Scrapie Isoform Research Articles

Bacteriophage-encoded chaperonins stimulate prion protein fibrillation in an ATP-dependent manner

The pathogenesis of the various prion diseases is based on the conformational conversion of the prion protein from its physiological cellular form to the insoluble scrapie isoform. Several chaperones, including the Hsp60 family of group I chaperonins, are known to contribute to this transformation, but data on their effects are scarce and conflicting. In this work, two GroEL-like phage chaperonins, the single-ring OBP and the double-ring EL, were found to stimulate monomeric prion protein fibrillation in an ATP-dependent manner. The resulting fibrils were characterised by thioflavin T fluorescence, electron microscopy, proteinase K digestion assay and other methods. In the presence of ATP, chaperonins were found to promote the conversion of prion protein monomers into short amyloid fibrils with their further aggregation into less toxic large clusters. Fibrils generated with the assistance of phage chaperonins differ in morphology and properties from those formed spontaneously from monomeric prion in the presence of denaturants at acidic pH.

Analysis of a large case series of fatal familial insomnia to determine tests with the highest diagnostic value.

Fatal familial insomnia (FFI) is a rare prionopathy with unusually high incidence in the Basque Country. We report detailed data on clinical, diagnostic, histopathological, and biochemical characteristics of a recent FFI case series. The Basque Brain Bank database was screened for patients diagnosed from 2010 to 2021 with standard genetic and/or neuropathological criteria. This series includes 16 patients, 25% without family history, with 12 cases from 9 unrelated (but geographically-linked, Basque country) kindreds, onset ranging from 36 to 70 years, and disease course from 7 to 11.5 months. Insomnia was the initial symptom in most cases, with consistent polysomnography in 92% of the cases. In contrast, 14-3-3 and RT-QuIC from cerebrospinal fluid were negative. Most patients were homozygous for methionine. Gliosis and neuronal loss in basal ganglia and thalamus were the main histopathological findings; Western blotting identified preferentially the protease-resistant prion protein (PrPres) type 2, although detection of the scrapie isoform of the prion protein (PrPSc) identified using brain tissue RT-QuIC was more successful. This is one of the largest current studies on FFI patients performed to provide improvements in diagnostic reliability. Among the analyzed tests, polysomnography and the genetic study show the highest diagnostic value in FFI.

Bifunctional carbazole derivatives for simultaneous therapy and fluorescence imaging in prion disease murine cell models

Prion diseases are characterized by the self-assembly of pathogenic misfolded scrapie isoforms (PrPSc) of the cellular prion protein (PrPC). In an effort to achieve a theranostic profile, symmetrical bifunctional carbazole derivatives were designed as fluorescent rigid analogues of GN8, a pharmacological chaperone that stabilizes the native PrPC conformation and prevents its pathogenic conversion. A focused library was synthesized via a four-step route, and a representative member was confirmed to have native fluorescence, including a band in the near-infrared region. After a cytotoxicity study, compounds were tested on the RML-infected ScGT1 neuronal cell line, by monitoring the levels of protease-resistant PrPSc. Small dialkylamino groups at the ends of the molecule were found to be optimal in terms of therapeutic index, and the bis-(dimethylaminoacetamido)carbazole derivative 2b was selected for further characterization. It showed activity in two cell lines infected with the mouse-adapted RML strain (ScGT1 and ScN2a). Unlike GN8, 2b did not affect PrPC levels, which represents a potential advantage in terms of toxicity. Amyloid Seeding Assay (ASA) experiments showed the capacity of 2b to delay the aggregation of recombinant mouse PrP. Its ability to interfere with the amplification of the scrapie RML strain by Protein Misfolding Cyclic Amplification (PMCA) was shown to be higher than that of GN8, although 2b did not inhibit the amplification of human vCJD prion. Fluorescent staining of PrPSc aggregates by 2b was confirmed in living cells. 2b emerges as an initial hit compound for further medicinal chemistry optimization towards strain-independent anti-prion compounds.

Fibrinogen Mitigates Prion-Mediated Platelet Activation and Neuronal Cell Toxicity.

