Horse Prion Protein NMR Structure and Comparisons with Related Variants of the Mouse Prion Protein

The NMR structure of the horse (Equus caballus) cellular prion protein at 25 °C exhibits the typical PrP^C [cellular form of prion protein (PrP)] global architecture, but in contrast to most other mammalian PrP^Cs, it contains a well-structured loop connecting the β2 strand with the α2 helix. Comparison with designed variants of the mouse prion protein resulted in the identification of a single amino acid exchange within the loop, D167S, which correlates with the high structural order of this loop in the solution structure at 25 °C and is unique to the PrP sequences of equine species. The β2-α2 loop and the α3 helix form a protein surface epitope that has been proposed to be the recognition area for a hypothetical chaperone, “protein X,” which would promote conversion of PrP^C into the disease-related scrapie form and thus mediate intermolecular interactions related to the transmission barrier for transmissible spongiform encephalopathies (TSEs) between different species. The present results are evaluated in light of recent indications from in vivo experiments that the local β2-α2 loop structure affects the susceptibility of transgenic mice to TSEs and the fact that there are no reports on TSE in horses.     

PrPC interacts with tetraspanin-7 through bovine PrP154-182 containing alpha-helix 1

The cellular prion protein (PrP^C) is highly conserved in the evolution of mammals, and therefore, thought to have important cellular functions. Despite decades of intensive research, the physiological function of PrP^C remains enigmatic. We carried out a yeast two-hybrid screen on a bovine brain cDNA expression library and identified the transmembrane protein tetraspanin-7 (CD231), as a PrP^C interacting protein. We confirmed the interaction between PrP^C and tetraspanin-7 by yeast two-hybrid assay, immunofluorescent co-localization, and immunocoprecipitation. Our mutational studies further demonstrated that PrP^C specifically binds tetraspanin-7 through the region corresponding to bovine PrP_154-182 containing alpha-helix 1.     

Inhibition of PrPSc formation in scrapie infected N2a cells by 5,7,8-trimethyl-3,4-dihydro-2H-1,4-benzoxazine derivatives

Prion diseases are fatal, neurodegenerative diseases characterized by the structural conversion of the normal, cellular prion protein, PrP^C into an abnormally structured, aggregated and partially protease-resistant isoform, termed PrP^Sc. Although substantial research has been directed toward development of therapeutics targeting prions, there is still no curative treatment for the disease. Benzoxazines are bicyclic heterocyclic compounds possessing several pharmaceutically important properties, including neuroprotection and reactive oxygen species scavenging. In an effort to identify novel inhibitors of prion formation, several 5,7,8-trimethyl-1,4-benzoxazine derivatives were evaluated in vitro for their effectiveness on the expression levels of normal PrP^C and its conversion to the abnormal isoforms of PrP^Sc in a scrapie-infected cell culture model. The most potent compound was 2-(4-methoxyphenyl)-5,7,8-trimethyl-3,4-dihydro-2H-1,4-benzoxazine, with a diminishing effect on the formation of PrP^Sc, thus establishing a class of compounds with a promising therapeutic use against prion diseases.     

Inhibition of PrPSc formation in scrapie infected N2a cells by 5,7,8-trimethyl-3,4-dihydro-2H-1,4-benzoxazine derivatives

Prion diseases are fatal, neurodegenerative diseases characterized by the structural conversion of the normal, cellular prion protein, PrP^C into an abnormally structured, aggregated and partially protease-resistant isoform, termed PrP^Sc. Although substantial research has been directed toward development of therapeutics targeting prions, there is still no curative treatment for the disease. Benzoxazines are bicyclic heterocyclic compounds possessing several pharmaceutically important properties, including neuroprotection and reactive oxygen species scavenging. In an effort to identify novel inhibitors of prion formation, several 5,7,8-trimethyl-1,4-benzoxazine derivatives were evaluated in vitro for their effectiveness on the expression levels of normal PrP^C and its conversion to the abnormal isoforms of PrP^Sc in a scrapie-infected cell culture model. The most potent compound was 2-(4-methoxyphenyl)-5,7,8-trimethyl-3,4-dihydro-2H-1,4-benzoxazine, with a diminishing effect on the formation of PrP^Sc, thus establishing a class of compounds with a promising therapeutic use against prion diseases.     

