Mapping of possible prion protein self-interaction domains using peptide arrays

Background  

The common event in transmissible spongiform encephalopathies (TSEs) or prion diseases is the conversion of host-encoded protease sensitive cellular prion protein (PrP^C) into strain dependent isoforms of scrapie associated protease resistant isoform (PrP^Sc) of prion protein (PrP). These processes are determined by similarities as well as strain dependent variations in the PrP structure. Selective self-interaction between PrP molecules is the most probable basis for initiation of these processes, potentially influenced by chaperone molecules, however the mechanisms behind these processes are far from understood. We previously determined that polymorphisms do not affect initial PrP^C to PrP^Sc binding but rather modulate a subsequent step in the conversion process. Determining possible sites of self-interaction could elucidate which amino acid(s) or amino acid sequences contribute to binding and further conversion into other isoforms. To this end, ovine – and bovine PrP peptide-arrays consisting of 15-mer overlapping peptides were probed with recombinant sheep PrP^C fused to maltose binding protein (MBP-PrP).     

Utilizing NMR and EPR spectroscopy to probe the role of copper in prion diseases

Copper is an essential nutrient for the normal development of the brain and nervous system, although the hallmark of several neurological diseases is a change in copper concentrations in the brain and central nervous system. Prion protein (PrP) is a copper‐binding, cell‐surface glycoprotein that exists in two alternatively folded conformations: a normal isoform (PrP^C) and a disease‐associated isoform (PrP^Sc). Prion diseases are a group of lethal neurodegenerative disorders that develop as a result of conformational conversion of PrP^C into PrP^Sc. The pathogenic mechanism that triggers this conformational transformation with the subsequent development of prion diseases remains unclear. It has, however, been shown repeatedly that copper plays a significant functional role in the conformational conversion of prion proteins. In this review, we focus on current research that seeks to clarify the conformational changes associated with prion diseases and the role of copper in this mechanism, with emphasis on the latest applications of NMR and EPR spectroscopy to probe the interactions of copper with prion proteins. Copyright © 2013 John Wiley & Sons, Ltd.     

Cross‐Talk Between the Octarepeat Domain and the Fifth Binding Site of Prion Protein Driven by the Interaction of Copper(II) with the N‐terminus

Prion diseases are a group of neurodegenerative diseases based on the conformational conversion of the normal form of the prion protein (PrP^C) to the disease‐related scrapie isoform (PrP^Sc). Copper(II) coordination to PrP^C has attracted considerable interest for almost 20 years, mainly due to the possibility that such an interaction would be an important event for the physiological function of PrP^C. In this work, we report the copper(II) coordination features of the peptide fragment Ac(PEG₁₁)₃PrP(60‐114) [Ac=acetyl] as a model for the whole N‐terminus of the PrP^C metal‐binding domain. We studied the complexation properties of the peptide by means of potentiometric, UV/Vis, circular dichroism and electrospray ionisation mass spectrometry techniques. The results revealed that the preferred histidyl binding sites largely depend on the pH and copper(II)/peptide ratio. Formation of macrochelate species occurs up to a 2:1 metal/peptide ratio in the physiological pH range and simultaneously involves the histidyl residues present both inside and outside the octarepeat domain. However, at increased copper(II)/peptide ratios amide‐bound species form, especially within the octarepeat domain. On the contrary, at basic pH the amide‐bound species predominate at any copper/peptide ratio and are formed preferably with the binding sites of His96 and His111, which is similar to the metal‐binding‐affinity order observed in our previous studies.     

