The 26S Proteasome Degrades the Soluble but Not the Fibrillar Form of the Yeast Prion Ure2p In Vitro

PubMed Central

Wang, Kai; Redeker, Virginie; Madiona, Karine; Melki, Ronald; Kabani, Mehdi

2015-01-01

Yeast prions are self-perpetuating protein aggregates that cause heritable and transmissible phenotypic traits. Among these, [PSI +] and [URE3] stand out as the most studied yeast prions, and result from the self-assembly of the translation terminator Sup35p and the nitrogen catabolism regulator Ure2p, respectively, into insoluble fibrillar aggregates. Protein quality control systems are well known to govern the formation, propagation and transmission of these prions. However, little is known about the implication of the cellular proteolytic machineries in their turnover. We previously showed that the 26S proteasome degrades both the soluble and fibrillar forms of Sup35p and affects [PSI +] propagation. Here, we show that soluble native Ure2p is degraded by the proteasome in an ubiquitin-independent manner. Proteasomal degradation of Ure2p yields amyloidogenic N-terminal peptides and a C-terminal resistant fragment. In contrast to Sup35p, fibrillar Ure2p resists proteasomal degradation. Thus, structural variability within prions may dictate their ability to be degraded by the cellular proteolytic systems. PMID:26115123

Prion-Associated Toxicity is Rescued by Elimination of Cotranslational Chaperones

PubMed Central

Keefer, Kathryn M.; True, Heather L.

2016-01-01

The nascent polypeptide-associated complex (NAC) is a highly conserved but poorly characterized triad of proteins that bind near the ribosome exit tunnel. The NAC is the first cotranslational factor to bind to polypeptides and assist with their proper folding. Surprisingly, we found that deletion of NAC subunits in Saccharomyces cerevisiae rescues toxicity associated with the strong [PSI+] prion. This counterintuitive finding can be explained by changes in chaperone balance and distribution whereby the folding of the prion protein is improved and the prion is rendered nontoxic. In particular, the ribosome-associated Hsp70 Ssb is redistributed away from Sup35 prion aggregates to the nascent chains, leading to an array of aggregation phenotypes that can mimic both overexpression and deletion of Ssb. This toxicity rescue demonstrates that chaperone modification can block key steps of the prion life cycle and has exciting implications for potential treatment of many human protein conformational disorders. PMID:27828954

The physical basis of how prion conformations determine strain phenotypes

NASA Astrophysics Data System (ADS)

Tanaka, Motomasa; Collins, Sean R.; Toyama, Brandon H.; Weissman, Jonathan S.

2006-08-01

A principle that has emerged from studies of protein aggregation is that proteins typically can misfold into a range of different aggregated forms. Moreover, the phenotypic and pathological consequences of protein aggregation depend critically on the specific misfolded form. A striking example of this is the prion strain phenomenon, in which prion particles composed of the same protein cause distinct heritable states. Accumulating evidence from yeast prions such as [PSI+] and mammalian prions argues that differences in the prion conformation underlie prion strain variants. Nonetheless, it remains poorly understood why changes in the conformation of misfolded proteins alter their physiological effects. Here we present and experimentally validate an analytical model describing how [PSI+] strain phenotypes arise from the dynamic interaction among the effects of prion dilution, competition for a limited pool of soluble protein, and conformation-dependent differences in prion growth and division rates. Analysis of three distinct prion conformations of yeast Sup35 (the [PSI+] protein determinant) and their in vivo phenotypes reveals that the Sup35 amyloid causing the strongest phenotype surprisingly shows the slowest growth. This slow growth, however, is more than compensated for by an increased brittleness that promotes prion division. The propensity of aggregates to undergo breakage, thereby generating new seeds, probably represents a key determinant of their physiological impact for both infectious (prion) and non-infectious amyloids.

Hsp104 Overexpression Cures Saccharomyces cerevisiae [PSI+] by Causing Dissolution of the Prion Seeds

PubMed Central

Park, Yang-Nim; Zhao, Xiaohong; Yim, Yang-In; Todor, Horia; Ellerbrock, Robyn; Reidy, Michael; Eisenberg, Evan; Masison, Daniel C.

2014-01-01

The [PSI+] yeast prion is formed when Sup35 misfolds into amyloid aggregates. [PSI+], like other yeast prions, is dependent on the molecular chaperone Hsp104, which severs the prion seeds so that they pass on as the yeast cells divide. Surprisingly, however, overexpression of Hsp104 also cures [PSI+]. Several models have been proposed to explain this effect: inhibition of severing, asymmetric segregation of the seeds between mother and daughter cells, and dissolution of the prion seeds. First, we found that neither the kinetics of curing nor the heterogeneity in the distribution of the green fluorescent protein (GFP)-labeled Sup35 foci in partially cured yeast cells is compatible with Hsp104 overexpression curing [PSI+] by inhibiting severing. Second, we ruled out the asymmetric segregation model by showing that the extent of curing was essentially the same in mother and daughter cells and that the fluorescent foci did not distribute asymmetrically, but rather, there was marked loss of foci in both mother and daughter cells. These results suggest that Hsp104 overexpression cures [PSI+] by dissolution of the prion seeds in a two-step process. First, trimming of the prion seeds by Hsp104 reduces their size, and second, their amyloid core is eliminated, most likely by proteolysis. PMID:24632242

Hsp104 overexpression cures Saccharomyces cerevisiae [PSI+] by causing dissolution of the prion seeds.

PubMed

Park, Yang-Nim; Zhao, Xiaohong; Yim, Yang-In; Todor, Horia; Ellerbrock, Robyn; Reidy, Michael; Eisenberg, Evan; Masison, Daniel C; Greene, Lois E

2014-05-01

The [PSI(+)] yeast prion is formed when Sup35 misfolds into amyloid aggregates. [PSI(+)], like other yeast prions, is dependent on the molecular chaperone Hsp104, which severs the prion seeds so that they pass on as the yeast cells divide. Surprisingly, however, overexpression of Hsp104 also cures [PSI(+)]. Several models have been proposed to explain this effect: inhibition of severing, asymmetric segregation of the seeds between mother and daughter cells, and dissolution of the prion seeds. First, we found that neither the kinetics of curing nor the heterogeneity in the distribution of the green fluorescent protein (GFP)-labeled Sup35 foci in partially cured yeast cells is compatible with Hsp104 overexpression curing [PSI(+)] by inhibiting severing. Second, we ruled out the asymmetric segregation model by showing that the extent of curing was essentially the same in mother and daughter cells and that the fluorescent foci did not distribute asymmetrically, but rather, there was marked loss of foci in both mother and daughter cells. These results suggest that Hsp104 overexpression cures [PSI(+)] by dissolution of the prion seeds in a two-step process. First, trimming of the prion seeds by Hsp104 reduces their size, and second, their amyloid core is eliminated, most likely by proteolysis.

Heterologous Gln/Asn-Rich Proteins Impede the Propagation of Yeast Prions by Altering Chaperone Availability

PubMed Central

Yang, Zi; Hong, Joo Y.; Derkatch, Irina L.; Liebman, Susan W.

2013-01-01

Prions are self-propagating conformations of proteins that can cause heritable phenotypic traits. Most yeast prions contain glutamine (Q)/asparagine (N)-rich domains that facilitate the accumulation of the protein into amyloid-like aggregates. Efficient transmission of these infectious aggregates to daughter cells requires that chaperones, including Hsp104 and Sis1, continually sever the aggregates into smaller “seeds.” We previously identified 11 proteins with Q/N-rich domains that, when overproduced, facilitate the de novo aggregation of the Sup35 protein into the [PSI +] prion state. Here, we show that overexpression of many of the same 11 Q/N-rich proteins can also destabilize pre-existing [PSI +] or [URE3] prions. We explore in detail the events leading to the loss (curing) of [PSI+] by the overexpression of one of these proteins, the Q/N-rich domain of Pin4, which causes Sup35 aggregates to increase in size and decrease in transmissibility to daughter cells. We show that the Pin4 Q/N-rich domain sequesters Hsp104 and Sis1 chaperones away from the diffuse cytoplasmic pool. Thus, a mechanism by which heterologous Q/N-rich proteins impair prion propagation appears to be the loss of cytoplasmic Hsp104 and Sis1 available to sever [PSI +]. PMID:23358669

Mutants of the Paf1 Complex Alter Phenotypic Expression of the Yeast Prion [PSI+

PubMed Central

Strawn, Lisa A.; Lin, Changyi A.; Tank, Elizabeth M.H.; Osman, Morwan M.; Simpson, Sarah A.

2009-01-01

The yeast [PSI+] prion is an epigenetic modifier of translation termination fidelity that causes nonsense suppression. The prion [PSI+] forms when the translation termination factor Sup35p adopts a self-propagating conformation. The presence of the [PSI+] prion modulates survivability in a variety of growth conditions. Nonsense suppression is essential for many [PSI+]-mediated phenotypes, but many do not appear to be due to read-through of a single stop codon, but instead are multigenic traits. We hypothesized that other global mechanisms act in concert with [PSI+] to influence [PSI+]-mediated phenotypes. We have identified one such global regulator, the Paf1 complex (Paf1C). Paf1C is conserved in eukaryotes and has been implicated in several aspects of transcriptional and posttranscriptional regulation. Mutations in Ctr9p and other Paf1C components reduced [PSI+]-mediated nonsense suppression. The CTR9 deletion also alters nonsense suppression afforded by other genetic mutations but not always to the same extent as the effects on [PSI+]-mediated read-through. Our data suggest that the Paf1 complex influences mRNA translatability but not solely through changes in transcript stability or abundance. Finally, we demonstrate that the CTR9 deletion alters several [PSI+]-dependent phenotypes. This provides one example of how [PSI+] and genetic modifiers can interact to uncover and regulate phenotypic variability. PMID:19225160

Prions, From Structure to Epigenetics and Neuronal Functions

NASA Astrophysics Data System (ADS)

Lindquist, Susan

2012-02-01

Prions are a unique type of protein that can misfold and convert other proteins to the same shape. The well-characterized yeast prion [PSI+] is formed from an inactive amyloid fiber conformation of the translation-termination factor, Sup35. This altered conformation is passed from mother cells to daughters, acting as a template to perpetuate the prion state and providing a mechanism of protein-based inheritance. We employed a variety of methods to determine the structure of Sup35 amyloid fibrils. First, using fluorescent tags and cross-linking we identified specific segments of the protein monomer that form intermolecular contacts in a ``Head-to-Head,'' ``Tail-to-Tail'' fashion while a central region forms intramolecular contacts. Then, using peptide arrays we mapped the region responsible for the prion transmission barrier between two different yeast species. We have also used optical tweezers to reveal that the non-covalent intermolecular contacts between monomers are unusually strong, and maintain fibril integrity even under forces that partially unfold individual monomers and extend fibril length. Based on the handful of known yeast prion proteins we predicted sequences that could be responsible for prion-like amyloid folding. Our screen identified 19 new candidate prions, whose protein-folding properties and diverse cellular functions we have characterized using a combination of genetic and biochemical techniques. Prion-driven phenotypic diversity increases under stress, and can be amplified by the dynamic maturation of prion-initiating states. These qualities allow prions to act as ``bet-hedging'' devices that facilitate the adaptation of yeast to stressful environments, and might speed the evolution of new traits. Together with Kandel and Si, we have also found that a regulatory protein that plays an important role in synaptic plasticity behaves as a prion in yeast. Cytoplasmic polyAdenylation element binding protein, CPEB, maintains synapses by promoting the local translation of mRNAs. We postulate that the self-perpetuating folding of the prion domain acts as a molecular memory. Thus yeast prions have provided evidence for the surprising possibility that amyloid protein folds can serve as the basis for memory and inheritance.

Amyloid cores in prion domains: Key regulators for prion conformational conversion.

PubMed

Fernández, María Rosario; Batlle, Cristina; Gil-García, Marcos; Ventura, Salvador

2017-01-02

Despite the significant efforts devoted to decipher the particular protein features that encode for a prion or prion-like behavior, they are still poorly understood. The well-characterized yeast prions constitute an ideal model system to address this question, because, in these proteins, the prion activity can be univocally assigned to a specific region of their sequence, known as the prion forming domain (PFD). These PFDs are intrinsically disordered, relatively long and, in many cases, of low complexity, being enriched in glutamine/asparagine residues. Computational analyses have identified a significant number of proteins having similar domains in the human proteome. The compositional bias of these regions plays an important role in the transition of the prions to the amyloid state. However, it is difficult to explain how composition alone can account for the formation of specific contacts that position correctly PFDs and provide the enthalpic force to compensate for the large entropic cost of immobilizing these domains in the initial assemblies. We have hypothesized that short, sequence-specific, amyloid cores embedded in PFDs can perform these functions and, accordingly, act as preferential nucleation centers in both spontaneous and seeded aggregation. We have shown that the implementation of this concept in a prediction algorithm allows to score the prion propensities of putative PFDs with high accuracy. Recently, we have provided experimental evidence for the existence of such amyloid cores in the PFDs of Sup35, Ure2, Swi1, and Mot3 yeast prions. The fibrils formed by these short stretches may recognize and promote the aggregation of the complete proteins inside cells, being thus a promising tool for targeted protein inactivation.

More than just trash bins? Potential roles for extracellular vesicles in the vertical and horizontal transmission of yeast prions.

PubMed

Kabani, Mehdi; Melki, Ronald

2016-05-01

In the yeast Saccharomyces cerevisiae, an ensemble of structurally and functionally diverse cytoplasmic proteins has the ability to form self-perpetuating protein aggregates (e.g. prions) which are the vectors of heritable non-Mendelian phenotypic traits. Whether harboring these prions is deleterious-akin to mammalian degenerative disorders-or beneficial-as epigenetic modifiers of gene expression-for yeasts has been intensely debated and strong arguments were made in support of both views. We recently reported that the yeast prion protein Sup35p is exported via extracellular vesicles (EV), both in its soluble and aggregated infectious states. Herein, we discuss the possible implications of this observation and propose several hypotheses regarding the roles of EV in both vertical and horizontal propagation of 'good' and 'bad' yeast prions.

Kinetic and Stochastic Models of 1D yeast ``prions"

NASA Astrophysics Data System (ADS)

Kunes, Kay

2005-03-01

Mammalian prion proteins (PrP) are of public health interest because of mad cow and chronic wasting diseases. Yeasts have proteins, which can undergo similar reconformation and aggregation processes to PrP; yeast ``prions" are simpler to experimentally study and model. Recent in vitro studies of the SUP35 protein (1), showed long aggregates and pure exponential growth of the misfolded form. To explain this data, we have extended a previous model of aggregation kinetics along with our own stochastic approach (2). Both models assume reconformation only upon aggregation, and include aggregate fissioning and an initial nucleation barrier. We find for sufficiently small nucleation rates or seeding by small dimer concentrations that we can achieve the requisite exponential growth and long aggregates.

Destabilization and recovery of a yeast prion after mild heat shock

PubMed Central

Newnam, Gary P.; Birchmore, Jennifer L.; Chernoff, Yury O.

2011-01-01

Yeast prion [PSI+] is a self-perpetuating amyloid of the translational termination factor Sup35. Although [PSI+] propagation is modulated by heat shock proteins (Hsps), high temperature was previously reported to have little or no effect on [PSI+]. Our results show that short-term exposure of exponentially growing yeast culture to mild heat shock, followed by immediate resumption of growth, leads to [PSI+] destabilization, sometimes persisting for several cell divisions after heat shock. Prion loss occurring in the first division after heat shock is preferentially detected in a daughter cell, indicating the impairment of prion segregation that results in asymmetric prion distribution between a mother cell and a bud. Longer heat shock or prolonged incubation in the absence of nutrients after heat shock lead to [PSI+] recovery. Both prion destabilization and recovery during heat shock depend on protein synthesis. Maximal prion destabilization coincides with maximal imbalance between Hsp104 and other Hsps such as Hsp70-Ssa. Deletions of individual SSA genes increase prion destabilization and/or counteract recovery. Dynamics of prion aggregation during destabilization and recovery is consistent with the notion that efficient prion fragmentation and segregation require a proper balance between Hsp104 and other (e. g. Hsp70-Ssa) chaperones. In contrast to heat shock, [PSI+] destabilization by osmotic stressors does not always depend on cell proliferation and/or protein synthesis, indicating that different stresses may impact the prion via different mechanisms. Our data demonstrate that heat stress causes asymmetric prion distribution in a cell division, and confirm that effects of Hsps on prions are physiologically relevant. PMID:21392508

A comparative molecular dynamics study on thermostability of human and chicken prion proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ji, Hong-Fang; Zhang, Hong-Yu

To compare the thermostabilities of human and chicken normal cellular prion proteins (HuPrP{sup C} and CkPrP{sup C}), molecular dynamics (MD) simulations were performed for both proteins at an ensemble level (10 parallel simulations at 400 K and 5 parallel simulations at 300 K as a control). It is found that the thermostability of HuPrP{sup C} is comparable with that of CkPrP{sup C}, which implicates that the non-occurrence of prion diseases in non-mammals cannot be completely attributed to the thermodynamic properties of non-mammalian PrP{sup C}.

Destabilization and recovery of a yeast prion after mild heat shock.

PubMed

Newnam, Gary P; Birchmore, Jennifer L; Chernoff, Yury O

2011-05-06

Yeast prion [PSI(+)] is a self-perpetuating amyloid of the translational termination factor Sup35. Although [PSI(+)] propagation is modulated by heat shock proteins (Hsps), high temperature was previously reported to have little or no effect on [PSI(+)]. Our results show that short-term exposure of exponentially growing yeast culture to mild heat shock, followed by immediate resumption of growth, leads to [PSI(+)] destabilization, sometimes persisting for several cell divisions after heat shock. Prion loss occurring in the first division after heat shock is preferentially detected in a daughter cell, indicating the impairment of prion segregation that results in asymmetric prion distribution between a mother cell and a bud. Longer heat shock or prolonged incubation in the absence of nutrients after heat shock led to [PSI(+)] recovery. Both prion destabilization and recovery during heat shock depend on protein synthesis. Maximal prion destabilization coincides with maximal imbalance between Hsp104 and other Hsps such as Hsp70-Ssa. Deletions of individual SSA genes increase prion destabilization and/or counteract recovery. The dynamics of prion aggregation during destabilization and recovery are consistent with the notion that efficient prion fragmentation and segregation require a proper balance between Hsp104 and other (e.g., Hsp70-Ssa) chaperones. In contrast to heat shock, [PSI(+)] destabilization by osmotic stressors does not always depend on cell proliferation and/or protein synthesis, indicating that different stresses may impact the prion via different mechanisms. Our data demonstrate that heat stress causes asymmetric prion distribution in a cell division and confirm that the effects of Hsps on prions are physiologically relevant. Copyright © 2011 Elsevier Ltd. All rights reserved.

Kinetic Model for 1D aggregation of yeast ``prions''

NASA Astrophysics Data System (ADS)

Kunes, Kay; Cox, Daniel; Singh, Rajiv

2004-03-01

Mammalian prion proteins (PrP) are of public health interest because of mad cow and chronic wasting diseases. Yeast have proteins which can undergo similar reconformation and aggregation processes to PrP; yeast forms are simpler to experimentally study and model. Recent in vitro studies of the SUP35 protein(1), showed long aggregates and pure exponential growth of the misfolded form. To explain this data, we have extended a previous model of aggregation kinetics(2). The model assumes reconformation only upon aggregation, and includes aggregate fissioning and an initial nucleation barrier. We find for sufficiently small nucleation rates or seeding by small dimer concentrations that we can achieve the requisite exponential growth and long aggregates. We will compare to a more realistic stochastic kinetics model and present prelimary attempts to describe recent experiments on SUP35 strains. *-Supported by U.S. Army Congressionally Mandated Research Fund. 1) P. Chien and J.S. Weissman, Nature 410, 223 (2001); http://online.kitp.ucsb.edu/online/bionet03/collins/. 2) J. Masel, V.A.> Jansen, M.A. Nowak, Biophys. Chem. 77, 139 (1999).

Prions on the run: How extracellular vesicles serve as delivery vehicles for self-templating protein aggregates.

PubMed

Liu, Shu; Hossinger, André; Göbbels, Sarah; Vorberg, Ina M

2017-03-04

Extracellular vesicles (EVs) are actively secreted, membrane-bound communication vehicles that exchange biomolecules between cells. EVs also serve as dissemination vehicles for pathogens, including prions, proteinaceous infectious agents that cause transmissible spongiform encephalopathies (TSEs) in mammals. Increasing evidence accumulates that diverse protein aggregates associated with common neurodegenerative diseases are packaged into EVs as well. Vesicle-mediated intercellular transmission of protein aggregates can induce aggregation of homotypic proteins in acceptor cells and might thereby contribute to disease progression. Our knowledge of how protein aggregates are sorted into EVs and how these vesicles adhere to and fuse with target cells is limited. Here we review how TSE prions exploit EVs for intercellular transmission and compare this to the transmission behavior of self-templating cytosolic protein aggregates derived from the yeast prion domain Sup 35 NM. Artificial NM prions are non-toxic to mammalian cell cultures and do not cause loss-of-function phenotypes. Importantly, NM particles are also secreted in association with exosomes that horizontally transmit the prion phenotype to naive bystander cells, a process that can be monitored with high accuracy by automated high throughput confocal microscopy. The high abundance of mammalian proteins with amino acid stretches compositionally similar to yeast prion domains makes the NM cell model an attractive model to study self-templating and dissemination properties of proteins with prion-like domains in the mammalian context.

Computational methods for yeast prion curing curves.

PubMed

Ridout, Martin S

2008-10-01

If the chemical guanidine hydrochloride is added to a dividing culture of yeast cells in which some of the protein Sup35p is in its prion form, the proportion of cells that carry replicating units of the prion, termed propagons, decreases gradually over time. Stochastic models to describe this process of 'curing' have been developed in earlier work. The present paper investigates the use of numerical methods of Laplace transform inversion to calculate curing curves and contrasts this with an alternative, more direct, approach that involves numerical integration. Transform inversion is found to provide a much more efficient computational approach that allows different models to be investigated with minimal programming effort. The method is used to investigate the robustness of the curing curve to changes in the assumed distribution of cell generation times. Matlab code is available for carrying out the calculations.

Mass analysis by scanning transmission electron microscopy and electron diffraction validate predictions of stacked beta-solenoid model of HET-s prion fibrils.

PubMed

Sen, Anindito; Baxa, Ulrich; Simon, Martha N; Wall, Joseph S; Sabate, Raimon; Saupe, Sven J; Steven, Alasdair C

2007-02-23

Fungal prions are infectious filamentous polymers of proteins that are soluble in uninfected cells. In its prion form, the HET-s protein of Podospora anserina participates in a fungal self/non-self recognition phenomenon called heterokaryon incompatibility. Like other prion proteins, HET-s has a so-called "prion domain" (its C-terminal region, HET-s-(218-289)) that is responsible for induction and propagation of the prion in vivo and for fibril formation in vitro. Prion fibrils are thought to have amyloid backbones of polymerized prion domains. A relatively detailed model has been proposed for prion domain fibrils of HET-s based on a variety of experimental constraints (Ritter, C., Maddelein, M. L., Siemer, A. B., Luhrs, T., Ernst, M., Meier, B. H., Saupe, S. J., and Riek, R. (2005) Nature 435, 844-848). To test specific predictions of this model, which envisages axial stacking of beta-solenoids with two coils per subunit, we examined fibrils by electron microscopy. Electron diffraction gave a prominent meridional reflection at (0.47 nm)(-1), indicative of cross-beta structure, as predicted. STEM (scanning transmission electron microscopy) mass-per-unit-length measurements yielded 1.02 +/- 0.16 subunits per 0.94 nm, in agreement with the model prediction (1 subunit per 0.94 nm). This is half the packing density of approximately 1 subunit per 0.47 nm previously obtained for fibrils of the yeast prion proteins, Ure2p and Sup35p, whence it follows that the respective amyloid architectures are basically different.

Yeast prion architecture explains how proteins can be genes

NASA Astrophysics Data System (ADS)

Wickner, Reed

2013-03-01

Prions (infectious proteins) transmit information without an accompanying DNA or RNA. Most yeast prions are self-propagating amyloids that inactivate a normally functional protein. A single protein can become any of several prion variants, with different manifestations due to different amyloid structures. We showed that the yeast prion amyloids of Ure2p, Sup35p and Rnq1p are folded in-register parallel beta sheets using solid state NMR dipolar recoupling experiments, mass-per-filament-length measurements, and filament diameter measurements. The extent of beta sheet structure, measured by chemical shifts in solid-state NMR and acquired protease-resistance on amyloid formation, combined with the measured filament diameters, imply that the beta sheets must be folded along the long axis of the filament. We speculate that prion variants of a single protein sequence differ in the location of these folds. Favorable interactions between identical side chains must hold these structures in-register. The same interactions must guide an unstructured monomer joining the end of a filament to assume the same conformation as molecules already in the filament, with the turns at the same locations. In this way, a protein can template its own conformation, in analogy to the ability of a DNA molecule to template its sequence by specific base-pairing. Bldg. 8, Room 225, NIH, 8 Center Drive MSC 0830, Bethesda, MD 20892-0830, wickner@helix.nih.gov, 301-496-3452

Exploring the role of hydration and confinement in the aggregation of amyloidogenic peptides Aβ16-22 and Sup357-13 in AOT reverse micelles

NASA Astrophysics Data System (ADS)

Martinez, Anna Victoria; Małolepsza, Edyta; Rivera, Eva; Lu, Qing; Straub, John E.

2014-12-01

Knowledge of how intermolecular interactions of amyloid-forming proteins cause protein aggregation and how those interactions are affected by sequence and solution conditions is essential to our understanding of the onset of many degenerative diseases. Of particular interest is the aggregation of the amyloid-β (Aβ) peptide, linked to Alzheimer's disease, and the aggregation of the Sup35 yeast prion peptide, which resembles the mammalian prion protein linked to spongiform encephalopathies. To facilitate the study of these important peptides, experimentalists have identified small peptide congeners of the full-length proteins that exhibit amyloidogenic behavior, including the KLVFFAE sub-sequence, Aβ16-22, and the GNNQQNY subsequence, Sup357-13. In this study, molecular dynamics simulations were used to examine these peptide fragments encapsulated in reverse micelles (RMs) in order to identify the fundamental principles that govern how sequence and solution environment influence peptide aggregation. Aβ16-22 and Sup357-13 are observed to organize into anti-parallel and parallel β-sheet arrangements. Confinement in the sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelles is shown to stabilize extended peptide conformations and enhance peptide aggregation. Substantial fluctuations in the reverse micelle shape are observed, in agreement with earlier studies. Shape fluctuations are found to facilitate peptide solvation through interactions between the peptide and AOT surfactant, including direct interaction between non-polar peptide residues and the aliphatic surfactant tails. Computed amide I IR spectra are compared with experimental spectra and found to reflect changes in the peptide structures induced by confinement in the RM environment. Furthermore, examination of the rotational anisotropy decay of water in the RM demonstrates that the water dynamics are sensitive to the presence of peptide as well as the peptide sequence. Overall, our results demonstrate that the RM is a complex confining environment where substantial direct interaction between the surfactant and peptides plays an important role in determining the resulting ensemble of peptide conformations. By extension the results suggest that similarly complex sequence-dependent interactions may determine conformational ensembles of amyloid-forming peptides in a cellular environment.

Extracellular environment modulates the formation and propagation of particular amyloid structures

PubMed Central

Westergard, Laura; True, Heather L.

2016-01-01

Summary Amyloidogenic proteins, including prions, assemble into multiple forms of structurally distinct fibres. The [PSI+] prion, endogenous to the yeast Saccharomyces cerevisiae, is a dominantly inherited, epigenetic modifier of phenotypes. [PSI+] formation relies on the coexistence of another prion, [RNQ+]. Here, in order to better define the role of amyloid diversity on cellular phenotypes, we investigated how physiological and environmental changes impact the generation and propagation of diverse protein conformations from a single polypeptide. Utilizing the yeast model system, we defined extracellular factors that influence the formation of a spectrum of alternative self-propagating amyloid structures of the Sup35 protein, called [PSI+] variants. Strikingly, exposure to specific stressful environments dramatically altered the variants of [PSI+] that formed de novo. Additionally, we found that stress also influenced the association between the [PSI+] and [RNQ+] prions in a way that it superceded their typical relationship. Furthermore, changing the growth environment modified both the biochemical properties and [PSI+]-inducing capabilities of the [RNQ+] template. These data suggest that the cellular environment contributes to both the generation and the selective propagation of specific amyloid structures, providing insight into a key feature that impacts phenotypic diversity in yeast and the cross-species transmission barriers characteristic of prion diseases. PMID:24628771

Single-particle tracking of quantum dot-conjugated prion proteins inside yeast cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsuji, Toshikazu; Kawai-Noma, Shigeko; Pack, Chan-Gi

2011-02-25

Research highlights: {yields} We develop a method to track a quantum dot-conjugated protein in yeast cells. {yields} We incorporate the conjugated quantum dot proteins into yeast spheroplasts. {yields} We track the motions by conventional or 3D tracking microscopy. -- Abstract: Yeast is a model eukaryote with a variety of biological resources. Here we developed a method to track a quantum dot (QD)-conjugated protein in the budding yeast Saccharomyces cerevisiae. We chemically conjugated QDs with the yeast prion Sup35, incorporated them into yeast spheroplasts, and tracked the motions by conventional two-dimensional or three-dimensional tracking microscopy. The method paves the way towardmore » the individual tracking of proteins of interest inside living yeast cells.« less

Hsp31 Is a Stress Response Chaperone That Intervenes in the Protein Misfolding Process*

PubMed Central

Tsai, Chai-jui; Aslam, Kiran; Drendel, Holli M.; Asiago, Josephat M.; Goode, Kourtney M.; Paul, Lake N.; Rochet, Jean-Christophe; Hazbun, Tony R.