Concentration of Disease-Associated Prion Protein with Silicon Dioxide

Reagents that can precipitate the disease-associated prion protein (PrP^Sc) are vital for the development of high sensitivity tests to detect low levels of this disease marker in biological material. Here, a range of minerals are shown to precipitate both ovine cellular prion protein (PrP^C) and ovine scrapie PrP^Sc. The precipitation of prion protein with silicon dioxide is unaffected by PrP^Sc strain or host species and the method can be used to precipitate bovine BSE. This method can reliably concentrate protease-resistant ovine PrP^Sc (PrP^res) derived from 1.69 μg of brain protein from a clinically infected animal diluted into either 50 ml of buffer or 15 ml of plasma. The introduction of a SiO₂ precipitation step into the immunological detection of PrP^res increased detection sensitivity by over 1,500-fold. Minerals such as SiO₂ are readily available, low cost reagents with generic application to the concentration of diseases-associated prion proteins.     

Prion Protein NMR Structure from Tammar Wallaby (Macropus eugenii) Shows that the β2-α2 Loop Is Modulated by Long-Range Sequence Effects

NMR structures are presented for the recombinant construct of residues 121-230 from the tammar wallaby (Macropus eugenii) prion protein (PrP) twPrP(121-230) and for the variant mouse PrPs mPrP[Y225A,Y226A](121-231) and mPrP[V166A](121-231) at 20 °C and pH 4.5. All three proteins exhibit the same global architecture as seen in other recombinant PrP^Cs (cellular isoforms of PrP) and shown to prevail in natural bovine PrP^C. Special interest was focused on a loop that connects the β2-strand with helix α2 in the PrP^C fold, since there are indications from in vivo experiments that this local structural feature affects the susceptibility of transgenic mice to transmissible spongiform encephalopathies. This β2-α2 loop and helix α3 form a solvent-accessible contiguous epitope, which has been proposed to be the recognition area for a hypothetical chaperone, the “protein X”. This hypothetical chaperone would affect the conversion of PrP^C into the disease-related scrapie form (PrP^Sc) by moderating intermolecular interactions related to the transmission barrier of transmissible spongiform encephalopathies between different species. In contrast to mPrP(121-231) and most other mammalian PrP^Cs, the β2-α2 loop is well defined at 20 °C in tammar wallaby PrP and in the two aforementioned variants of mPrP, showing that long-range interactions with helix α3 can have an overriding influence on the structural definition of the β2-α2 loop. Further NMR studies with two variant mPrPs, mPrP[Y225A](121-231) and mPrP[Y226A](121-231), showed that these interactions are dominantly mediated by close contacts between residues 166 and 225. The results of the present study then lead to the intriguing indication that well-defined long-range intramolecular interactions could act as regulators of the functional specificity of PrP^C.     

Prion Protein mPrP[F175A](121-231): Structure and Stability in Solution

The three-dimensional structures of prion proteins (PrPs) in the cellular form (PrP^C) include a stacking interaction between the aromatic rings of the residues Y169 and F175, where F175 is conserved in all but two so far analyzed mammalian PrP sequences and where Y169 is strictly conserved. To investigate the structural role of F175, we characterized the variant mouse prion protein mPrP[F175A](121-231). The NMR solution structure represents a typical PrP^C-fold, and it contains a 3₁₀-helical β2-α2 loop conformation, which is well defined because all amide group signals in this loop are observed at 20 °C. With this “rigid‐loop PrP^C” behavior, mPrP[F175A](121-231) differs from the previously studied mPrP[Y169A](121-231), which contains a type I β-turn β2-α2 loop structure. When compared to other rigid‐loop variants of mPrP(121-231), mPrP[F175A](121-231) is unique in that the thermal unfolding temperature is lowered by 8 °C. These observations enable further refined dissection of the effects of different single-residue exchanges on the PrP^C conformation and their implications for the PrP^C physiological function.     