Prion peptide (PrP) misfolds to infectious scrapie isoform, the β pleat-rich insoluble fibrils responsible for neurodegeneration and fatal conformational diseases in humans. The amino acid sequence 106–126 from prion proteins, PrP(106–126), is highly amyloidogenic and implicated in prion-induced pathologies. Here, we report a novel interaction between PrP(106–126) and the thrombogenic plasma protein fibrinogen that can lead to mitigation of prion-mediated pro-thrombotic responses in human platelets as well as significant decline in neuronal toxicity. Thus, prior exposure to fibrinogen-restrained PrP-induced rise in cytosolic calcium, calpain activation, and shedding of extracellular vesicles in platelets while it, too, averted cytotoxicity of neuronal cells triggered by prion peptide. Interestingly, PrP was found to accelerate fibrin-rich clot formation, which was resistant to plasmin-mediated fibrinolysis, consistent with enhanced thrombus stability provoked by PrP. We propose that PrP-fibrinogen interaction can be clinically exploited further for prevention and management of infectious prion related disorders. Small molecules or peptides mimicking PrP-binding sites on fibrinogen can potentially mitigate PrP-induced cellular toxicity while also preventing the negative impact of PrP on fibrin clot formation and lysis.

Therapeutic development of polymers for prion disease.

Prion diseases, also known as transmissible spongiform encephalopathies, are caused by the accumulation of abnormal isoforms of the prion protein (scrapie isoform of the prion protein, PrPSc) in the central nervous system. Many compounds with anti-prion activities have been found using in silico screening, in vitro models, persistently prion-infected cell models, and prion-infected rodent models. Some of these compounds include several types of polymers. Although the inhibition or removal of PrPSc production is the main target of therapy, the unique features of prions, namely protein aggregation and assembly accompanied by steric structural transformation, may require different strategies for the development of anti-prion drugs than those for conventional therapeutics targeting enzyme inhibition, agonist ligands, or modulation of signaling. In this paper, we first overview the history of the application of polymers to prion disease research. Next, we describe the characteristics of each type of polymer with anti-prion activity. Finally, we discuss the common features of these polymers. Although drug delivery of these polymers to the brain is a challenge, they are useful not only as leads for therapeutic drugs but also as tools to explore the structure of PrPSc and are indispensable for prion disease research.

Zn(II) binding causes interdomain changes in the structure and flexibility of the human prion protein

The cellular prion protein (PrPC) is a mainly α-helical 208-residue protein located in the pre- and postsynaptic membranes. For unknown reasons, PrPC can undergo a structural transition into a toxic, β-sheet rich scrapie isoform (PrPSc) that is responsible for transmissible spongiform encephalopathies (TSEs). Metal ions seem to play an important role in the structural conversion. PrPC binds Zn(II) ions and may be involved in metal ion transport and zinc homeostasis. Here, we use multiple biophysical techniques including optical and NMR spectroscopy, molecular dynamics simulations, and small angle X-ray scattering to characterize interactions between human PrPC and Zn(II) ions. Binding of a single Zn(II) ion to the PrPC N-terminal domain via four His residues from the octarepeat region induces a structural transition in the C-terminal α-helices 2 and 3, promotes interaction between the N-terminal and C-terminal domains, reduces the folded protein size, and modifies the internal structural dynamics. As our results suggest that PrPC can bind Zn(II) under physiological conditions, these effects could be important for the physiological function of PrPC.

Biosemiotics comprehension of PrP code and prion disease

Prions or PrPSc (prion protein, Scrapie isoform) are proteins with an aberrant three-dimensional conformation that present the ability to alter the three-dimensional structure of natively folded PrPC (prion protein, cellular isoform) inducing its abnormal folding, giving raise to neurological diseases known as Transmissible spongiforms encephalopathies (TSEs) or prion diseases. In this work, through a biosemiotic study, we will analyze the molecular code of meanings that are known in the molecular pathway of PrPC and how it is altered in prion diseases. This biosemiotic code presents a socio-semiotic correlate in organisms that could be unraveled with the ultimate goal of understanding the code of signs that mediates the process. Finally, we will study recent works that indicate possible relationships in the code between prion proteins and other proteins such as the tau protein and alpha-synuclein to evaluate if it is possible that there is a semiotic expansion of the PrP code and prion diseases in the meaning recently expounded by Prusiner, winner of the Nobel Prize for describing these unusual pathological processes.