2015-01-01

The Saccharomyces cerevisiae heat shock protein Hsp31 is a stress-inducible homodimeric protein that is involved in diauxic shift reprogramming and has glyoxalase activity. We show that substoichiometric concentrations of Hsp31 can abrogate aggregation of a broad array of substrates in vitro. Hsp31 also modulates the aggregation of α-synuclein (αSyn), a target of the chaperone activity of human DJ-1, an Hsp31 homolog. We demonstrate that Hsp31 is able to suppress the in vitro fibrillization or aggregation of αSyn, citrate synthase and insulin. Chaperone activity was also observed in vivo because constitutive overexpression of Hsp31 reduced the incidence of αSyn cytoplasmic foci, and yeast cells were rescued from αSyn-generated proteotoxicity upon Hsp31 overexpression. Moreover, we showed that Hsp31 protein levels are increased by H2O2, in the diauxic phase of normal growth conditions, and in cells under αSyn-mediated proteotoxic stress. We show that Hsp31 chaperone activity and not the methylglyoxalase activity or the autophagy pathway drives the protective effects. We also demonstrate reduced aggregation of the Sup35 prion domain, PrD-Sup35, as visualized by fluorescent protein fusions. In addition, Hsp31 acts on its substrates prior to the formation of large aggregates because Hsp31 does not mutually localize with prion aggregates, and it prevents the formation of detectable in vitro αSyn fibrils. These studies establish that the protective role of Hsp31 against cellular stress is achieved by chaperone activity that intervenes early in the protein misfolding process and is effective on a wide spectrum of substrate proteins, including αSyn and prion proteins. PMID:26306045

Animal models for testing anti-prion drugs.

PubMed

Fernández-Borges, Natalia; Elezgarai, Saioa R; Eraña, Hasier; Castilla, Joaquín

2013-01-01

Prion diseases belong to a group of fatal infectious diseases with no effective therapies available. Throughout the last 35 years, less than 50 different drugs have been tested in different experimental animal models without hopeful results. An important limitation when searching for new drugs is the existence of appropriate models of the disease. The three different possible origins of prion diseases require the existence of different animal models for testing anti-prion compounds. Wild type, over-expressing transgenic mice and other more sophisticated animal models have been used to evaluate a diversity of compounds which some of them were previously tested in different in vitro experimental models. The complexity of prion diseases will require more pre-screening studies, reliable sporadic (or spontaneous) animal models and accurate chemical modifications of the selected compounds before having an effective therapy against human prion diseases. This review is intended to put on display the more relevant animal models that have been used in the search of new antiprion therapies and describe some possible procedures when handling chemical compounds presumed to have anti-prion activity prior to testing them in animal models.

Ssb1 chaperone is a [PSI+] prion-curing factor.

PubMed

Chacinska, A; Szczesniak, B; Kochneva-Pervukhova, N V; Kushnirov, V V; Ter-Avanesyan, M D; Boguta, M

2001-04-01

Yeast SUP7' or SUP11 nonsense suppressors have no phenotypic expression in strains deficient in the isopentenylation of A37 in tRNA. Here we show that such strains spontaneously produce cells with a nonsense suppressor phenotype which is related to the cytoplasmically inherited determinant and manifests all the key features of the [PSI+] prion. A screen of a multicopy yeast genomic library for genes that inactivate the [PSI+]-related suppressor phenotype resulted in the isolation of the SSB1 gene. Moreover, we demonstrate that multicopy plasmid encoding the Ssb1 chaperone cures cells of the [PSI+] prion.

Prion protein induced signaling cascades in monocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krebs, Bjarne; Dorner-Ciossek, Cornelia; Schmalzbauer, Ruediger

2006-02-03

Prion proteins play a central role in transmission and pathogenesis of transmissible spongiform encephalopathies. The cellular prion protein (PrP{sup C}), whose physiological function remains elusive, is anchored to the surface of a variety of cell types including neurons and cells of the lymphoreticular system. In this study, we investigated the response of a mouse monocyte/macrophage cell line to exposure with PrP{sup C} fusion proteins synthesized with a human Fc-tag. PrP{sup C} fusion proteins showed an attachment to the surface of monocyte/macrophages in nanomolar concentrations. This was accompanied by an increase of cellular tyrosine phosphorylation as a result of activated signalingmore » pathways. Detailed investigations exhibited activation of downstream pathways through a stimulation with PrP fusion proteins, which include phosphorylation of ERK{sub 1,2} and Akt kinase. Macrophages opsonize and present antigenic structures, contact lymphocytes, and deliver cytokines. The findings reported here may become the basis of understanding the molecular function of PrP{sup C} in monocytes and macrophages.« less

Ab initio structure determination from prion nanocrystals at atomic resolution by MicroED

PubMed Central

Sawaya, Michael R.; Rodriguez, Jose; Cascio, Duilio; Collazo, Michael J.; Shi, Dan; Reyes, Francis E.; Gonen, Tamir; Eisenberg, David S.

2016-01-01

Electrons, because of their strong interaction with matter, produce high-resolution diffraction patterns from tiny 3D crystals only a few hundred nanometers thick in a frozen-hydrated state. This discovery offers the prospect of facile structure determination of complex biological macromolecules, which cannot be coaxed to form crystals large enough for conventional crystallography or cannot easily be produced in sufficient quantities. Two potential obstacles stand in the way. The first is a phenomenon known as dynamical scattering, in which multiple scattering events scramble the recorded electron diffraction intensities so that they are no longer informative of the crystallized molecule. The second obstacle is the lack of a proven means of de novo phase determination, as is required if the molecule crystallized is insufficiently similar to one that has been previously determined. We show with four structures of the amyloid core of the Sup35 prion protein that, if the diffraction resolution is high enough, sufficiently accurate phases can be obtained by direct methods with the cryo-EM method microelectron diffraction (MicroED), just as in X-ray diffraction. The success of these four experiments dispels the concern that dynamical scattering is an obstacle to ab initio phasing by MicroED and suggests that structures of novel macromolecules can also be determined by direct methods. PMID:27647903

Binding of anti-prion agents to glycosaminoglycans: Evidence from electronic absorption and circular dichroism spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zsila, Ferenc; Gedeon, Gabor

2006-08-11

The polyanionic glycosaminoglycans (GAGs) are intimately involved in the pathogenesis of protein conformational disorders such as amyloidosis and prion diseases. Several cationic agents are known to exhibit anti-prion activity but their mechanism of action is poorly understood. In this study, UV absorption and circular dichroism (CD) spectroscopic techniques were used to investigate the interaction between heparin and chondroitin-6-sulfate and anti-prion drugs including acridine, quinoline, and phenothiazine derivatives. UV band hypochromism of ({+-})-quinacrine, ({+-})-primaquine, tacrine, quinidine, chlorpromazine, and induced CD spectra of ({+-})-quinacrine upon addition of GAGs provided evidence for the GAG binding of these compounds. The association constants ({approx}10{sup 6}-10{supmore » 7} M{sup -1}) estimated from the UV titration curves show high-affinity drug-heparin interactions. Ionic strength-dependence of the absorption spectra suggested that the interaction between GAGs and the cationic drugs is principally electrostatic in nature. Drug binding differences of heparin and chondroitin-6-sulfate were attributed to their different negative charge density. These results call the attention to the alteration of GAG-prion/GAG-amyloid interactions by which these compounds might exert their anti-prion/anti-amyloidogenic activities.« less

Structural characterization of POM6 Fab and mouse prion protein complex identifies key regions for prions conformational conversion.

PubMed

Baral, Pravas Kumar; Swayampakula, Mridula; Aguzzi, Adriano; James, Michael N G

2018-05-01

Conversion of the cellular prion protein PrP C into its pathogenic isoform PrP S c is the hallmark of prion diseases, fatal neurodegenerative diseases affecting many mammalian species including humans. Anti-prion monoclonal antibodies can arrest the progression of prion diseases by stabilizing the cellular form of the prion protein. Here, we present the crystal structure of the POM6 Fab fragment, in complex with the mouse prion protein (moPrP). The prion epitope of POM6 is in close proximity to the epitope recognized by the purportedly toxic antibody fragment, POM1 Fab also complexed with moPrP. The POM6 Fab recognizes a larger binding interface indicating a likely stronger binding compared to POM1. POM6 and POM1 exhibit distinct biological responses. Structural comparisons of the bound mouse prion proteins from the POM6 Fab:moPrP and POM1 Fab:moPrP complexes reveal several key regions of the prion protein that might be involved in initiating mis-folding events. The structural data of moPrP:POM6 Fab complex are available in the PDB under the accession number www.rcsb.org/pdb/search/structidSearch.do?structureId=6AQ7. © 2018 Federation of European Biochemical Societies.

Ab initio structure determination from prion nanocrystals at atomic resolution by MicroED

DOE PAGES

Sawaya, Michael R.; Rodriguez, Jose; Cascio, Duilio; ...

2016-09-19

Electrons, because of their strong interaction with matter, produce high-resolution diffraction patterns from tiny 3D crystals only a few hundred nanometers thick in a frozen-hydrated state. This discovery offers the prospect of facile structure determination of complex biological macromolecules, which cannot be coaxed to form crystals large enough for conventional crystallography or cannot easily be produced in sufficient quantities. Two potential obstacles stand in the way. The first is a phenomenon known as dynamical scattering, in which multiple scattering events scramble the recorded electron diffraction intensities so that they are no longer informative of the crystallized molecule. The second obstaclemore » is the lack of a proven means of de novo phase determination, as is required if the molecule crystallized is insufficiently similar to one that has been previously determined.We showwith four structures of the amyloid core of the Sup35 prion protein that, if the diffraction resolution is high enough, sufficiently accurate phases can be obtained by direct methods with the cryo-EM method microelectron diffraction (MicroED), just as in X-ray diffraction. The success of these four experiments dispels the concern that dynamical scattering is an obstacle to ab initio phasing by MicroED and suggests that structures of novel macromolecules can also be determined by direct methods.« less

Ab initio structure determination from prion nanocrystals at atomic resolution by MicroED

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sawaya, Michael R.; Rodriguez, Jose; Cascio, Duilio

Electrons, because of their strong interaction with matter, produce high-resolution diffraction patterns from tiny 3D crystals only a few hundred nanometers thick in a frozen-hydrated state. This discovery offers the prospect of facile structure determination of complex biological macromolecules, which cannot be coaxed to form crystals large enough for conventional crystallography or cannot easily be produced in sufficient quantities. Two potential obstacles stand in the way. The first is a phenomenon known as dynamical scattering, in which multiple scattering events scramble the recorded electron diffraction intensities so that they are no longer informative of the crystallized molecule. The second obstaclemore » is the lack of a proven means of de novo phase determination, as is required if the molecule crystallized is insufficiently similar to one that has been previously determined.We showwith four structures of the amyloid core of the Sup35 prion protein that, if the diffraction resolution is high enough, sufficiently accurate phases can be obtained by direct methods with the cryo-EM method microelectron diffraction (MicroED), just as in X-ray diffraction. The success of these four experiments dispels the concern that dynamical scattering is an obstacle to ab initio phasing by MicroED and suggests that structures of novel macromolecules can also be determined by direct methods.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marín-Moreno, Alba; Espinosa, Juan-Carlos; Fernánd

The environment plays a key role in horizontal transmission of prion diseases, since prions are extremely resistant to classical inactivation procedures. In prior work, we observed the high stability of bovine spongiform encephalopathy (BSE) infectivity when these prions were incubated in aqueous media such as phosphate-buffered saline (PBS) or wastewater for nearly nine months. As a continuation of this experiment, the same samples were maintained in PBS or wastewater for five additional years and residual BSE infectivity was assessed in bovine PrP{sup C} transgenic mice. Over this long time period (more than six years), BSE infectivity was reduced by threemore » and one orders of magnitude in wastewater and PBS respectively. To rule out a possible agent specific effect, sheep scrapie prions were subjected to the same experimental protocol, using eight years as the experimental end-point. No significant reduction in scrapie infectivity was observed over the first nine months of wastewater incubation while PBS incubation for eight years only produced a two logarithmic unit reduction in infectivity. By contrast, the dynamics of PrP{sup Res} persistence was different, disappearing progressively over the first year. The long persistence of prion infectivity observed in this study for two different agents provides supporting evidence of the assumed high stability of these agents in aquatic environments and that environmental processes or conventional wastewater treatments with low retention times would have little impact on prion infectivity. These results could have great repercussions in terms of risk assessment and safety for animals and human populations. - Highlights: • Prion infectivity resists long term incubations in aquatic environments. • Infectivity persistence in wastewater is reduced when compared to PBS. • In this study PrPRes fails as a marker for prion detection. • Mice bioassay is the most powerful tool for assessing prion presence. • Wastewater conventional treatment would not eliminate prion infectivity.« less

Truncated forms of the prion protein PrP demonstrate the need for complexity in prion structure.

PubMed

Wan, William; Stöhr, Jan; Kendall, Amy; Stubbs, Gerald

2015-01-01

Self-propagation of aberrant protein folds is the defining characteristic of prions. Knowing the structural basis of self-propagation is essential to understanding prions and their related diseases. Prion rods are amyloid fibrils, but not all amyloids are prions. Prions have been remarkably intractable to structural studies, so many investigators have preferred to work with peptide fragments, particularly in the case of the mammalian prion protein PrP. We compared the structures of a number of fragments of PrP by X-ray fiber diffraction, and found that although all of the peptides adopted amyloid conformations, only the larger fragments adopted conformations that modeled the complexity of self-propagating prions, and even these fragments did not always adopt the PrP structure. It appears that the relatively complex structure of the prion form of PrP is not accessible to short model peptides, and that self-propagation may be tied to a level of structural complexity unobtainable in simple model systems. The larger fragments of PrP, however, are useful to illustrate the phenomenon of deformed templating (heterogeneous seeding), which has important biological consequences.

Truncated forms of the prion protein PrP demonstrate the need for complexity in prion structure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wan, William; Stöhr, Jan; Kendall, Amy

2015-09-01

Self-propagation of aberrant protein folds is the defining characteristic of prions. Knowing the structural basis of self-propagation is essential to understanding prions and their related diseases. Prion rods are amyloid fibrils, but not all amyloids are prions. Prions have been remarkably intractable to structural studies, so many investigators have preferred to work with peptide fragments, particularly in the case of the mammalian prion protein PrP. We compared the structures of a number of fragments of PrP by X-ray fiber diffraction, and found that although all of the peptides adopted amyloid conformations, only the larger fragments adopted conformations that modeled themore » complexity of self-propagating prions, and even these fragments did not always adopt the PrP structure. It appears that the relatively complex structure of the prion form of PrP is not accessible to short model peptides, and that self-propagation may be tied to a level of structural complexity unobtainable in simple model systems. The larger fragments of PrP, however, are useful to illustrate the phenomenon of deformed templating (heterogeneous seeding), which has important biological consequences.« less

STITCHER: Dynamic assembly of likely amyloid and prion β-structures from secondary structure predictions

PubMed Central

Bryan, Allen W; O’Donnell, Charles W; Menke, Matthew; Cowen, Lenore J; Lindquist, Susan; Berger, Bonnie

2012-01-01

The supersecondary structure of amyloids and prions, proteins of intense clinical and biological interest, are difficult to determine by standard experimental or computational means. In addition, significant conformational heterogeneity is known or suspected to exist in many amyloid fibrils. Previous work has demonstrated that probability-based prediction of discrete β-strand pairs can offer insight into these structures. Here, we devise a system of energetic rules that can be used to dynamically assemble these discrete β-strand pairs into complete amyloid β-structures. The STITCHER algorithm progressively ‘stitches’ strand-pairs into full β-sheets based on a novel free-energy model, incorporating experimentally observed amino-acid side-chain stacking contributions, entropic estimates, and steric restrictions for amyloidal parallel β-sheet construction. A dynamic program computes the top 50 structures and returns both the highest scoring structure and a consensus structure taken by polling this list for common discrete elements. Putative structural heterogeneity can be inferred from sequence regions that compose poorly. Predictions show agreement with experimental models of Alzheimer’s amyloid beta peptide and the Podospora anserina Het-s prion. Predictions of the HET-s homolog HET-S also reflect experimental observations of poor amyloid formation. We put forward predicted structures for the yeast prion Sup35, suggesting N-terminal structural stability enabled by tyrosine ladders, and C-terminal heterogeneity. Predictions for the Rnq1 prion and alpha-synuclein are also given, identifying a similar mix of homogenous and heterogeneous secondary structure elements. STITCHER provides novel insight into the energetic basis of amyloid structure, provides accurate structure predictions, and can help guide future experimental studies. Proteins 2012. © 2011 Wiley Periodicals, Inc. PMID:22095906

STITCHER: Dynamic assembly of likely amyloid and prion β-structures from secondary structure predictions.

PubMed

Bryan, Allen W; O'Donnell, Charles W; Menke, Matthew; Cowen, Lenore J; Lindquist, Susan; Berger, Bonnie

2012-02-01

The supersecondary structure of amyloids and prions, proteins of intense clinical and biological interest, are difficult to determine by standard experimental or computational means. In addition, significant conformational heterogeneity is known or suspected to exist in many amyloid fibrils. Previous work has demonstrated that probability-based prediction of discrete β-strand pairs can offer insight into these structures. Here, we devise a system of energetic rules that can be used to dynamically assemble these discrete β-strand pairs into complete amyloid β-structures. The STITCHER algorithm progressively 'stitches' strand-pairs into full β-sheets based on a novel free-energy model, incorporating experimentally observed amino-acid side-chain stacking contributions, entropic estimates, and steric restrictions for amyloidal parallel β-sheet construction. A dynamic program computes the top 50 structures and returns both the highest scoring structure and a consensus structure taken by polling this list for common discrete elements. Putative structural heterogeneity can be inferred from sequence regions that compose poorly. Predictions show agreement with experimental models of Alzheimer's amyloid beta peptide and the Podospora anserina Het-s prion. Predictions of the HET-s homolog HET-S also reflect experimental observations of poor amyloid formation. We put forward predicted structures for the yeast prion Sup35, suggesting N-terminal structural stability enabled by tyrosine ladders, and C-terminal heterogeneity. Predictions for the Rnq1 prion and alpha-synuclein are also given, identifying a similar mix of homogenous and heterogeneous secondary structure elements. STITCHER provides novel insight into the energetic basis of amyloid structure, provides accurate structure predictions, and can help guide future experimental studies. Copyright © 2011 Wiley Periodicals, Inc.

Signaling induced by hop/STI-1 depends on endocytosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Americo, Tatiana A.; Chiarini, Luciana B.; Linden, Rafael

The co-chaperone hop/STI-1 is a ligand of the cell surface prion protein (PrP{sup C}), and their interaction leads to signaling and biological effects. Among these, hop/STI-1 induces proliferation of A172 glioblastoma cells, dependent on both PrP{sup C} and activation of the Erk pathway. We tested whether clathrin-mediated endocytosis affects signaling induced by hop/STI-1. Both hyperosmolarity induced by sucrose and monodansyl-cadaverine blocked Erk activity induced by hop/STI-1, without affecting the high basal Akt activity typical of A172. The endocytosis inhibitors also affected the sub-cellular distribution of phosphorylated Erk, consistent with blockade of the latter's activity. The data indicate that signaling inducedmore » by hop/STI-1 depends on endocytosis. These findings are consistent with a role of sub-cellular trafficking in signal transduction following engagement by PrP{sup C} by ligands such as hop/STI-1, and may help help unravel both the functions of the prion protein, as well as possible loss-of-function components of prion diseases.« less

Prions, prionoid complexes and amyloids: the bad, the good and something in between.

PubMed

Hafner Bratkovič, Iva

2017-04-19

Prions are infectious agents causing transmissible spongiform encephalopathies in humans and animals. These protein-based particles template conformational changes in a host-encoded prion protein to an insoluble self-like conformation. Prions are also present in yeast, where they support protein-based epigenetic inheritance. There is emerging evidence that prion-like (prionoid) particles can support a variety of pathological and beneficial functions. The recent data on the prionoid spread of other pathological amyloids are discussed in light of differences between prions and prion-like aggregates. On the other hand, prion-like action has also been found to support important functions such as memory, and amyloids were shown to have a variety of physiological roles from storage to scaffolding in simple organisms and in humans. Higher-order protein complexes play important roles in signalling. Many death-fold domains can polymerise upon nucleation to enhance sensitivity and induce a robust response. Although these polymers are structurally different from amyloids, some of them are characterised by prionoid activities, such as intercellular spread. The initial activation of these complexes is vital for organismal health, whereas prolonged activation leading to unresolved inflammation underlies autoinflammatory and other diseases. Prionoid complexes play important roles far beyond prion diseases and neurodegeneration.

The complexity and implications of yeast prion domains

PubMed Central

2011-01-01

Prions are infectious proteins with altered conformations converted from otherwise normal host proteins. While there is only one known mammalian prion protein, PrP, a handful of prion proteins have been identified in the yeast Saccharomyces cerevisiae. Yeast prion proteins usually have a defined region called prion domain (PrD) essential for prion properties, which are typically rich in glutamine (Q) and asparagine (N). Despite sharing several common features, individual yeast PrDs are generally intricate and divergent in their compositional characteristics, which potentially implicates their prion phenotypes, such as prion-mediated transcriptional regulations. PMID:22156731

Operational Plasticity Enables Hsp104 to Disaggregate Diverse Amyloid and Non-Amyloid Clients

PubMed Central

DeSantis, Morgan E.; Leung, Eunice H.; Sweeny, Elizabeth A.; Jackrel, Meredith E.; Cushman-Nick, Mimi; Neuhaus-Follini, Alexandra; Vashist, Shilpa; Sochor, Matthew A.; Knight, M. Noelle; Shorter, James

2012-01-01

Summary It is not understood how Hsp104, a hexameric AAA+ ATPase from yeast, disaggregates diverse structures including stress-induced aggregates, prions, and α-synuclein conformers connected to Parkinson disease. Here, we establish that Hsp104 hexamers adapt different mechanisms of intersubunit collaboration to disaggregate stress-induced aggregates versus amyloid. To resolve disordered aggregates, Hsp104 subunits collaborate non-co-operatively via probabilistic substrate binding and ATP hydrolysis. To disaggregate amyloid, several subunits co-operatively engage substrate and hydrolyze ATP. Importantly, Hsp104 variants with impaired intersubunit communication dissolve disordered aggregates but not amyloid. Unexpectedly, prokaryotic ClpB subunits collaborate differently than Hsp104 and couple probabilistic substrate binding to cooperative ATP hydrolysis, which enhances disordered aggregate dissolution but sensitizes ClpB to inhibition and diminishes amyloid disaggregation. Finally, we establish that Hsp104 hexamers deploy more subunits to disaggregate Sup35 prion strains with more stable ‘cross-β’ cores. Thus, operational plasticity enables Hsp104 to robustly dissolve amyloid and non-amyloid clients, which impose distinct mechanical demands. PMID:23141537

Estimating Prion Adsorption Capacity of Soil by BioAssay of Subtracted Infectivity from Complex Solutions (BASICS)

PubMed Central

Wyckoff, A. Christy; Lockwood, Krista L.; Meyerett-Reid, Crystal; Michel, Brady A.; Bender, Heather; VerCauteren, Kurt C.; Zabel, Mark D.

2013-01-01

Prions, the infectious agent of scrapie, chronic wasting disease and other transmissible spongiform encephalopathies, are misfolded proteins that are highly stable and resistant to degradation. Prions are known to associate with clay and other soil components, enhancing their persistence and surprisingly, transmissibility. Currently, few detection and quantification methods exist for prions in soil, hindering an understanding of prion persistence and infectivity in the environment. Variability in apparent infectious titers of prions when bound to soil has complicated attempts to quantify the binding capacity of soil for prion infectivity. Here, we quantify the prion adsorption capacity of whole, sandy loam soil (SLS) typically found in CWD endemic areas in Colorado; and purified montmorillonite clay (Mte), previously shown to bind prions, by BioAssay of Subtracted Infectivity in Complex Solutions (BASICS). We incubated prion positive 10% brain homogenate from terminally sick mice infected with the Rocky Mountain Lab strain of mouse-adapted prions (RML) with 10% SLS or Mte. After 24 hours samples were centrifuged five minutes at 200×g and soil-free supernatant was intracerebrally inoculated into prion susceptible indicator mice. We used the number of days post inoculation to clinical disease to calculate the infectious titer remaining in the supernatant, which we subtracted from the starting titer to determine the infectious prion binding capacity of SLS and Mte. BASICS indicated SLS bound and removed ≥ 95% of infectivity. Mte bound and removed lethal doses (99.98%) of prions from inocula, effectively preventing disease in the mice. Our data reveal significant prion-binding capacity of soil and the utility of BASICS to estimate prion loads and investigate persistence and decomposition in the environment. Additionally, since Mte successfully rescued the mice from prion disease, Mte might be used for remediation and decontamination protocols. PMID:23484043

Estimating prion adsorption capacity of soil by BioAssay of Subtracted Infectivity from Complex Solutions (BASICS).

PubMed

Wyckoff, A Christy; Lockwood, Krista L; Meyerett-Reid, Crystal; Michel, Brady A; Bender, Heather; VerCauteren, Kurt C; Zabel, Mark D

2013-01-01

Prions, the infectious agent of scrapie, chronic wasting disease and other transmissible spongiform encephalopathies, are misfolded proteins that are highly stable and resistant to degradation. Prions are known to associate with clay and other soil components, enhancing their persistence and surprisingly, transmissibility. Currently, few detection and quantification methods exist for prions in soil, hindering an understanding of prion persistence and infectivity in the environment. Variability in apparent infectious titers of prions when bound to soil has complicated attempts to quantify the binding capacity of soil for prion infectivity. Here, we quantify the prion adsorption capacity of whole, sandy loam soil (SLS) typically found in CWD endemic areas in Colorado; and purified montmorillonite clay (Mte), previously shown to bind prions, by BioAssay of Subtracted Infectivity in Complex Solutions (BASICS). We incubated prion positive 10% brain homogenate from terminally sick mice infected with the Rocky Mountain Lab strain of mouse-adapted prions (RML) with 10% SLS or Mte. After 24 hours samples were centrifuged five minutes at 200 × g and soil-free supernatant was intracerebrally inoculated into prion susceptible indicator mice. We used the number of days post inoculation to clinical disease to calculate the infectious titer remaining in the supernatant, which we subtracted from the starting titer to determine the infectious prion binding capacity of SLS and Mte. BASICS indicated SLS bound and removed ≥ 95% of infectivity. Mte bound and removed lethal doses (99.98%) of prions from inocula, effectively preventing disease in the mice. Our data reveal significant prion-binding capacity of soil and the utility of BASICS to estimate prion loads and investigate persistence and decomposition in the environment. Additionally, since Mte successfully rescued the mice from prion disease, Mte might be used for remediation and decontamination protocols.

The many shades of prion strain adaptation.

PubMed

Baskakov, Ilia V

2014-01-01

In several recent studies transmissible prion disease was induced in animals by inoculation with recombinant prion protein amyloid fibrils produced in vitro. Serial transmission of amyloid fibrils gave rise to a new class of prion strains of synthetic origin. Gradual transformation of disease phenotypes and PrP(Sc) properties was observed during serial transmission of synthetic prions, a process that resembled the phenomenon of prion strain adaptation. The current article discusses the remarkable parallels between phenomena of prion strain adaptation that accompanies cross-species transmission and the evolution of synthetic prions occurring within the same host. Two alternative mechanisms underlying prion strain adaptation and synthetic strain evolution are discussed. The current article highlights the complexity of the prion transmission barrier and strain adaptation and proposes that the phenomenon of prion adaptation is more common than previously thought.

Hop/STI1 modulates retinal proliferation and cell death independent of PrP{sup C}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arruda-Carvalho, Maithe; Njaine, Brian; Silveira, Mariana S.