Structural analysis of prion proteins by means of drift cell and traveling wave ion mobility mass spectrometry

The prion protein (PrP) is implicitly involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs). The conversion of normal cellular PrP (PrP^C), a protein that is predominantly α-helical, to a β-sheet-rich isoform (PrP^Sc), which has a propensity to aggregate, is the key molecular event in prion diseases. During its short life span, PrP can experience two different pH environments; a mildly acidic environment, whilst cycling within the cell, and a neutral pH when it is glycosyl phosphatidylinositol (GPI)-anchored to the cell membrane. Ion mobility (IM) combined with mass spectrometry has been employed to differentiate between two conformational isoforms of recombinant Syrian hamster prion protein (SHaPrP). The recombinant proteins studied were α-helical SHaPrP(90-231) and β-sheet-rich SHaPrP(90-231) at pH 5.5 and pH 7.0. The recombinant proteins have the same nominal mass-to-charge ratio (m/z) but differ in their secondary and tertiary structures. A comparison of traveling-wave (T-Wave) ion mobility and drift cell ion mobility (DCIM) mass spectrometry estimated and absolute cross-sections showed an excellent agreement between the two techniques. The use of T-Wave ion mobility as a shape-selective separation technique enabled differentiation between the estimated cross-sections and arrival time distributions (ATDs) of α-helical SHaPrP(90-231) and β-sheet-rich SHaPrP(90-231) at pH 5.5. No differences in cross-section or ATD profiles were observed between the protein isoforms at pH 7.0. The findings have potential implications for a new ante-mortem screening assay, in bodily fluids, for prion misfolding diseases such as TSEs.     

Kinetics and mechanism of amyloid formation by the prion protein H1 peptide as determined by time-dependent ESR

Background: Peptides derived from three of four putative α-helical regions of the prion protein (PrP) form amyloid in solution. These peptides serve as models for amyloidogenesis and for understanding the α helix → β strand conformational change that is responsible for the development of disease. Kinetic studies of amyloid formation usually rely on the detection of fibrils. No study has yet explored the rate of monomer peptide uptake or the presence of nonfibrillar intermediate species. We present a new electron spin resonance (ESR) method for probing the kinetics of amyloid formation. A spin label was covalently attached to a highly amyloidogenic peptide and kinetic trials were monitored by ESR.Results: Electron microscopy shows that the spin-labeled peptide forms amyloid, and ESR reveals the kinetic decay of free peptide monomer during amyloid formation. The combination of electron microscopy and ESR suggests that there are three kinetically relevant species: monomer peptide, amyloid and amorphous aggregate (peptide aggregates devoid of fibrils or other structures with long-range order). A rather surprising result is that amyloid formation requires the presence of this amorphous aggregate. This is particularly interesting because PrP^Sc the form of PrP associated with scrapie, is often found as an aggregate and amyloid formation is not a necessary component of prion replication or pathogenesis.Conclusions: Kinetic analysis of the time-dependent data suggests a model whereby the amorphous aggregate has a previously unsuspected dual role: it releases monomer into solution and also provides initiation sites for fibril growth. These findings suggest that the β-sheet-rich PrP^Sc may be stabilized by aggregation.     

Discriminant analysis of prion sequences for prediction of susceptibility

Prion diseases, including ovine scrapie, bovine spongiform encephalopathy (BSE), human kuru and Creutzfeldt–Jakob disease (CJD), originate from a conformational change of the normal cellular prion protein (PrP^C) into abnormal protease-resistant prion protein (PrP^Sc). There is concern regarding these prion diseases because of the possibility of their zoonotic infections across species. Mutations and polymorphisms of prion sequences may influence prion-disease susceptibility through the modified expression and conformation of proteins. Rapid determination of susceptibility based on prion-sequence polymorphism information without complex structural and molecular biological analyses may be possible. Information regarding the effects of mutations and polymorphisms on prion-disease susceptibility was collected based on previous studies to classify the susceptibilities of sequences, whereas the BLOSUM62 scoring matrix and the position-specific scoring matrix were utilised to determine the distance of target sequences. The k-nearest neighbour analysis was validated with cross-validation methods. The results indicated that the number of polymorphisms did not influence prion-disease susceptibility, and three and four k-objects showed the best accuracy in identifying the susceptible group. Although sequences with negative polymorphisms showed relatively high accuracy for determination, polymorphisms may still not be an appropriate factor for estimating variation in susceptibility. Discriminant analysis of prion sequences with scoring matrices was attempted as a possible means of determining susceptibility to prion diseases. Further research is required to improve the utility of this method.     

