggered by the binding of RNA. Here, we study how this process is modulated by the prion sequence. We focus on residues 129 and 178, which are connected to the hereditary neurodegenerative disease fatal familial insomnia.

Differential effects of divalent cations on elk prion protein fibril formation and stability

ABSTRACTMisfolding of the normally folded prion protein of mammals (PrPC) into infectious fibrils causes a variety of diseases, from scrapie in sheep to chronic wasting disease (CWD) in cervids. The misfolded form of PrPC, termed PrPSc, or in this case PrPCWD, interacts with PrPC to create more PrPCWD. This process is not clearly defined but is affected by the presence and interactions of biotic and abiotic cofactors. These include nucleic acids, lipids, glycosylation, pH, and ionic character. PrPC has been shown to act as a copper-binding protein in vivo, though it also binds to other divalents as well. The significance of this action has not been conclusively elucidated. Previous reports have shown that metal binding sites occur throughout the N-terminal region of PrPC. Other cations like manganese have also been shown to affect PrPC abundance in a transcript-independent fashion. Here, we examined the ability of different divalent cations to influence the stability and in vitro conversion of two variants of PrP from elk (L/M132, 26–234). We find that copper and zinc de-stabilize PrP. We also find that PrP M132 exhibits a greater degree of divalent cation induced destabilization than L132. This supports findings that leucine at position 132 confers resistance to CWD, while M132 is susceptible. However, in vitro conversion of PrP is equally suppressed by either copper or zinc, in both L132 and M132 backgrounds. This report demonstrates the complex importance of ionic character on the PrPC folding pathway selection on the route to PrPSc formation.

Flow Cytometric Detection of PrPSc in Neurons and Glial Cells from Prion-Infected Mouse Brains.

In prion diseases, an abnormal isoform of prion protein (PrPSc) accumulates in neurons, astrocytes, and microglia in the brains of animals affected by prions. Detailed analyses of PrPSc-positive neurons and glial cells are required to clarify their pathophysiological roles in the disease. Here, we report a novel method for the detection of PrPSc in neurons and glial cells from the brains of prion-infected mice by flow cytometry using PrPSc-specific staining with monoclonal antibody (MAb) 132. The combination of PrPSc staining and immunolabeling of neural cell markers clearly distinguished neurons, astrocytes, and microglia that were positive for PrPSc from those that were PrPSc negative. The flow cytometric analysis of PrPSc revealed the appearance of PrPSc-positive neurons, astrocytes, and microglia at 60 days after intracerebral prion inoculation, suggesting the presence of PrPSc in the glial cells, as well as in neurons, from an early stage of infection. Moreover, the kinetic analysis of PrPSc revealed a continuous increase in the proportion of PrPSc-positive cells for all cell types with disease progression. Finally, we applied this method to isolate neurons, astrocytes, and microglia positive for PrPSc from a prion-infected mouse brain by florescence-activated cell sorting. The method described here enables comprehensive analyses specific to PrPSc-positive neurons, astrocytes, and microglia that will contribute to the understanding of the pathophysiological roles of neurons and glial cells in PrPSc-associated pathogenesis.IMPORTANCE Although formation of PrPSc in neurons is associated closely with neurodegeneration in prion diseases, the mechanism of neurodegeneration is not understood completely. On the other hand, recent studies proposed the important roles of glial cells in PrPSc-associated pathogenesis, such as the intracerebral spread of PrPSc and clearance of PrPSc from the brain. Despite the great need for detailed analyses of PrPSc-positive neurons and glial cells, methods available for cell type-specific analysis of PrPSc have been limited thus far to microscopic observations. Here, we have established a novel high-throughput method for flow cytometric detection of PrPSc in cells with more accurate quantitative performance. By applying this method, we succeeded in isolating PrPSc-positive cells from the prion-infected mouse brains via fluorescence-activated cell sorting. This allows us to perform further detailed analysis specific to PrPSc-positive neurons and glial cells for the clarification of pathological changes in neurons and pathophysiological roles of glial cells.