Hop/STI1 is a co-chaperone adaptor protein for Hsp70/Hsp90 complexes. Hop/STI1 is found extracellularly and modulates cell death and differentiation through interaction with the prion protein (PrP{sup C}). Here, we investigated the expression of hop/STI1 and its role upon cell proliferation and cell death in the developing retina. Hop/STI1 is more expressed in developing rat retina than in the mature tissue. Hop/STI1 blocks retinal cell death in the neuroblastic layer (NBL) in a PrP{sup C} dependent manner, but failed to protect ganglion cells against axotomy-induced cell death. An antibody raised against hop/STI1 ({alpha}-STI1) blocked both ganglion cell and NBL cell deathmore » independent of PrP{sup C}. cAMP/PKA, ERK, PI3K and PKC signaling pathways were not involved in these effects. Hop/STI1 treatment reduced proliferation, while {alpha}-STI1 increased proliferation in the developing retina, both independent of PrP{sup C}. We conclude that hop/STI1 can modulate both proliferation and cell death in the developing retina independent of PrP{sup C}.« less

Pyrethroid insecticides and radioligand displacement from the GABA receptor chloride ionophore complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crofton, K.M.; Reiter, L.W.; Mailman, R.B.

1987-01-01

Radioligand binding displacement studies were conducted to determine the effects of Type I and II pyrethroids on /sup 3/H-flunitrazepam (FLU), /sup 3/H-muscimol (MUS), and (/sup 35/S-t-butylbicyclophosphorothionate (TBPS) binding. Competition experiments with /sup 3/H-FLU and /sup 3/H-MUS indicate a lack of competition for binding by the pyrethroids. Type I pyrethroids failed to compete for the binding of (/sup 35/S-TBPS at concentrations as high as 50 pM. Type II pyrethroids inhibited (/sup 35/S-TBPS binding to rat brain synaptosomes with Ki values ranging from 5-10 pM. The data presented suggest that the interaction of Type II pyrethroids with the GABA receptor-ionophore complex ismore » restricted to a site near the TBPS/picrotoxinin binding site.« less

Spontaneous generation of rapidly transmissible prions in transgenic mice expressing wild-type bank vole prion protein.

PubMed

Watts, Joel C; Giles, Kurt; Stöhr, Jan; Oehler, Abby; Bhardwaj, Sumita; Grillo, Sunny K; Patel, Smita; DeArmond, Stephen J; Prusiner, Stanley B

2012-02-28

Currently, there are no animal models of the most common human prion disorder, sporadic Creutzfeldt-Jakob disease (CJD), in which prions are formed spontaneously from wild-type (WT) prion protein (PrP). Interestingly, bank voles (BV) exhibit an unprecedented promiscuity for diverse prion isolates, arguing that bank vole PrP (BVPrP) may be inherently prone to adopting misfolded conformations. Therefore, we constructed transgenic (Tg) mice expressing WT BVPrP. Tg(BVPrP) mice developed spontaneous CNS dysfunction between 108 and 340 d of age and recapitulated the hallmarks of prion disease, including spongiform degeneration, pronounced astrogliosis, and deposition of alternatively folded PrP in the brain. Brain homogenates of ill Tg(BVPrP) mice transmitted disease to Tg(BVPrP) mice in ∼35 d, to Tg mice overexpressing mouse PrP in under 100 d, and to WT mice in ∼185 d. Our studies demonstrate experimentally that WT PrP can spontaneously form infectious prions in vivo. Thus, Tg(BVPrP) mice may be useful for studying the spontaneous formation of prions, and thus may provide insight into the etiology of sporadic CJD.

β-sheet-like formation during the mechanical unfolding of prion protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tao, Weiwei; Cao, Penghui; Park, Harold S., E-mail: parkhs@bu.edu

2015-09-28

Single molecule experiments and simulations have been widely used to characterize the unfolding and folding pathways of different proteins. However, with few exceptions, these tools have not been applied to study prion protein, PrP{sup C}, whose misfolded form PrP{sup Sc} can induce a group of fatal neurodegenerative diseases. Here, we apply novel atomistic modeling based on potential energy surface exploration to study the constant force unfolding of human PrP at time scales inaccessible with standard molecular dynamics. We demonstrate for forces around 100 pN, prion forms a stable, three-stranded β-sheet-like intermediate configuration containing residues 155-214 with a lifetime exceeding hundredsmore » of nanoseconds. A mutant without the disulfide bridge shows lower stability during the unfolding process but still forms the three-stranded structure. The simulations thus not only show the atomistic details of the mechanically induced structural conversion from the native α-helical structure to the β-rich-like form but also lend support to the structural theory that there is a core of the recombinant PrP amyloid, a misfolded form reported to induce transmissible disease, mapping to C-terminal residues ≈160-220.« less

Fungal prion HET-s as a model for structural complexity and self-propagation in prions.

PubMed

Wan, William; Stubbs, Gerald

2014-04-08

The highly ordered and reproducible structure of the fungal prion HET-s makes it an excellent model system for studying the inherent properties of prions, self-propagating infectious proteins that have been implicated in a number of fatal diseases. In particular, the HET-s prion-forming domain readily folds into a relatively complex two-rung β-solenoid amyloid. The faithful self-propagation of this fold involves a diverse array of inter- and intramolecular structural features. These features include a long flexible loop connecting the two rungs, buried polar residues, salt bridges, and asparagine ladders. We have used site-directed mutagenesis and X-ray fiber diffraction to probe the relative importance of these features for the formation of β-solenoid structure, as well as the cumulative effects of multiple mutations. Using fibrillization kinetics and chemical stability assays, we have determined the biophysical effects of our mutations on the assembly and stability of the prion-forming domain. We have found that a diversity of structural features provides a level of redundancy that allows robust folding and stability even in the face of significant sequence alterations and suboptimal environmental conditions. Our findings provide fundamental insights into the structural interactions necessary for self-propagation. Propagation of prion structure seems to require an obligatory level of complexity that may not be reproducible in short peptide models.

Assessing transmissible spongiform encephalopathy species barriers with an in vitro prion protein conversion assay

USGS Publications Warehouse

Johnson, Christopher J.; Carlson, Christina M.; Morawski, Aaron R.; Manthei, Alyson; Cashman, Neil R.

2015-01-01

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitroprion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host’s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host’s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Oligomers of Amyloid β Prevent Physiological Activation of the Cellular Prion Protein-Metabotropic Glutamate Receptor 5 Complex by Glutamate in Alzheimer Disease.

PubMed

Haas, Laura T; Strittmatter, Stephen M

2016-08-12

The dysfunction and loss of synapses in Alzheimer disease are central to dementia symptoms. We have recently demonstrated that pathological Amyloid β oligomer (Aβo) regulates the association between intracellular protein mediators and the synaptic receptor complex composed of cellular prion protein (PrP(C)) and metabotropic glutamate receptor 5 (mGluR5). Here we sought to determine whether Aβo alters the physiological signaling of the PrP(C)-mGluR5 complex upon glutamate activation. We provide evidence that acute exposure to Aβo as well as chronic expression of familial Alzheimer disease mutant transgenes in model mice prevents protein-protein interaction changes of the complex induced by the glutamate analog 3,5-dihydroxyphenylglycine. We further show that 3,5-dihydroxyphenylglycine triggers the phosphorylation and activation of protein-tyrosine kinase 2-β (PTK2B, also referred to as Pyk2) and of calcium/calmodulin-dependent protein kinase II in wild-type brain slices but not in Alzheimer disease transgenic brain slices or wild-type slices incubated with Aβo. This study further distinguishes two separate Aβo-dependent signaling cascades, one dependent on extracellular Ca(2+) and Fyn kinase activation and the other dependent on the release of Ca(2+) from intracellular stores. Thus, Aβo triggers multiple distinct PrP(C)-mGluR5-dependent events implicated in neurodegeneration and dementia. We propose that targeting the PrP(C)-mGluR5 complex will reverse aberrant Aβo-triggered states of the complex to allow physiological fluctuations of glutamate signaling. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ma, Biao; Yamaguchi, Keiichi; Fukuoka, Mayuko

To accelerate the logical drug design procedure, we created the program “NAGARA,” a plugin for PyMOL, and applied it to the discovery of small compounds called medical chaperones (MCs) that stabilize the cellular form of a prion protein (PrP{sup C}). In NAGARA, we constructed a single platform to unify the docking simulation (DS), free energy calculation by molecular dynamics (MD) simulation, and interfragment interaction energy (IFIE) calculation by quantum chemistry (QC) calculation. NAGARA also enables large-scale parallel computing via a convenient graphical user interface. Here, we demonstrated its performance and its broad applicability from drug discovery to lead optimization withmore » full compatibility with various experimental methods including Western blotting (WB) analysis, surface plasmon resonance (SPR), and nuclear magnetic resonance (NMR) measurements. Combining DS and WB, we discovered anti-prion activities for two compounds and tegobuvir (TGV), a non-nucleoside non-structural protein NS5B polymerase inhibitor showing activity against hepatitis C virus genotype 1. Binding profiles predicted by MD and QC are consistent with those obtained by SPR and NMR. Free energy analyses showed that these compounds stabilize the PrP{sup C} conformation by decreasing the conformational fluctuation of the PrP{sup C}. Because TGV has been already approved as a medicine, its extension to prion diseases is straightforward. Finally, we evaluated the affinities of the fragmented regions of TGV using QC and found a clue for its further optimization. By repeating WB, MD, and QC recursively, we were able to obtain the optimum lead structure. - Highlights: • NAGARA integrates docking simulation, molecular dynamics, and quantum chemistry. • We found many compounds, e.g., tegobuvir (TGV), that exhibit anti-prion activities. • We obtained insights into the action mechanism of TGV as a medical chaperone. • Using QC, we obtained useful information for optimization of the lead compound, TGV. • NAGARA is a convenient platform for drug discovery and lead optimization.« less

Prion protein gene analysis in three kindreds with fatal familial insomnia (FFI): Codon 178 mutation and codon 129 polymorphism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Medori, R.; Tritschler, H.J.

1993-10-01

Fatal familial insomnia (FFI) is a disease linked to a GAC(Asp) [yields] AAC(Asn) mutation in codon 178 of the prion protein (PrP) gene. FFI is characterized clinically by untreatable progressive insomnia, dysautonomia, and motor dysfunctions and is characterized pathologically by selective thalamic atrophy. The authors confirmed the 178[sup Asn] mutation in the PrP gene of a third FFI family of French ancestry. Three family members who are under 40 years of age and who inherited the mutation showed only reduced perfusion in the basal ganglia on single photon emission computerized tomography. Some FFI features differ from the clinical and neuropathologicmore » findings associated with 178[sup Asn] reported elsewhere. However, additional intragenic mutations accounting for the phenotypic differences were not observed in two affected individuals. In other sporadic and familial forms of Creutzfeldt-Jakob disease and Gerstmann-Straeussler syndrome, Met or Val homozygosity at polymorphic codon 129 is associated with a more severe phenotype, younger age at onset, and faster progression. In FFI, young and old individuals at disease onset had 129[sup Met/Val]. Moreover, of five 178[sup Asn] individuals who are above age-at-onset range and who are well, two have 129[sup Met] and three have 129[sup Met/Val], suggesting that polymorphic site 129 does not modulate FFI phenotypic expression. Genetic heterogeneity and environment may play an important role in inter- and intrafamilial variability of the 178[sup Asn] mutation. 32 refs., 5 figs., 1 tab.« less

Current and future molecular diagnostics for prion diseases.

PubMed

Lehto, Marty T; Peery, Harry E; Cashman, Neil R

2006-07-01

It is now widely held that the infectious agents underlying the transmissible spongiform encephalopathies are prions, which are primarily composed of a misfolded, protease-resistant isoform of the host prion protein. Untreatable prion disorders include some human diseases, such as Creutzfeldt-Jakob disease, and diseases of economically important animals, such as bovine spongiform encephalopathy (cattle) and chronic wasting disease (deer and elk). Detection and diagnosis of prion disease (and presymptomatic incubation) is contingent upon developing novel assays, which exploit properties uniquely possessed by this misfolded protein complex, rather than targeting an agent-specific nucleic acid. This review highlights some of the conventional and disruptive technologies developed to respond to this challenge.

A novel and rapid method for obtaining high titre intact prion strains from mammalian brain.

PubMed

Wenborn, Adam; Terry, Cassandra; Gros, Nathalie; Joiner, Susan; D'Castro, Laura; Panico, Silvia; Sells, Jessica; Cronier, Sabrina; Linehan, Jacqueline M; Brandner, Sebastian; Saibil, Helen R; Collinge, John; Wadsworth, Jonathan D F

2015-05-07

Mammalian prions exist as multiple strains which produce characteristic and highly reproducible phenotypes in defined hosts. How this strain diversity is encoded by a protein-only agent remains one of the most interesting and challenging questions in biology with wide relevance to understanding other diseases involving the aggregation or polymerisation of misfolded host proteins. Progress in understanding mammalian prion strains has however been severely limited by the complexity and variability of the methods used for their isolation from infected tissue and no high resolution structures have yet been reported. Using high-throughput cell-based prion bioassay to re-examine prion purification from first principles we now report the isolation of prion strains to exceptional levels of purity from small quantities of infected brain and demonstrate faithful retention of biological and biochemical strain properties. The method's effectiveness and simplicity should facilitate its wide application and expedite structural studies of prions.

A novel and rapid method for obtaining high titre intact prion strains from mammalian brain

PubMed Central

Wenborn, Adam; Terry, Cassandra; Gros, Nathalie; Joiner, Susan; D’Castro, Laura; Panico, Silvia; Sells, Jessica; Cronier, Sabrina; Linehan, Jacqueline M.; Brandner, Sebastian; Saibil, Helen R.; Collinge, John; Wadsworth, Jonathan D. F.

2015-01-01

Mammalian prions exist as multiple strains which produce characteristic and highly reproducible phenotypes in defined hosts. How this strain diversity is encoded by a protein-only agent remains one of the most interesting and challenging questions in biology with wide relevance to understanding other diseases involving the aggregation or polymerisation of misfolded host proteins. Progress in understanding mammalian prion strains has however been severely limited by the complexity and variability of the methods used for their isolation from infected tissue and no high resolution structures have yet been reported. Using high-throughput cell-based prion bioassay to re-examine prion purification from first principles we now report the isolation of prion strains to exceptional levels of purity from small quantities of infected brain and demonstrate faithful retention of biological and biochemical strain properties. The method’s effectiveness and simplicity should facilitate its wide application and expedite structural studies of prions. PMID:25950908

Presence of voltage-gated potassium channel complex antibody in a case of genetic prion disease

PubMed Central

Jammoul, Adham; Lederman, Richard J; Tavee, Jinny; Li, Yuebing

2014-01-01

Voltage-gated potassium channel (VGKC) complex antibody-mediated encephalitis is a recently recognised entity which has been reported to mimic the clinical presentation of Creutzfeldt-Jakob disease (CJD). Testing for the presence of this neuronal surface autoantibody in patients presenting with subacute encephalopathy is therefore crucial as it may both revoke the bleak diagnosis of prion disease and allow institution of potentially life-saving immunotherapy. Tempering this optimistic view is the rare instance when a positive VGKC complex antibody titre occurs in a definite case of prion disease. We present a pathologically and genetically confirmed case of CJD with elevated serum VGKC complex antibody titres. This case highlights the importance of interpreting the result of a positive VGKC complex antibody with caution and in the context of the overall clinical manifestation. PMID:24903967

Presence of voltage-gated potassium channel complex antibody in a case of genetic prion disease.

PubMed

Jammoul, Adham; Lederman, Richard J; Tavee, Jinny; Li, Yuebing

2014-06-05

Voltage-gated potassium channel (VGKC) complex antibody-mediated encephalitis is a recently recognised entity which has been reported to mimic the clinical presentation of Creutzfeldt-Jakob disease (CJD). Testing for the presence of this neuronal surface autoantibody in patients presenting with subacute encephalopathy is therefore crucial as it may both revoke the bleak diagnosis of prion disease and allow institution of potentially life-saving immunotherapy. Tempering this optimistic view is the rare instance when a positive VGKC complex antibody titre occurs in a definite case of prion disease. We present a pathologically and genetically confirmed case of CJD with elevated serum VGKC complex antibody titres. This case highlights the importance of interpreting the result of a positive VGKC complex antibody with caution and in the context of the overall clinical manifestation. 2014 BMJ Publishing Group Ltd.

[PSI+] prion propagation is controlled by inositol polyphosphates

PubMed Central

Wickner, Reed B.; Kelly, Amy C.; Bezsonov, Evgeny E.; Edskes, Herman K.

2017-01-01

The yeast prions [PSI+] and [URE3] are folded in-register parallel β-sheet amyloids of Sup35p and Ure2p, respectively. In a screen for antiprion systems curing [PSI+] without protein overproduction, we detected Siw14p as an antiprion element. An array of genetic tests confirmed that many variants of [PSI+] arising in the absence of Siw14p are cured by restoring normal levels of the protein. Siw14p is a pyrophosphatase specifically cleaving the β phosphate from 5-diphosphoinositol pentakisphosphate (5PP-IP5), suggesting that increased levels of this or some other inositol polyphosphate favors [PSI+] propagation. In support of this notion, we found that nearly all variants of [PSI+] isolated in a WT strain were lost upon loss of ARG82, which encodes inositol polyphosphate multikinase. Inactivation of the Arg82p kinase by D131A and K133A mutations (preserving Arg82p’s nonkinase transcription regulation functions) resulted the loss of its ability to support [PSI+] propagation. The loss of [PSI+] in arg82Δ is independent of Hsp104’s antiprion activity. [PSI+] variants requiring Arg82p could propagate in ipk1Δ (IP5 kinase), kcs1Δ (IP6 5-kinase), vip1Δ (IP6 1-kinase), ddp1Δ (inositol pyrophosphatase), or kcs1Δ vip1Δ mutants but not in ipk1Δ kcs1Δ or ddp1Δ kcs1Δ double mutants. Thus, nearly all [PSI+] prion variants require inositol poly-/pyrophosphates for their propagation, and at least IP6 or 5PP-IP4 can support [PSI+] propagation. PMID:28923943

Domain-Specific Activation of Death-Associated Intracellular Signalling Cascades by the Cellular Prion Protein in Neuroblastoma Cells.

PubMed

Vilches, Silvia; Vergara, Cristina; Nicolás, Oriol; Mata, Ágata; Del Río, José A; Gavín, Rosalina

2016-09-01

The biological functions of the cellular prion protein remain poorly understood. In fact, numerous studies have aimed to determine specific functions for the different protein domains. Studies of cellular prion protein (PrP(C)) domains through in vivo expression of molecules carrying internal deletions in a mouse Prnp null background have provided helpful data on the implication of the protein in signalling cascades in affected neurons. Nevertheless, understanding of the mechanisms underlying the neurotoxicity induced by these PrP(C) deleted forms is far from complete. To better define the neurotoxic or neuroprotective potential of PrP(C) N-terminal domains, and to overcome the heterogeneity of results due to the lack of a standardized model, we used neuroblastoma cells to analyse the effects of overexpressing PrP(C) deleted forms. Results indicate that PrP(C) N-terminal deleted forms were properly processed through the secretory pathway. However, PrPΔF35 and PrPΔCD mutants led to death by different mechanisms sharing loss of alpha-cleavage and activation of caspase-3. Our data suggest that both gain-of-function and loss-of-function pathogenic mechanisms may be associated with N-terminal domains and may therefore contribute to neurotoxicity in prion disease. Dissecting the molecular response induced by PrPΔF35 may be the key to unravelling the physiological and pathological functions of the prion protein.

Catalytic hydrogenation using complexes of base metals with tridentate ligands

DOEpatents

Hanson, Susan K.; Zhang, Guoqi; Vasudevan, Kalyan V.

2017-02-14

Complexes of cobalt and nickel with tridentate ligand PNHP.sup.R are effective for hydrogenation of unsaturated compounds. Cobalt complex [(PNHP.sup.Cy)Co(CH.sub.2SiMe.sub.3)]BAr.sup.F.sub.4 (PNHP.sup.Cy=bis[2-(dicyclohexylphosphino)ethyl]amine, BAr.sup.F.sub.4=B(3,5-(CF.sub.3).sub.2C.sub.6H.sub.3).sub.4)) was prepared and used with hydrogen for hydrogenation of alkenes, aldehydes, ketones, and imines under mild conditions (25-60.degree. C., 1-4 atm H.sub.2). Nickel complex [(PNHP.sup.Cy)Ni(H)]BPh.sub.4 was used for hydrogenation of styrene and 1-octene under mild conditions. (PNP.sup.Cy)Ni(H) was used for hydrogenating alkenes.

Catalytic hydrogenation using complexes of base metals with tridentate ligands

DOEpatents

Vasudevan, Kalyan V.; Zhang, Guoqi; Hanson, Susan K.

2016-09-06

Complexes of cobalt and nickel with tridentate ligand PNHP.sup.R are effective for hydrogenation of unsaturated compounds. Cobalt complex [(PNHP.sup.Cy)Co(CH.sub.2SiMe.sub.3)]BAr.sup.F.sub.4 (PNHP.sup.Cy=bis[2-(dicyclohexylphosphino)ethyl]amine, BAr.sup.F.sub.4=B(3,5-(CF.sub.3).sub.2C.sub.6H.sub.3).sub.4)) was prepared and used with hydrogen for hydrogenation of alkenes, aldehydes, ketones, and imines under mild conditions (25-60.degree. C., 1-4 atm H.sub.2). Nickel complex [(PNHP.sup.Cy)Ni(H)]BPh.sub.4 was used for hydrogenation of styrene and 1-octene under mild conditions. (PNP.sup.Cy)Ni(H) was used for hydrogenating alkenes.

Hsp40 function in yeast prion propagation: Amyloid diversity necessitates chaperone functional complexity.

PubMed

Sporn, Zachary A; Hines, Justin K

2015-01-01

Yeast prions are heritable protein-based elements, most of which are formed of amyloid aggregates that rely on the action of molecular chaperones for transmission to progeny. Prions can form distinct amyloid structures, known as 'strains' in mammalian systems, that dictate both pathological progression and cross-species infection barriers. In yeast these same amyloid structural polymorphisms, called 'variants', dictate the intensity of prion-associated phenotypes and stability in mitosis. We recently reported that [PSI(+)] prion variants differ in the fundamental domain requirements for one chaperone, the Hsp40/J-protein Sis1, which are mutually exclusive between 2 different yeast prions, demonstrating a functional plurality for Sis1. Here we extend that analysis to incorporate additional data that collectively support the hypothesis that Sis1 has multiple functional roles that can be accomplished by distinct sets of domains. These functions are differentially required by distinct prions and prion variants. We also present new data regarding Hsp104-mediated prion elimination and show that some Sis1 functions, but not all, are conserved in the human homolog Hdj1/DNAJB1. Importantly, of the 10 amyloid-based prions indentified to date in Saccharomyces cerevisiae, the chaperone requirements of only 4 are known, leaving a great diversity of amyloid structures, and likely modes of amyloid-chaperone interaction, largely unexplored.

Identification of clinical target areas in the brainstem of prion‐infected mice

PubMed Central

Mirabile, Ilaria; Jat, Parmjit S.; Brandner, Sebastian

2015-01-01

Aims While prion infection ultimately involves the entire brain, it has long been thought that the abrupt clinical onset and rapid neurological decline in laboratory rodents relates to involvement of specific critical neuroanatomical target areas. The severity and type of clinical signs, together with the rapid progression, suggest the brainstem as a candidate location for such critical areas. In this study we aimed to correlate prion pathology with clinical phenotype in order to identify clinical target areas. Method We conducted a comprehensive survey of brainstem pathology in mice infected with two distinct prion strains, which produce different patterns of pathology, in mice overexpressing prion protein (with accelerated clinical onset) and in mice in which neuronal expression was reduced by gene targeting (which greatly delays clinical onset). Results We identified specific brainstem areas that are affected by prion pathology during the progression of the disease. In the early phase of disease the locus coeruleus, the nucleus of the solitary tract, and the pre‐Bötzinger complex were affected by prion protein deposition. This was followed by involvement of the motor and autonomic centres of the brainstem. Conclusions Neurodegeneration in the locus coeruleus, the nucleus of the solitary tract and the pre‐Bötzinger complex predominated and corresponded to the manifestation of the clinical phenotype. Because of their fundamental role in controlling autonomic function and the overlap with clinical signs in sporadic Creutzfeldt–Jakob disease, we suggest that these nuclei represent key clinical target areas in prion diseases. PMID:25311251

The mechanisms of humic substances self-assembly with biological molecules: The case study of the prion protein

PubMed Central

Nepravishta, Ridvan; Mandaliti, Walter; Melino, Sonia; Margon, Alja; Scaini, Denis; Mazzei, Pierluigi; Piccolo, Alessandro; Legname, Giuseppe; Paci, Maurizio; Leita, Liviana

2017-01-01

Humic substances (HS) are the largest constituent of soil organic matter and are considered as a key component of the terrestrial ecosystem. HS may facilitate the transport of organic and inorganic molecules, as well as the sorption interactions with environmentally relevant proteins such as prions. Prions enter the environment through shedding from live hosts, facilitating a sustained incidence of animal prion diseases such as Chronic Wasting Disease and scrapie in cervid and ovine populations, respectively. Changes in prion structure upon environmental exposure may be significant as they can affect prion infectivity and disease pathology. Despite its relevance, the mechanisms of prion interaction with HS are still not completely understood. The goal of this work is to advance a structural-level picture of the encapsulation of recombinant, non-infectious, prion protein (PrP) into different natural HS. We observed that PrP precipitation upon addition of HS is mainly driven by a mechanism of “salting-out” whereby PrP molecules are rapidly removed from the solution and aggregate in insoluble adducts with humic molecules. Importantly, this process does not alter the protein folding since insoluble PrP retains its α-helical content when in complex with HS. The observed ability of HS to promote PrP insolubilization without altering its secondary structure may have potential relevance in the context of “prion ecology”. These results suggest that soil organic matter interacts with prions possibly without altering the protein structures. This may facilitate prions preservation from biotic and abiotic degradation leading to their accumulation in the environment. PMID:29161325

Extraction of cesium, strontium and the platinium group metals from acidic high activity nuclear waste using a Purex process compatible organic extractant. Final report, December 15, 1980-August 15, 1983

DOE Office of Scientific and Technical Information (OSTI.GOV)

Davis, M.W. Jr.; Van Brunt, V.

1984-09-14

Purex process compatible organic systems which selectively and reversibly extract cesium, strontium, and palladium from synthetic mixed fission product solutions containing 3M HNO/sub 3/ have been developed. This advance makes the development of continuous solvent extraction processes for their recovery more likely. The most favorable cesium and strontium complexing solutions have been tested for radiation stability to 10/sup 7/ rad using a 0.4 x 10/sup 7/ rad/h /sup 60/Co source. The distribution coefficients dropped somewhat but remained above unity. For cesium the complexing organic solution is 5 vol % (0.1M) NNS, 27 vol % TBP and 68 vol % kerosenemore » containing 0.05m Bis 4,4',(5')(1-hydroxy 2-ethylhexyl)-benzo 18-crown-6 (Crown XVII). The NNS is a sulfonic acid cation exchanger. With an aqueous phase containing 0.006M Cs/sup +1/ in contact with an equal volume of extractant the D org/aq = 1.6 at a temperature of 25 to 35/sup 0/C. For strontium the complexing organic solution is 5 vol % (0.1M) NNS, 27 vol % TBP and 68 vol % Kerosene containing 0.02M Bis 4,4'(5') (1-hydroxyheptyl)cyclohexo 18-crown-6 (Crown XVI). With an aqueous phase containing 0.003M Sr/sup +2/ in contact with an equal volume of extractant the D org/aq = 1.98 at a temperature of 25 to 35/sup 0/C. For palladium the complexing organic solution consisted of a ratio of TBP/kerosene of 0.667 containing 0.3M Alamine 336 which is a tertiary amine anion exchanger. With an aqueous phase containing 0.0045M Pd/sup +/ in contact with an equal volume of extractant the D org/aq = 1.95 at a temperature of 25 to 35/sup 0/C.« less

Extensive Diversity of Prion Strains Is Defined by Differential Chaperone Interactions and Distinct Amyloidogenic Regions

PubMed Central

Stein, Kevin C.; True, Heather L.