Prion protein insertional mutations increase aggregation propensity but not fiber stability

Background  

Mutations in the PRNPgene account for ~15% of all prion disease cases. Little is understood about the mechanism of how some of these mutations in PRNPcause the protein to aggregate into amyloid fibers or cause disease. We have taken advantage of a chimeric protein system to study the oligopeptide repeat domain (ORD) expansions of the prion protein, PrP, and their effect on protein aggregation and amyloid fiber formation. We replaced the ORD of the yeast prion protein Sup35p with that from wild type and expanded ORDs of PrP and compared their biochemical properties in vitro. We previously determined that these chimeric proteins maintain the [PSI⁺] yeast prion phenotype in vivo. Interestingly, we noted that the repeat expanded chimeric prions seemed to be able to maintain a stronger strain of [PSI ⁺] and convert from [psi ^-] to [PSI ⁺] with a much higher frequency. In this study we have attempted to understand the biochemical properties of these chimeric proteins and to establish a system to study the properties of the ORD of PrP both in vivoand in vitro.     

Prion replication and secondary nucleation

The infectious agents responsible for the prion diseases appear to be aggregates of a modified form of the prion protein, PrP^Sc, that grow at the expense of the normal form, PrP^c. The mechanism of the transformation of PrP^c to PrP^sc is the subject of intense research interest. The way in which PrP^sc aggregates generate new nuclei also deserves attention.     

Hydrogen/deuterium exchange mass spectrometry identifies two highly protected regions in recombinant full‐length prion protein amyloid fibrils

Understanding the structural basis that distinguishes the amyloid form of the prion protein from its monomeric homologue is of crucial importance to elucidate the mechanism of the lethal diseases related to this protein. Recently, an in vitro conversion system was established which reproduces the transition of recombinant prion protein PrP(23–230) from its native α‐helical rich form into an aggregated amyloid β‐sheet rich form with physicochemical properties reminiscent to those of the disease‐related isoform of the prion protein, PrP^Sc. To study the tertiary and quaternary structural organization within recombinant amyloid fibrils from mouse, mPrP(23–231)^βf; bovine, bPrP(23–230)^βf; and elk, ePrP(23–230)^βf; we utilized hydrogen/deuterium (H/D) exchange analyzed by matrix‐assisted laser desorption/ionization (MALDI) and nano‐electrospray (nano‐ESI) mass spectrometry. No significant differences were found by measuring the deuterium exchange kinetics of the aggregated fibrillar forms for mPrP(23–231)^βf, bPrP(23–230)^βf and ePrP(23–230)^βf, indicating a similar overall structural organization of the fibrils from all three species. Next, we characterized the solvent accessibility for the soluble and fibrillar forms of the mouse prion protein by hydrogen exchange, pepsin proteolysis and nano‐ESI ion trap mass spectrometry analysis. In its amyloid form, two highly protected regions of mPrP(23–231) comprising residues [24–98] and [182–212] were identified. The residues between the two highly protected stretches were found to be more solvent exposed, but less than in the soluble protein, and might therefore rather form part of a fibrillar interface. Copyright © 2009 John Wiley & Sons, Ltd.     

Chemistry and Molecular Biology of Transmissible Spongiform Encephalopathies

Prion diseases are currently in the spotlight. Among them, the Creutzfeldt–Jakob disease in humans, scrapie in sheep, and bovine spongiform encephalopathy, or mad cow disease, are most commonly known. The term “spongiform” refers to the characteristic appearance of the lesions found in affected brains. It is likely that prion diseases originate from a causative agent that replicates independently of nucleic acids. Current research assumes that a structural isoform of prion protein, the scrapie form PrP^Sc, is the responsible pathogen. The three-dimensional structure, but not the amino acid sequence of the isoform differs from that of the normal cellular isoform, PrP^c. According to a widely accepted hypothesis, the normal isoform of the protein is converted by an autocatalytic process into the scrapie form upon contact with the latter. This hypothesis has not yet been proven. However, considerable progress has been made in the last few years, which might provide answers to many open questions about prion diseases, the subject of this review.     