The compound (3-{5-[(2,5-dimethoxyphenyl)amino]-1,3,4-thiadiazolidin-2-yl}-5,8-methoxy-2H-chromen-2-one) inhibits the prion protein conversion from PrPC to PrPSc with lower IC50 in ScN2a cells

Prion diseases are fatal neurodegenerative disorders of the central nervous system characterized by the accumulation of a protease resistant form (PrPSc) of the cellular prion protein (PrPC) in the brain. Two types of cellular prion (PrPC) compounds have been identified that appear to affect prion conversion are known as Effective Binders (EBs) and Accelerators (ACCs). Effective binders shift the balance in favour of PrPC, whereas Accelerators favour the formation of PrPSc. Molecular docking indicates EBs and ACCs both bind to pocket-D of the SHaPrPC molecule. However, EBs and ACCs may have opposing effects on the stability of the salt bridge between Arg156 and Glu196/Glu200. Computational docking data indicate that the hydrophobic benzamide group of the EB, GFP23 and the 1-(3,3-dimethylcyclohexylidene)piperidinium group of the ACC, GFP22 play an important role in inhibition and conversion from SHaPrPC to SHaPrPSc, respectively. Experimentally, NMR confirmed the amide chemical shift perturbations observed upon the binding of GFP23 to pocket-D of SHaPrPC. Consistent with its role as an ACC, titration of GFP22 resulted in widespread chemical shift changes and signal intensity loss due to protein unfolding. Virtual screening of a ligand database using the molecular scaffold developed from the set of EBs identified six of our compounds (previously studied using fluorescence quenching) as being among the top 100 best binders. Among them, compounds 5 and 6 were found to be particularly potent in decreasing the accumulation SHaPrPSc in ScN2a cells with an IC50 of ∼35µM and 20µM.

Biochemical Analysis of Interaction between Kringle Domains of Plasminogen and Prion Proteins with Q167R Mutation.

The conformational change of cellular prion protein (PrPC) to its misfolded counterpart, termed PrPSc, is mediated by a hypothesized cellular cofactor. This cofactor is believed to interact directly with certain amino acid residues of PrPC. When these are mutated into cationic amino acid residues, PrPSc formation and prion replication halt in a dominant negative (DN) manner, presumably due to strong binding of the cofactor to mutated PrPC, designated as DN PrP mutants. Previous studies demonstrated that plasminogen and its kringle domains bind to PrP and accelerate PrPSc generation. In this study, in vitro binding analysis of kringle domains of plasminogen to Q167R DN mutant PrP (PrPQ167R) was performed in parallel with the wild type (WT) and Q218K DN mutant PrP (PrPQ218K). The binding affinity of PrPQ167R was higher than that of WT PrP, but lower than that of PrPQ218K. Scatchard analysis further indicated that, like PrPQ218K and WT PrP, PrPQ167R interaction with plasminogen occurred at multiple sites, suggesting cooperativity in this interaction. Competitive binding analysis using L-lysine or L-arginine confirmed the increase of the specificity and binding affinity of the interaction as PrP acquired DN mutations. Circular dichroism spectroscopy demonstrated that the recombinant PrPs used in this study retained the α-helix-rich structure. The α-helix unfolding study revealed similar conformational stability for WT and DN-mutated PrPs. This study provides an additional piece of biochemical evidence concerning the interaction of plasminogen with DN mutant PrPs.

PrPSc formation and clearance as determinants of prion tropism.

Prion strains are characterized by strain-specific differences in neuropathology but can also differ in incubation period, clinical disease, host-range and tissue tropism. The hyper (HY) and drowsy (DY) strains of hamster-adapted transmissible mink encephalopathy (TME) differ in tissue tropism and susceptibility to infection by extraneural routes of infection. Notably, DY TME is not detected in the secondary lymphoreticular system (LRS) tissues of infected hosts regardless of the route of inoculation. We found that similar to the lymphotropic strain HY TME, DY TME crosses mucosal epithelia, enters draining lymphatic vessels in underlying laminae propriae, and is transported to LRS tissues. Since DY TME causes disease once it enters the peripheral nervous system, the restriction in DY TME pathogenesis is due to its inability to establish infection in LRS tissues, not a failure of transport. To determine if LRS tissues can support DY TME formation, we performed protein misfolding cyclic amplification using DY PrPSc as the seed and spleen homogenate as the source of PrPC. We found that the spleen environment can support DY PrPSc formation, although at lower rates compared to lymphotropic strains, suggesting that the failure of DY TME to establish infection in the spleen is not due to the absence of a strain-specific conversion cofactor. Finally, we provide evidence that DY PrPSc is more susceptible to degradation when compared to PrPSc from other lymphotrophic strains. We hypothesize that the relative rates of PrPSc formation and clearance can influence prion tropism.