Taking advantage of physiological proteolytic processing of the prion protein for a therapeutic perspective in prion and Alzheimer diseases

Prion and Alzheimer diseases are fatal neurodegenerative diseases caused by misfolding and aggregation of the cellular prion protein (PrP^C) and the β-amyloid peptide, respectively. Soluble oligomeric species rather than large aggregates are now believed to be neurotoxic. PrP^C undergoes three proteolytic cleavages as part of its natural life cycle, α-cleavage, β-cleavage, and ectodomain shedding. Recent evidences demonstrate that the resulting secreted PrP^C molecules might represent natural inhibitors against soluble toxic species. In this mini-review, we summarize recent observations suggesting the potential benefit of using PrP^C-derived molecules as therapeutic agents in prion and Alzheimer diseases.     

Nascent β Structure in the Elongated Hydrophobic Region of a Gerstmann–Sträussler–Scheinker PrP Allele

Prion diseases are neurodegenerative disorders caused by the misfolding of the cellular prion protein (PrP^C). Gerstmann–Sträussler–Scheinker syndrome is an inherited prion disease with one early-onset allele (HRdup) containing an eight-amino-acid insertion; this LGGLGGYV insert is positioned after valine 129 (human PrP^C sequence) in a hydrophobic tract in the natively disordered region. Here we have characterized the structure and explored the molecular motions and dynamics of HRdup PrP and a control allele. High-resolution NMR data suggest that the core of HRdup has a canonical PrP^C structure, yet a nascent β-structure is observed in the flexible elongated hydrophobic region of HRdup. In addition, using mouse PrP^C sequence, we observed that a methionine/valine polymorphism at codon 128 (equivalent of methionine/valine 129 in human sequence) and oligomerization caused by high protein concentration affects conformational exchange dynamics at residue G130. We hypothesize that with the β-structure at the N-terminus, the hydrophobic region of HRdup can adopt a fully extended configuration and fold back to form an extended β-sheet with the existing β-sheet. We propose that these structures are early chemical events in disease pathogenesis.     

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 PrP^Sc) that occur throughout the brain. Formation of synthetic or in vitro PrP^Sc 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 PrP^Sc. 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 PrP^c (spanning residues 90–231). We then obtained two-dimensional solution NMR spectra of the PrP^c monomer, a 40% converted oligomer, and a 94% converted oligomer. Heteronuclear single-quantum correlation (¹H–¹⁵N) studies revealed that, in comparison to monomeric PrP^c, 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 PrP^c monomer structure. Using high-resolution solution-state NMR, this work reveals detailed and divergent residue-specific changes in soluble oligomeric models of PrP.     

Induction of ligand-specific PrPC signaling in human neuronal cells

Cellular prion protein (PrP^C) has attracted considerable attention for its role in transmissible spongiform encephalopathies (TSEs). In spite of being a point of intense research effort critical questions still remain regarding the physiological function of PrP^C and how these functions may change with the conversion of the protein into the infectious and pathological conformation (PrP^Sc). While emerging evidence suggests PrP^C/Sc are involved in signal transduction there is little consensus on the signaling pathways associated with the normal and diseased states. The purported involvement of PrP^C in signal transduction, and the association of TSEs with neural pathology, makes kinome analysis of human neurons an interesting and appropriate model to characterize patterns of signal transduction following activation of PrP^C by two commonly employed experimental ligands; antibody-induced dimerization by 6H4 and the amino acids 106-126 PrP peptide fragment (PrP 106–126). Analysis of the induced kinome responses reveals distinct patterns of signaling activity following each treatment. Specifically, stimulation of human neurons with the 6H4 antibody results in alterations in mitogen activated protein kinase (MAPK) signaling pathways while the 106-126 peptide activates growth factor related signaling pathways including vascular endothelial growth factor (VEGF) signaling and the phosphoinositide-3 kinase (PI3K) pathway. These pathways were validated through independent functional assays. Collectively these results indicate that stimulation of PrP^C with distinct ligands, even within the same cell type, results in unique patterns of signaling. While this investigation highlights the apparent functional versatility of PrP^C as a signaling molecule and may offer insight into cellular mechanisms of TSE pathology it also emphasizes the potential dangers associated with attributing activation of specific intracellular events to particular receptors through artificial models of receptor activation.     