High‐resolution differentiation of transmissible spongiform encephalopathy strains by quantitative N‐terminal amino acid profiling (N‐TAAP) of PK‐digested abnormal prion protein

New forms of transmissible spongiform encephalopathy (TSE) continue to be identified, and consequently sensitive differential diagnosis is increasingly important both for the management of disease in humans and livestock and in providing confidence in the safety of the food chain. TSE diseases are associated with accumulation of protease‐resistant prion protein (PrP^Sc) and detection of this marker protein is central to diagnosis. Proteolysis by proteinase K (PK) generates protease‐resistant products (PrP^res) with partially variable N‐termini. The conformation(s) of PrP^Sc and thus the points of PK cleavage are thought to be dependent on the strain of prion disease. Western blot (WB) analysis of PrP^res gives characteristic migration patterns that can be used to diagnose TSEs, but the relatively low resolution of this technique limits its ability to differentiate certain disease strains. Mass spectrometry (MS) has the capability to resolve these various PK cleavage sites to the level of individual amino acid residues. In the present study multiple selected reaction monitoring (mSRM) was used to detect and quantify PrP^res N‐terminal tryptic peptides by MS and thus to define the N‐terminal amino acid profiles (N‐TAAPs) of PrP^res characteristic for various TSEs in sheep. The fragmentation behaviour of the N‐terminal tryptic peptides was studied to allow selection of the transitions specific for each peptide. Different PrP^res preparation methods were evaluated and the most effective approach applied to differentiate the N‐TAAPs corresponding to various sheep TSE isolates. Marked differences were identified between the N‐TAAPs of bovine spongiform encephalopathy (BSE) and classical scrapie, and between classical scrapie and the experimental strains SSBP/1 and CH1641, thereby validating this approach as a means of TSE‐strain specific diagnosis. Copyright © 2008 John Wiley & Sons, Ltd.     

Remarkable difference of phospholipid molecular chirality in regulating PrP aggregation and cell responses

Prion diseases are fatal neurodegenerative diseases that can cause severe dementia.The misfolding and accumulation of the prion peptide (Pr P)_106–126is crucial,and this process is closely relevant to biological membranes.However,how Pr P_106–126aggregation is affected by the molecular chirality of phospholipid membrane is unknown.Thus,in this study,a pair of L-and D-aspartic acid (Asp)-modified 1,2-dipalmitoyl-sn–glycero-3-phosphoethanolamine (DPPE) were synthesized to const...     

Single-Chain Fv Antibody Fragments Retain Binding Properties of the Monoclonal Antibody Raised Against Peptide P1 of the Human Prion Protein

Prion diseases are incurable neurodegenerative diseases that affect both humans and animals. The infectious agent is a pathogenic form of the prion protein that accumulates in brain as amyloids. Currently, there is neither cure nor reliable preclinical diagnostics on the market available. The growing number of reports shows that passive immunisation is one of the most promising strategies for prion disease therapy, where antibodies against prions may prevent and even cure the infection. Since antibodies are large molecules and, thus, might not be suitable for the therapy, different antibody fragments are a good alternative. Therefore, we have designed and prepared single-chain antibody fragments (scFvs) derived from the PrP^Sc-specific murine monoclonal antibody V5B2. Using a new expression vector pMD204, we produced scFvs in two opposing chain orientations in the periplasm of Escherichia coli. Both recombinant antibody fragments retained the specificity of the parent antibody and one of these exhibited binding properties comparable to the corresponding murine Fab fragments with the affinity in nM range. Our monovalent antibody fragments are of special interest in view of possible therapeutic reagents for prion diseases as well as for development of a new generation of diagnostics.     