Novel strain properties distinguishing sporadic prion diseases sharing prion protein genotype and prion type

In most human sporadic prion diseases the phenotype is consistently associated with specific pairings of the genotype at codon 129 of the prion protein gene and conformational properties of the scrapie PrP (PrPSc) grossly identified types 1 and 2. This association suggests that the 129 genotype favours the selection of a distinct strain that in turn determines the phenotype. However, this mechanism cannot play a role in the phenotype determination of sporadic fatal insomnia (sFI) and a subtype of sporadic Creutzfeldt-Jakob disease (sCJD) identified as sCJDMM2, which share 129 MM genotype and PrPSc type 2 but are associated with quite distinct phenotypes. Our detailed comparative study of the PrPSc conformers has revealed major differences between the two diseases, which preferentially involve the PrPSc component that is sensitive to digestion with proteases (senPrPSc) and to a lesser extent the resistant component (resPrPSc). We conclude that these variations are consistent with two distinct strains in sFI and sCJDMM2, and that the rarer sFI is the result of a variant strain selection pathway that might be favoured by a different brain site of initial PrPSc formation in the two diseases.

Mechanism of Unfolding of Human Prion Protein.

Misfolding and aggregation of prion proteins are associated with several neurodegenerative diseases. Therefore, understanding the mechanism of the misfolding process is of enormous interest in the scientific community. It has been speculated and widely discussed that the native cellular prion protein (PrPC) form needs to undergo substantial unfolding to a more stable PrPC* state, which may further oligomerize into the toxic scrapie (PrPSc) form. Here, we have studied the mechanism of the unfolding of the human prion protein (huPrP) using a set of extensive well-tempered metadynamics simulations. Through multiple microsecond-long metadynamics simulations, we find several possible unfolding pathways. We show that each pathway leads to an unfolded state of lower free energy than the native state. Thus, our study may point to the signature of a PrPC* form that corresponds to a global minimum on the conformational free-energy landscape. Moreover, we find that these global minima states do not involve an increased β-sheet content, as was assumed to be a signature of PrPSc formation in previous simulation studies. We have further analyzed the origin of metastability of the PrPC form through free-energy surfaces of the chopped helical segments to show that the helices, particularly H2 and H3 of the prion protein, have the tendency to form either a random coil or a β-structure. Therefore, the secondary structural elements of the prion protein are only weakly stabilized by tertiary contacts and solvation forces so that relatively weak perturbations induced by temperature, pressure, pH, and so forth can lead to substantial unfolding with characteristics of intrinsically disordered proteins.

Quiescin-sulfhydryl oxidase inhibits prion formation in vitro.

Prions are infectious proteins that cause a group of fatal transmissible diseases in animals and humans. The scrapie isoform (PrPSc) of the cellular prion protein (PrPC) is the only known component of the prion. Several lines of evidence have suggested that the formation and molecular features of PrPSc are associated with an abnormal unfolding/refolding process. Quiescin-sulfhydryl oxidase (QSOX) plays a role in protein folding by introducing disulfides into unfolded reduced proteins. Here we report that QSOX inhibits human prion propagation in protein misfolding cyclic amplification reactions and murine prion propagation in scrapie-infected neuroblastoma cells. Moreover, QSOX preferentially binds PrPSc from prion-infected human or animal brains, but not PrPC from uninfected brains. Surface plasmon resonance of the recombinant mouse PrP (moPrP) demonstrates that the affinity of QSOX for monomer is significantly lower than that for octamer (312 nM vs 1.7 nM). QSOX exhibits much lower affinity for N-terminally truncated moPrP (PrP89-230) than for the full-length moPrP (PrP23-231) (312 nM vs 2 nM), suggesting that the N-terminal region of PrP is critical for the interaction of PrP with QSOX. Our study indicates that QSOX may play a role in prion formation, which may open new therapeutic avenues for treating prion diseases.

