Recombinant Prion Protein Research Articles

Self-propagating, protease-resistant, recombinant prion protein conformers with or without in vivo pathogenicity.

Prions, characterized by self-propagating protease-resistant prion protein (PrP) conformations, are agents causing prion disease. Recent studies generated several such self-propagating protease-resistant recombinant PrP (rPrP-res) conformers. While some cause prion disease, others fail to induce any pathology. Here we showed that although distinctly different, the pathogenic and non-pathogenic rPrP-res conformers were similarly recognized by a group of conformational antibodies against prions and shared a similar guanidine hydrochloride denaturation profile, suggesting a similar overall architecture. Interestingly, two independently generated non-pathogenic rPrP-res were almost identical, indicating that the particular rPrP-res resulted from cofactor-guided PrP misfolding, rather than stochastic PrP aggregation. Consistent with the notion that cofactors influence rPrP-res conformation, the propagation of all rPrP-res formed with phosphatidylglycerol/RNA was cofactor-dependent, which is different from rPrP-res generated with a single cofactor, phosphatidylethanolamine. Unexpectedly, despite the dramatic difference in disease-causing capability, RT-QuIC assays detected large increases in seeding activity in both pathogenic and non-pathogenic rPrP-res inoculated mice, indicating that the non-pathogenic rPrP-res is not completely inert in vivo. Together, our study supported a role of cofactors in guiding PrP misfolding, indicated that relatively small structural features determine rPrP-res’ pathogenicity, and revealed that the in vivo seeding ability of rPrP-res does not necessarily result in pathogenicity.

Ovine recombinant PrP as an inhibitor of ruminant prion propagation in vitro

ABSTRACTPrion diseases are fatal and incurable neurodegenerative diseases of humans and animals. Despite years of research, no therapeutic agents have been developed that can effectively manage or reverse disease progression. Recently it has been identified that recombinant prion proteins (rPrP) expressed in bacteria can act as inhibitors of prion replication within the in vitro prion replication system protein misfolding cyclic amplification (PMCA). Here, within PMCA reactions amplifying a range of ruminant prions including distinct Prnp genotypes/host species and distinct prion strains, recombinant ovine VRQ PrP displayed consistent inhibition of prion replication and produced IC50 values of 122 and 171 nM for ovine scrapie and bovine BSE replication, respectively. These findings illustrate the therapeutic potential of rPrPs with distinct TSE diseases.

Use of bovine recombinant prion protein and real-time quaking-induced conversion to detect cattle transmissible mink encephalopathy prions and discriminate classical and atypical L- and H-Type bovine spongiform encephalopathy.

Prions are amyloid-forming proteins that cause transmissible spongiform encephalopathies through a process involving conversion from the normal cellular prion protein to the pathogenic misfolded conformation (PrPSc). This conversion has been used for in vitro assays including serial protein misfolding amplification and real-time quaking induced conversion (RT-QuIC). RT-QuIC can be used for the detection of prions in a variety of biological tissues from humans and animals. Extensive work has been done to demonstrate that RT-QuIC is a rapid, specific, and highly sensitive prion detection assay. RT-QuIC uses recombinant prion protein to detect minute amounts of PrPSc. RT-QuIC has been successfully used to detect PrPSc from different prion diseases with a variety of substrates including hamster, human, sheep, bank vole, bovine and chimeric forms of prion protein. However, recombinant bovine prion protein has not been used to detect transmissible mink encephalopathy (TME) or to differentiate types of bovine spongiform encephalopathy (BSE) in samples from cattle. We evaluated whether PrPSc from TME and BSE infected cattle can be detected with RT-QuIC using recombinant bovine prion proteins, and optimized the reaction conditions to specifically detect cattle TME and to discriminate between classical and atypical BSE by conversion efficiency. We also found that substrate composed of the disease associated E211K mutant protein can be effective for the detection of TME in cattle and that wild type prion protein appears to be a practical substrate to discriminate between the different types of BSEs.