2014-01-01

Amyloidogenic proteins associated with a variety of unrelated diseases are typically capable of forming several distinct self-templating conformers. In prion diseases, these different structures, called prion strains (or variants), confer dramatic variation in disease pathology and transmission. Aggregate stability has been found to be a key determinant of the diverse pathological consequences of different prion strains. Yet, it remains largely unclear what other factors might account for the widespread phenotypic variation seen with aggregation-prone proteins. Here, we examined a set of yeast prion variants of the [RNQ+] prion that differ in their ability to induce the formation of another yeast prion called [PSI+]. Remarkably, we found that the [RNQ+] variants require different, non-contiguous regions of the Rnq1 protein for both prion propagation and [PSI+] induction. This included regions outside of the canonical prion-forming domain of Rnq1. Remarkably, such differences did not result in variation in aggregate stability. Our analysis also revealed a striking difference in the ability of these [RNQ+] variants to interact with the chaperone Sis1. Thus, our work shows that the differential influence of various amyloidogenic regions and interactions with host cofactors are critical determinants of the phenotypic consequences of distinct aggregate structures. This helps reveal the complex interdependent factors that influence how a particular amyloid structure may dictate disease pathology and progression. PMID:24811344

Thermodynamics of Protein Aggregation

NASA Astrophysics Data System (ADS)

Osborne, Kenneth L.; Barz, Bogdan; Bachmann, Michael; Strodel, Birgit

Amyloid protein aggregation characterizes many neurodegenerative disorders, including Alzheimer's, Parkinson's, and Creutz- feldt-Jakob disease. Evidence suggests that amyloid aggregates may share similar aggregation pathways, implying simulation of full-length amyloid proteins is not necessary for understanding amyloid formation. In this study we simulate GNNQQNY, the N-terminal prion-determining domain of the yeast protein Sup35 to investigate the thermodynamics of structural transitions during aggregation. We use a coarse-grained model with replica-exchange molecular dynamics to investigate the association of 3-, 6-, and 12-chain GNNQQNY systems and we determine the aggregation pathway by studying aggregation states of GN- NQQNY. We find that the aggregation of the hydrophilic GNNQQNY sequence is mainly driven by H-bond formation, leading to the formation of /3-sheets from the very beginning of the assembly process. Condensation (aggregation) and ordering take place simultaneously, which is underpinned by the occurrence of a single heat capacity peak only.

Synthesis of Mikto-Arm Star Peptide Conjugates.

PubMed

Koo, Jin Mo; Su, Hao; Lin, Yi-An; Cui, Honggang

2018-01-01

Mikto-arm star peptide conjugates are an emerging class of self-assembling peptide-based structural units that contain three or more auxiliary segments of different chemical compositions and/or functionalities. This group of molecules exhibit interesting self-assembly behavior in solution due to their chemically asymmetric topology. Here we describe the detailed procedure for synthesis of an ABC Mikto-arm star peptide conjugate in which two immiscible entities (a saturated hydrocarbon and a hydrophobic and lipophobic fluorocarbon) are conjugated onto a short β-sheet forming peptide sequence, GNNQQNY, derived from the Sup35 prion, through a lysine junction. Automated and manual Fmoc-solid phase synthesis techniques are used to synthesize the Mikto-arm star peptide conjugates, followed by HPLC purification. We envision that this set of protocols can afford a versatile platform to synthesize a new class of peptidic building units for diverse applications.

PrionScan: an online database of predicted prion domains in complete proteomes.

PubMed

Espinosa Angarica, Vladimir; Angulo, Alfonso; Giner, Arturo; Losilla, Guillermo; Ventura, Salvador; Sancho, Javier

2014-02-05

Prions are a particular type of amyloids related to a large variety of important processes in cells, but also responsible for serious diseases in mammals and humans. The number of experimentally characterized prions is still low and corresponds to a handful of examples in microorganisms and mammals. Prion aggregation is mediated by specific protein domains with a remarkable compositional bias towards glutamine/asparagine and against charged residues and prolines. These compositional features have been used to predict new prion proteins in the genomes of different organisms. Despite these efforts, there are only a few available data sources containing prion predictions at a genomic scale. Here we present PrionScan, a new database of predicted prion-like domains in complete proteomes. We have previously developed a predictive methodology to identify and score prionogenic stretches in protein sequences. In the present work, we exploit this approach to scan all the protein sequences in public databases and compile a repository containing relevant information of proteins bearing prion-like domains. The database is updated regularly alongside UniprotKB and in its present version contains approximately 28000 predictions in proteins from different functional categories in more than 3200 organisms from all the taxonomic subdivisions. PrionScan can be used in two different ways: database query and analysis of protein sequences submitted by the users. In the first mode, simple queries allow to retrieve a detailed description of the properties of a defined protein. Queries can also be combined to generate more complex and specific searching patterns. In the second mode, users can submit and analyze their own sequences. It is expected that this database would provide relevant insights on prion functions and regulation from a genome-wide perspective, allowing researches performing cross-species prion biology studies. Our database might also be useful for guiding experimentalists in the identification of new candidates for further experimental characterization.

Double-Edge Sword of Sustained ROCK Activation in Prion Diseases through Neuritogenesis Defects and Prion Accumulation

PubMed Central

Alleaume-Butaux, Aurélie; Nicot, Simon; Pietri, Mathéa; Baudry, Anne; Dakowski, Caroline; Tixador, Philippe; Ardila-Osorio, Hector; Haeberlé, Anne-Marie; Bailly, Yannick; Peyrin, Jean-Michel; Launay, Jean-Marie; Kellermann, Odile; Schneider, Benoit

2015-01-01

In prion diseases, synapse dysfunction, axon retraction and loss of neuronal polarity precede neuronal death. The mechanisms driving such polarization defects, however, remain unclear. Here, we examined the contribution of RhoA-associated coiled-coil containing kinases (ROCK), key players in neuritogenesis, to prion diseases. We found that overactivation of ROCK signaling occurred in neuronal stem cells infected by pathogenic prions (PrPSc) and impaired the sprouting of neurites. In reconstructed networks of mature neurons, PrPSc-induced ROCK overactivation provoked synapse disconnection and dendrite/axon degeneration. This overactivation of ROCK also disturbed overall neurotransmitter-associated functions. Importantly, we demonstrated that beyond its impact on neuronal polarity ROCK overactivity favored the production of PrPSc through a ROCK-dependent control of 3-phosphoinositide-dependent kinase 1 (PDK1) activity. In non-infectious conditions, ROCK and PDK1 associated within a complex and ROCK phosphorylated PDK1, conferring basal activity to PDK1. In prion-infected neurons, exacerbated ROCK activity increased the pool of PDK1 molecules physically interacting with and phosphorylated by ROCK. ROCK-induced PDK1 overstimulation then canceled the neuroprotective α-cleavage of normal cellular prion protein PrPC by TACE α-secretase, which physiologically precludes PrPSc production. In prion-infected cells, inhibition of ROCK rescued neurite sprouting, preserved neuronal architecture, restored neuronal functions and reduced the amount of PrPSc. In mice challenged with prions, inhibition of ROCK also lowered brain PrPSc accumulation, reduced motor impairment and extended survival. We conclude that ROCK overactivation exerts a double detrimental effect in prion diseases by altering neuronal polarity and triggering PrPSc accumulation. Eventually ROCK emerges as therapeutic target to combat prion diseases. PMID:26241960

Does the central dogma still stand?

PubMed

Koonin, Eugene V

2012-08-23

Prions are agents of analog, protein conformation-based inheritance that can confer beneficial phenotypes to cells, especially under stress. Combined with genetic variation, prion-mediated inheritance can be channeled into prion-independent genomic inheritance. Latest screening shows that prions are common, at least in fungi. Thus, there is non-negligible flow of information from proteins to the genome in modern cells, in a direct violation of the Central Dogma of molecular biology. The prion-mediated heredity that violates the Central Dogma appears to be a specific, most radical manifestation of the widespread assimilation of protein (epigenetic) variation into genetic variation. The epigenetic variation precedes and facilitates genetic adaptation through a general 'look-ahead effect' of phenotypic mutations. This direction of the information flow is likely to be one of the important routes of environment-genome interaction and could substantially contribute to the evolution of complex adaptive traits.

Metabotropic glutamate receptor 5 couples cellular prion protein to intracellular signalling in Alzheimer’s disease

PubMed Central

Haas, Laura T.; Salazar, Santiago V.; Kostylev, Mikhail A.; Um, Ji Won; Kaufman, Adam C.

2016-01-01

Alzheimer’s disease-related phenotypes in mice can be rescued by blockade of either cellular prion protein or metabotropic glutamate receptor 5. We sought genetic and biochemical evidence that these proteins function cooperatively as an obligate complex in the brain. We show that cellular prion protein associates via transmembrane metabotropic glutamate receptor 5 with the intracellular protein mediators Homer1b/c, calcium/calmodulin-dependent protein kinase II, and the Alzheimer’s disease risk gene product protein tyrosine kinase 2 beta. Coupling of cellular prion protein to these intracellular proteins is modified by soluble amyloid-β oligomers, by mouse brain Alzheimer’s disease transgenes or by human Alzheimer’s disease pathology. Amyloid-β oligomer-triggered phosphorylation of intracellular protein mediators and impairment of synaptic plasticity in vitro requires Prnp–Grm5 genetic interaction, being absent in transheterozygous loss-of-function, but present in either single heterozygote. Importantly, genetic coupling between Prnp and Grm5 is also responsible for signalling, for survival and for synapse loss in Alzheimer’s disease transgenic model mice. Thus, the interaction between metabotropic glutamate receptor 5 and cellular prion protein has a central role in Alzheimer’s disease pathogenesis, and the complex is a potential target for disease-modifying intervention. PMID:26667279

Prion subcellular fractionation reveals infectivity spectrum, with a high titre-low PrPres level disparity

PubMed Central

2012-01-01

Background Prion disease transmission and pathogenesis are linked to misfolded, typically protease resistant (PrPres) conformers of the normal cellular prion protein (PrPC), with the former posited to be the principal constituent of the infectious 'prion'. Unexplained discrepancies observed between detectable PrPres and infectivity levels exemplify the complexity in deciphering the exact biophysical nature of prions and those host cell factors, if any, which contribute to transmission efficiency. In order to improve our understanding of these important issues, this study utilized a bioassay validated cell culture model of prion infection to investigate discordance between PrPres levels and infectivity titres at a subcellular resolution. Findings Subcellular fractions enriched in lipid rafts or endoplasmic reticulum/mitochondrial marker proteins were equally highly efficient at prion transmission, despite lipid raft fractions containing up to eight times the levels of detectable PrPres. Brain homogenate infectivity was not differentially enhanced by subcellular fraction-specific co-factors, and proteinase K pre-treatment of selected fractions modestly, but equally reduced infectivity. Only lipid raft associated infectivity was enhanced by sonication. Conclusions This study authenticates a subcellular disparity in PrPres and infectivity levels, and eliminates simultaneous divergence of prion strains as the explanation for this phenomenon. On balance, the results align best with the concept that transmission efficiency is influenced more by intrinsic characteristics of the infectious prion, rather than cellular microenvironment conditions or absolute PrPres levels. PMID:22534096

Mono- and binuclear molybdenum and tungsten complexes containing asymmetric bridging ligands: Effects of ligand conjugation and conformation on metal-metal interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Das, A.; Jeffery, J.C.; Maher, J.P.

The authors have prepared the new monodentate ligands 4-(4-methoxyphenyl)pyridine, 1-(4-pyridyl)-2-(4-methoxyphenyl)ethene, 1-(4-pyridyl)-2-(3-methoxyphenyl)ethene, and 1-(3-pyridyl)-2-(4-methoxyphenyl)ethene (L[sup 5]-L[sup 8]); demethylation of the methoxy group in each case afforded the new bridging bidentate ligands HL[sup 1]-HL[sup 4], which contain one pyridyl and one phenolate donor. Attachment of a MoL*(NO)Cl [L* = hydrotris(3,5-dimethylpyrazolyl)borate] moiety to the pyridyl groups of L[sup 5]-L[sup 8] gave the 17-electron complexes [Mo(NO)L*ClL[prime

Exploring Anti-Prion Glyco-Based and Aromatic Scaffolds: A Chemical Strategy for the Quality of Life.

PubMed

Blázquez-Sánchez, María Teresa; de Matos, Ana M; Rauter, Amélia P

2017-05-24

Prion diseases are fatal neurodegenerative disorders caused by protein misfolding and aggregation, affecting the brain progressively and consequently the quality of life. Alzheimer's is also a protein misfolding disease, causing dementia in over 40 million people worldwide. There are no therapeutics able to cure these diseases. Cellular prion protein is a high-affinity binding partner of amyloid β (Aβ) oligomers, the most toxic species in Alzheimer's pathology. These findings motivate the development of new chemicals for a better understanding of the events involved. Disease control is far from being reached by the presently known therapeutics. In this review we describe the synthesis and mode of action of molecular entities with intervention in prion diseases' biological processes and, if known, their role in Alzheimer's. A diversity of structures is covered, based on glycans, steroids and terpenes, heterocycles, polyphenols, most of them embodying aromatics and a structural complexity. These molecules may be regarded as chemical tools to foster the understanding of the complex mechanisms involved, and to encourage the scientific community towards further developments for the cure of these devastating diseases.

Cholesterol Balance in Prion Diseases and Alzheimer’s Disease

PubMed Central

Hannaoui, Samia; Shim, Su Yeon; Cheng, Yo Ching; Corda, Erica; Gilch, Sabine

2014-01-01

Prion diseases are transmissible and fatal neurodegenerative disorders of humans and animals. They are characterized by the accumulation of PrPSc, an aberrantly folded isoform of the cellular prion protein PrPC, in the brains of affected individuals. PrPC is a cell surface glycoprotein attached to the outer leaflet of the plasma membrane by a glycosyl-phosphatidyl-inositol (GPI) anchor. Specifically, it is associated with lipid rafts, membrane microdomains enriched in cholesterol and sphinoglipids. It has been established that inhibition of endogenous cholesterol synthesis disturbs lipid raft association of PrPC and prevents PrPSc accumulation in neuronal cells. Additionally, prion conversion is reduced upon interference with cellular cholesterol uptake, endosomal export, or complexation at the plasma membrane. Altogether, these results demonstrate on the one hand the importance of cholesterol for prion propagation. On the other hand, growing evidence suggests that prion infection modulates neuronal cholesterol metabolism. Similar results were reported in Alzheimer’s disease (AD): whereas amyloid β peptide formation is influenced by cellular cholesterol, levels of cholesterol in the brains of affected individuals increase during the clinical course of the disease. In this review, we summarize commonalities of alterations in cholesterol homeostasis and discuss consequences for neuronal function and therapy of prion diseases and AD. PMID:25419621

Detection of chronic wasting disease prion seeding activity in deer and elk feces by real-time quaking-induced conversion

PubMed Central

Tennant, Joanne M.; Haley, Nicholas J.; Denkers, Nathaniel D.; Mathiason, Candace K.; Hoover, Edward A.

2017-01-01

Chronic wasting disease (CWD) is an emergent prion disease affecting cervid species in North America, Canada, South Korea, and recently, Norway. Detection of CWD has been advanced by techniques that rely on amplification of low levels of prion amyloid to a detectable level. However, the increased sensitivity of amplification assays is often compromised by inhibitors and/or activators in complex biologic samples including body fluids, excreta, or the environment. Here, we adapt real-time quaking-induced conversion conditions to specifically detect CWD prions in fecal samples from both experimentally infected deer and naturally infected elk and estimate environmental contamination. The results have application to detection, surveillance and management of CWD, and potentially to other protein-misfolding diseases. PMID:28703697

Does the central dogma still stand?

PubMed Central

2012-01-01

Abstract Prions are agents of analog, protein conformation-based inheritance that can confer beneficial phenotypes to cells, especially under stress. Combined with genetic variation, prion-mediated inheritance can be channeled into prion-independent genomic inheritance. Latest screening shows that prions are common, at least in fungi. Thus, there is non-negligible flow of information from proteins to the genome in modern cells, in a direct violation of the Central Dogma of molecular biology. The prion-mediated heredity that violates the Central Dogma appears to be a specific, most radical manifestation of the widespread assimilation of protein (epigenetic) variation into genetic variation. The epigenetic variation precedes and facilitates genetic adaptation through a general ‘look-ahead effect’ of phenotypic mutations. This direction of the information flow is likely to be one of the important routes of environment-genome interaction and could substantially contribute to the evolution of complex adaptive traits. Reviewers This article was reviewed by Jerzy Jurka, Pierre Pontarotti and Juergen Brosius. For the complete reviews, see the Reviewers’ Reports section. PMID:22913395

Interaction between a recombinant prion protein and organo-mineral complexes as evidenced by CPMAS 13C-NMR

NASA Astrophysics Data System (ADS)

Russo, F.; Scotti, R.; Gianfreda, L.; Conte, P.; Rao, M. A.

2009-04-01

Prion proteins (PrP) are the main responsible for Transmissible Spongiform Encephalopathies (TSE). The TSE etiological agent is a misfolded form of the normal cellular prion protein. The amyloidal aggregates accumulated in the brain of infected animals and mainly composed of PrPSc exhibit resistance to protease attack and many conventional inactivating procedures. The prion protein diseases cause an environmental issue because the environment and in particular the soil compartment can be contaminated and then become a potential reservoir and diffuser of TSEs infectivity as a consequence of (i) accidental dispersion from storage plants of meat and bone meal, (ii) incorporation of contaminated material in fertilizers, (iii) possible natural contamination of pasture soils by grazing herds, and (v) burial of carcasses. The environmental problem can be even more relevant because very low amounts of PrPSc are able to propagate the disease. Several studies evidenced that infectious prion protein remains active in soils for years. Contaminated soils result, thus, a possible critical route of TSE transmission in wild animals. Soil can also protect prion protein toward degradation processes due to the presence of humic substances and inorganic components such as clays. Mineral and organic colloids and the more common association between clay minerals and humic substances can contribute to the adsorption/entrapment of molecules and macromolecules. The polymerization of organic monomeric humic precursors occurring in soil in the presence of oxidative enzymes or manganese and iron oxides, is considered one of the most important processes contributing to the formation of humic substances. The process is very fast and produces a population of polymeric products of different molecular structures, sizes, shapes and complexity. Other molecules and possibly biomacromolecules such as proteins may be involved. The aim of the present work was to study by CPMAS 13C-NMR the interactions between a non pathogenic ovine recombinant prion protein and a model soil system represented by a manganese oxide in the form of birnessite (δ-MnO2), coated with a polymerized catechol. To better understand the effect of the polymerization process, PrP was added to the birnessite-cathecol system either before or after the polymerization processes. The NMR spectra of the prion protein interacting directly with birnessite revealed disappearance of the signals due to the paramagnetic nature of manganese oxide or abiotic degradation. Conversely, the signal pattern of the protein re-appeared as it is mixed to the soil-like system either during or after the catechol polymerization process. Results suggested that the possible interactions of the prion protein on soil systems can be mediated by natural organic matter. However, deeper studies on more complex real soil systems are needed to definitely confirm such hypothesis.

Relaxometry, luminescence measurements, electrophoresis, and animal biodistribution of lanthanide(III) complexes of some polyaza macrocyclic acetates containing pyridine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, W.D.; Sherry, A.D.; Kiefer, G.E.

Four Gd{sup 3+} complexes [Gd(BP2A){sup +}, Gd(PC2A){sup +}, Gd(PCTA){sup 0}, and Gd(BPO4A){sup {minus}}] with polyazamacrocyclic ligands that contain a pyridine moiety were prepared and examined for possible use as MRI contrast enhancement agents. The authors estimated the number of inner sphere water molecules (q{sub Gd}) for the Gd{sup 3+} complexes from the values of q found for the Tb{sup 3+} and/or Eu{sup 3+} complexes by luminescence lifetime measurements. It was estimated that q{sub Gd} = 3.5, 3.3, 2.4, and 0.2 for Gd(BP2A){sup +}, Gd(PC2A){sup +}, Gd(PCTA){sup 0}, and Gd(BPO4A){sup {minus}}, respectively. The inner sphere relaxivities (r{sub 1,inner}) of these tetraazamore » macrocyclic complexes were higher than that of Gd(DOTA){sup {minus}} [i.e. 6.79 for Gd(BP2A){sup +}, 6.21 for Gd(PC2A){sup +}, and 4.60 for Gd(PCTA){sup 0} mM{sup {minus}1}s{sup {minus}1} at 40 MHz and 25{degrees}C], but the normalized relaxivities per q{sub Gd} (1.94, 1.88, and 1.92 mM{sup {minus}1}s{sup {minus}1}, respectively) were comparable to that of Gd(DOTA){sup {minus}}. A quantitative treatment of the NMRD profiles based on Solomon-Bloembergen-Morgan theory, using the NMRD profile of Gd(BPO4A){sup {minus}} to correct for an outer sphere contribution, showed that the complexes exhibit characteristics similar to that of Gd(DOTA){sup {minus}} but with shorter electronic relaxation times. Tissue biodistribution results using radioactive {sup 153}Sm and {sup 159}Gd complexes in rats indicate that the cationic [{sup 153}Sm-(BP2A){sup +} and {sup 153}Sm(PC2A){sup +}] complexes accumulate preferably in the bone tissue while the neutral [{sup 153}Sm-(PCTA){sup 0}] and anionic [{sup 153}Sm(BPO4A){sup {minus}}] complexes appear to have renal clearances similar to those of other low molecular weight contrast agents [i.e. Gd(DTPA){sup 2{minus}} and Gd(DOTA){sup {minus}}].« less

The NLRP3-Caspase 1 Inflammasome Negatively Regulates Autophagy via TLR4-TRIF in Prion Peptide-Infected Microglia

PubMed Central

Lai, Mengyu; Yao, Hao; Shah, Syed Zahid Ali; Wu, Wei; Wang, Di; Zhao, Ying; Wang, Lu; Zhou, Xiangmei; Zhao, Deming; Yang, Lifeng

2018-01-01

Prion diseases are neurodegenerative disorders characterized by the accumulation of misfolded prion protein, spongiform changes in the brain, and brain inflammation as a result of the wide-spread activation of microglia. Autophagy is a highly conserved catabolic process for the clearance of cytoplasmic components, including protein aggregates and damaged organelles; this process also eliminates pathological PrPSc as it accumulates during prion infection. The NALP3 inflammasome is a multiprotein complex that is a component of the innate immune system and is responsible for the release of pro-inflammatory cytokines. Our previous study showed that the neurotoxic prion peptide PrP106-126 induces NALP3 inflammasome activation and subsequent IL-1β release in microglia. Autophagy is involved in the regulation of the immune responses and inflammation in many diseases including neurodegenerative diseases. However, the relationship between autophagy and NALP3 inflammasome in prion diseases has not been investigated. In this study, we demonstrated that the processing and release of mature IL-1β is significantly enhanced by the inhibition of autophagy. Conversely, gene-silencing of the NALP3 inflammasome promotes autophagy. Suppression of TRIF or TLR4 by siRNA attenuated PrP106-126-induced autophagy, which is indicating that the TLR4-TRIF signaling pathway is involved in PrP106-26-induced autophagy. Caspase 1 directly cleaved TRIF to diminish TLR-4-TRIF mediated autophagy. Our findings suggest that the inhibition of autophagy by NALP3 inflammasome is probably mediated by activated Caspase-1-induced TRIF cleavage. This is the first study reporting that the NALP3 inflammasome complex negatively regulates autophagy in response to PrP106-126 stimulation in microglia, and partly explains the mechanism of autophagy inhibition by Caspase-1 in PrP106-126-induced BV2 cell activation. Our findings suggest that autophagy up-regulation and inhibition of Caspase-1 may protect against prion-induced neuroinflammation and accelerate misfolded protein degradation and are potential therapeutic approaches for prion diseases. PMID:29720937

Application of “omics” to Prion Biomarker Discovery

PubMed Central

Huzarewich, Rhiannon L. C. H.; Siemens, Christine G.; Booth, Stephanie A.

2010-01-01

The advent of genomics and proteomics has been a catalyst for the discovery of biomarkers able to discriminate biological processes such as the pathogenesis of complex diseases. Prompt detection of prion diseases is particularly desirable given their transmissibility, which is responsible for a number of human health risks stemming from exogenous sources of prion protein. Diagnosis relies on the ability to detect the biomarker PrPSc, a pathological isoform of the host protein PrPC, which is an essential component of the infectious prion. Immunochemical detection of PrPSc is specific and sensitive enough for antemortem testing of brain tissue, however, this is not the case in accessible biological fluids or for the detection of recently identified novel prions with unique biochemical properties. A complementary approach to the detection of PrPSc itself is to identify alternative, “surrogate” gene or protein biomarkers indicative of disease. Biomarkers are also useful to track the progress of disease, especially important in the assessment of therapies, or to identify individuals “at risk”. In this review we provide perspective on current progress and pitfalls in the use of “omics” technologies to screen body fluids and tissues for biomarker discovery in prion diseases. PMID:20224650

Experimental approaches to the interaction of the prion protein with nucleic acids and glycosaminoglycans: Modulators of the pathogenic conversion.

PubMed

Silva, Jerson L; Vieira, Tuane C R G; Gomes, Mariana P B; Rangel, Luciana P; Scapin, Sandra M N; Cordeiro, Yraima

2011-03-01

The concept that transmissible spongiform encephalopathies (TSEs) are caused only by proteins has changed the traditional paradigm that disease transmission is due solely to an agent that carries genetic information. The central hypothesis for prion diseases proposes that the conversion of a cellular prion protein (PrP(C)) into a misfolded, β-sheet-rich isoform (PrP(Sc)) accounts for the development of (TSE). There is substantial evidence that the infectious material consists chiefly of a protein, PrP(Sc), with no genomic coding material, unlike a virus particle, which has both. However, prions seem to have other partners that chaperone their activities in converting the PrP(C) into the disease-causing isoform. Nucleic acids (NAs) and glycosaminoglycans (GAGs) are the most probable accomplices of prion conversion. Here, we review the recent experimental approaches that have been employed to characterize the interaction of prion proteins with nucleic acids and glycosaminoglycans. A PrP recognizes many nucleic acids and GAGs with high affinities, and this seems to be related to a pathophysiological role for this interaction. A PrP binds nucleic acids and GAGs with structural selectivity, and some PrP:NA complexes can become proteinase K-resistant, undergoing amyloid oligomerization and conversion to a β-sheet-rich structure. These results are consistent with the hypothesis that endogenous polyanions (such as NAs and GAGs) may accelerate the rate of prion disease progression by acting as scaffolds or lattices that mediate the interaction between PrP(C) and PrP(Sc) molecules. In addition to a still-possible hypothesis that nucleic acids and GAGs, especially those from the host, may modulate the conversion, the recent structural characterization of the complexes has raised the possibility of developing new diagnostic and therapeutic strategies. Copyright © 2010 Elsevier Inc. All rights reserved.

Indexing amyloid peptide diffraction from serial femtosecond crystallography: New algorithms for sparse patterns

DOE PAGES

Brewster, Aaron S.; Sawaya, Michael R.; Rodriguez, Jose; ...

2015-01-23

Still diffraction patterns from peptide nanocrystals with small unit cells are challenging to index using conventional methods owing to the limited number of spots and the lack of crystal orientation information for individual images. New indexing algorithms have been developed as part of the Computational Crystallography Toolbox( cctbx) to overcome these challenges. Accurate unit-cell information derived from an aggregate data set from thousands of diffraction patterns can be used to determine a crystal orientation matrix for individual images with as few as five reflections. These algorithms are potentially applicable not only to amyloid peptides but also to any set ofmore » diffraction patterns with sparse properties, such as low-resolution virus structures or high-throughput screening of still images captured by raster-scanning at synchrotron sources. As a proof of concept for this technique, successful integration of X-ray free-electron laser (XFEL) data to 2.5 Å resolution for the amyloid segment GNNQQNY from the Sup35 yeast prion is presented.« less

Ethics in Prion Disease

PubMed Central

Bechtel, Kendra; Geschwind, Michael D.

2013-01-01

This paper is intended to discuss some of the scientific and ethical issues that are created by increased research efforts towards earlier diagnosis, as well as to treatment of, human prion diseases (and related dementias), including the resulting consequences for individuals, their families, and society. Most patients with prion disease currently are diagnosed when they are about 2/3 of the way through their disease course (Geschwind, Kuo et al. 2010; Paterson, Torres-Chae et al. 2012), when the disease has progressed so far that even treatments that stop the disease process would probably have little benefit. Although there are currently no treatments available for prion diseases, we and others have realized that we must diagnose patients earlier and with greater accuracy so that future treatments have hope of success. As approximately 15% of prion diseases have a autosomal dominant genetic etiology, this further adds to the complexity of ethical issues, particularly regarding when to conduct genetic testing, release of genetic results, and when or if to implement experimental therapies. Human prion diseases are both infectious and transmissible; great care is required to balance the needs of the family and individual with both public health needs and strained hospital budgets. It is essential to proactively examine and address the ethical issues involved, as well as to define and in turn provide best standards of care. PMID:23906487

Nanoengineered analytical immobilized metal affinity chromatography stationary phase by atom transfer radical polymerization: Separation of synthetic prion peptides

PubMed Central

McCarthy, P.; Chattopadhyay, M.; Millhauser, G.L.; Tsarevsky, N.V.; Bombalski, L.; Matyjaszewski, K.; Shimmin, D.; Avdalovic, N.; Pohl, C.