Decrease in Skin Prion-Seeding Activity of Prion-Infected Mice Treated with a Compound Against Human and Animal Prions: a First Possible Biomarker for Prion Therapeutics

Previous studies have revealed that the infectious scrapie isoform of prion protein (PrPSc) harbored in the skin tissue of patients or animals with prion diseases can be amplified and detected through the serial protein misfolding cyclic amplification (sPMCA) or real-time quaking-induced conversion (RT-QuIC) assays. These findings suggest that skin PrPSc-seeding activity may serve as a biomarker for the diagnosis of prion diseases; however, its utility as a biomarker for prion therapeutics remains largely unknown. Cellulose ethers (CEs, such as TC-5RW), widely used as food and pharmaceutical additives, have recently been shown to prolong the lifespan of prion-infected mice and hamsters. Here we report that in transgenic (Tg) mice expressing hamster cellular prion protein (PrPC) infected with the 263K prion, the prion-seeding activity becomes undetectable in the skin tissues of TC-5RW-treated Tg mice by both sPMCA and RT-QuIC assays, whereas such prion-seeding activity is readily detectable in the skin of untreated mice. Notably, TC-5RW exhibits an inhibitory effect on the in vitro amplification of PrPSc in both skin and brain tissues by sPMCA and RT-QuIC. Moreover, we reveal that TC-5RW is able to directly decrease protease-resistant PrPSc and inhibit the seeding activity of PrPSc from chronic wasting disease and various human prion diseases. Our results suggest that the level of prion-seeding activity in the skin may serve as a useful biomarker for assessing the therapeutic efficacy of compounds in a clinical trial of prion diseases and that TC-5RW may have the potential for the prevention/treatment of human prion diseases.

A rare case of sporadic Creutzfeldt-Jakob disease in an 83 years old female

Sporadic Creutzfeldt-Jakob disease is a rare invariably fatal neurodegenerative prion disease. Prion disease are associated with the conversion of alpha-helix rich cellular prion protein (PrPC) into a beta-structure rich insoluble conformer scrapie isoform (PrPSc) thought to be infectious isoform. Here we present a case of an 83 years old woman with findings of rapidly progressive dementia, cognitive disturbance, myoclonic jerks and extrapyramidal signs (cogwheel rigidity). Following a series of clinical and diagnostic (diffusion-weighted magnetic resonance imaging (MRI) brain, electroencephalogram (EEG)) examination she was diagnosed with sporadic Creutzfeldt-Jakob disease based on Centers for disease control and prevention (CDC) criteria.

Posttranslational modifications define course of prion strain adaptation and disease phenotype.

Posttranslational modifications are a common feature of proteins associated with neurodegenerative diseases including prion protein (PrPC), tau, and α-synuclein. Alternative self-propagating protein states or strains give rise to different disease phenotypes and display strain-specific subsets of posttranslational modifications. The relationships between strain-specific structure, posttranslational modifications, and disease phenotype are poorly understood. We previously reported that among hundreds of PrPC sialoglycoforms expressed by a cell, individual prion strains recruited PrPC molecules selectively, according to the sialylation status of their N-linked glycans. Here we report that transmission of a prion strain to a new host is accompanied by a dramatic shift in the selectivity of recruitment of PrPC sialoglycoforms, giving rise to a self-propagating scrapie isoform (PrPSc) with a unique sialoglycoform signature and disease phenotype. The newly emerged strain has the shortest incubation time to disease and is characterized by colocalization of PrPSc with microglia and a very profound proinflammatory response, features that are linked to a unique sialoglycoform composition of PrPSc. The current work provides experimental support for the hypothesis that strain-specific patterns of PrPSc sialoglycoforms formed as a result of selective recruitment dictate strain-specific disease phenotypes. This work suggests a causative relationship between a strain-specific structure, posttranslational modifications, and disease phenotype.