Ultra-efficient Amplification of Abnormal Prion Protein by Modified Protein Misfolding Cyclic Amplification with Electric Current

Prion diseases are clinically diagnosed and confirmed upon post-mortem histopathological examination of brain tissue. The only reliable molecular marker for prion diseases is abnormal prion protein (PrPSc), a pathologically conformed prion protein that primarily accumulates in the central nervous system and to a lesser extent in lymphoreticular tissues. However, the use of PrPSc as a marker for preclinical diagnoses is limited because the concentration of PrPSc in easily accessible body fluids is extremely low. Hence, one of the most promising approaches would be the development of an efficient in vitro amplification method for PrPSc. Indeed, protein misfolding cyclic amplification (PMCA) has become an important diagnostic tool for prion diseases. Here, we first describe a new superior PMCA device that employs electricity (referred to as ePMCA) to amplify PrPSc. The ePMCA device markedly improved the detection limit for PrPSc by amplifying trace amounts of pathogenic prion protein by applying electricity to improve PMCA. To increase the cavitation of sonication, a glass sample tube was used, and the upper side of the horn was shaped such that it had a curved cross-section. The ePMCA device enabled PrPSc to be amplified even from a sample seeded with 10–28-fold diluted 263K scrapie-infected brain homogenates with recombinant hamster prion protein (rHaPrP). In addition, the efficiency of prion amplification was best when 50 mM HEPES and 1% Triton X-100 were used as a PMCA conversion buffer in the various conditions that we applied. These results indicate that ePMCA would be very valuable for the rapid and specific diagnosis of human prion diseases and, thus, may provide a practically improved method for antemortem diagnoses using the body fluids of patients and animals with prion disease.

Purification and Fibrillation of Full-Length Recombinant PrP.

Misfolding and aggregation of prion protein are related to several neurodegenerative diseases in humans such as Creutzfeldt-Jakob disease, fatal familial insomnia, and Gerstmann-Straussler-Scheinker disease. A growing number of applications in the prion field including assays for detection of PrPSc and methods for production of PrPSc de novo require recombinant prion protein (PrP) of high purity and quality. Here, we report an experimental procedure for expression and purification of full-length mammalian prion protein. This protocol has been proved to yield PrP of extremely high purity that lacks PrP adducts, oxidative modifications, or truncation, which is typically generated as a result of spontaneous oxidation or degradation. We also describe methods for preparation of amyloid fibrils from recombinant PrP in vitro. Recombinant PrP fibrils can be used as a noninfectious synthetic surrogate of PrPSc for development of prion diagnostics including generation of PrPSc-specific antibody.

RT-QuIC Assays for Prion Disease Detection and Diagnostics.

In coping with prion diseases, it is important to have tests that are practical enough for routine applications in medicine, agriculture, wildlife biology, and research, yet sensitive enough to detect minimal amounts of infectivity. Real-time quaking-induced conversion (RT-QuIC) assays have evolved to the point where they fulfill these criteria in applications to various human and animal prion diseases. For example, RT-QuIC assays of cerebrospinal fluid and nasal brushings allow for highly sensitive (77-97%) and specific (99-100%) identification of human sCJD patients. Recent improvements have markedly enhanced sensitivity andreduced the assay time required for many samples to a matter of hours rather than days. By combining analyses of cerebrospinal fluid and nasal brushings, diagnostic sensitivities and specificities of nearly 100% can be achieved. RT-QuIC assays are based on prion-seeded amyloid fibril formation by recombinant prion protein (rPrPSen) in multiwell plates using a Thioflavin T fluorescence readout. Here we describe our current RT-QuIC methodologies as well as technical considerations in executing, troubleshooting, and adapting the assay to new strains of prions and sample types.

Amplified Detection of Prions and Other Amyloids by RT-QuIC in Diagnostics and the Evaluation of Therapeutics and Disinfectants.

Among the most sensitive, specific and practical of methods for detecting prions are the real-time quaking-induced conversion (RT-QuIC) assays. These assays exploit the fundamental self-propagating activity of prions to amplify the presence of prion seeds by as much as a trillion-fold. The reactions can detect most of the known mammalian prion diseases, often with sensitivities greater than those of animal bioassays. RT-QuIC assays are performed in multiwell plates with fluorescence detection and have now reached the sensitivity and practicality required for routine prion disease diagnostics. Some key strains of prions within particular host species, e.g., humans, cattle, and sheep, can be discriminated by comparison of RT-QuIC responses with different recombinant prion protein substrates. The most thoroughly validated diagnostic application of RT-QuIC is in the diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD) using cerebrospinal fluid. Diagnostic sensitivities as high as 96% can be achieved in less than 24h with specificities of 98%-100%. The ability, if needed, to also test nasal swab samples can increase the RT-QuIC sensitivity for sCJD to virtually 100%. In addition to diagnostic applications, RT-QuIC has also been used in the testing of prion disinfectants and potential therapeutics. Mechanistically related assays are also now being developed for other protein misfolding diseases.