Aptamer-mediated turn-on fluorescence assay for prion protein based on guanine quenched fluophor

An aptamer-participated haprin structure was designed by employing cellular prion protein (PrP^C) as a model protein, and thus an aptamer-mediated turn-on fluorescence assay for proteins was developed in this contribution. The designed aptamer-participated haprin structure consists of three segments. Namely, an aptamer sequence located in the loop, three guanine bases at 3′-terminal, and a fluophor modified at 5′-terminal. It was found that the guanine bases at the 3′-terminal could quench the fluorescence of the fluophor such as tetramethyl-6-carboxyrhodamine (TAMRA) at the 5′-terminal about 76.6% via electron transfer if the guanine bases are close enough to the fluophor, and the quenched fluorescence could get restored when the target protein is present since the interaction, which could be confirmed by measuring fluorescence lifetime, between TAMRA-aptamer and the target protein forces the guanines away from TAMRA so that TAMRA-modified aptamer changes into turn-on state. A linear relationship was then constructed between the turn-on fluorescence intensity and the concentration of PrP^C in the range from 1.1 to 44.7 μg/mL with a limit of detection of 0.3 μg/mL (3σ).     

Recombinant Single-Chain Antibody with the Trojan Peptide Penetratin Positioned in the Linker Region Enables Cargo Transfer Across the Blood–Brain Barrier

Delivery of therapeutic proteins into tissues and across the blood–brain barrier (BBB) is limited by the size and biochemical properties of the proteins. Efficient delivery across BBB is generally restricted to small, highly lipophilic molecules. However, in the last decades, several peptides that can pass cell membranes have been identified. It has been shown that these peptides are also capable of delivering large hydrophilic cargoes into cells and are therefore a powerful biological tool for transporting drugs across cell membranes and even into the brain. We designed and prepared a single-chain antibody fragment (scFvs), specific for the pathological form of the prion protein (PrP^Sc), where a cell-penetrating peptide (CPP) was used as a linker between the two variable domains of the scFv. The intravenously administered recombinant scFv-CPP was successfully targeted to and delivered into mouse brain cells. Our single-chain antibody fragments are of special interest in view of possible therapeutic reagents design not only for prion diseases but also for other neurodegenerative diseases.     

Binding kinetics of human cellular prion detection by DNA aptamers immobilized on a conducting polypyrrole

We developed a biosensor based on the surface plasmon resonance (SPR) method for the study of the binding kinetics and detection of human cellular prions (PrP^C) using DNA aptamers as bioreceptors. The biosensor was formed by immobilization of various biotinylated DNA aptamers on a surface of conducting polypyrrole modified by streptavidin. We demonstrated that PrP^C interaction with DNA aptamers could be followed by measuring the variation of the resonance angle. This was studied using DNA aptamers of various configurations, including conventional single-stranded aptamers that contained a rigid double-stranded supporting part and aptamer dimers containing two binding sites. The kinetic constants determined by the SPR method suggest strong interaction of PrP^C with various DNA aptamers depending on their configuration. SPR aptasensors have a high selectivity to PrP^C and were regenerable by a brief wash in 0.1 M NaOH. The best limit of detection (4 nM) has been achieved with this biosensor based on DNA aptamers with one binding site but containing a double-stranded supporting part.

Amyloid binding and beyond: a new approach for Alzheimer's disease drug discovery targeting Aβo–PrPC binding and downstream pathways

Amyloid β oligomers (Aβo) are the main toxic species in Alzheimer''s disease, which have been targeted for single drug treatment with very little success. In this work we report a new approach for identifying functional Aβo binding compounds. A tailored library of 971 fluorine containing compounds was selected by a computational method, developed to generate molecular diversity. These compounds were screened for Aβo binding by a combined ¹⁹F and STD NMR technique. Six hits were evaluated in three parallel biochemical and functional assays. Two compounds disrupted Aβo binding to its receptor PrP^C in HEK293 cells. They reduced the pFyn levels triggered by Aβo treatment in neuroprogenitor cells derived from human induced pluripotent stem cells (hiPSC). Inhibitory effects on pTau production in cortical neurons derived from hiPSC were also observed. These drug-like compounds connect three of the pillars in Alzheimer''s disease pathology, i.e. prion, Aβ and Tau, affecting three different pathways through specific binding to Aβo and are, indeed, promising candidates for further development.

A new approach combining virtual screening, ¹⁹F and STD NMR, and biochemical assays using hiPSC and targetting multiple pathways involving Aβ, PrP^C and Tau provides a more effective strategy for Alzheimer''s disease drug discovery than Aβ only approach.     