BMD42-2910, a Novel Benzoxazole Derivative, Shows a Potent Anti-prion Activity and Prolongs the Mean Survival in an Animal Model of Prion Disease.

Prion diseases are a group of neurodegenerative and fatal central nervous system disorders. The pathogenic mechanism involves the conversion of cellular prion protein (PrPC) to an altered scrapie isoform (PrPSc), which accumulates in amyloid deposits in the brain. However, no therapeutic drugs have demonstrated efficacy in clinical trials. We previously reported that BMD42-29, a synthetic compound discovered in silico, is a novel anti-prion compound that inhibits the conversion of PrPC to protease K (PK)-resistant PrPSc fragments (PrPres). In the present study, 14 derivatives of BMD42-29 were obtained from BMD42-29 by modifying in the side chain by in silico feedback, with the aim to determine whether they improve anti-prion activity. These derivatives were assessed in a PrPSc-infected cell model and some derivatives were further tested using real time-quaking induced conversion (RT-QuIC). Among them, BMD42-2910 showed high anti-prion activity at low concentrations in vitro and also no toxic effects in a mouse model. Interestingly, abundant PrPres was reduced in brains of mice infected with prion strain when treated with BMD42-2910, and the mice survived longer than control mice and even that treated with BMD42-29. Finally, high binding affinity was predicted in the virtual binding sites (Asn159, Gln 160, Lys194, and Glu196) when PrPC was combined with BMD-42-2910. Our findings showed that BMD42-2910 sufficiently reduces PrPres generation in vitro and in vivo and may be a promising novel anti-prion compound.

Structural Mechanism of Barriers to Interspecies Seeding Transmissibility of Full-Length Prion Protein Amyloid.

A puzzling feature of prion diseases is the cross-species barriers. The detailed molecular mechanisms underlying these interspecies barriers remain poorly understood because of a lack of high-resolution structural information on the scrapie isoform of the prion protein (PrPSc ). In this study we identified the critical role of the residues 165/167 in the barrier to seeding mouse PrP (mPrP) fibril seeds to human cellular prion protein (PrPC ). Solid-state NMR revealed a C-terminal β-sheet core spanning residues 165-230 and the packing arrangement of mPrP fibrils. Residues 165/167 are located on one end of the fibril core. Molecular dynamics simulations demonstrated that the stabilities of the seeding-induced β-strand structures are significantly impacted by hydrogen bonds involving the side chain of residue 167 and steric resistance involving residue 165. These findings suggest that the α2-β2 loop containing residues 165/167 could be the initial site of seed-template conformational conversion.

Full atomistic model of prion structure and conversion.

Prions are unusual protein assemblies that propagate their conformationally-encoded information in absence of nucleic acids. The first prion identified, the scrapie isoform (PrPSc) of the cellular prion protein (PrPC), caused epidemic and epizootic episodes [1]. Most aggregates of other misfolding-prone proteins are amyloids, often arranged in a Parallel-In-Register-β-Sheet (PIRIBS) [2] or β-solenoid conformations [3]. Similar folding models have also been proposed for PrPSc, although none of these have been confirmed experimentally. Recent cryo-electron microscopy (cryo-EM) and X-ray fiber-diffraction studies provided evidence that PrPSc is structured as a 4-rung β-solenoid (4RβS) [4, 5]. Here, we combined different experimental data and computational techniques to build the first physically-plausible, atomic resolution model of mouse PrPSc, based on the 4RβS architecture. The stability of this new PrPSc model, as assessed by Molecular Dynamics (MD) simulations, was found to be comparable to that of the prion forming domain of Het-s, a naturally-occurring β-solenoid. Importantly, the 4RβS arrangement allowed the first simulation of the sequence of events underlying PrPC conversion into PrPSc. This study provides the most updated, experimentally-driven and physically-coherent model of PrPSc, together with an unprecedented reconstruction of the mechanism underlying the self-catalytic propagation of prions.

Epigenetic Control of the Notch and Eph Signaling Pathways by the Prion Protein: Implications for Prion Diseases.