Smad3 contributes to positioning of proliferating cells in colonic crypts by inducing EphB receptor protein expression

In prion diseases cellular prion protein (PrP^C) undergoes conformational transition into the β-sheet-rich form (PrP^Sc). PrP^C consists of the disordered N-terminal part and a C-terminal globular domain containing three α-helices (H1, H2, H3) and an antiparallel beta sheet (B1, B2). B2–H2 loop, which has a focal role in the species barrier, contains the highest density of asparagine (N) and glutamine (Q) residues in the whole sequence. Q/N-rich domains are essential for the conversion of yeast prions. We investigated the role of Q/N residues in the B2–H2 loop in PrP conversion. We prepared mouse PrP mutants with increasing number of consecutive Q/N residues in the B2–H2 loop. Stability of the mutants decreased with the increasing number of inserted glutamines. In vitro conversion of mutants yielded fibrils of similar morphology as the wild-type PrP. Q/N mutants accelerated fibrillization in comparison to the wild-type PrP, with mutant containing the most glutamines having the shortest lag phase. The effect of Q/N residues was specific for the B2–H2 loop and was not due to simple increase in flexibility as the introduction of Gly-Ser or Ala residues slowed the conversion despite their decreased stability. Our results thus suggest that Q/N residues in the B2–H2 loop of PrP promote protein conversion and may represent a link to conversion of Q/N-rich prions.     

Prion protein and Alzheimer disease

Alzheimer and prion diseases are neurodegenerative disorders characterised by the abnormal processing of amyloid-b (Ab) peptide and prion protein (PrP^C), respectively. Recent evidence indicates that PrP^C may play a critical role in the pathogenesis of Alzheimer disease. PrP^C interacts with and inhibits the b-secretase BACE1, the rate-limiting enzyme in the production of Ab. More recently PrP^C was identified as a receptor for Ab oligomers and the expression of PrP^C appears to be controlled by the amyloid intracellular domain (AICD). Here we review these observations and propose a feedback loop in the normal brain where PrP^C exerts an inhibitory effect on BACE1 to decrease both Ab and AICD production. In turn, the AICD upregulates PrP^C expression, thus maintaining the inhibitory effect of PrP^C on BACE1. In Alzheimer disease, this feedback loop is disrupted, and the increased level of Ab oligomers bind to PrP^C and prevent it from regulating BACE1 activity.     

The PrPC C1 fragment derived from the ovine A136R154R171 PRNP allele is highly abundant in sheep brain and inhibits fibrillisation of full-length PrPC protein in vitro

Expression of the cellular prion protein (PrP^C) is crucial for the development of prion diseases. Resistance to prion diseases can result from reduced availability of the prion protein or from amino acid changes in the prion protein sequence. We propose here that increased production of a natural PrP α-cleavage fragment, C1, is also associated with resistance to disease. We show, in brain tissue, that ARR homozygous sheep, associated with resistance to disease, produced PrP^C comprised of 25% more C1 fragment than PrP^C from the disease-susceptible ARQ homozygous and highly susceptible VRQ homozygous animals. Only the C1 fragment derived from the ARR allele inhibits in-vitro fibrillisation of other allelic PrP^C variants. We propose that the increased α-cleavage of ovine ARR PrP^C contributes to a dominant negative effect of this polymorphism on disease susceptibility. Furthermore, the significant reduction in PrP^C β-cleavage product C2 in sheep of the ARR/ARR genotype compared to ARQ/ARQ and VRQ/VRQ genotypes, may add to the complexity of genetic determinants of prion disease susceptibility.     