Development of radioiodinated acridine derivatives for in vivo imaging of prion deposits in the brain

Prion diseases are caused by deposition of abnormal prion protein aggregates (PrPSc) in the central nervous system. This study aimed to develop in vivo imaging probes that can detect cerebral PrPSc deposits. We synthesized several quinacrine-based acridine (AC) derivatives with 2,9-substitution and radioiodinated them. The AC derivatives were evaluated as prion-imaging probes using recombinant mouse prion protein (rMoPrP) aggregates and brain sections of mouse-adapted bovine spongiform encephalopathy (mBSE)-infected mice. The distribution of these compounds in mice was also evaluated. The 2-methoxy derivative [125I]2 exhibited the highest binding affinity for rMoPrP aggregates with an equilibrium dissociation constant (Kd) value of 43.4nM. Fluorescence imaging with 2 showed clear signals at the thioflavin T (ThT)-positive amyloid deposits in the mBSE-infected mouse brain. Although a discrepancy was observed between the in vitro binding of AC derivatives to the aggregates and in vivo distribution of these compounds in the brain and we failed to identify prospective prion-imaging probes in this study, the AC derivatives may be considered a useful scaffold for the development of in vivo imaging probes. Further chemical modification of these AC derivatives may discover clinically applicable prion imaging probes.

Multiple affinity purification of a baculovirus-derived recombinant prion protein with in vitro ability to convert to its pathogenic form

ABSTRACTWe previously showed that baculovirus-derived recombinant prion protein (Bac-PrP) can be converted to the misfolded infectious form (PrPSc) by protein misfolding cyclic amplification, an in vitro conversion technique. Bac-PrP, with post-translational modifications, would be useful for various applications such as using PrP as an immunogen for generating anti-PrP antibody, developing anti-prion drugs or diagnostic assays using in vitro conversion systems, and establishing an in vitro prion propagation model. For this purpose, highly purified Bac-PrP with in vitro conversion activity is necessary for use as a PrPC source, to minimize contamination. Furthermore, an exogenous affinity tag-free form is desirable to avoid potential steric interference by the affinity tags during the conversion process. In this study, we established purification methods for the untagged Bac-PrP under native conditions by combining exogenous double-affinity tags, namely, a polyhistidine-tag and a profinity eXact tag, with an octarepeat sequence of the N-terminal region of PrP, which has metal ion-binding affinity. The untagged Bac-PrP with near-homogeneity was obtained by three-step affinity purification, and it was shown that the final, purified Bac-PrP could convert to its pathogenic form. The presented purification procedure could be applied not only to PrP but also to other eukaryotic, recombinant proteins that require high purity and intact physiological activity.

The real-time quaking-induced conversion assay for detection of human prion disease and study of other protein misfolding diseases

The development and adaption of in vitro misfolded protein amplification systems has been a major innovation in the detection of abnormally folded prion protein scrapie (PrPSc) in human brain and cerebrospinal fluid (CSF) samples. Herein, we describe a fast and efficient protein amplification technique, real-time quaking-induced conversion (RT-QuIC), for the detection of a PrPSc seed in human brain and CSF. In contrast to other in vitro misfolded protein amplification assays-such as protein misfolding cyclic amplification (PMCA)-which are based on sonication, the RT-QuIC technique is based on prion seed-induced misfolding and aggregation of recombinant prion protein substrate, accelerated by alternating cycles of shaking and rest in fluorescence plate readers. A single RT-QuIC assay typically analyzes up to 32 samples in triplicate, using a 96-well-plate format. From sample preparation to analysis of results, the protocol takes ∼87 h to complete. In addition to diagnostics, this technique has substantial generic analytical applications, including drug screening, prion strain discrimination, biohazard screening (e.g., to reduce transmission risk related to prion diseases) and the study of protein misfolding; in addition, it can potentially be used for the investigation of other protein misfolding diseases such as Alzheimer's and Parkinson's disease.

An acoustic prion assay

An acoustic prion assay has been demonstrated for sheep brain samples. Only five false positives and no false negatives were observed in a test of 45 positive and 45 negative samples. The acoustic prion sensor was constructed using a thickness shear mode quartz resonator coated with a covalently bound recombinant prion protein. The characteristic indicator of a scrapie infected sheep brain sample was an observed shoulder in the frequency decrease in response to a sample.The response of the sensor aligns with a conformational shift in the surface protein and with the propagation mechanism of the disease. This alignment is evident in the response timing and shape, dependence on concentration, cross species behaviour and impact of blood plasma. This alignment is far from sufficient to prove the mechanism of the sensor but it does offer the possibility of a rapid and inexpensive additional tool to explore prion disease.

The Anti-Prion Antibody 15B3 Detects Toxic Amyloid-β Oligomers.