Cytotoxicity of prion protein-derived cell-penetrating peptides is modulated by pH but independent of amyloid formation

Prion diseases are associated with conversion of cellular prion protein (PrPC) into an abnormally folded and infectious scrapie isoform (PrPSc). We previously showed that peptides derived from the unprocessed N-termini of mouse and bovine prion proteins, mPrP1-28 and bPrP1-30, function as cell-penetrating peptides (CPPs), and destabilize model membrane systems, which could explain the infectivity and toxicity of prion diseases. However, subsequent studies revealed that treatment with mPrP1-28 or bPrP1-30 significantly reduce PrPSc levels in prion-infected cells. To explain these seemingly contradictory results, we correlated the aggregation, membrane perturbation and cytotoxicity of the peptides with their cellular uptake and intracellular localization. Although the peptides have a similar primary sequence, mPrP1-28 is amyloidogenic, whereas bPrP1-30 forms smaller oligomeric or non-fibrillar aggregates. Surprisingly, bPrP1-30 induces much higher cytotoxicity than mPrP1-28, indicating that amyloid formation and toxicity are independent. The toxicity is correlated with prolonged residence at the plasma membrane and membrane perturbation. Both ordered aggregation and toxicity of the peptides are inhibited by low pH. Under non-toxic conditions, the peptides are internalized by lipid-raft dependent macropinocytosis and localize to acidic lysosomal compartments. Our results shed light on the antiprion mechanism of the prion protein-derived CPPs and identify a potential site for PrPSc formation.

Role of polysaccharide and lipid in lipopolysaccharide induced prion protein conversion

ABSTRACTConversion of native cellular prion protein (PrPc) from an α-helical structure to a toxic and infectious β-sheet structure (PrPSc) is a critical step in the development of prion disease. There are some indications that the formation of PrPSc is preceded by a β-sheet rich PrP (PrPβ) form which is non-infectious, but is an intermediate in the formation of infectious PrPSc. Furthermore the presence of lipid cofactors is thought to be critical in the formation of both intermediate-PrPβ and lethal, infectious PrPSc. We previously discovered that the endotoxin, lipopolysaccharide (LPS), interacts with recombinant PrPc and induces rapid conformational change to a β-sheet rich structure. This LPS induced PrPβ structure exhibits PrPSc-like features including proteinase K (PK) resistance and the capacity to form large oligomers and rod-like fibrils. LPS is a large, complex molecule with lipid, polysaccharide, 2-keto-3-deoxyoctonate (Kdo) and glucosamine components. To learn more about which LPS chemical constituents are critical for binding PrPc and inducing β-sheet conversion we systematically investigated which chemical components of LPS either bind or induce PrP conversion to PrPβ. We analyzed this PrP conversion using resolution enhanced native acidic gel electrophoresis (RENAGE), tryptophan fluorescence, circular dichroism, electron microscopy and PK resistance. Our results indicate that a minimal version of LPS (called detoxified and partially de-acylated LPS or dLPS) containing a portion of the polysaccharide and a portion of the lipid component is sufficient for PrP conversion. Lipid components, alone, and saccharide components, alone, are insufficient for conversion.

Glycoform-independent prion conversion by highly efficient, cell-based, protein misfolding cyclic amplification.

Prions are formed of misfolded assemblies (PrPSc) of the variably N-glycosylated cellular prion protein (PrPC). In infected species, prions replicate by seeding the conversion and polymerization of host PrPC. Distinct prion strains can be recognized, exhibiting defined PrPSc biochemical properties such as the glycotype and specific biological traits. While strain information is encoded within the conformation of PrPSc assemblies, the storage of the structural information and the molecular requirements for self-perpetuation remain uncertain. Here, we investigated the specific role of PrPC glycosylation status. First, we developed an efficient protein misfolding cyclic amplification method using cells expressing the PrPC species of interest as substrate. Applying the technique to PrPC glycosylation mutants expressing cells revealed that neither PrPC nor PrPSc glycoform stoichiometry was instrumental to PrPSc formation and strainness perpetuation. Our study supports the view that strain properties, including PrPSc glycotype are enciphered within PrPSc structural backbone, not in the attached glycans.