Among the ever-growing number of self-replicating proteins involved in neurodegenerative diseases, the prion protein PrP remains the most infamous for its central role in transmissible spongiform encephalopathies (TSEs). In these diseases, pathogenic prions propagate through a seeding mechanism, where normal PrPC molecules are converted into abnormally folded scrapie isoforms termed PrPSc. Since its discovery over 30years ago, much advance has contributed to define the host-encoded cellular prion protein PrPC as a critical relay of prion-induced neuronal cell demise. A current consensual view is that the conversion of PrPC into PrPSc in neuronal cells diverts the former from its normal function with subsequent molecular alterations affecting synaptic plasticity. Here, we report that prion infection is associated with reduced expression of key effectors of the Notch pathway in vitro and in vivo, recapitulating changes fostered by the absence of PrPC. We further show that both prion infection and PrPC depletion promote drastic alterations in the expression of a defined set of Eph receptors and their ephrin ligands, which represent important players in synaptic function. Our data indicate that defects in the Notch and Eph axes can be mitigated in response to histone deacetylase inhibition in PrPC-depleted as well as prion-infected cells. We thus conclude that infectious prions cause a loss-of-function phenotype with respect to Notch and Eph signaling and that these alterations are sustained by epigenetic mechanisms.

Dimerization of the cellular prion protein inhibits propagation of scrapie prions

A central step in the pathogenesis of prion diseases is the conformational transition of the cellular prion protein (PrPC) into the scrapie isoform, denoted PrPSc Studies in transgenic mice have indicated that this conversion requires a direct interaction between PrPC and PrPSc; however, insights into the underlying mechanisms are still missing. Interestingly, only a subfraction of PrPC is converted in scrapie-infected cells, suggesting that not all PrPC species are suitable substrates for the conversion. On the basis of the observation that PrPC can form homodimers under physiological conditions with the internal hydrophobic domain (HD) serving as a putative dimerization domain, we wondered whether PrP dimerization is involved in the formation of neurotoxic and/or infectious PrP conformers. Here, we analyzed the possible impact on dimerization of pathogenic mutations in the HD that induce a spontaneous neurodegenerative disease in transgenic mice. Similarly to wildtype (WT) PrPC, the neurotoxic variant PrP(AV3) formed homodimers as well as heterodimers with WTPrPC Notably, forced PrP dimerization via an intermolecular disulfide bond did not interfere with its maturation and intracellular trafficking. Covalently linked PrP dimers were complex glycosylated, GPI-anchored, and sorted to the outer leaflet of the plasma membrane. However, forced PrPC dimerization completely blocked its conversion into PrPSc in chronically scrapie-infected mouse neuroblastoma cells. Moreover, PrPC dimers had a dominant-negative inhibition effect on the conversion of monomeric PrPC Our findings suggest that PrPC monomers are the major substrates for PrPSc propagation and that it may be possible to halt prion formation by stabilizing PrPC dimers.

The Structure of PrPSc Prions.

PrPSc (scrapie isoform of the prion protein) prions are the infectious agent behind diseases such as Creutzfeldt–Jakob disease in humans, bovine spongiform encephalopathy in cattle, chronic wasting disease in cervids (deer, elk, moose, and reindeer), as well as goat and sheep scrapie. PrPSc is an alternatively folded variant of the cellular prion protein, PrPC, which is a regular, GPI-anchored protein that is present on the cell surface of neurons and other cell types. While the structure of PrPC is well studied, the structure of PrPSc resisted high-resolution determination due to its general insolubility and propensity to aggregate. Cryo-electron microscopy, X-ray fiber diffraction, and a variety of other approaches defined the structure of PrPSc as a four-rung β-solenoid. A high-resolution structure of PrPSc still remains to be solved, but the four-rung β-solenoid architecture provides a molecular framework for the autocatalytic propagation mechanism that gives rise to the alternative conformation of PrPSc. Here, we summarize the current knowledge regarding the structure of PrPSc and speculate about the molecular conversion mechanisms that leads from PrPC to PrPSc.

A Sequence-Dependent DNA Condensation Induced by Prion Protein.