15B3 is a monoclonal IgM antibody that selectively detects pathological aggregates of the prion protein (PrP). We report the unexpected finding that 15B3 also recognizes oligomeric but not monomeric forms of amyloid-β (Aβ)42, an aggregating peptide implicated in the pathogenesis of Alzheimer’s disease (AD). The 15B3 antibody: i) inhibits the binding of synthetic Aβ42 oligomers to recombinant PrP and neuronal membranes; ii) prevents oligomer-induced membrane depolarization; iii) antagonizes the inhibitory effects of oligomers on the physiological pharyngeal contractions of the nematode Caenorhabditis elegans; and iv) counteracts the memory deficits induced by intracerebroventricular injection of Aβ42 oligomers in mice. Thus this antibody binds to pathologically relevant forms of Aβ, and offers a potential research, diagnostic, and therapeutic tool for AD.

PrP aggregation can be seeded by pre-formed recombinant PrP amyloid fibrils without the replication of infectious prions.

Mammalian prions are unusual infectious agents, as they are thought to consist solely of aggregates of misfolded prion protein (PrP). Generation of synthetic prions, composed of recombinant PrP (recPrP) refolded into fibrils, has been utilised to address whether PrP aggregates are, indeed, infectious prions. In several reports, neurological disease similar to transmissible spongiform encephalopathy (TSE) has been described following inoculation and passage of various forms of fibrils in transgenic mice and hamsters. However, in studies described here, we show that inoculation of recPrP fibrils does not cause TSE disease, but, instead, seeds the formation of PrP amyloid plaques in PrP-P101L knock-in transgenic mice (101LL). Importantly, both WT-recPrP fibrils and 101L-recPrP fibrils can seed plaque formation, indicating that the fibrillar conformation, and not the primary sequence of PrP in the inoculum, is important in initiating seeding. No replication of infectious prions or TSE disease was observed following both primary inoculation and subsequent subpassage. These data, therefore, argue against recPrP fibrils being infectious prions and, instead, indicate that these pre-formed seeds are acting to accelerate the formation of PrP amyloid plaques in 101LL Tg mice. In addition, these data reproduce a phenotype which was previously observed in 101LL mice following inoculation with brain extract containing in vivo-generated PrP amyloid fibrils, which has not been shown for other synthetic prion models. These data are reminiscent of the “prion-like” spread of aggregated forms of the beta-amyloid peptide (Aβ), α-synuclein and tau observed following inoculation of transgenic mice with pre-formed seeds of each misfolded protein. Hence, even when the protein is PrP, misfolding and aggregation do not reproduce the full clinicopathological phenotype of disease. The initiation and spread of protein aggregation in transgenic mouse lines following inoculation with pre-formed fibrils may, therefore, more closely resemble a seeded proteinopathy than an infectious TSE disease.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-016-1594-5) contains supplementary material, which is available to authorized users.

ChemInform Abstract: Cyclodextrins as Protective Agents of Protein Aggregation: An Overview

AbstractReview: 72 refs.

Advanced tests for early and accurate diagnosis of Creutzfeldt-Jakob disease.

Nature Reviews Neurology 12, 325–333 (2016); 10.1038/nrneurol.2016.65 In the version of this article initially published online and in print, the Figure 2 erroneously depicted recombinant prion protein with attached glycans, and Table 2 listed an erroneous glycotype for sporadic Creutzfeldt–Jakob disease.

Cerebrospinal fluid real-time quaking-induced conversion is a robust and reliable test for sporadic creutzfeldt-jakob disease: An international study.

Real‐time quaking‐induced conversion (RT‐QuIC) has been proposed as a sensitive diagnostic test for sporadic Creutzfeldt–Jakob disease; however, before this assay can be introduced into clinical practice, its reliability and reproducibility need to be demonstrated. Two international ring trials were undertaken in which a set of 25 cerebrospinal fluid samples were analyzed by a total of 11 different centers using a range of recombinant prion protein substrates and instrumentation. The results show almost complete concordance between the centers and demonstrate that RT‐QuIC is a suitably reliable and robust technique for clinical practice. Ann Neurol 2016;80:160–165

Ex vivo mammalian prions are formed of paired double helical prion protein fibrils.

Mammalian prions are hypothesized to be fibrillar or amyloid forms of prion protein (PrP), but structures observed to date have not been definitively correlated with infectivity and the three-dimensional structure of infectious prions has remained obscure. Recently, we developed novel methods to obtain exceptionally pure preparations of prions from mouse brain and showed that pathogenic PrP in these high-titre preparations is assembled into rod-like assemblies. Here, we have used precise cell culture-based prion infectivity assays to define the physical relationship between the PrP rods and prion infectivity and have used electron tomography to define their architecture. We show that infectious PrP rods isolated from multiple prion strains have a common hierarchical assembly comprising twisted pairs of short fibres with repeating substructure. The architecture of the PrP rods provides a new structural basis for understanding prion infectivity and can explain the inability to systematically generate high-titre synthetic prions from recombinant PrP.