Identification of the Most Stable Sc2C80 Isomers: Structure,Electronic Property,and Molecular Spectra Investigations

A systematic density functional theory investigation has been carried out to explore the possible structures of Sc₂C₈₀ at the BMK/6‐31G(d) level. The results clearly show that Sc₂@C₈₀‐I_h, Sc₂@C₈₀‐D_5h, and Sc₂C₂@C₇₈‐C_2v can be identified as three isomers of Sc₂C₈₀ metallofullerene with the lowest energy. Frontier molecular orbital analysis indicates that the two Sc₂@C₈₀ isomers have a charge state as (Sc³⁺)₂@C₈₀^6?and the Sc₂C₂@C₇₈ has a charge state of (Sc³⁺)₂C₂^2?@C₇₈^4?. Moreover, the metal‐cage covalent interactions have been studied to reveal the dynamics of endohedral moiety. The vertical electron affinity, vertical ionization potential, infrared spectra and ¹³C nuclear magnetic resonance spectra have been also computed to further disclose the molecular structures and properties.     

Prion protein detection in serum using micromechanical resonator arrays

Prion proteins that have transformed from their normal cellular counterparts (PrP^c) into infectious form (PrP^res) are responsible for causing progressive neurodegenerative diseases in numerous species, such as bovine spongiform encephalopathy (BSE) in cattle (also known as mad cow disease), scrapie in sheep, and Creutzfeldt-Jakob disease (CJD) in humans. Due to a possible link between BSE and CJD it is highly desirable to develop non-invasive and ante mortem tests for the detection of prion proteins in bovine samples. Such ante mortem tests of all cows prior to slaughter will help to prevent the introduction of PrP^res into the human food supply. Furthermore, detection of PrP^res in donated blood will also help to prevent the transmission of CJD among humans through blood transfusion. In this study, we have continued development of a micromechanical resonator array that is capable of detecting PrP^c in bovine blood serum. The sensitivity of the resonators for the detection of PrP^c is further enhanced by the use of secondary mass labels. A pair of antibodies is used in a sandwich immunoassay format to immobilize PrP^c on the surface of resonators and attach nanoparticles as secondary mass labels to PrP^c. Secondary mass labeling is optimized in terms of incubation time to maximize the frequency shifts that correspond to the presence of PrP^c on the surface of resonators. Our results show that a minimum of 200 pg mL⁻¹ of PrP^c in blood serum can be detected using micromechanical resonator arrays.     

Construction and Characterization of an Anti‐Prion scFv Fusion Protein Pair for Detection of Prion Protein on Antibody Chip

Abstract

A pair of single chain Fv fragment (scFv) fusion proteins were constructed and characterized. Antibody chips using the pair were designed for sensitive detection of prion protein. Phage displayed antibody library was synthesized by immunizing mice with thioredoxin‐mature bovine prion fusion protein (TrxA‐bPrP^c). After five rounds of panning against recombinant bovine prion protein (rb‐PrP^c) and ELISA test, two positive clones with high affinity to rb‐PrP^c, named Z163 and Z186, were obtained. They were conjugated with a linker‐streptavidin binding protein (SBP) or human IgG1 constant fragment (Fc) to form the scFv fusion protein pair Z186‐L‐SBP/Z163‐Fc. Western blot experiments showed that the scFv fusion pair specifically interacted with the line epitopes of the protease resistant core region bPrP27‐30. Surface plasmon resonance (SPR) sensorgrams revealed that the equilibrium dissociation constants of the interactions with rb‐PrP^c were 3.24×10^?8 M, 8.82×10^?8M, and 8.10×10^?9 M for Z186‐L‐SBP, Z163, and Z163‐Fc, respectively. All binding reactions followed rapid association and slow dissociation kinetics. As a detection pair, Z186‐L‐SBP functioned as a capture probe and was immobilized on the streptavidin coated slides to form reactive layer of the antibody chip, and Z163‐Fc labeled with fluorescence dye Cy3 functioned as a detection probe generating fluorescence signal. The antibody chip could detect existence of rb‐PrP^c with detection limit of 1 pg/ml.