2010-01-01

Atom transfer radical polymerization (ATRP) was employed to create isolated, metal-containing nanoparticles on the surface of non-porous polymeric beads with the goal of developing a new immobilized metal affnity chromatography (IMAC) stationary phase for separating prion peptides and proteins. Transmission electron microscopy was used to visualize nanoparticles on the substrate surface. Individual ferritin molecules were also visualized as ferritin–nanoparticle complexes. The column's resolving power was tested by synthesizing peptide analogs to the copper binding region of prion protein and injecting mixtures of these analogs onto the column. As expected, the column was capable of separating prion-related peptides differing in number of octapeptide repeat units (PHGGGWGQ), (PHGGGWGQ)2, and (PHGGGWGQ)4. Unexpectedly, the column could also resolve peptides containing the same number of repeats but differing only in the presence of a hydrophilic tail, Q → A substitution, or amide nitrogen methylation. PMID:17481564

Soil humic substances hinder the propagation of prions

NASA Astrophysics Data System (ADS)

Leita, Liviana; Giachin, Gabriele; Margon, Alja; Narkiewicz, Joanna; Legname, Giuseppe

2013-04-01

Prions are infectious pathogens causing fatal neurodegenerative disorders, known as transmissible spongiform encephalopathies (TSEs), or prion diseases, which affect different mammalian species. TSEs include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in mule deer, elk, and moose (cervids), and Creutzfeldt-Jakob disease (CJD) in humans. The prominent, if not only, component of prions is a misfolded conformer (PrPSc) of a constitutive sialoglycoprotein, the cellular prion protein (PrPC). A notable feature of prion diseases is horizontal transmission between grazing animals, implying that contaminated soil may serve to propagate the disease. In this respect, it has been reported that grazing animals ingest from tens to hundreds grams of soil per day, either incidentally through the diet, or deliberately in answering salt needs, and that mule deer can develop CWD after grazing in locations that previously housed infected animals. Prions may enter the environment through different routes, including animal excreta and secreta which mainly contribute to soil contamination. Recent studies have proven that prions can be retained in soil, which could act as a carrier of infectivity even several years after the contamination. However, within the large spread of potentially infected lands, prion diseases have become endemic only in geographically limited regions. The reasons for this geographical distribution remain unknown, but it suggests a role of the different kinds of soil in either enhancing or attenuating prion infectivity. The extent of prion transmission from the contaminated environment is unknown. Several studies have tried to address the issue of prion interaction with soil, but, at the present, different approaches show several drawbacks and technical difficulties, as soil is a complex, multi-component system of both mineral and organic interacting substances. Most research has focused on the adsorption capacity of clay minerals; however the contribution of soil organic components in adsorption has so far been neglected, as they represent a minor soil fraction on a weight basis. Among organic molecules, humic substances (HSs) are natural polyanions that result among the most reactive compounds in the soil and possess the largest specific surface area. Humic substances make up a large portion of the dark matter in humus and consist of heterogeneous mixtures of transformed biomolecules exhibiting a supramolecular structure. HSs are classified as humic acids (HAs), which are soluble only in alkaline solutions, and fulvic acids (FAs), which are soluble in both alkaline and acid solutions. The amphiphilic characteristics confer to HAs and FAs great versatility to interact with xenobiotics and reasonably also with prion proteins and/or prions too, leading to the formation of adducts with peculiar chemical and biophysical characteristics, thus affecting the transport, fixation and toxicity of prion. Results from our chemical, biophysical and biochemical investigation will be presented and results on anti-prion activity exerted by HAs and FAs will be provided, thus suggesting that amendment of contaminated soil with humic substances could be a strategy to contrast prion diffusion.

[A case of Creutzfeldt-Jakob in the Mexican north-east and review of current concepts on prion disease].

PubMed

Calderón-Garcidueñas, A L; Sagastegui-Rodríguez, J A; Canales-Ibarra, C; Farías-García, R

2001-01-01

The case reported here is that of a 50-year-old man from Saltillo, Coahuila, Mexico, who during the previous 15 months developed a demential syndrome and myoclonia. The brain biopsy led to establish a diagnosis of spongiform encephalopathy. The EEG showed periodic sharp wave complexes over the right hemisphere. A review on about prion diseases is included.

Complex folding and misfolding effects of deer-specific amino acid substitutions in the β2-α2 loop of murine prion protein

NASA Astrophysics Data System (ADS)

Agarwal, Sonya; Döring, Kristina; Gierusz, Leszek A.; Iyer, Pooja; Lane, Fiona M.; Graham, James F.; Goldmann, Wilfred; Pinheiro, Teresa J. T.; Gill, Andrew C.

2015-10-01

The β2-α2 loop of PrPC is a key modulator of disease-associated prion protein misfolding. Amino acids that differentiate mouse (Ser169, Asn173) and deer (Asn169, Thr173) PrPC appear to confer dramatically different structural properties in this region and it has been suggested that amino acid sequences associated with structural rigidity of the loop also confer susceptibility to prion disease. Using mouse recombinant PrP, we show that mutating residue 173 from Asn to Thr alters protein stability and misfolding only subtly, whilst changing Ser to Asn at codon 169 causes instability in the protein, promotes oligomer formation and dramatically potentiates fibril formation. The doubly mutated protein exhibits more complex folding and misfolding behaviour than either single mutant, suggestive of differential effects of the β2-α2 loop sequence on both protein stability and on specific misfolding pathways. Molecular dynamics simulation of protein structure suggests a key role for the solvent accessibility of Tyr168 in promoting molecular interactions that may lead to prion protein misfolding. Thus, we conclude that ‘rigidity’ in the β2-α2 loop region of the normal conformer of PrP has less effect on misfolding than other sequence-related effects in this region.

Structure-activity correlations for interactions of bicyclophosphorus esters and some polychlorocycloalkane and pyrethroid insecticides with the brain-specific t-butylbicyclophosphorothionate receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Casida, J.E.; Lawrence, L.J.

1985-09-01

(/sup 35/S)t-Butylbicyclophosphorothionate or (/sup 35/S)TBPS is an improved radioligand for the picrotoxinin binding site in rat brain synaptic membranes. The toxic isomers of the hexachlorocyclohexanes, polychlorobornanes, and chlorinated cyclodienes displace (/sup 35/S)TBPS with a stereospecificity and potency generally correlated with their mammalian toxicity. In a few cases this correlation is improved by correction for metabolic activation or detoxification on using a coupled brain receptor/liver microsomal oxidase system. The alpha-cyano-3-phenoxybenzyl pyrethroids, although less potent, inhibit (/sup 35/S)TBPS binding in a stereospecific manner correlated with their toxicity. Scatchard analyses indicate that these three classes of polychlorocycloalkane insecticides act at the TBPS bindingmore » site within the gamma-aminobutyric acid (GABA) receptor-ionophore complex whereas the alpha-cyano pyrethroids interact with a closely associated site. These insecticides and TBPS analogs may serve as useful probes further to elucidate the topography of the TBPS binding site and its relationship to the chloride channel. 46 references.« less

Prion Propagation in Cells Expressing PrP Glycosylation Mutants ▿

PubMed Central

Salamat, Muhammad K.; Dron, Michel; Chapuis, Jérôme; Langevin, Christelle; Laude, Hubert

2011-01-01

Infection by prions involves conversion of a host-encoded cell surface protein (PrPC) to a disease-related isoform (PrPSc). PrPC carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrPC glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrPSc and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrPSc, while PrPC with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrPC, were able to form infectious PrPSc. Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection. PMID:21248032

Development of a bifunctional filter for prion protein and leukoreduction of red blood cell components.

PubMed

Yokomizo, Tomo; Kai, Takako; Miura, Morikazu; Ohto, Hitoshi

2015-02-01

Leukofiltration of blood components is currently implemented worldwide as a precautionary measure against white blood cell-associated adverse effects and the potential transmission of variant Creutzfeldt-Jakob disease (vCJD). A newly developed bifunctional filter (Sepacell Prima, Asahi Kasei Medical) was assessed for prion removal, leukoreduction (LR), and whether the filter significantly affected red blood cells (RBCs). Sepacell Prima's postfiltration effects on RBCs, including hemolysis, complement activation, and RBC chemistry, were compared with those of a conventional LR filter (Sepacell Pure RC). Prion removal was measured by Western blot after spiking RBCs with microsomal fractions derived from scrapie-infected hamster brain homogenate. Serially diluted exogenous prion solutions (0.05 mL), with or without filtration, were injected intracerebrally into Golden Syrian hamsters. LR efficiency of 4.44 log with the Sepacell Prima was comparable to 4.11 log with the conventional LR filter. There were no significant differences between the two filters in hemoglobin loss, hemolysis, complement activation, and RBC biomarkers. In vitro reduction of exogenously spiked prions by the filter exceeded 3 log. The titer, 6.63 (log ID50 /mL), of prefiltration infectivity of healthy hamsters was reduced to 2.52 (log ID50 /mL) after filtration. The reduction factor was calculated as 4.20 (log ID50 ). With confirmed removal efficacy for exogenous prion protein, this new bifunctional prion and LR filter should reduce the residual risk of vCJD transmission through blood transfusion without adding complexity to component processing. © 2014 AABB.

The Neutral Sphingomyelinase Pathway Regulates Packaging of the Prion Protein into Exosomes*

PubMed Central

Guo, Belinda B.; Bellingham, Shayne A.; Hill, Andrew F.

2015-01-01

Prion diseases are a group of transmissible, fatal neurodegenerative disorders associated with the misfolding of the host-encoded prion protein, PrPC, into a disease-associated form, PrPSc. The transmissible prion agent is principally formed of PrPSc itself and is associated with extracellular vesicles known as exosomes. Exosomes are released from cells both in vitro and in vivo, and have been proposed as a mechanism by which prions spread intercellularly. The biogenesis of exosomes occurs within the endosomal system, through formation of intraluminal vesicles (ILVs), which are subsequently released from cells as exosomes. ILV formation is known to be regulated by the endosomal sorting complexes required for transport (ESCRT) machinery, although an alternative neutral sphingomyelinase (nSMase) pathway has been suggested to also regulate this process. Here, we investigate a role for the nSMase pathway in exosome biogenesis and packaging of PrP into these vesicles. Inhibition of the nSMase pathway using GW4869 revealed a role for the nSMase pathway in both exosome formation and PrP packaging. In agreement, targeted knockdown of nSMase1 and nSMase2 in mouse neurons using lentivirus-mediated RNAi also decreases exosome release, demonstrating the nSMase pathway regulates the biogenesis and release of exosomes. We also demonstrate that PrPC packaging is dependent on nSMase2, whereas the packaging of disease-associated PrPSc into exosomes occurs independently of nSMase2. These findings provide further insight into prion transmission and identify a pathway which directly assists exosome-mediated transmission of prions. PMID:25505180

Phosphorus-31, sup 15 N, and sup 13 C NMR of glyphosate: Comparison of pH titrations to the herbicidal dead-end complex with 5-enolpyruvoylshikimate-3-phosphate synthase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Castellino, S.; Leo, G.C.; Sammons, R.D.

1989-05-02

The herbicidal dead-end ternary complex (E{sup S3P}{sub Glyph}) of glyphosate (N-(phosphonomethyl)glycine) with 5-enolpyruvoylshikimate-3-phosphate synthase (EPSPS) and the substrate shikimate 3-phosphate (S3P) has been characterized by {sup 31}P, {sup 15}N, and {sup 13}C NMR. The NMR spectra of EPSPS-bound glyphosate show unique chemical shifts ({delta}) for each of the three nuclei. By {sup 31}P NMR, glyphosate in the dead-end complex is a distinct species 3.5 ppm downfield from free glyphosate. The {sup 13}C signal of glyphosate in the dead-end complex is shifted 4 ppm downfield from that of free glyphosate. The {sup 15}N signal for glyphosate (99%) in the dead-end complexmore » is 5 ppm further downfield than that of any free zwitterionic species and 10 ppm downfield from that of the average free species at pH 10.1. The structures of each ionic state of glyphosate are modeled with force field calculations by using MacroModel. A correlation is made for the {sup 31}P {delta} and the C-P-O bond angle, and the {sup 13}C and {sup 15}N {delta} values are postulated to be related to C-C-O and C-N-C bond angles, respectively. The downfield {sup 31}P chemical shift perturbation for S3P in the EPSPS binary complex is consistent with ionization of the 3-phosphate of S3P upon binding. Comparison with the S3P {sup 31}P {delta} vs pH titration curve specifies predominantly the dianion of the 3-phosphate in the E{sup S3P} binary complex, while the E{sup S3P}{sub Glyph} complex indicates net protonation at the 3-phosphate. Chemical shift perturbations of this latter type may be explained by changes in the O-P-O bond angle.« less

Minimalist Prion-Inspired Polar Self-Assembling Peptides.

PubMed

Díaz-Caballero, Marta; Navarro, Susanna; Fuentes, Isabel; Teixidor, Francesc; Ventura, Salvador

2018-06-04

Nature provides copious examples of self-assembling supramolecular nanofibers. Among them, amyloid structures have found amazing applications as advanced materials in fields such as biomedicine and nanotechnology. Prions are a singular subset of proteins able to switch between a soluble conformation and an amyloid state. The ability to transit between these two conformations is encoded in the so-called prion domains (PrDs), which are long and disordered regions of low complexity, enriched in polar and uncharged amino acids such as Gln, Asn, Tyr, Ser, and Gly. The polar nature of PrDs results in slow amyloid formation, which allows kinetic control of fiber assembly. This approach has been exploited for fabrication of multifunctional materials because in contrast to most amyloids, PrDs lack hydrophobic stretches that can nucleate their aggregation, their assembly depends on the establishment of a large number of weak interactions along the complete domain. The length and low complexity of PrDs make their chemical synthesis for applied purposed hardly affordable. Here, we designed four minimalist polar binary patterned peptides inspired in PrDs, which include the [Q/N/G/S]-Y-[Q/N/G/S] motif frequently observed in these domains: NYNYNYN, QYQYQYQ, SYSYSYS, and GYGYGYG. Despite their small size, they all recapitulate the properties of full-length PrDs, self-assembling into nontoxic amyloids under physiological conditions. Thus, they constitute small building blocks for the construction of tailored prion-inspired nanostructures. We exploited Tyr residues in these peptides to generate highly stable dityrosine cross-linked assemblies for the immobilization of metal nanoparticles in the fibrils surface and to develop an electrocatalytic amyloid scaffold. Moreover, we show that the shorter and more polar NYNNYN, QYQQYQ, and SYSSYS hexapeptides also self-assemble into amyloid-like structures, consistent with the presence of these tandem motifs in human prion-like proteins.

The neutral sphingomyelinase pathway regulates packaging of the prion protein into exosomes.

PubMed

Guo, Belinda B; Bellingham, Shayne A; Hill, Andrew F

2015-02-06

Prion diseases are a group of transmissible, fatal neurodegenerative disorders associated with the misfolding of the host-encoded prion protein, PrP(C), into a disease-associated form, PrP(Sc). The transmissible prion agent is principally formed of PrP(Sc) itself and is associated with extracellular vesicles known as exosomes. Exosomes are released from cells both in vitro and in vivo, and have been proposed as a mechanism by which prions spread intercellularly. The biogenesis of exosomes occurs within the endosomal system, through formation of intraluminal vesicles (ILVs), which are subsequently released from cells as exosomes. ILV formation is known to be regulated by the endosomal sorting complexes required for transport (ESCRT) machinery, although an alternative neutral sphingomyelinase (nSMase) pathway has been suggested to also regulate this process. Here, we investigate a role for the nSMase pathway in exosome biogenesis and packaging of PrP into these vesicles. Inhibition of the nSMase pathway using GW4869 revealed a role for the nSMase pathway in both exosome formation and PrP packaging. In agreement, targeted knockdown of nSMase1 and nSMase2 in mouse neurons using lentivirus-mediated RNAi also decreases exosome release, demonstrating the nSMase pathway regulates the biogenesis and release of exosomes. We also demonstrate that PrP(C) packaging is dependent on nSMase2, whereas the packaging of disease-associated PrP(Sc) into exosomes occurs independently of nSMase2. These findings provide further insight into prion transmission and identify a pathway which directly assists exosome-mediated transmission of prions. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Prion propagation in cells expressing PrP glycosylation mutants.

PubMed

Salamat, Muhammad K; Dron, Michel; Chapuis, Jérôme; Langevin, Christelle; Laude, Hubert

2011-04-01

Infection by prions involves conversion of a host-encoded cell surface protein (PrP(C)) to a disease-related isoform (PrP(Sc)). PrP(C) carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrP(C) glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrP(Sc) and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrP(Sc), while PrP(C) with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrP(C), were able to form infectious PrP(Sc). Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection.

Quantitative Detection and Biological Propagation of Scrapie Seeding Activity In Vitro Facilitate Use of Prions as Model Pathogens for Disinfection

PubMed Central

Pritzkow, Sandra; Wagenführ, Katja; Daus, Martin L.; Boerner, Susann; Lemmer, Karin; Thomzig, Achim; Mielke, Martin; Beekes, Michael

2011-01-01

Prions are pathogens with an unusually high tolerance to inactivation and constitute a complex challenge to the re-processing of surgical instruments. On the other hand, however, they provide an informative paradigm which has been exploited successfully for the development of novel broad-range disinfectants simultaneously active also against bacteria, viruses and fungi. Here we report on the development of a methodological platform that further facilitates the use of scrapie prions as model pathogens for disinfection. We used specifically adapted serial protein misfolding cyclic amplification (PMCA) for the quantitative detection, on steel wires providing model carriers for decontamination, of 263K scrapie seeding activity converting normal protease-sensitive into abnormal protease-resistant prion protein. Reference steel wires carrying defined amounts of scrapie infectivity were used for assay calibration, while scrapie-contaminated test steel wires were subjected to fifteen different procedures for disinfection that yielded scrapie titre reductions of ≤101- to ≥105.5-fold. As confirmed by titration in hamsters the residual scrapie infectivity on test wires could be reliably deduced for all examined disinfection procedures, from our quantitative seeding activity assay. Furthermore, we found that scrapie seeding activity present in 263K hamster brain homogenate or multiplied by PMCA of scrapie-contaminated steel wires both triggered accumulation of protease-resistant prion protein and was further propagated in a novel cell assay for 263K scrapie prions, i.e., cerebral glial cell cultures from hamsters. The findings from our PMCA- and glial cell culture assays revealed scrapie seeding activity as a biochemically and biologically replicative principle in vitro, with the former being quantitatively linked to prion infectivity detected on steel wires in vivo. When combined, our in vitro assays provide an alternative to titrations of biological scrapie infectivity in animals that substantially facilitates the use of prions as potentially highly indicative test agents in the search for novel broad-range disinfectants. PMID:21647368

Controlling the prion propensity of glutamine/asparagine-rich proteins

PubMed Central

Paul, Kacy R; Ross, Eric D

2015-01-01

ABSTRACT The yeast Saccharomyces cerevisiae can harbor a number of distinct prions. Most of the yeast prion proteins contain a glutamine/asparagine (Q/N) rich region that drives prion formation. Prion-like domains, defined as regions with high compositional similarity to yeast prion domains, are common in eukaryotic proteomes, and mutations in various human proteins containing prion-like domains have been linked to degenerative diseases, including amyotrophic lateral sclerosis. Here, we discuss a recent study in which we utilized two strategies to generate prion activity in non-prion Q/N-rich domains. First, we made targeted mutations in four non-prion Q/N-rich domains, replacing predicted prion-inhibiting amino acids with prion-promoting amino acids. All four mutants formed foci when expressed in yeast, and two acquired bona fide prion activity. Prion activity could be generated with as few as two mutations, suggesting that many non-prion Q/N-rich proteins may be just a small number of mutations from acquiring aggregation or prion activity. Second, we created tandem repeats of short prion-prone segments, and observed length-dependent prion activity. These studies demonstrate the considerable progress that has been made in understanding the sequence basis for aggregation of prion and prion-like domains, and suggest possible mechanisms by which new prion domains could evolve. PMID:26555096

Controlling the prion propensity of glutamine/asparagine-rich proteins.

PubMed

Paul, Kacy R; Ross, Eric D

2015-01-01

The yeast Saccharomyces cerevisiae can harbor a number of distinct prions. Most of the yeast prion proteins contain a glutamine/asparagine (Q/N) rich region that drives prion formation. Prion-like domains, defined as regions with high compositional similarity to yeast prion domains, are common in eukaryotic proteomes, and mutations in various human proteins containing prion-like domains have been linked to degenerative diseases, including amyotrophic lateral sclerosis. Here, we discuss a recent study in which we utilized two strategies to generate prion activity in non-prion Q/N-rich domains. First, we made targeted mutations in four non-prion Q/N-rich domains, replacing predicted prion-inhibiting amino acids with prion-promoting amino acids. All four mutants formed foci when expressed in yeast, and two acquired bona fide prion activity. Prion activity could be generated with as few as two mutations, suggesting that many non-prion Q/N-rich proteins may be just a small number of mutations from acquiring aggregation or prion activity. Second, we created tandem repeats of short prion-prone segments, and observed length-dependent prion activity. These studies demonstrate the considerable progress that has been made in understanding the sequence basis for aggregation of prion and prion-like domains, and suggest possible mechanisms by which new prion domains could evolve.

Prions in Yeast

PubMed Central

Liebman, Susan W.; Chernoff, Yury O.

2012-01-01

The concept of a prion as an infectious self-propagating protein isoform was initially proposed to explain certain mammalian diseases. It is now clear that yeast also has heritable elements transmitted via protein. Indeed, the “protein only” model of prion transmission was first proven using a yeast prion. Typically, known prions are ordered cross-β aggregates (amyloids). Recently, there has been an explosion in the number of recognized prions in yeast. Yeast continues to lead the way in understanding cellular control of prion propagation, prion structure, mechanisms of de novo prion formation, specificity of prion transmission, and the biological roles of prions. This review summarizes what has been learned from yeast prions. PMID:22879407

Molecular structures of amyloid and prion fibrils: consensus versus controversy.

PubMed

Tycko, Robert; Wickner, Reed B

2013-07-16

Many peptides and proteins self-assemble into amyloid fibrils. Examples include mammalian and fungal prion proteins, polypeptides associated with human amyloid diseases, and proteins that may have biologically functional amyloid states. To understand the propensity for polypeptides to form amyloid fibrils and to facilitate rational design of amyloid inhibitors and imaging agents, it is necessary to elucidate the molecular structures of these fibrils. Although fibril structures were largely mysterious 15 years ago, a considerable body of reliable structural information about amyloid fibril structures now exists, with essential contributions from solid state nuclear magnetic resonance (NMR) measurements. This Account reviews results from our laboratories and discusses several structural issues that have been controversial. In many cases, the amino acid sequences of amyloid fibrils do not uniquely determine their molecular structures. Self-propagating, molecular-level polymorphism complicates the structure determination problem and can lead to apparent disagreements between results from different laboratories, particularly when different laboratories study different polymorphs. For 40-residue β-amyloid (Aβ₁₋₄₀) fibrils associated with Alzheimer's disease, we have developed detailed structural models from solid state NMR and electron microscopy data for two polymorphs. These polymorphs have similar peptide conformations, identical in-register parallel β-sheet organizations, but different overall symmetry. Other polymorphs have also been partially characterized by solid state NMR and appear to have similar structures. In contrast, cryo-electron microscopy studies that use significantly different fibril growth conditions have identified structures that appear (at low resolution) to be different from those examined by solid state NMR. Based on solid state NMR and electron paramagnetic resonance (EPR) measurements, the in-register parallel β-sheet organization found in β-amyloid fibrils also occurs in many other fibril-forming systems. We attribute this common structural motif to the stabilization of amyloid structures by intermolecular interactions among like amino acids, including hydrophobic interactions and polar zippers. Surprisingly, we have recently identified and characterized antiparallel β-sheets in certain fibrils that are formed by the D23N mutant of Aβ₁₋₄₀, a mutant that is associated with early-onset, familial neurodegenerative disease. Antiparallel D23N-Aβ₁₋₄₀ fibrils are metastable with respect to parallel structures and, therefore, represent an off-pathway intermediate in the amyloid fibril formation process. Other methods have recently produced additional evidence for antiparallel β-sheets in other amyloid-formation intermediates. As an alternative to simple parallel and antiparallel β-sheet structures, researchers have proposed β-helical structural models for some fibrils, especially those formed by mammalian and fungal prion proteins. Solid state NMR and EPR data show that fibrils formed in vitro by recombinant PrP have in-register parallel β-sheet structures. However, the structure of infectious PrP aggregates is not yet known. The fungal HET-s prion protein has been shown to contain a β-helical structure. However, all yeast prions studied by solid state NMR (Sup35p, Ure2p, and Rnq1p) have in-register parallel β-sheet structures, with their Gln- and Asn-rich N-terminal segments forming the fibril core.

Classifying prion and prion-like phenomena.

PubMed

Harbi, Djamel; Harrison, Paul M

2014-01-01

The universe of prion and prion-like phenomena has expanded significantly in the past several years. Here, we overview the challenges in classifying this data informatically, given that terms such as "prion-like", "prion-related" or "prion-forming" do not have a stable meaning in the scientific literature. We examine the spectrum of proteins that have been described in the literature as forming prions, and discuss how "prion" can have a range of meaning, with a strict definition being for demonstration of infection with in vitro-derived recombinant prions. We suggest that although prion/prion-like phenomena can largely be apportioned into a small number of broad groups dependent on the type of transmissibility evidence for them, as new phenomena are discovered in the coming years, a detailed ontological approach might be necessary that allows for subtle definition of different "flavors" of prion / prion-like phenomena.

PrionHome: a database of prions and other sequences relevant to prion phenomena.

PubMed

Harbi, Djamel; Parthiban, Marimuthu; Gendoo, Deena M A; Ehsani, Sepehr; Kumar, Manish; Schmitt-Ulms, Gerold; Sowdhamini, Ramanathan; Harrison, Paul M

2012-01-01

Prions are units of propagation of an altered state of a protein or proteins; prions can propagate from organism to organism, through cooption of other protein copies. Prions contain no necessary nucleic acids, and are important both as both pathogenic agents, and as a potential force in epigenetic phenomena. The original prions were derived from a misfolded form of the mammalian Prion Protein PrP. Infection by these prions causes neurodegenerative diseases. Other prions cause non-Mendelian inheritance in budding yeast, and sometimes act as diseases of yeast. We report the bioinformatic construction of the PrionHome, a database of >2000 prion-related sequences. The data was collated from various public and private resources and filtered for redundancy. The data was then processed according to a transparent classification system of prionogenic sequences (i.e., sequences that can make prions), prionoids (i.e., proteins that propagate like prions between individual cells), and other prion-related phenomena. There are eight PrionHome classifications for sequences. The first four classifications are derived from experimental observations: prionogenic sequences, prionoids, other prion-related phenomena, and prion interactors. The second four classifications are derived from sequence analysis: orthologs, paralogs, pseudogenes, and candidate-prionogenic sequences. Database entries list: supporting information for PrionHome classifications, prion-determinant areas (where relevant), and disordered and compositionally-biased regions. Also included are literature references for the PrionHome classifications, transcripts and genomic coordinates, and structural data (including comparative models made for the PrionHome from manually curated alignments). We provide database usage examples for both vertebrate and fungal prion contexts. Using the database data, we have performed a detailed analysis of the compositional biases in known budding-yeast prionogenic sequences, showing that the only abundant bias pattern is for asparagine bias with subsidiary serine bias. We anticipate that this database will be a useful experimental aid and reference resource. It is freely available at: http://libaio.biol.mcgill.ca/prion.

PrionHome: A Database of Prions and Other Sequences Relevant to Prion Phenomena

PubMed Central

Harbi, Djamel; Parthiban, Marimuthu; Gendoo, Deena M. A.; Ehsani, Sepehr; Kumar, Manish; Schmitt-Ulms, Gerold; Sowdhamini, Ramanathan; Harrison, Paul M.