Nanopore Analysis of Wild-Type and Mutant Prion Protein (PrPC): Single Molecule Discrimination and PrPC Kinetics

Prion diseases are fatal neurodegenerative diseases associated with the conversion of cellular prion protein (PrP^C) in the central nervous system into the infectious isoform (PrP^Sc). The mechanics of conversion are almost entirely unknown, with understanding stymied by the lack of an atomic-level structure for PrP^Sc. A number of pathogenic PrP^C mutants exist that are characterized by an increased propensity for conversion into PrP^Sc and that differ from wild-type by only a single amino-acid point mutation in their primary structure. These mutations are known to perturb the stability and conformational dynamics of the protein. Understanding of how this occurs may provide insight into the mechanism of PrP^C conversion. In this work we sought to explore wild-type and pathogenic mutant prion protein structure and dynamics by analysis of the current fluctuations through an organic α-hemolysin nanometer-scale pore (nanopore) in which a single prion protein has been captured electrophoretically. In doing this, we find that wild-type and D178N mutant PrP^C, (a PrP^C mutant associated with both Fatal Familial Insomnia and Creutzfeldt-Jakob disease), exhibit easily distinguishable current signatures and kinetics inside the pore and we further demonstrate, with the use of Hidden Markov Model signal processing, accurate discrimination between these two proteins at the single molecule level based on the kinetics of a single PrP^C capture event. Moreover, we present a four-state model to describe wild-type PrP^C kinetics in the pore as a first step in our investigation on characterizing the differences in kinetics and conformational dynamics between wild-type and D178N mutant PrP^C. These results demonstrate the potential of nanopore analysis for highly sensitive, real-time protein and small molecule detection based on single molecule kinetics inside a nanopore, and show the utility of this technique as an assay to probe differences in stability between wild-type and mutant prion proteins at the single molecule level.     

Anti-prion activity of an RNA aptamer and its structural basis

Prion proteins (PrPs) cause prion diseases, such as bovine spongiform encephalopathy. The conversion of a normal cellular form (PrP^C) of PrP into an abnormal form (PrP^Sc) is thought to be associated with the pathogenesis. An RNA aptamer that tightly binds to and stabilizes PrP^C is expected to block this conversion and to thereby prevent prion diseases. Here, we show that an RNA aptamer comprising only 12 residues, r(GGAGGAGGAGGA) (R12), reduces the PrP^Sc level in mouse neuronal cells persistently infected with the transmissible spongiform encephalopathy agent. Nuclear magnetic resonance analysis revealed that R12, folded into a unique quadruplex structure, forms a dimer and that each monomer simultaneously binds to two portions of the N-terminal half of PrP^C, resulting in tight binding. Electrostatic and stacking interactions contribute to the affinity of each portion. Our results demonstrate the therapeutic potential of an RNA aptamer as to prion diseases.     

Novel Aspects of Prions,Their Receptor Molecules,and Innovative Approaches for TSE Therapy

1. Prion diseases are a group of rare, fatal neurodegenerative diseases, also known as transmissible spongiform encephalopathies (TSEs), that affect both animals and humans and include bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, chronic wasting disease (CWD) in deer and elk, and Creutzfeldt–Jakob disease (CJD) in humans. TSEs are usually rapidly progressive and clinical symptoms comprise dementia and loss of movement coordination due to the accumulation of an abnormal isoform (PrP^Sc) of the host-encoded prion protein (PrP^c). 2. This article reviews the current knowledge on PrP^c and PrP^Sc, prion replication mechanisms, interaction partners of prions, and their cell surface receptors. Several strategies, summarized in this article, have been investigated for an effective antiprion treatment including development of a vaccination therapy and screening for potent chemical compounds. Currently, no effective treatment for prion diseases is available. 3. The identification of the 37 kDa/67 kDa laminin receptor (LRP/LR) and heparan sulfate as cell surface receptors for prions, however, opens new avenues for the development of alternative TSE therapies.     