Distinctive properties of plaque-type dura mater graft-associated Creutzfeldt–Jakob disease in cell-protein misfolding cyclic amplification

There are two distinct subtypes of dura mater graft-associated Creutzfeldt–Jakob disease (dCJD) with methionine homozygosity at codon 129 of the PRNP gene. The majority of cases is represented by a non-plaque-type (np-dCJD) resembling sporadic CJD (sCJD)-MM1 or -MV1, while the minority by a plaque-type (p-dCJD). p-dCJD shows distinctive phenotypic features, namely numerous kuru plaques and an abnormal isoform of prion protein (PrPSc) intermediate in size between types 1 and 2. Transmission studies have shown that the unusual phenotypic features of p-dCJD are linked to the V2 prion strain that is associated with sCJD subtypes VV2 or -MV2. In this study, we applied protein misfolding cyclic amplification (PMCA) using recombinant human prion protein as a substrate and demonstrated that p-dCJD prions show amplification features that are distinct from those of np-dCJD. Although no amplification of np-dCJD prions was observed with either 129 M or 129 V substrate, p-dCJD prions were drastically amplified with the 129 V substrates, despite the PRNP codon 129 incompatibility between seed and substrate. Moreover, by using a type 2 PrPSc-specific antibody not recognizing PrPSc in p-dCJD, we found that type 2 products are generated de novo from p-dCJD prions during PMCA with the 129 V substrates. These findings suggest that our cell-PMCA is a useful tool for easily and rapidly identifying acquired CJD associated with the transmission of the V2 CJD strain to codon 129 methionine homozygotes, based on the preference for the 129 V substrate and the type of the amplified products.

Cyclodextrins as Protective Agents of Protein Aggregation: An Overview.

Cyclodextrins are extensively used in different fields (e.g., catalysis, chromatography, pharma, supramolecular chemistry, bioorganic chemistry, and bioinorganic chemistry), and their applications have been widely reviewed. Their main application in the field of pharmaceutical is as a drug carrier. This review overviews, for the first time, the use of cyclodextrins and their derivatives as antiaggregant agents in a number of proteins (e.g., amyloid-β, insulin, recombinant human growth hormone, prion protein, transthyretin, and α-synuclein) and some multimeric enzymes. There are many diseases that are correlated to protein misfolding and amyloid formation processes affecting numerous organs and tissues. There are over 30 different amyloid proteins and a number of corresponding diseases. Alzheimer's disease is the most common neurodegenerative disease. Treatment of these diseases is still a goal to reach, and many molecules are studied in this perspective. Cyclodextrins have also been studied, and they show great potential; as such, further studies could be very promising. This review aims to be a stimulus for the design of new cyclodextrin derivatives to obtain multifunctional systems with antiaggregant activity.

Defining and Assessing Analytical Performance Criteria for Transmissible Spongiform Encephalopathy–Detecting Amyloid Seeding Assays

Transmissible spongiform encephalopathies (TSEs) are infectious, fatal neurodegenerative diseases that affect production animal health, and thus human food safety. Enhanced TSE detection methods mimic the conjectured basis for prion replication, invitro; biological matrices can be tested for prion activity via their ability to convert recombinant cellular prion protein (PrP) into amyloid fibrils; fluorescent spectra changes of amyloid-binding fluorophores in the reaction vessel detect fibril formation. Invitro PrP conversion techniques have high analytical sensitivity for prions, comparable with that of bioassays, yet no such protocol has gained regulatory approval for use in animal TSE surveillance programs. This study describes a timed invitro PrP conversion protocol with accurate, well-defined analytical criteria based on probability density and mass functions of TSE(+) and TSE(-) associated thioflavin T signal times, a new approach within this field. The prion detection model used is elk chronic wasting disease (CWD) in brain tissues. The protocol and analytical criteria proved as sensitive for elk CWD as two bioassay models, and upward of approximately 1.2 log10 more sensitive than the most sensitive TSE rapid test we assessed. Furthermore, we substantiate that timing invitro PrP conversion may be used to titrate TSEinfectivity, and, as a result, provide a comprehensive extrapolation of analytical sensitivity differences between bioassay, TSE rapid tests, and invitro PrP conversion for elk CWD.