2012-01-01

Prions are units of propagation of an altered state of a protein or proteins; prions can propagate from organism to organism, through cooption of other protein copies. Prions contain no necessary nucleic acids, and are important both as both pathogenic agents, and as a potential force in epigenetic phenomena. The original prions were derived from a misfolded form of the mammalian Prion Protein PrP. Infection by these prions causes neurodegenerative diseases. Other prions cause non-Mendelian inheritance in budding yeast, and sometimes act as diseases of yeast. We report the bioinformatic construction of the PrionHome, a database of >2000 prion-related sequences. The data was collated from various public and private resources and filtered for redundancy. The data was then processed according to a transparent classification system of prionogenic sequences (i.e., sequences that can make prions), prionoids (i.e., proteins that propagate like prions between individual cells), and other prion-related phenomena. There are eight PrionHome classifications for sequences. The first four classifications are derived from experimental observations: prionogenic sequences, prionoids, other prion-related phenomena, and prion interactors. The second four classifications are derived from sequence analysis: orthologs, paralogs, pseudogenes, and candidate-prionogenic sequences. Database entries list: supporting information for PrionHome classifications, prion-determinant areas (where relevant), and disordered and compositionally-biased regions. Also included are literature references for the PrionHome classifications, transcripts and genomic coordinates, and structural data (including comparative models made for the PrionHome from manually curated alignments). We provide database usage examples for both vertebrate and fungal prion contexts. Using the database data, we have performed a detailed analysis of the compositional biases in known budding-yeast prionogenic sequences, showing that the only abundant bias pattern is for asparagine bias with subsidiary serine bias. We anticipate that this database will be a useful experimental aid and reference resource. It is freely available at: http://libaio.biol.mcgill.ca/prion. PMID:22363733

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seong, Yeon-Jae; Hafis Clinic, Seoul; Sung, Pil Soo

Cellular prion protein (PrP{sup C}) is widely expressed in various cell types, including cells of the immune system. However, the specific roles of PrP{sup C} in the immune system have not been clearly elucidated. In the present study, we investigated the effects of a soluble form of recombinant PrP{sup C} protein on human natural killer (NK) cells. Recombinant soluble PrP{sup C} protein was generated by fusion of human PrP{sup C} with the Fc portion of human IgG{sub 1} (PrP{sup C}-Fc). PrP{sup C}-Fc binds to the surface of human NK cells, particularly to CD56{sup dim} NK cells. PrP{sup C}-Fc induced themore » production of cytokines and chemokines and the degranulation of granzyme B from NK cells. In addition, PrP{sup C}-Fc facilitated the IL-15-induced proliferation of NK cells. PrP{sup C}-Fc induced phosphorylation of ERK-1/2 and JNK in NK cells, and inhibitors of the ERK or the JNK pathways abrogated PrP{sup C}-Fc-induced cytokine production in NK cells. In conclusion, the soluble form of recombinant PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways. - Highlights: • Recombinant soluble PrP{sup C} (PrP{sup C}-Fc) was generated by fusion of human PrP{sup C} with IgG1 Fc portion. • PrP{sup C}-Fc protein induces the production of cytokines and degranulation from human NK cells. • PrP{sup C}-Fc protein enhances the IL-15-induced proliferation of human NK cells. • PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways.« less

Prion replication without host adaptation during interspecies transmissions.

PubMed

Bian, Jifeng; Khaychuk, Vadim; Angers, Rachel C; Fernández-Borges, Natalia; Vidal, Enric; Meyerett-Reid, Crystal; Kim, Sehun; Calvi, Carla L; Bartz, Jason C; Hoover, Edward A; Agrimi, Umberto; Richt, Jürgen A; Castilla, Joaquín; Telling, Glenn C

2017-01-31

Adaptation of prions to new species is thought to reflect the capacity of the host-encoded cellular form of the prion protein (PrP C ) to selectively propagate optimized prion conformations from larger ensembles generated in the species of origin. Here we describe an alternate replicative process, termed nonadaptive prion amplification (NAPA), in which dominant conformers bypass this requirement during particular interspecies transmissions. To model susceptibility of horses to prions, we produced transgenic (Tg) mice expressing cognate PrP C Although disease transmission to only a subset of infected TgEq indicated a significant barrier to EqPrP C conversion, the resulting horse prions unexpectedly failed to cause disease upon further passage to TgEq. TgD expressing deer PrP C was similarly refractory to deer prions from diseased TgD infected with mink prions. In both cases, the resulting prions transmitted to mice expressing PrP C from the species of prion origin, demonstrating that transmission barrier eradication of the originating prions was ephemeral and adaptation superficial in TgEq and TgD. Horse prions produced in vitro by protein misfolding cyclic amplification of mouse prions using horse PrP C also failed to infect TgEq but retained tropism for wild-type mice. Concordant patterns of neuropathology and prion deposition in susceptible mice infected with NAPA prions and the corresponding prion of origin confirmed preservation of strain properties. The comparable responses of both prion types to guanidine hydrochloride denaturation indicated this occurs because NAPA precludes selection of novel prion conformations. Our findings provide insights into mechanisms regulating interspecies prion transmission and a framework to reconcile puzzling epidemiological features of certain prion disorders.

Prion replication without host adaptation during interspecies transmissions

PubMed Central

Bian, Jifeng; Khaychuk, Vadim; Angers, Rachel C.; Fernández-Borges, Natalia; Meyerett-Reid, Crystal; Kim, Sehun; Calvi, Carla L.; Bartz, Jason C.; Hoover, Edward A.; Agrimi, Umberto; Richt, Jürgen A.; Castilla, Joaquín; Telling, Glenn C.

2017-01-01

Adaptation of prions to new species is thought to reflect the capacity of the host-encoded cellular form of the prion protein (PrPC) to selectively propagate optimized prion conformations from larger ensembles generated in the species of origin. Here we describe an alternate replicative process, termed nonadaptive prion amplification (NAPA), in which dominant conformers bypass this requirement during particular interspecies transmissions. To model susceptibility of horses to prions, we produced transgenic (Tg) mice expressing cognate PrPC. Although disease transmission to only a subset of infected TgEq indicated a significant barrier to EqPrPC conversion, the resulting horse prions unexpectedly failed to cause disease upon further passage to TgEq. TgD expressing deer PrPC was similarly refractory to deer prions from diseased TgD infected with mink prions. In both cases, the resulting prions transmitted to mice expressing PrPC from the species of prion origin, demonstrating that transmission barrier eradication of the originating prions was ephemeral and adaptation superficial in TgEq and TgD. Horse prions produced in vitro by protein misfolding cyclic amplification of mouse prions using horse PrPC also failed to infect TgEq but retained tropism for wild-type mice. Concordant patterns of neuropathology and prion deposition in susceptible mice infected with NAPA prions and the corresponding prion of origin confirmed preservation of strain properties. The comparable responses of both prion types to guanidine hydrochloride denaturation indicated this occurs because NAPA precludes selection of novel prion conformations. Our findings provide insights into mechanisms regulating interspecies prion transmission and a framework to reconcile puzzling epidemiological features of certain prion disorders. PMID:28096357

Resistance of Bovine Spongiform Encephalopathy (BSE) Prions to Inactivation

PubMed Central

Giles, Kurt; Glidden, David V.; Beckwith, Robyn; Seoanes, Rose; Peretz, David; DeArmond, Stephen J.; Prusiner, Stanley B.

2008-01-01

Distinct prion strains often exhibit different incubation periods and patterns of neuropathological lesions. Strain characteristics are generally retained upon intraspecies transmission, but may change on transmission to another species. We investigated the inactivation of two related prions strains: BSE prions from cattle and mouse-passaged BSE prions, termed 301V. Inactivation was manipulated by exposure to sodium dodecyl sulfate (SDS), variations in pH, and different temperatures. Infectivity was measured using transgenic mouse lines that are highly susceptible to either BSE or 301V prions. Bioassays demonstrated that BSE prions are up to 1,000-fold more resistant to inactivation than 301V prions while Western immunoblotting showed that short acidic SDS treatments reduced protease-resistant PrPSc from BSE prions and 301V prions at similar rates. Our findings argue that despite being derived from BSE prions, mouse 301V prions are not necessarily a reliable model for cattle BSE prions. Extending these comparisons to human sporadic Creutzfeldt-Jakob disease and hamster Sc237 prions, we found that BSE prions were 10- and 106-fold more resistant to inactivation, respectively. Our studies contend that any prion inactivation procedures must be validated by bioassay against the prion strain for which they are intended to be used. PMID:19008948

Prion-specific Hsp40 function: The role of the auxilin homolog Swa2

PubMed Central

Oliver, Emily E.; Troisi, Elizabeth M.; Hines, Justin K.

2017-01-01

ABSTRACT Yeast prions are protein-based genetic elements that propagate through cell populations via cytosolic transfer from mother to daughter cell. Molecular chaperone proteins including Hsp70, the Hsp40/J-protein Sis1, and Hsp104 are required for continued prion propagation, however the specific requirements of chaperone proteins differ for various prions. We recently reported that Swa2, the yeast homolog of the mammalian protein auxilin, is specifically required for the propagation of the prion [URE3].1 [URE3] propagation requires both a functional J-domain and the tetratricopeptide repeat (TPR) domain of Swa2, but does not require Swa2 clathrin binding. We concluded that the TPR domain determines the specificity of the genetic interaction between Swa2 and [URE3], and that this domain likely interacts with one or more proteins with a C-terminal EEVD motif. Here we extend that analysis to incorporate additional data that supports this hypothesis. We also present new data eliminating Hsp104 as the relevant Swa2 binding partner and discuss our findings in the context of other recent work involving Hsp90. Based on these findings, we propose a new model for Swa2's involvement in [URE3] propagation in which Swa2 and Hsp90 mediate the formation of a multi-protein complex that increases the number of sites available for Hsp104 disaggregation. PMID:28574745

Prions spread via the autonomic nervous system from the gut to the central nervous system in cattle incubating bovine spongiform encephalopathy.

PubMed

Hoffmann, Christine; Ziegler, Ute; Buschmann, Anne; Weber, Artur; Kupfer, Leila; Oelschlegel, Anja; Hammerschmidt, Baerbel; Groschup, Martin H

2007-03-01

To elucidate the still-unknown pathogenesis of bovine spongiform encephalopathy (BSE), an oral BSE challenge and sequential kill study was carried out on 56 calves. Relevant tissues belonging to the peripheral and central nervous system, as well as to the lymphoreticular tract, from necropsied animals were analysed by highly sensitive immunohistochemistry and immunoblotting techniques to reveal the presence of BSE-associated pathological prion protein (PrPSc) depositions. Our results demonstrate two routes involving the autonomic nervous system through which BSE prions spread by anterograde pathways from the gastrointestinal tract (GIT) to the central nervous system (CNS): (i) via the coeliac and mesenteric ganglion complex, splanchnic nerves and the lumbal/caudal thoracic spinal cord (representing the sympathetic GIT innervation); and (ii) via the Nervus vagus (parasympathetic GIT innervation). The dorsal root ganglia seem to be subsequently affected, so it is likely that BSE prion invasion of the non-autonomic peripheral nervous system (e.g. sciatic nerve) is a secondary retrograde event following prion replication in the CNS. Moreover, BSE-associated PrPSc was already detected in the brainstem of an animal 24 months post-infection, which is 8 months earlier than reported previously. These findings are important for the understanding of BSE pathogenesis and for the development of new diagnostic strategies for this infectious disease.

Incunabular Immunological Events in Prion Trafficking

PubMed Central

Michel, Brady; Meyerett-Reid, Crystal; Johnson, Theodore; Ferguson, Adam; Wyckoff, Christy; Pulford, Bruce; Bender, Heather; Avery, Anne; Telling, Glenn; Dow, Steven; Zabel, Mark D.

2012-01-01

While prions probably interact with the innate immune system immediately following infection, little is known about this initial confrontation. Here we investigated incunabular events in lymphotropic and intranodal prion trafficking by following highly enriched, fluorescent prions from infection sites to draining lymph nodes. We detected biphasic lymphotropic transport of prions from the initial entry site upon peripheral prion inoculation. Prions arrived in draining lymph nodes cell autonomously within two hours of intraperitoneal administration. Monocytes and dendritic cells (DCs) required Complement for optimal prion delivery to lymph nodes hours later in a second wave of prion trafficking. B cells constituted the majority of prion-bearing cells in the mediastinal lymph node by six hours, indicating intranodal prion reception from resident DCs or subcapsulary sinus macrophages or directly from follicular conduits. These data reveal novel, cell autonomous prion lymphotropism, and a prominent role for B cells in intranodal prion movement. PMID:22679554

Prions are affected by evolution at two levels.

PubMed

Wickner, Reed B; Kelly, Amy C

2016-03-01

Prions, infectious proteins, can transmit diseases or be the basis of heritable traits (or both), mostly based on amyloid forms of the prion protein. A single protein sequence can be the basis for many prion strains/variants, with different biological properties based on different amyloid conformations, each rather stably propagating. Prions are unique in that evolution and selection work at both the level of the chromosomal gene encoding the protein, and on the prion itself selecting prion variants. Here, we summarize what is known about the evolution of prion proteins, both the genes and the prions themselves. We contrast the one known functional prion, [Het-s] of Podospora anserina, with the known disease prions, the yeast prions [PSI+] and [URE3] and the transmissible spongiform encephalopathies of mammals.

Neurobehavioral toxicology of pyrethroid insecticides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crofton, K.M.

1986-01-01

Pyrethroid insecticides are classified as either Type I or Type II based upon in vivo toxic signs, and neurophysiological and biochemical data. Both axonal sodium channels and the ..gamma..-aminobutyric acid (GABA) receptor complex have been proposed as the major site of action of the Type II pyrethroids. This investigation characterized the behavior and biochemical effects of low dosages of pyrethroids in rats. Type I and II pyrethroids were tested for effects on figure-eight maze activity and the acoustic startle response (ASR). All compounds decreased figure-eight maze activity. Interactions of Type I and II pyrethroids with the three major binding sitesmore » on the GABA complex were determined in vivo. Radioligand binding experiments assessed in vitro interactions of pyrethroids with the three major GABA-complex binding sites. None of the pyrethroids competed for (/sup 3/H)-muscimol or (/sup 3/H)-flunitrazepam binding. Only Type II pyrethroids inhibited binding of (/sup 35/S)-t-butylbicyclophosphorothionate (TBPS) in cortical synaptosome preparations with K/sub i/ values of 5 to 10 ..mu..M. The (/sup 35/S)-TBPS data implicate the TBPS/picrotoxinin binding site in the mechanism of Type II pyrethroid toxicity. The results of these experiments support the classification of pyrethroids into two classes, and demonstrate the utility of the figure-eight maze and the ASR in studies to elucidate neurotoxic mechanisms. The interaction of the Type II pyrethroids is probably restricted to the TBPS/picrotoxinin binding domain on the GABA complex as shown by both the in vivo and in vitro studies.« less

Electron transfer reactions of excited dyes with metal complexes. Progress report, May 1, 1976--January 31, 1977. [Fe(III)--thionine reaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lichtin, N.N.

1977-02-01

A study was initiated of the factors which determine quantum efficiency of transfer of reducing equivalents between excited dye molecules and metal complexes in their ground state and composition and dynamics of formation and decay of related photostationary states. A ruby laser capable of delivering a 3.6 J, 19 nsec flash was acquired and assembly of an apparatus for laser flash photolysis begun. At the same time, conventional flash photolysis was used to pursue investigation of the dependence upon solvent, anions, pH, and ionic strength of the kinetics of the spontaneous dark reaction of Fe(H/sub 2/O)/sup 3 +//sub 6/ withmore » leucothionine and with semithionine, reactions which contribute to the composition and dynamics of formation and decay of the photostationary state of the iron-thionine photoredox reaction. Results are consistent with formation of an intermediate complex between leucothionine and Fe(III), K/sub A/ = 380 M/sup -1/ and k(elec. transfer) = 0.88 s/sup -1/ at approximately 22/sup 0/ in water solution at pH2, with sulfate as anion and ..mu.. = .05 - .1 M. Under similar conditions in 50 v/v percent aqueous CH/sub 3/CN, K/sub A/ = 780 M/sup -1/, k(elec. transfer) = 0.55 s/sup -1/. In both solvents, sulfate produces a large positive salt effect. Intermediacy of a complex was not established for the faster reaction of Fe(III) with semithionine under similar conditions: K/sub A/ . k(elec. transfer) approximately 3.5 x 10/sup 5/ M/sup -1/s/sup -1/ in H/sub 2/O, approximately 1.0 x 10/sup 4/ in 50 v/v percent aqueous CH/sub 3/CN.« less

Physiological and environmental control of yeast prions

PubMed Central

Chernova, Tatiana A.; Wilkinson, Keith D.; Chernoff, Yury O.

2014-01-01

Prions are self-perpetuating protein isoforms that cause fatal and incurable neurodegenerative disease in mammals. Recent evidence indicates that a majority of human proteins involved in amyloid and neural inclusion disorders possess at least some prion properties. In lower eukaryotes, such as yeast, prions act as epigenetic elements, which increase phenotypic diversity by altering a range of cellular processes. While some yeast prions are clearly pathogenic, it is also postulated that prion formation could be beneficial in variable environmental conditions. Yeast and mammalian prions have similar molecular properties. Crucial cellular factors and conditions influencing prion formation and propagation were uncovered in the yeast models. Stress-related chaperones, protein quality control deposits, degradation pathways and cytoskeletal networks control prion formation and propagation in yeast. Environmental stresses trigger prion formation and loss, supposedly acting via influencing intracellular concentrations of the prion-inducing proteins, and/or by localizing prionogenic proteins to the prion induction sites via heterologous ancillary helpers. Physiological and environmental modulation of yeast prions points to new opportunities for pharmacological intervention and/or prophylactic measures targeting general cellular systems rather than the properties of individual amyloids and prions. PMID:24236638

Gerstmann-Straüssler-Scheinker disease: novel PRNP mutation and VGKC-complex antibodies.

PubMed

Jones, Matthew; Odunsi, Sola; du Plessis, Daniel; Vincent, Angela; Bishop, Matthew; Head, Mark W; Ironside, James W; Gow, David

2014-06-10

To describe a unique case of Gerstmann-Straüssler-Scheinker (GSS) disease caused by a novel prion protein (PRNP) gene mutation and associated with strongly positive voltage-gated potassium channel (VGKC)-complex antibodies (Abs). Clinical data were gathered from retrospective review of the case notes. Postmortem neuropathologic examination was performed, and DNA was extracted from frozen brain tissue for full sequence analysis of the PRNP gene. The patient was diagnosed in life with VGKC-complex Ab-associated encephalitis based on strongly positive VGKC-complex Ab titers but no detectable LGI1 or CASPR2 Abs. He died despite 1 year of aggressive immunosuppressive treatment. The neuropathologic diagnosis was GSS disease, and a novel mutation, P84S, in the PRNP gene was found. VGKC-complex Abs are described in an increasingly broad range of clinical syndromes, including progressive encephalopathies, and may be amenable to treatment with immunosuppression. However, the failure to respond to aggressive immunotherapy warns against VGKC-complex Abs being pathogenic, and their presence does not preclude the possibility of prion disease. © 2014 American Academy of Neurology.

Investigation on the grain boundaries electrical characteristics of perovskite lithium ion conductors by derivative of tanδ approach

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mariappan, C.R., E-mail: crmari2005@yahoo.com; Bohnke, O.

2016-02-15

Graphical abstract: Cole–Cole impedance spectra for Li{sub 0.30}La{sub 0.56}TiO{sub 3}-SSR and Li{sub 0.30}La{sub 0.56}TiO{sub 3}-PC at 310 K and for Li{sub 0.30}Ca{sub 0.35}TaO{sub 3}-SSR and Li{sub 0.30}Ca{sub 0.35}TaO{sub 3}-PC at 473 K. - Highlights: • Li{sub 0.30}La{sub 0.56}TiO{sub 3} & Li{sub 0.30}Ca{sub 0.35}TaO{sub 3} perovskites made by Pechini polymerization route. • Relaxation processes of perovskites are analyzed by derivative of tanδ (α{sub θ}) method. • α{sub θ} (ν) graph resolves the two closer relaxation processes at grain boundary region. • SEM/TEM study supports the resolving ability of α{sub θ} vs log (ν) method. • χ{sup 2} improves from ∼10{sup −3}more » to ∼10{sup −5} for equivalent circuit fitting based on α{sub θ} peaks. - Abstract: We report on the grain boundaries electrical relaxation processes of Li{sub 0.30}La{sub 0.56}TiO{sub 3} and Li{sub 0.30}Ca{sub 0.35}TaO{sub 3} perovskites by derivative of the tanδ (α{sub θ}) method. The α{sub θ} vs log(ν) representation is clearly resolved the two grain boundary relaxation processes at grain boundary region for perovskites synthesized via the modified Pechini-type polymerization complex route in comparison to perovskites obtained by solid state reaction method. The resolving ability of α{sub θ} vs log(ν) representation is much higher than other complex impedance spectroscopic representations. The goodness of the fitting χ{sup 2} is improved from ∼10{sup −3} to ∼10{sup −5} for the electrical equivalent circuit fitting based on α{sub θ} vs log(ν) peaks.« less

Increasing Prion Propensity by Hydrophobic Insertion

PubMed Central

Petri, Michelina; Flores, Noe; Rogge, Ryan A.; Cascarina, Sean M.; Ross, Eric D.

2014-01-01

Prion formation involves the conversion of proteins from a soluble form into an infectious amyloid form. Most yeast prion proteins contain glutamine/asparagine-rich regions that are responsible for prion aggregation. Prion formation by these domains is driven primarily by amino acid composition, not primary sequence, yet there is a surprising disconnect between the amino acids thought to have the highest aggregation propensity and those that are actually found in yeast prion domains. Specifically, a recent mutagenic screen suggested that both aromatic and non-aromatic hydrophobic residues strongly promote prion formation. However, while aromatic residues are common in yeast prion domains, non-aromatic hydrophobic residues are strongly under-represented. Here, we directly test the effects of hydrophobic and aromatic residues on prion formation. Remarkably, we found that insertion of as few as two hydrophobic residues resulted in a multiple orders-of-magnitude increase in prion formation, and significant acceleration of in vitro amyloid formation. Thus, insertion or deletion of hydrophobic residues provides a simple tool to control the prion activity of a protein. These data, combined with bioinformatics analysis, suggest a limit on the number of strongly prion-promoting residues tolerated in glutamine/asparagine-rich domains. This limit may explain the under-representation of non-aromatic hydrophobic residues in yeast prion domains. Prion activity requires not only that a protein be able to form prion fibers, but also that these fibers be cleaved to generate new independently-segregating aggregates to offset dilution by cell division. Recent studies suggest that aromatic residues, but not non-aromatic hydrophobic residues, support the fiber cleavage step. Therefore, we propose that while both aromatic and non-aromatic hydrophobic residues promote prion formation, aromatic residues are favored in yeast prion domains because they serve a dual function, promoting both prion formation and chaperone-dependent prion propagation. PMID:24586661

Chronic Lymphocytic Inflammation Specifies the Organ Tropism of Prions

NASA Astrophysics Data System (ADS)

Heikenwalder, Mathias; Zeller, Nicolas; Seeger, Harald; Prinz, Marco; Klöhn, Peter-Christian; Schwarz, Petra; Ruddle, Nancy H.; Weissmann, Charles; Aguzzi, Adriano

2005-02-01

Prions typically accumulate in nervous and lymphoid tissues. Because proinflammatory cytokines and immune cells are required for lymphoid prion replication, we tested whether inflammatory conditions affect prion pathogenesis. We administered prions to mice with five inflammatory diseases of the kidney, pancreas, or liver. In all cases, chronic lymphocytic inflammation enabled prion accumulation in otherwise prion-free organs. Inflammatory foci consistently correlated with lymphotoxin up-regulation and ectopic induction of FDC-M1+ cells expressing the normal cellular prion protein PrPC. By contrast, inflamed organs of mice lacking lymphotoxin-α or its receptor did not accumulate the abnormal isoform PrPSc, nor did they display infectivity upon prion inoculation. By expanding the tissue distribution of prions, chronic inflammatory conditions may act as modifiers of natural and iatrogenic prion transmission.

Life cycle of cytosolic prions.

PubMed

Hofmann, Julia; Vorberg, Ina

2013-01-01

Prions are self-templating protein aggregates that were originally identified as the causative agent of prion diseases in mammals, but have since been discovered in other kingdoms. Mammalian prions represent a unique class of infectious agents that are composed of misfolded prion protein. Prion proteins usually exist as soluble proteins but can refold and assemble into highly ordered, self-propagating prion polymers. The prion concept is also applicable to a growing number of non-Mendelian elements of inheritance in lower eukaryotes. While prions identified in mammals are clearly pathogens, prions in lower eukaryotes can be either detrimental or beneficial to the host. Prion phenotypes in fungi are transmitted vertically from mother to daughter cells during cell division and horizontally during mating or abortive mating, but extracellular phases have not been reported. Recent findings now demonstrate that in a mammalian cell environment, protein aggregates derived from yeast prion domains exhibit a prion life cycle similar to mammalian prions propagated ex vivo. This life cycle includes a soluble state of the protein, an induction phase by exogenous prion fibrils, stable replication of prion entities, vertical transmission to progeny and natural horizontal transmission to neighboring cells. Our data reveal that mammalian cells contain all co-factors required for cytosolic prion propagation and dissemination. This has important implications for understanding prion-like properties of disease-related protein aggregates. In light of the growing number of identified functional amyloids, cell-to-cell propagation of cytosolic protein conformers might not only be relevant for the spreading of disease-associated proteins, but might also be of more general relevance under non-disease conditions.

Analysis of the [RNQ+] Prion Reveals Stability of Amyloid Fibers as the Key Determinant of Yeast Prion Variant Propagation*

PubMed Central

Kalastavadi, Tejas; True, Heather L.

2010-01-01

Variation in pathology of human prion disease is believed to be caused, in part, by distinct conformations of aggregated protein resulting in different prion strains. Several prions also exist in yeast and maintain different self-propagating structures, referred to as prion variants. Investigation of the yeast prion [PSI+] has been instrumental in deciphering properties of prion variants and modeling the physical basis of their formation. Here, we describe the generation of specific variants of the [RNQ+] prion in yeast transformed with fibers formed in vitro in different conditions. The fibers of the Rnq1p prion-forming domain (PFD) that induce different variants in vivo have distinct biochemical properties. The physical basis of propagation of prion variants has been previously correlated to rates of aggregation and disaggregation. With [RNQ+] prion variants, we found that the prion variant does not correlate with the rate of aggregation as anticipated but does correlate with stability. Interestingly, we found that there are differences in the ability of the [RNQ+] prion variants to faithfully propagate themselves and to template the aggregation of other proteins. Incorporating the mechanism of variant formation elucidated in this study with that previously proposed for [PSI+] variants has provided a framework to separate general characteristics of prion variant properties from those specific to individual prion proteins. PMID:20442412

Levels of dissolved zinc and cadmium in some surface waters of western Nigeria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fatoki, O.S.

1993-12-31

Dissolved zinc and cadmium in some surface waters of Western Nigeria were separated and quantified using anion exchange of their chloro-complexes and detected by atomic absorption spectrophotometry. Concentrations of zinc and cadmium found in tested water samples ranged from 0.99 to 2.97 mg L{sup {minus}1} and 0.13 to 0.17 mg L{sup {minus}1}, respectively. 35 refs., 2 tabs.

The multivesicular body is the major internal site of prion conversion

PubMed Central

Yim, Yang-In; Park, Bum-Chan; Yadavalli, Rajgopal; Zhao, Xiaohong; Eisenberg, Evan; Greene, Lois E.

2015-01-01

ABSTRACT The conversion of the properly folded prion protein, PrPc, to its misfolded amyloid form, PrPsc, occurs as the two proteins traffic along the endocytic pathway and PrPc is exposed to PrPsc. To determine the specific site of prion conversion, we knocked down various proteins in the endocytic pathway including Rab7a, Tsg101 and Hrs (also known as HGS). PrPsc was markedly reduced in two chronically infected cell lines by preventing the maturation of the multivesicular body, a process that begins in the early endosome and ends with the sorting of cargo to the lysosome. By contrast, knocking down proteins in the retromer complex, which diverts cargo away from the multivesicular body caused an increase in PrPsc levels. These results suggest that the multivesicular body is the major site for intracellular conversion of PrPc to PrPsc. PMID:25663703

The inhibition of prions through blocking prion conversion by permanently charged branched polyamines of low cytotoxicity.

PubMed

Lim, Yong-beom; Mays, Charles E; Kim, Younghwan; Titlow, William B; Ryou, Chongsuk

2010-03-01

Branched polyamines are effective in inhibiting prions in a cationic surface charge density dependent manner. However, toxicity associated with branched polyamines, in general, often hampers the successful application of the compounds to treat prion diseases. Here, we report that constitutively maintained cationic properties in branched polyamines reduced the intrinsic toxicity of the compounds while retaining the anti-prion activities. In prion-infected neuroblastoma cells, quaternization of amines in polyethyleneimine (PEI) and polyamidoamine (PAMAM) dendrimers markedly increased the nontoxic concentration ranges of the compounds and still supported, albeit reduced, an appreciable level of anti-prion activity in clearing prions from the infected cells. Furthermore, quaternized PEI was able to degrade prions at acidic pH conditions and inhibit the in vitro prion propagation facilitated by conversion of the normal prion protein isoform to its misfolded counterpart, although such activities were decreased by quaternization. Quaternized PAMAM was least effective in degrading prions but efficiently inhibited prion conversion with the same efficacy as unmodified PAMAM. Our results suggest that quaternization represents an effective strategy for developing nontoxic branched polyamines with potent anti-prion activity. This study highlights the importance of polyamine structural control for developing polyamine-based anti-prion agents and understanding of an action mechanism of quaternized branched polyamines. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

Yeast prions: structure, biology, and prion-handling systems.