Prion Propagation is Dependent on Key Amino Acids in Charge Cluster 2 within the Prion Protein

To dissect the N-terminal residues within the cellular prion protein (PrP^C) that are critical for efficient prion propagation, we generated a library of point, double, or triple alanine replacements within residues 23–111 of PrP, stably expressed them in cells silenced for endogenous mouse PrP^C and challenged the reconstituted cells with four common but biologically diverse mouse prion strains. Amino acids (aa) 105–111 of Charge Cluster 2 (CC2), which is disordered in PrP^C, were found to be required for propagation of all four prion strains; other residues had no effect or exhibited strain-specific effects. Replacements in CC2, including aa105-111, dominantly inhibited prion propagation in the presence of endogenous wild type PrP^C whilst other changes were not inhibitory. Single alanine replacements within aa105-111 identified leucine 108 and valine 111 or the cluster of lysine 105, threonine 106 and asparagine 107 as critical for prion propagation. These residues mediate specific ordering of unstructured CC2 into β-sheets in the infectious prion fibrils from Rocky Mountain Laboratory (RML) and ME7 mouse prion strains.     

Induction of ligand-specific PrPC signaling in human neuronal cells

Cellular prion protein (PrP^C) has attracted considerable attention for its role in transmissible spongiform encephalopathies (TSEs). In spite of being a point of intense research effort critical questions still remain regarding the physiological function of PrP^C and how these functions may change with the conversion of the protein into the infectious and pathological conformation (PrP^Sc). While emerging evidence suggests PrP^C/Sc are involved in signal transduction there is little consensus on the signaling pathways associated with the normal and diseased states. The purported involvement of PrP^C in signal transduction, and the association of TSEs with neural pathology, makes kinome analysis of human neurons an interesting and appropriate model to characterize patterns of signal transduction following activation of PrP^C by two commonly employed experimental ligands; antibody-induced dimerization by 6H4 and the amino acids 106-126 PrP peptide fragment (PrP 106–126). Analysis of the induced kinome responses reveals distinct patterns of signaling activity following each treatment. Specifically, stimulation of human neurons with the 6H4 antibody results in alterations in mitogen activated protein kinase (MAPK) signaling pathways while the 106-126 peptide activates growth factor related signaling pathways including vascular endothelial growth factor (VEGF) signaling and the phosphoinositide-3 kinase (PI3K) pathway. These pathways were validated through independent functional assays. Collectively these results indicate that stimulation of PrP^C with distinct ligands, even within the same cell type, results in unique patterns of signaling. While this investigation highlights the apparent functional versatility of PrP^C as a signaling molecule and may offer insight into cellular mechanisms of TSE pathology it also emphasizes the potential dangers associated with attributing activation of specific intracellular events to particular receptors through artificial models of receptor activation.     

Prion protein and Alzheimer disease

Alzheimer and prion diseases are neurodegenerative disorders characterised by the abnormal processing of amyloid-β (Aβ) peptide and prion protein (PrP^C), respectively. Recent evidence indicates that PrP^C may play a critical role in the pathogenesis of Alzheimer disease. PrP^C interacts with and inhibits the β-secretase BACE1, the rate-limiting enzyme in the production of Aβ. More recently PrP^C was identified as a receptor for Aβ oligomers and the expression of PrP^C appears to be controlled by the amyloid intracellular domain (AICD). Here we review these observations and propose a feedback loop in the normal brain where PrP^C exerts an inhibitory effect on BACE1 to decrease both Aβ and AICD production. In turn, the AICD upregulates PrP^C expression, thus maintaining the inhibitory effect of PrP^C on BACE1. In Alzheimer disease, this feedback loop is disrupted, and the increased level of Aβ oligomers bind to PrP^C and prevent it from regulating BACE1 activity.Key words: alzheimer disease, amyloid-β, Aβ oligomers, amyloid intracellular domain, BACE1, presenilin, prion protein