Different studies indicated that the prion protein induces hybridization of complementary DNA strands. Cell culture studies showed that the scrapie isoform of prion protein remained bound with the chromosome. In present work, we used an oxazole dye, YOYO, as a reporter to quantitative characterization of the DNA condensation by prion protein. We observe that the prion protein induces greater fluorescence quenching of YOYO intercalated in DNA containing only GC bases compared to the DNA containing four bases whereas the effect of dye bound to DNA containing only AT bases is marginal. DNA-condensing biological polyamines are less effective than prion protein in quenching of DNA-bound YOYO fluorescence. The prion protein induces marginal quenching of fluorescence of the dye bound to oligonucleotides, which are resistant to condensation. The ultrastructural studies with electron microscope also validate the biophysical data. The GC bases of the target DNA are probably responsible for increased condensation in the presence of prion protein. To our knowledge, this is the first report of a human cellular protein inducing a sequence-dependent DNA condensation. The increased condensation of GC-rich DNA by prion protein may suggest a biological function of the prion protein and a role in its pathogenesis.

Role of Prion protein in premature senescence of human fibroblasts

Prion protein (PrP) is essentially known for its capacity to induce neurodegenerative prion diseases in mammals caused by a conformational change in its normal cellular isoform (PrPC) into an infectious and disease-associated misfolded form, called scrapie isoform (PrPSc). Although its sequence is highly conserved, less information is available on its physiological role under normal conditions. However, increasing evidence supports a role for PrPC in the cellular response to oxidative stress. In the present study, a new link between PrP and senescence is highlighted. The role of PrP in premature senescence induced by copper was investigated. WI-38 human fibroblasts were incubated with copper sulfate (CuSO4) to trigger premature senescence. This induced an increase of PrP mRNA level, an increase of protein abundance of the normal form of PrP and a nuclear localization of the protein. Knockdown of PrP expression using specific small interfering RNA (siRNA) gave rise to appearance of several biomarkers of senescence as a senescent morphology, an increase of senescence associated β-galactosidase activity and a decrease of the cellular proliferative potential. Overall these data suggest that PrP protects cells against premature senescence induced by copper.

Designed Cell-Penetrating Peptides for Inhibition of Amyloid-Beta (Aβ) Amyloid Formation and Cytotoxicity

Several neurodegenerative diseases are characterized by self-assembly of proteins or peptides into aggregates termed amyloid, which share various physicochemical features: a fibrillar morphology, a predominantly β-sheet secondary structure, insolubility in common solvents and detergents, and protease resistance. These so-called amyloid diseases include Alzheimer's disease (AD), Huntington's disease, Parkinson's disease and prion diseases. Despite intense research efforts over several decades, the pathophysiology and interlinkage of these diseases remains unresolved. This is mainly due to the transient nature of the different aggregation states along the amyloid formation pathway, which impedes identification of the cytotoxic species. Recently, a peptide derived from the unprocessed N-terminus of cellular prion protein (PrPC) was shown to function as a cell-penetrating peptide (CPP) and to significantly reduce levels of the disease-associated scrapie isoform (PrPSc) in prion-infected cells. This peptide comprises two segments: a hydrophobic signal sequence, to which the CPP property is attributed, followed by a polycationic nuclear localization signal (NLS)-like sequence that interacts with PrPSc. Interestingly, the anti-prion efficiency of the peptide could be enhanced by substitution of the PrPC signal sequence with a more flexible signal sequence from the neural cell adhesion molecule-1 (NCAM-1). Here, we extended the study towards amyloid-beta (Aβ) aggregation, which is associated with AD. We investigated the amyloid formation and cytotoxicity of the Aβ-inhibitor complexes using thioflavin T fluorescence assays, AFM, NMR, cell proliferation assays and fluorescence imaging methods. We report that the NCAM-1-based inhibitor stabilizes Aβ in a non-amyloid state and protects neuronal cells against Aβ-induced neurotoxicity. Moreover, we show that the replacement of the PrPC NLS-like sequence with a NLS-like segment from Aβ results in similar CPP-functionality and anti-Aβ effects. Our findings reveal a general underlying principle for the inhibition of pathogenic protein aggregation that will allow for the design of a ‘universal’ peptide-based inhibitor.

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.