PubMed

Wickner, Reed B; Shewmaker, Frank P; Bateman, David A; Edskes, Herman K; Gorkovskiy, Anton; Dayani, Yaron; Bezsonov, Evgeny E

2015-03-01

A prion is an infectious protein horizontally transmitting a disease or trait without a required nucleic acid. Yeast and fungal prions are nonchromosomal genes composed of protein, generally an altered form of a protein that catalyzes the same alteration of the protein. Yeast prions are thus transmitted both vertically (as genes composed of protein) and horizontally (as infectious proteins, or prions). Formation of amyloids (linear ordered β-sheet-rich protein aggregates with β-strands perpendicular to the long axis of the filament) underlies most yeast and fungal prions, and a single prion protein can have any of several distinct self-propagating amyloid forms with different biological properties (prion variants). Here we review the mechanism of faithful templating of protein conformation, the biological roles of these prions, and their interactions with cellular chaperones, the Btn2 and Cur1 aggregate-handling systems, and other cellular factors governing prion generation and propagation. Human amyloidoses include the PrP-based prion conditions and many other, more common amyloid-based diseases, several of which show prion-like features. Yeast prions increasingly are serving as models for the understanding and treatment of many mammalian amyloidoses. Patients with different clinical pictures of the same amyloidosis may be the equivalent of yeasts with different prion variants. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Yeast Prions: Structure, Biology, and Prion-Handling Systems

PubMed Central

Shewmaker, Frank P.; Bateman, David A.; Edskes, Herman K.; Gorkovskiy, Anton; Dayani, Yaron; Bezsonov, Evgeny E.

2015-01-01

SUMMARY A prion is an infectious protein horizontally transmitting a disease or trait without a required nucleic acid. Yeast and fungal prions are nonchromosomal genes composed of protein, generally an altered form of a protein that catalyzes the same alteration of the protein. Yeast prions are thus transmitted both vertically (as genes composed of protein) and horizontally (as infectious proteins, or prions). Formation of amyloids (linear ordered β-sheet-rich protein aggregates with β-strands perpendicular to the long axis of the filament) underlies most yeast and fungal prions, and a single prion protein can have any of several distinct self-propagating amyloid forms with different biological properties (prion variants). Here we review the mechanism of faithful templating of protein conformation, the biological roles of these prions, and their interactions with cellular chaperones, the Btn2 and Cur1 aggregate-handling systems, and other cellular factors governing prion generation and propagation. Human amyloidoses include the PrP-based prion conditions and many other, more common amyloid-based diseases, several of which show prion-like features. Yeast prions increasingly are serving as models for the understanding and treatment of many mammalian amyloidoses. Patients with different clinical pictures of the same amyloidosis may be the equivalent of yeasts with different prion variants. PMID:25631286

Yeast and Fungal Prions: Amyloid-Handling Systems, Amyloid Structure, and Prion Biology.

PubMed

Wickner, R B; Edskes, H K; Gorkovskiy, A; Bezsonov, E E; Stroobant, E E

2016-01-01

Yeast prions (infectious proteins) were discovered by their outré genetic properties and have become important models for an array of human prion and amyloid diseases. A single prion protein can become any of many distinct amyloid forms (called prion variants or strains), each of which is self-propagating, but with different biological properties (eg, lethal vs mild). The folded in-register parallel β sheet architecture of the yeast prion amyloids naturally suggests a mechanism by which prion variant information can be faithfully transmitted for many generations. The yeast prions rely on cellular chaperones for their propagation, but can be cured by various chaperone imbalances. The Btn2/Cur1 system normally cures most variants of the [URE3] prion that arise. Although most variants of the [PSI+] and [URE3] prions are toxic or lethal, some are mild in their effects. Even the most mild forms of these prions are rare in the wild, indicating that they too are detrimental to yeast. The beneficial [Het-s] prion of Podospora anserina poses an important contrast in its structure, biology, and evolution to the yeast prions characterized thus far. Copyright © 2016 Elsevier Inc. All rights reserved.

Mechanical Deformation Mechanisms and Properties of Prion Fibrils Probed by Atomistic Simulations

NASA Astrophysics Data System (ADS)

Choi, Bumjoon; Kim, Taehee; Ahn, Eue Soo; Lee, Sang Woo; Eom, Kilho

2017-03-01

Prion fibrils, which are a hallmark for neurodegenerative diseases, have recently been found to exhibit the structural diversity that governs disease pathology. Despite our recent finding concerning the role of the disease-specific structure of prion fibrils in determining their elastic properties, the mechanical deformation mechanisms and fracture properties of prion fibrils depending on their structures have not been fully characterized. In this work, we have studied the tensile deformation mechanisms of prion and non-prion amyloid fibrils by using steered molecular dynamics simulations. Our simulation results show that the elastic modulus of prion fibril, which is formed based on left-handed β-helical structure, is larger than that of non-prion fibril constructed based on right-handed β-helix. However, the mechanical toughness of prion fibril is found to be less than that of non-prion fibril, which indicates that infectious prion fibril is more fragile than non-infectious (non-prion) fibril. Our study sheds light on the role of the helical structure of amyloid fibrils, which is related to prion infectivity, in determining their mechanical deformation mechanisms and properties.

Prions amplify through degradation of the VPS10P sorting receptor sortilin.

PubMed

Uchiyama, Keiji; Tomita, Mitsuru; Yano, Masashi; Chida, Junji; Hara, Hideyuki; Das, Nandita Rani; Nykjaer, Anders; Sakaguchi, Suehiro

2017-06-01

Prion diseases are a group of fatal neurodegenerative disorders caused by prions, which consist mainly of the abnormally folded isoform of prion protein, PrPSc. A pivotal pathogenic event in prion disease is progressive accumulation of prions, or PrPSc, in brains through constitutive conformational conversion of the cellular prion protein, PrPC, into PrPSc. However, the cellular mechanism by which PrPSc is progressively accumulated in prion-infected neurons remains unknown. Here, we show that PrPSc is progressively accumulated in prion-infected cells through degradation of the VPS10P sorting receptor sortilin. We first show that sortilin interacts with PrPC and PrPSc and sorts them to lysosomes for degradation. Consistently, sortilin-knockdown increased PrPSc accumulation in prion-infected cells. In contrast, overexpression of sortilin reduced PrPSc accumulation in prion-infected cells. These results indicate that sortilin negatively regulates PrPSc accumulation in prion-infected cells. The negative role of sortilin in PrPSc accumulation was further confirmed in sortilin-knockout mice infected with prions. The infected mice had accelerated prion disease with early accumulation of PrPSc in their brains. Interestingly, sortilin was reduced in prion-infected cells and mouse brains. Treatment of prion-infected cells with lysosomal inhibitors, but not proteasomal inhibitors, increased the levels of sortilin. Moreover, sortilin was reduced following PrPSc becoming detectable in cells after infection with prions. These results indicate that PrPSc accumulation stimulates sortilin degradation in lysosomes. Taken together, these results show that PrPSc accumulation of itself could impair the sortilin-mediated sorting of PrPC and PrPSc to lysosomes for degradation by stimulating lysosomal degradation of sortilin, eventually leading to progressive accumulation of PrPSc in prion-infected cells.

Comparison of physical and photophysical properties of monometallic and bimetallic ruthenium(II) complexes containing structurally altered diimine ligands

DOE Office of Scientific and Technical Information (OSTI.GOV)

Macatangay, A.; Jackman, D.C.; Merkert, J.W.

1996-11-06

The physical and photophysical properties of a series of monometallic, [Ru(bpy){sub 2}(dmb)]{sup 2+}, [Ru(bpy){sub 2}(BPY)]{sup 2+}, [Ru(bpy)(Obpy)]{sup 2+} and [Ru(bpy){sub 2}(Obpy)] {sup 2+}, and bimetallic, [(Ru(bpy){sub 2}){sub 2}(BPY)]{sup 4+} and [(Ru(bpy){sub 2}){sub 2}(Obpy)]{sup 4+}, complexes are examined, where bpy is 2,2{prime}-bipyridine, dmb is 4,4{prime}-dimethyl-2,2{prime}-bipyridine, BPY is 1,2-bis(4-methyl-2,2{prime}-bipyridin-4{prime}-yl)ethane, and Obpy is 1,2-bis(2,2{prime}-bipyridin-6-yl)ethane. The complexes display metal-to-ligand charge transfer transitions in the 450 nmn region, intraligand {pi}{yields}{pi}* transitions at energies greater than 300 nm, a reversible oxidation of the ruthenium(II) center in the 1.25-1.40 V vs SSCE region, a series of three reductions associated with each coordinated ligand commencing at {minus}1.3 Vmore » and ending at {approximately}{minus}1.9 V, and emission from a {sup 3}MLCT state having energy maxima between 598 and 610 nm. The Ru{sup III}/Ru{sup II} oxidation of the two bimetallic complexes is a single, two one-electron process. Relative to [Ru(bpy){sub 2}(BPY)]{sup 2+}, the Ru{sup III}/Ru{sup II} potential for [Ru-(bpy){sub 2}(Obpy)]{sup 2+} increases from 1.24 to 1.35 V, the room temperature emission lifetime decreases from 740 to 3ns, and the emission quantum yield decreases from 0.078 to 0.000 23. Similarly, relative to [(Ru(bpy){sub 2}){sub 2}(BPY)]{sup 4+}, the Ru{sup III}/Ru{sup II} potential for [(Ru(bpy){sub 2}){sub 2}(Obpy)]{sup 4+} increases from 1.28 to 1.32 V, the room temperature emission lifetime decreases from 770 to 3 ns, and the room temperature emission quantum yield decreases from 0.079 to 0.000 26.« less

Preparation, structure, and luminescence of dinuclear lanthanide complexes of a novel imine-amine phenolate macrocycle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matthews, K.D.; Kahwa, I.A.; Williams, D.J.

1994-03-30

Metal-free condensation of 2,6-diformyl-p-cresol with 3,6-dioxa-1,8-octanediamine followed by reduction with sodium tetrahydroborate and addition of lanthanide(III) nitrate salts, in that order, yield (slowly) crystalline dinuclear complexes of a novel imine-amine phenolate macrocycle 2. The decacoordination geometry of the identical Pr[sup 3+] ions in a C[sub 2v] 4A,6B-extended dodecahedron made up of two bidentate NO[sub 3]-ions, two phenolate and two either oxygens, and one imine and one amine nitrogens. Dinuclear lanthanide complexes of 2 appear to be more stable than those of the totally reduced chelate 2 in alcoholic media. The Tb[sub 2]2(NO[sub 3])[sub 4][center dot]1.2CH[sub 3]-OH and (La[sub 0.97]Tb[sub 0.03])[submore » 2]2(NO[sub 3])[sub 4][center dot]1.2CH[sub 3]OH compounds exhibit strong Tb[sup 3+] ([sup 5]D[sub 4] [yields] [sup 7]F[sub J]) emission sensitized by the single state of 2 at both 77 and 295 K. No Tb[sup 3+]-Tb[sup 3+] self-quenching or N-H trapping effects are observed at 77 K (decay rate is 598 s[sup [minus]1]); the coordination cavities of 2 are therefore potentially good hosts for Tb[sup 3+] in luminescent diagnostic agents. At room temperature the complex decay kinetics of Tb[sup 3+] in Tb[sub 2]2(NO[sub 3])[sub 4][center dot]1.2CH[sub 3]OH are similar to those of Tb[sub 2]1(NO[sub 3])[sub 4][center dot]H[sub 2]O. But for the dilute complex, (La[sub 0.97]-Tb[sub 0.03])[sub 2]2(NO[sub 3])[sub 4][center dot]1.35CH[sub 3]OH, unusual thermal equilibration of the ligand triplet and Tb[sup 3+] [sup 5]D[sub 4] states occurs at room temperature; the ligand-to-Tb[sup 3+] energy-transfer rate is [approx]4.36 x 10[sup 4] s[sup [minus]1], while Tb[sup 3+]-to-ligand back-energy-transfer is [approx]7.1 x 10[sup 4] s[sup [minus]1].« less

Enhanced neuroinvasion by smaller, soluble prions.

PubMed

Bett, Cyrus; Lawrence, Jessica; Kurt, Timothy D; Orru, Christina; Aguilar-Calvo, Patricia; Kincaid, Anthony E; Surewicz, Witold K; Caughey, Byron; Wu, Chengbiao; Sigurdson, Christina J

2017-04-21

Infectious prion aggregates can propagate from extraneural sites into the brain with remarkable efficiency, likely transported via peripheral nerves. Yet not all prions spread into the brain, and the physical properties of a prion that is capable of transit within neurons remain unclear. We hypothesized that small, diffusible aggregates spread into the CNS via peripheral nerves. Here we used a structurally diverse panel of prion strains to analyze how the prion conformation impacts transit into the brain. Two prion strains form fibrils visible ultrastructurally in the brain in situ, whereas three strains form diffuse, subfibrillar prion deposits and no visible fibrils. The subfibrillar strains had significantly higher levels of soluble prion aggregates than the fibrillar strains. Primary neurons internalized both the subfibrillar and fibril-forming prion strains by macropinocytosis, and both strain types were transported from the axon terminal to the cell body in vitro. However in mice, only the predominantly soluble, subfibrillar prions, and not the fibrillar prions, were efficiently transported from the tongue to the brain. Sonicating a fibrillar prion strain increased the solubility and enabled prions to spread into the brain in mice, as evident by a 40% increase in the attack rate, indicating that an increase in smaller particles enhances prion neuroinvasion. Our data suggest that the small, highly soluble prion particles have a higher capacity for transport via nerves. These findings help explain how prions that predominantly assemble into subfibrillar states can more effectively traverse into and out of the CNS, and suggest that promoting fibril assembly may slow the neuron-to-neuron spread of protein aggregates.

Strategies for identifying new prions in yeast

PubMed Central

MacLea, Kyle S

2011-01-01

The unexpected discovery of two prions, [URE3] and [PSI+], in Saccharomyces cerevisiae led to questions about how many other proteins could undergo similar prion-based structural conversions. However, [URE3] and [PSI+] were discovered by serendipity in genetic screens. Cataloging the full range of prions in yeast or in other organisms will therefore require more systematic search methods. Taking advantage of some of the unique features of prions, various researchers have developed bioinformatic and experimental methods for identifying novel prion proteins. These methods have generated long lists of prion candidates. The systematic testing of some of these prion candidates has led to notable successes; however, even in yeast, where rapid growth rate and ease of genetic manipulation aid in testing for prion activity, such candidate testing is laborious. Development of better methods to winnow the field of prion candidates will greatly aid in the discovery of new prions, both in yeast and in other organisms, and help us to better understand the role of prions in biology. PMID:22052351

New insights into structural determinants of prion protein folding and stability.

PubMed

Benetti, Federico; Legname, Giuseppe

2015-01-01

Prions are the etiological agent of fatal neurodegenerative diseases called prion diseases or transmissible spongiform encephalopathies. These maladies can be sporadic, genetic or infectious disorders. Prions are due to post-translational modifications of the cellular prion protein leading to the formation of a β-sheet enriched conformer with altered biochemical properties. The molecular events causing prion formation in sporadic prion diseases are still elusive. Recently, we published a research elucidating the contribution of major structural determinants and environmental factors in prion protein folding and stability. Our study highlighted the crucial role of octarepeats in stabilizing prion protein; the presence of a highly enthalpically stable intermediate state in prion-susceptible species; and the role of disulfide bridge in preserving native fold thus avoiding the misfolding to a β-sheet enriched isoform. Taking advantage from these findings, in this work we present new insights into structural determinants of prion protein folding and stability.

Glutathione Complex Formation With Mercury(Ii) in Aqueous Solution at Physiological Ph

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mah, V.; Jalilehvand, F.; /SLAC

2012-08-23

The mercury(II) complexes formed in neutral aqueous solution with glutathione (GSH, here denoted AH{sub 3} in its triprotonated form) were studied using Hg L{sub III}-edge extended X-ray absorption fine structure (EXAFS) and {sup 199}Hg NMR spectroscopy, complemented with electrospray ionization mass spectrometric (ESI-MS) analyses. The [Hg(AH){sub 2}]{sup 2-} complex, with the Hg-S bond distances at 2.325 {+-} 0.01 {angstrom} in linear S-Hg-S coordination, and the {sup 199}Hg NMR chemical shift at -984 ppm, dominates except at high excess of glutathione. In a series of solutions with C{sub Hg(II)} {approx} 17 mM and GSH/Hg(II) mole ratios rising from 2.4 to 11.8,more » the gradually increasing mean Hg-S bond distance corresponds to an increasing amount of the [Hg(AH){sub 3}]{sup 4-} complex. ESI-MS peaks appear at -m/z values of 1208 and 1230 corresponding to the [Na{sub 4}Hg(AH){sub 2}(A)]{sup -} and [Na{sub 5}Hg(AH)(A){sub 2}]{sup -} species, respectively. In another series of solutions at pH 7.0 with CHg(II) 50 mM and GSH/Hg(II) ratios from 2.0 to 10.0, the Hg L{sub III}-edge EXAFS and {sup 199}Hg NMR spectra show that at high excess of glutathione (0.35 M) about 70% of the total mercury(II) concentration is present as the [Hg(AH){sub 3}]4- complex, with the average Hg-S bond distance 2.42 {+-} 0.02 {angstrom} in trigonal HgS{sub 3} coordination. The proportions of HgSn species, n = 2, 3, and 4, quantified by fitting linear combinations of model EXAFS oscillations to the experimental EXAFS data in our present and previous studies were used to obtain stability constants for the [Hg(AH){sub 3}]{sup 4-} complex and also for the [Hg(A){sub 4}]{sup 10-} complex that is present at high pH. For Hg(II) in low concentration at physiological conditions (pH 7.4, C{sub GSH} = 2.2 mM), the relative amounts of the HgS{sub 2} species [Hg(AH){sub 2}]{sup 2-}, [Hg(AH)(A)]{sup 3-}, and the HgS{sub 3} complex [Hg(AH){sub 3}]{sup 4-} were calculated to be 95:2:3. Our results are not consistent with the formation of dimeric Hg(II)-GSH complexes proposed in a recent EXAFS study.« less

Chronic wasting disease prions are not transmissible to transgenic mice overexpressing human prion protein.

PubMed

Sandberg, Malin K; Al-Doujaily, Huda; Sigurdson, Christina J; Glatzel, Markus; O'Malley, Catherine; Powell, Caroline; Asante, Emmanuel A; Linehan, Jacqueline M; Brandner, Sebastian; Wadsworth, Jonathan D F; Collinge, John

2010-10-01

Chronic wasting disease (CWD) is a prion disease that affects free-ranging and captive cervids, including mule deer, white-tailed deer, Rocky Mountain elk and moose. CWD-infected cervids have been reported in 14 USA states, two Canadian provinces and in South Korea. The possibility of a zoonotic transmission of CWD prions via diet is of particular concern in North America where hunting of cervids is a popular sport. To investigate the potential public health risks posed by CWD prions, we have investigated whether intracerebral inoculation of brain and spinal cord from CWD-infected mule deer transmits prion infection to transgenic mice overexpressing human prion protein with methionine or valine at polymorphic residue 129. These transgenic mice have been utilized in extensive transmission studies of human and animal prion disease and are susceptible to BSE and vCJD prions, allowing comparison with CWD. Here, we show that these mice proved entirely resistant to infection with mule deer CWD prions arguing that the transmission barrier associated with this prion strain/host combination is greater than that observed with classical BSE prions. However, it is possible that CWD may be caused by multiple prion strains. Further studies will be required to evaluate the transmission properties of distinct cervid prion strains as they are characterized.

Gerstmann-Straüssler-Scheinker disease

PubMed Central

Jones, Matthew; Odunsi, Sola; du Plessis, Daniel; Vincent, Angela; Bishop, Matthew; Head, Mark W.; Ironside, James W.

2014-01-01

Objective: To describe a unique case of Gerstmann-Straüssler-Scheinker (GSS) disease caused by a novel prion protein (PRNP) gene mutation and associated with strongly positive voltage-gated potassium channel (VGKC)-complex antibodies (Abs). Methods: Clinical data were gathered from retrospective review of the case notes. Postmortem neuropathologic examination was performed, and DNA was extracted from frozen brain tissue for full sequence analysis of the PRNP gene. Results: The patient was diagnosed in life with VGKC-complex Ab–associated encephalitis based on strongly positive VGKC-complex Ab titers but no detectable LGI1 or CASPR2 Abs. He died despite 1 year of aggressive immunosuppressive treatment. The neuropathologic diagnosis was GSS disease, and a novel mutation, P84S, in the PRNP gene was found. Conclusion: VGKC-complex Abs are described in an increasingly broad range of clinical syndromes, including progressive encephalopathies, and may be amenable to treatment with immunosuppression. However, the failure to respond to aggressive immunotherapy warns against VGKC-complex Abs being pathogenic, and their presence does not preclude the possibility of prion disease. PMID:24814844

Guinea Pig Prion Protein Supports Rapid Propagation of Bovine Spongiform Encephalopathy and Variant Creutzfeldt-Jakob Disease Prions.

PubMed

Watts, Joel C; Giles, Kurt; Saltzberg, Daniel J; Dugger, Brittany N; Patel, Smita; Oehler, Abby; Bhardwaj, Sumita; Sali, Andrej; Prusiner, Stanley B

2016-11-01

The biochemical and neuropathological properties of bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (vCJD) prions are faithfully maintained upon transmission to guinea pigs. However, primary and secondary transmissions of BSE and vCJD in guinea pigs result in long incubation periods of ∼450 and ∼350 days, respectively. To determine if the incubation periods of BSE and vCJD prions could be shortened, we generated transgenic (Tg) mice expressing guinea pig prion protein (GPPrP). Inoculation of Tg(GPPrP) mice with BSE and vCJD prions resulted in mean incubation periods of 210 and 199 days, respectively, which shortened to 137 and 122 days upon serial transmission. In contrast, three different isolates of sporadic CJD prions failed to transmit disease to Tg(GPPrP) mice. Many of the strain-specified biochemical and neuropathological properties of BSE and vCJD prions, including the presence of type 2 protease-resistant PrP Sc , were preserved upon propagation in Tg(GPPrP) mice. Structural modeling revealed that two residues near the N-terminal region of α-helix 1 in GPPrP might mediate its susceptibility to BSE and vCJD prions. Our results demonstrate that expression of GPPrP in Tg mice supports the rapid propagation of BSE and vCJD prions and suggest that Tg(GPPrP) mice may serve as a useful paradigm for bioassaying these prion isolates. Variant Creutzfeldt-Jakob disease (vCJD) and bovine spongiform encephalopathy (BSE) prions are two of the prion strains most relevant to human health. However, propagating these strains in mice expressing human or bovine prion protein has been difficult because of prolonged incubation periods or inefficient transmission. Here, we show that transgenic mice expressing guinea pig prion protein are fully susceptible to vCJD and BSE prions but not to sporadic CJD prions. Our results suggest that the guinea pig prion protein is a better, more rapid substrate than either bovine or human prion protein for propagating BSE and vCJD prions. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Sulfate-bicarbonate exchange in brush-border membranes from rat renal cortex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pritchard, J.B.

1987-02-01

Under Na/sup +/-free conditions /sup 35/SO/sub 4//sup 2 -/ uptake by rat renal brush-border membrane (BBM) vesicles could be driven by imposition of a HCO/sup -//sub 3/ gradient (in greater than out). The initial rate of /sup 35/SO/sub 4//sup 2 -/ uptake was stimulated 10-fold, and peak overshoot exceeded equilibrium uptake by 2-3 times. Cl/sup -/, SCN/sup -/, NO/sub 3//sup -/, I/sup -/, and OH/sup -/ were able to substitute for HCO/sub 3//sup -/. Divalent anions, including /sup 35/SO/sub 4//sup 2 -/ itself, were less effective as counterions. HCO/sub 3//sup -/-SO/sub 4//sup 2 -/ exchange was cis-inhibited by disulfonic stilbenes,more » ((SITS)(DIDS)), phloretin, Hg, and S/sub 2/O/sub 3//sup 2 -/. HCO/sub 3//sup -/-driven /sup 35/SO/sub 4//sup 2 -/ uptake was saturable, with an apparent K/sub m/ of 0.4 mM for SO/sub 4//sup 2 -/. Simultaneous imposition of Na/sup +/ and HCO/sub 3//sup -/ gradients produced approximately additive stimulation of /sup 35/SO/sub 4//sup 2 -/ uptake. The HCO/sub 3//sup -/-driven component of /sup 35/SO/sub 4//sup 2 -/ uptake, but not the component driven by Na/sup +/, was inhibited by SITS. Finally, Na/sup +/-driven SO/sub 4//sup 2 -/ accumulation could be reduced by imposing an out greater than in HCO/sub 3//sup +/ gradient, conditions accelerating exchange driven SO/sub 4//sup 2 -/ efflux. These findings indicate the presence of separate Na/sup +/-SO/sub 4//sup 2 -/ cotransport and SO /sub 4//sup 2 -/-anion exchange pathways in the same BBM vesicles.« less

The Prion Concept and Synthetic Prions.

PubMed

Legname, Giuseppe; Moda, Fabio

2017-01-01

Transmissible spongiform encephalopathies or prion diseases are a group of fatal neurodegenerative diseases caused by unconventional infectious agents, known as prions (PrP Sc ). Prions derive from a conformational conversion of the normally folded prion protein (PrP C ), which acquires pathological and infectious features. Moreover, PrP Sc is able to transmit the pathological conformation to PrP C through a mechanism that is still not well understood. The generation of synthetic prions, which behave like natural prions, is of fundamental importance to study the process of PrP C conversion and to assess the efficacy of therapeutic strategies to interfere with this process. Moreover, the ability of synthetic prions to induce pathology in animals confirms that the pathological properties of the prion strains are all enciphered in abnormal conformations, characterizing these infectious agents. © 2017 Elsevier Inc. All rights reserved.

Insights into mechanisms of transmission and pathogenesis from transgenic mouse models of prion diseases

PubMed Central

Moreno, Julie A.; Telling, Glenn C.

2018-01-01

Prions represent a new paradigm of protein-mediated information transfer. In the case of mammals, prions are the cause of fatal, transmissible neurodegenerative diseases, sometimes referred to as transmissible spongiform encephalopathies (TSE’s), which frequently occur as epidemics. An increasing body of evidence indicates that the canonical mechanism of conformational corruption of cellular prion protein (PrPC) by the pathogenic isoform (PrPSc) that is the basis of prion formation in TSE’s, is common to a spectrum of proteins associated with various additional human neurodegenerative disorders, including the more common Alzheimer’s and Parkinson’s diseases. The peerless infectious properties of TSE prions, and the unparalleled tools for their study, therefore enable elucidation of mechanisms of template-mediated conformational propagation that are generally applicable to these related disease states. Many unresolved issues remain including the exact molecular nature of the prion, the detailed cellular and molecular mechanisms of prion propagation, and the means by which prion diseases can be both genetic and infectious. In addition, we know little about the mechanism by which neurons degenerate during prion diseases. Tied to this, the physiological role of the normal form of the prion protein remains unclear and it is uncertain whether or not loss of this function contributes to prion pathogenesis. The factors governing the transmission of prions between species remain unclear, in particular the means by which prion strains and PrP primary structure interact to affect inter-species prion transmission. Despite all these unknowns, advances in our understanding of prions have occurred because of their transmissibility to experimental animals and the development of transgenic (Tg) mouse models has done much to further our understanding about various aspects of prion biology. In this review we will focus on advances in our understanding of prion biology that occurred in the past eight years since our last review of this topic. PMID:28861793

Conserved properties of human and bovine prion strains on transmission to guinea pigs

PubMed Central

Safar, Jiri G.; Giles, Kurt; Lessard, Pierre; Letessier, Frederic; Patel, Smita; Serban, Ana; DeArmond, Stephen J.; Prusiner, Stanley B.

2011-01-01

The first transmissions of human prion diseases to rodents used guinea pigs (Gps, Cavia porcellus). Later, transgenic (Tg) mice expressing human or chimeric human/mouse PrP replaced Gps, but the small size of the mouse limits some investigations. To investigate the fidelity of strain-specific prion transmission to Gps, we inoculated “type 1” and “type 2” prion strains into Gps: we measured the incubation times and determined the strain-specified size of the unglycosylated, protease-resistant (r) PrPSc fragment. Prions passaged once in Gps from cases of sporadic (s) Creutzfeldt–Jakob disease (CJD) and Gerstmann-Sträussler-Scheinker (GSS) disease caused by the P102L mutation were used as well as human prions from a variant (v) CJD case, bovine prions from bovine spongiform encephalopathy (BSE), and mouse-passaged scrapie prions. Variant CJD and BSE prions transmitted to all the inoculated Gps with incubation times of 367 ± 4 d and 436 ± 28 d, respectively. On second passage in Gps, vCJD and BSE prions caused disease in 287 ± 4 d and 310 ± 4 d, while sCJD and GSS prions transmitted in 237 ± 4 d and 279 ± 19 d, respectively. Although hamster Sc237 prions transmitted to 2 of 3 Gps after 574 and 792 d, mouse-passaged RML and 301V prion strains, the latter derived from BSE prions, failed to transmit disease to Gps. Those Gps inoculated with vCJD or BSE prions exhibited “type 2” unglycosylated, rPrPSc (19 kDa) while those receiving sCJD or GSS prions displayed “type 1” prions (21 kDa), as determined by Western blotting. Such strain-specific properties were maintained in Gps as well as mice expressing a chimeric human/mouse transgene. Gps may prove particularly useful in further studies of novel human prions such as those causing vCJD. PMID:21727894

Human stem cell–derived astrocytes replicate human prions in a PRNP genotype–dependent manner

PubMed Central

Krejciova, Zuzana; Alibhai, James; Zhao, Chen; Rzechorzek, Nina M.; Ullian, Erik M.; Manson, Jean

2017-01-01

Prions are infectious agents that cause neurodegenerative diseases such as Creutzfeldt–Jakob disease (CJD). The absence of a human cell culture model that replicates human prions has hampered prion disease research for decades. In this paper, we show that astrocytes derived from human induced pluripotent stem cells (iPSCs) support the replication of prions from brain samples of CJD patients. For experimental exposure of astrocytes to variant CJD (vCJD), the kinetics of prion replication occur in a prion protein codon 129 genotype–dependent manner, reflecting the genotype-dependent susceptibility to clinical vCJD found in patients. Furthermore, iPSC-derived astrocytes can replicate prions associated with the major sporadic CJD strains found in human patients. Lastly, we demonstrate the subpassage of prions from infected to naive astrocyte cultures, indicating the generation of prion infectivity in vitro. Our study addresses a long-standing gap in the repertoire of human prion disease research, providing a new in vitro system for accelerated mechanistic studies and drug discovery. PMID:29141869

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

PubMed

Terry, Cassandra; Wenborn, Adam; Gros, Nathalie; Sells, Jessica; Joiner, Susan; Hosszu, Laszlo L P; Tattum, M Howard; Panico, Silvia; Clare, Daniel K; Collinge, John; Saibil, Helen R; Wadsworth, Jonathan D F

2016-05-01

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. © 2016 The Authors.

A closer look at prion strains

PubMed Central

Solforosi, Laura; Milani, Michela; Mancini, Nicasio; Clementi, Massimo; Burioni, Roberto

2013-01-01

Prions are infectious proteins that are responsible for transmissible spongiform encephalopathies (TSEs) and consist primarily of scrapie prion protein (PrPSc), a pathogenic isoform of the host-encoded cellular prion protein (PrPC). The absence of nucleic acids as essential components of the infectious prions is the most striking feature associated to these diseases. Additionally, different prion strains have been isolated from animal diseases despite the lack of DNA or RNA molecules. Mounting evidence suggests that prion-strain-specific features segregate with different PrPSc conformational and aggregation states. Strains are of practical relevance in prion diseases as they can drastically differ in many aspects, such as incubation period, PrPSc biochemical profile (e.g., electrophoretic mobility and glycoform ratio) and distribution of brain lesions. Importantly, such different features are maintained after inoculation of a prion strain into genetically identical hosts and are relatively stable across serial passages. This review focuses on the characterization of prion strains and on the wide range of important implications that the study of prion strains involves. PMID:23357828

Atypical scrapie prions from sheep and lack of disease in transgenic mice overexpressing human prion protein.

PubMed

Wadsworth, Jonathan D F; Joiner, Susan; Linehan, Jacqueline M; Balkema-Buschmann, Anne; Spiropoulos, John; Simmons, Marion M; Griffiths, Peter C; Groschup, Martin H; Hope, James; Brandner, Sebastian; Asante, Emmanuel A; Collinge, John

2013-11-01

Public and animal health controls to limit human exposure to animal prions are focused on bovine spongiform encephalopathy (BSE), but other prion strains in ruminants may also have zoonotic potential. One example is atypical/Nor98 scrapie, which evaded statutory diagnostic methods worldwide until the early 2000s. To investigate whether sheep infected with scrapie prions could be another source of infection, we inoculated transgenic mice that overexpressed human prion protein with brain tissue from sheep with natural field cases of classical and atypical scrapie, sheep with experimental BSE, and cattle with BSE. We found that these mice were susceptible to BSE prions, but disease did not develop after prolonged postinoculation periods when mice were inoculated with classical or atypical scrapie prions. These data are consistent with the conclusion that prion disease is less likely to develop in humans after exposure to naturally occurring prions of sheep than after exposure to epizootic BSE prions of ruminants.

Atypical Scrapie Prions from Sheep and Lack of Disease in Transgenic Mice Overexpressing Human Prion Protein

PubMed Central

Joiner, Susan; Linehan, Jacqueline M.; Balkema-Buschmann, Anne; Spiropoulos, John; Simmons, Marion M.; Griffiths, Peter C.; Groschup, Martin H.; Hope, James; Brandner, Sebastian; Asante, Emmanuel A.; Collinge, John

2013-01-01

Public and animal health controls to limit human exposure to animal prions are focused on bovine spongiform encephalopathy (BSE), but other prion strains in ruminants may also have zoonotic potential. One example is atypical/Nor98 scrapie, which evaded statutory diagnostic methods worldwide until the early 2000s. To investigate whether sheep infected with scrapie prions could be another source of infection, we inoculated transgenic mice that overexpressed human prion protein with brain tissue from sheep with natural field cases of classical and atypical scrapie, sheep with experimental BSE, and cattle with BSE. We found that these mice were susceptible to BSE prions, but disease did not develop after prolonged postinoculation periods when mice were inoculated with classical or atypical scrapie prions. These data are consistent with the conclusion that prion disease is less likely to develop in humans after exposure to naturally occurring prions of sheep than after exposure to epizootic BSE prions of ruminants. PMID:24188521

Early increase and late decrease of purkinje cell dendritic spine density in prion-infected organotypic mouse cerebellar cultures.

PubMed

Campeau, Jody L; Wu, Gengshu; Bell, John R; Rasmussen, Jay; Sim, Valerie L

2013-01-01

Prion diseases are infectious neurodegenerative diseases associated with the accumulation of protease-resistant prion protein, neuronal loss, spongiform change and astrogliosis. In the mouse model, the loss of dendritic spines is one of the earliest pathological changes observed in vivo, occurring 4-5 weeks after the first detection of protease-resistant prion protein in the brain. While there are cell culture models of prion infection, most do not recapitulate the neuropathology seen in vivo. Only the recently developed prion organotypic slice culture assay has been reported to undergo neuronal loss and the development of some aspects of prion pathology, namely small vacuolar degeneration and tubulovesicular bodies. Given the rapid replication of prions in this system, with protease-resistant prion protein detectable by 21 days, we investigated whether the dendritic spine loss and altered dendritic morphology seen in prion disease might also develop within the lifetime of this culture system. Indeed, six weeks after first detection of protease-resistant prion protein in tga20 mouse cerebellar slice cultures infected with RML prion strain, we found a statistically significant loss of Purkinje cell dendritic spines and altered dendritic morphology in infected cultures, analogous to that seen in vivo. In addition, we found a transient but statistically significant increase in Purkinje cell dendritic spine density during infection, at the time when protease-resistant prion protein was first detectable in culture. Our findings support the use of this slice culture system as one which recapitulates prion disease pathology and one which may facilitate study of the earliest stages of prion disease pathogenesis.

Covalent surface modification of prions: a mass spectrometry-based means of detecting distinctive structural features of prion strains

USDA-ARS?s Scientific Manuscript database

Prions (PrPSc) are molecular pathogens that are able to convert the isosequential normal cellular prion protein (PrPC) into a prion. The only demonstrated differences between PrPC and PrPSc is conformational, they are isoforms. A given host can be infected by more than one kind or strain of prion. F...

Functional diversification of hsp40: distinct j-protein functional requirements for two prions allow for chaperone-dependent prion selection.

PubMed

Harris, Julia M; Nguyen, Phil P; Patel, Milan J; Sporn, Zachary A; Hines, Justin K

2014-07-01

Yeast prions are heritable amyloid aggregates of functional yeast proteins; their propagation to subsequent cell generations is dependent upon fragmentation of prion protein aggregates by molecular chaperone proteins. Mounting evidence indicates the J-protein Sis1 may act as an amyloid specificity factor, recognizing prion and other amyloid aggregates and enabling Ssa and Hsp104 to act in prion fragmentation. Chaperone interactions with prions, however, can be affected by variations in amyloid-core structure resulting in distinct prion variants or 'strains'. Our genetic analysis revealed that Sis1 domain requirements by distinct variants of [PSI+] are strongly dependent upon overall variant stability. Notably, multiple strong [PSI+] variants can be maintained by a minimal construct of Sis1 consisting of only the J-domain and glycine/phenylalanine-rich (G/F) region that was previously shown to be sufficient for cell viability and [RNQ+] prion propagation. In contrast, weak [PSI+] variants are lost under the same conditions but maintained by the expression of an Sis1 construct that lacks only the G/F region and cannot support [RNQ+] propagation, revealing mutually exclusive requirements for Sis1 function between these two prions. Prion loss is not due to [PSI+]-dependent toxicity or dependent upon a particular yeast genetic background. These observations necessitate that Sis1 must have at least two distinct functional roles that individual prions differentially require for propagation and which are localized to the glycine-rich domains of the Sis1. Based on these distinctions, Sis1 plasmid-shuffling in a [PSI+]/[RNQ+] strain permitted J-protein-dependent prion selection for either prion. We also found that, despite an initial report to the contrary, the human homolog of Sis1, Hdj1, is capable of [PSI+] prion propagation in place of Sis1. This conservation of function is also prion-variant dependent, indicating that only one of the two Sis1-prion functions may have been maintained in eukaryotic chaperone evolution.

Functional Diversification of Hsp40: Distinct J-Protein Functional Requirements for Two Prions Allow for Chaperone-Dependent Prion Selection

PubMed Central

Patel, Milan J.; Sporn, Zachary A.; Hines, Justin K.

2014-01-01

Yeast prions are heritable amyloid aggregates of functional yeast proteins; their propagation to subsequent cell generations is dependent upon fragmentation of prion protein aggregates by molecular chaperone proteins. Mounting evidence indicates the J-protein Sis1 may act as an amyloid specificity factor, recognizing prion and other amyloid aggregates and enabling Ssa and Hsp104 to act in prion fragmentation. Chaperone interactions with prions, however, can be affected by variations in amyloid-core structure resulting in distinct prion variants or ‘strains’. Our genetic analysis revealed that Sis1 domain requirements by distinct variants of [PSI +] are strongly dependent upon overall variant stability. Notably, multiple strong [PSI +] variants can be maintained by a minimal construct of Sis1 consisting of only the J-domain and glycine/phenylalanine-rich (G/F) region that was previously shown to be sufficient for cell viability and [RNQ +] prion propagation. In contrast, weak [PSI +] variants are lost under the same conditions but maintained by the expression of an Sis1 construct that lacks only the G/F region and cannot support [RNQ +] propagation, revealing mutually exclusive requirements for Sis1 function between these two prions. Prion loss is not due to [PSI +]-dependent toxicity or dependent upon a particular yeast genetic background. These observations necessitate that Sis1 must have at least two distinct functional roles that individual prions differentially require for propagation and which are localized to the glycine-rich domains of the Sis1. Based on these distinctions, Sis1 plasmid-shuffling in a [PSI +]/[RNQ +] strain permitted J-protein-dependent prion selection for either prion. We also found that, despite an initial report to the contrary, the human homolog of Sis1, Hdj1, is capable of [PSI +] prion propagation in place of Sis1. This conservation of function is also prion-variant dependent, indicating that only one of the two Sis1-prion functions may have been maintained in eukaryotic chaperone evolution. PMID:25058638

Infective dementias.

PubMed

Ironside, J W; Bell, J E

2007-12-01

A wide range of infectious diseases can result in dementia, although the identity and nature of these diseases has changed over time. Two of the most significant current groups in terms of scientific complexity are HIV/AIDS and prion diseases. In these disorders, dementia occurs either as a consequence of targeting the brain and selectively damaging neurones, or by an indirect effect of neuroinflammation. In prion diseases, both direct neurotoxicity and neuroinflammation may act to result in neuronal damage. In HIV encephalitis, the progression of the dementia is slower, perhaps reflecting indirect damage that appears to result from neuroinflammation as a main cause of neuronal death. An ever-increasing range of model systems is now available to study the neuronal damage in infectious dementias, ranging from cell culture systems to animal models, some of which, particularly in the case of prion diseases, are very well characterised and amenable to controlled manipulation in terms of both host and agent parameters. As valuable as these experimental models are, they do not allow a direct approach to an understanding of dementia, the complexities of which cannot readily be studied in vitro or in animal models, but they do allow studies of interventions and therapeutic strategies. This review summarises the current state of knowledge regarding the major infective dementias.

Human stem cell-derived astrocytes replicate human prions in a PRNP genotype-dependent manner.

PubMed

Krejciova, Zuzana; Alibhai, James; Zhao, Chen; Krencik, Robert; Rzechorzek, Nina M; Ullian, Erik M; Manson, Jean; Ironside, James W; Head, Mark W; Chandran, Siddharthan

2017-12-04

Prions are infectious agents that cause neurodegenerative diseases such as Creutzfeldt-Jakob disease (CJD). The absence of a human cell culture model that replicates human prions has hampered prion disease research for decades. In this paper, we show that astrocytes derived from human induced pluripotent stem cells (iPSCs) support the replication of prions from brain samples of CJD patients. For experimental exposure of astrocytes to variant CJD (vCJD), the kinetics of prion replication occur in a prion protein codon 129 genotype-dependent manner, reflecting the genotype-dependent susceptibility to clinical vCJD found in patients. Furthermore, iPSC-derived astrocytes can replicate prions associated with the major sporadic CJD strains found in human patients. Lastly, we demonstrate the subpassage of prions from infected to naive astrocyte cultures, indicating the generation of prion infectivity in vitro. Our study addresses a long-standing gap in the repertoire of human prion disease research, providing a new in vitro system for accelerated mechanistic studies and drug discovery. © 2017 Krejciova et al.

A closer look at prion strains: characterization and important implications.

PubMed

Solforosi, Laura; Milani, Michela; Mancini, Nicasio; Clementi, Massimo; Burioni, Roberto

2013-01-01

Prions are infectious proteins that are responsible for transmissible spongiform encephalopathies (TSEs) and consist primarily of scrapie prion protein (PrP (Sc) ), a pathogenic isoform of the host-encoded cellular prion protein (PrP (C) ). The absence of nucleic acids as essential components of the infectious prions is the most striking feature associated to these diseases. Additionally, different prion strains have been isolated from animal diseases despite the lack of DNA or RNA molecules. Mounting evidence suggests that prion-strain-specific features segregate with different PrP (Sc) conformational and aggregation states. Strains are of practical relevance in prion diseases as they can drastically differ in many aspects, such as incubation period, PrP (Sc) biochemical profile (e.g., electrophoretic mobility and glycoform ratio) and distribution of brain lesions. Importantly, such different features are maintained after inoculation of a prion strain into genetically identical hosts and are relatively stable across serial passages. This review focuses on the characterization of prion strains and on the wide range of important implications that the study of prion strains involves.

Cellular Aspects of Prion Replication In Vitro

PubMed Central

Grassmann, Andrea; Wolf, Hanna; Hofmann, Julia; Graham, James; Vorberg, Ina

2013-01-01

Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative disorders in mammals that are caused by unconventional agents predominantly composed of aggregated misfolded prion protein (PrP). Prions self-propagate by recruitment of host-encoded PrP into highly ordered β-sheet rich aggregates. Prion strains differ in their clinical, pathological and biochemical characteristics and are likely to be the consequence of distinct abnormal prion protein conformers that stably replicate their alternate states in the host cell. Understanding prion cell biology is fundamental for identifying potential drug targets for disease intervention. The development of permissive cell culture models has greatly enhanced our knowledge on entry, propagation and dissemination of TSE agents. However, despite extensive research, the precise mechanism of prion infection and potential strain effects remain enigmatic. This review summarizes our current knowledge of the cell biology and propagation of prions derived from cell culture experiments. We discuss recent findings on the trafficking of cellular and pathologic PrP, the potential sites of abnormal prion protein synthesis and potential co-factors involved in prion entry and propagation. PMID:23340381

Use of ade1 and ade2 mutations for development of a versatile red/white colour assay of amyloid-induced oxidative stress in saccharomyces cerevisiae.

PubMed

Bharathi, Vidhya; Girdhar, Amandeep; Prasad, Archana; Verma, Meenkshi; Taneja, Vibha; Patel, Basant K

2016-12-01

Mutations in adenine biosynthesis pathway genes ADE1 and ADE2 have been conventionally used to score for prion [PSI + ] in yeast. If ade1-14 mutant allele is present, which contains a premature stop codon, [psi - ] yeast appear red on YPD medium owing to accumulation of a red intermediate compound in vacuoles. In [PSI + ] yeast, partial inactivation of the translation termination factor, Sup35 protein, owing to its amyloid aggregation allows for read-through of the ade1-14 stop codon and the yeast appears white as the red intermediate pigment is not accumulated. The red colour development in ade1 and ade2 mutant yeast requires reduced-glutathione, which helps in transport of the intermediate metabolite P-ribosylaminoimidazole carboxylate into vacuoles, which develops the red colour. Here, we hypothesize that amyloid-induced oxidative stress would deplete reduced-glutathione levels and thus thwart the development of red colour in ade1 or ade2 yeast. Indeed, when we overexpressed amyloid-forming human proteins TDP-43, Aβ-42 and Poly-Gln-103 and the yeast prion protein Rnq1, the otherwise red ade1 yeast yielded some white colonies. Further, the white colour eventually reverted back to red upon turning off the amyloid protein's expression. Also, the aggregate-bearing yeast have increased oxidative stress and white phenotype yeast revert to red when grown on media with reducing agent. Furthermore, the red/white assay could also be emulated in ade2-1, ade2Δ, and ade1Δ mutant yeast and also in an ade1-14 mutant with erg6 gene deletion that increases cell-wall permeability. This model would be useful tool for drug-screening against general amyloid-induced oxidative stress and toxicity. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

A prion primer

PubMed Central

Cashman, N R

1997-01-01

By biological and medical criteria, prions are infectious agents; however, many of their properties differ profoundly from those of conventional microbes. Prions are "encoded" by alterations in protein conformation rather than in nucleic acid or amino acid sequence. New epidemic prion diseases (bovine spongiform encephalopathy and new variant Creutzfeldt-Jakob disease) have recently emerged under the active surveillance of the modern world. The risk of contracting prion disease from blood products or other biologicals is now a focus of worldwide concern. Much has been discovered about prions and prion diseases, but much remains to be done. PMID:9371069

Live-cell FRET imaging reveals clustering of the prion protein at the cell surface induced by infectious prions.

PubMed

Tavares, Evandro; Macedo, Joana A; Paulo, Pedro M R; Tavares, Catarina; Lopes, Carlos; Melo, Eduardo P

2014-07-01

Prion diseases are associated to the conversion of the prion protein into a misfolded pathological isoform. The mechanism of propagation of protein misfolding by protein templating remains largely unknown. Neuroblastoma cells were transfected with constructs of the prion protein fused to both CFP-GPI-anchored and to YFP-GPI-anchored and directed to its cell membrane location. Live-cell FRET imaging between the prion protein fused to CFP or YFP was measured giving consistent values of 10±2%. This result was confirmed by fluorescence lifetime imaging microscopy and indicates intermolecular interactions between neighbor prion proteins. In particular, considering that a maximum FRET efficiency of 17±2% was determined from a positive control consisting of a fusion CFP-YFP-GPI-anchored. A stable cell clone expressing the two fusions containing the prion protein was also selected to minimize cell-to-cell variability. In both, stable and transiently transfected cells, the FRET efficiency consistently increased in the presence of infectious prions - from 4±1% to 7±1% in the stable clone and from 10±2% to 16±1% in transiently transfected cells. These results clearly reflect an increased clustering of the prion protein on the membrane in the presence of infectious prions, which was not observed in negative control using constructs without the prion protein and upon addition of non-infected brain. Our data corroborates the recent view that the primary site for prion conversion is the cell membrane. Since our fluorescent cell clone is not susceptible to propagate infectivity, we hypothesize that the initial event of prion infectivity might be the clustering of the GPI-anchored prion protein. Copyright © 2014 Elsevier B.V. All rights reserved.

Prions, prionoids and pathogenic proteins in Alzheimer disease.

PubMed

Ashe, Karen H; Aguzzi, Adriano

2013-01-01

Like patients with prion disease, Alzheimer patients suffer from a fatal, progressive form of dementia. There is growing evidence that amyloid-β (Aβ) aggregates may be transmissible similar to prions, at least under extreme experimental conditions. However, unlike mice infected with prion protein (PrP) prions, those inoculated with Aβ do not die. The transmission of Aβ and PrP thus differs conspicuously in the neurological effects they induce in their hosts, the difference being no less than a matter of life and death. Far from being a mere academic nuance, this distinction between Aβ and PrP begs the crucial questions of what, exactly, controls prion toxicity and how prion toxicity relates to prion infectivity.

Generation of Infectious Prions and Detection with the Prion-Infected Cell Assay.

PubMed

Vella, Laura J; Coleman, Bradley; Hill, Andrew F

2017-01-01

Cell lines propagating prions are an efficient and useful means for studying the cellular and molecular mechanisms implicated in prion disease. Utilization of cell-based models has led to the finding that PrP C and PrP Sc are released from cells in association with extracellular vesicles known as exosomes. Exosomes have been shown to act as vehicles for infectivity, transferring infectivity between cell lines and providing a mechanism for prion spread between tissues. Here, we describe the methods for generating a prion-propagating cell line with prion-infected brain homogenate, cell lysate, conditioned media, and exosomes and also detection of protease-resistant PrP with the prion-infected cell assay.

Pressure-assisted dissociation and degradation of "proteinase K-resistant" fibrils prepared by seeding with scrapie-infected hamster prion protein.

PubMed

Akasaka, Kazuyuki; Maeno, Akihiro; Murayama, Taichi; Tachibana, Hideki; Fujita, Yuzo; Yamanaka, Hitoki; Nishida, Noriyuki; Atarashi, Ryuichiro

2014-01-01

The crucial step for the fatal neurodegenerative prion diseases involves the conversion of a normal cellular protein, PrP(C), into a fibrous pathogenic form, PrP(Sc), which has an unusual stability against heat and resistance against proteinase K digestion. A successful challenge to reverse the reaction from PrP(Sc) into PrP(C) is considered valuable, as it would give a key to dissolving the complex molecular events into thermodynamic and kinetic analyses and may also provide a means to prevent the formation of PrP(Sc) from PrP(C) eventually in vivo. Here we show that, by applying pressures at kbar range, the "proteinase K-resistant" fibrils (rHaPrP(res)) prepared from hamster prion protein (rHaPrP [23-231]) by seeding with brain homogenate of scrapie-infected hamster, becomes easily digestible. The result is consistent with the notion that rHaPrP(res) fibrils are dissociated into rHaPrP monomers under pressure and that the formation of PrP(Sc) from PrP(C) is thermodynamically controlled. Moreover, the efficient degradation of prion fibrils under pressure provides a novel means of eliminating infectious PrP(Sc) from various systems of pathogenic concern.

Yeast Prions and Human Prion-like Proteins: Sequence Features and Prediction Methods

PubMed Central

Cascarina, Sean; Ross, Eric D.

2014-01-01

Prions are self-propagating infectious protein isoforms. A growing number of prions have been identified in yeast, each resulting from the conversion of soluble proteins into an insoluble amyloid form. These yeast prions have served as a powerful model system for studying the causes and consequences of prion aggregation. Remarkably, a number of human proteins containing prion-like domains, defined as domains with compositional similarity to yeast prion domains, have recently been linked to various human degenerative diseases, including amyotrophic lateral sclerosis (ALS). This suggests that the lessons learned from yeast prions may help in understanding these human diseases. In this review, we examine what has been learned about the amino acid sequence basis for prion aggregation in yeast, and how this information has been used to develop methods to predict aggregation propensity. We then discuss how this information is being applied to understand human disease, and the challenges involved in applying yeast prediction methods to higher organisms. PMID:24390581

Biology and Genetics of Prions Causing Neurodegeneration

PubMed Central

Prusiner, Stanley B.

2014-01-01

Prions are proteins that acquire alternative conformations that become self-propagating. Transformation of proteins into prions is generally accompanied by an increase in β-sheet structure and a propensity to aggregate into oligomers. Some prions are beneficial and perform cellular functions, whereas others cause neurodegeneration. In mammals, more than a dozen proteins that become prions have been identified and a similar number has been found in fungi. In both mammals and fungi, variations in the prion conformation encipher the biological properties of distinct prion strains. Increasing evidence argues that prions cause many neurodegenerative diseases (NDs), including Alzheimer’s, Parkinson’s, Creutzfeldt-Jakob, and Lou Gehrig’s diseases, as well as the tauopathies. The majority of NDs are sporadic, and 10% to 20% are inherited. The late onset of heritable NDs, like their sporadic counterparts, may reflect the stochastic nature of prion formation; the pathogenesis of such illnesses seems to require prion accumulation to exceed some critical threshold before neurological dysfunction manifests. PMID:24274755

Yeast prions and human prion-like proteins: sequence features and prediction methods.

PubMed

Cascarina, Sean M; Ross, Eric D

2014-06-01

Prions are self-propagating infectious protein isoforms. A growing number of prions have been identified in yeast, each resulting from the conversion of soluble proteins into an insoluble amyloid form. These yeast prions have served as a powerful model system for studying the causes and consequences of prion aggregation. Remarkably, a number of human proteins containing prion-like domains, defined as domains with compositional similarity to yeast prion domains, have recently been linked to various human degenerative diseases, including amyotrophic lateral sclerosis. This suggests that the lessons learned from yeast prions may help in understanding these human diseases. In this review, we examine what has been learned about the amino acid sequence basis for prion aggregation in yeast, and how this information has been used to develop methods to predict aggregation propensity. We then discuss how this information is being applied to understand human disease, and the challenges involved in applying yeast prediction methods to higher organisms.

Accelerating Yeast Prion Biology using Droplet Microfluidics

NASA Astrophysics Data System (ADS)

Ung, Lloyd; Rotem, Assaf; Jarosz, Daniel; Datta, Manoshi; Lindquist, Susan; Weitz, David

2012-02-01

Prions are infectious proteins in a misfolded form, that can induce normal proteins to take the misfolded state. Yeast prions are relevant, as a model of human prion diseases, and interesting from an evolutionary standpoint. Prions may also be a form of epigenetic inheritance, which allow yeast to adapt to stressful conditions at rates exceeding those of random mutations and propagate that adaptation to their offspring. Encapsulation of yeast in droplet microfluidic devices enables high-throughput measurements with single cell resolution, which would not be feasible using bulk methods. Millions of populations of yeast can be screened to obtain reliable measurements of prion induction and loss rates. The population dynamics of clonal yeast, when a fraction of the cells are prion expressing, can be elucidated. Furthermore, the mechanism by which certain strains of bacteria induce yeast to express prions in the wild can be deduced. Integrating the disparate fields of prion biology and droplet microfluidics reveals a more complete picture of how prions may be more than just diseases and play a functional role in yeast.

Quinacrine promotes replication and conformational mutation of chronic wasting disease prions.

PubMed

Bian, Jifeng; Kang, Hae-Eun; Telling, Glenn C

2014-04-22

Quinacrine's ability to reduce levels of pathogenic prion protein (PrP(Sc)) in mouse cells infected with experimentally adapted prions led to several unsuccessful clinical studies in patients with prion diseases, a 10-y investment to understand its mechanism of action, and the production of related compounds with expectations of greater efficacy. We show here, in stark contrast to this reported inhibitory effect, that quinacrine enhances deer and elk PrP(Sc) accumulation and promotes propagation of prions causing chronic wasting disease (CWD), a fatal, transmissible, neurodegenerative disorder of cervids of uncertain zoonotic potential. Surprisingly, despite increased prion titers in quinacrine-treated cells, transmission of the resulting prions produced prolonged incubation times and altered PrP(Sc) deposition patterns in the brains of diseased transgenic mice. This unexpected outcome is consistent with quinacrine affecting the intrinsic properties of the CWD prion. Accordingly, quinacrine-treated CWD prions were comprised of an altered PrP(Sc) conformation. Our findings provide convincing evidence for drug-induced conformational mutation of prions without the prerequisite of generating drug-resistant variants of the original strain. More specifically, they show that a drug capable of restraining prions in one species/strain setting, and consequently used to treat human prion diseases, improves replicative ability in another and therefore force reconsideration of current strategies to screen antiprion compounds.

Prions and lymphoid organs

PubMed Central

O’Connor, Tracy; Aguzzi, Adriano

2013-01-01

Prion colonization of secondary lymphoid organs (SLOs) is a critical step preceding neuroinvasion in prion pathogenesis. Follicular dendritic cells (FDCs), which depend on both tumor necrosis factor receptor 1 (TNFR1) and lymphotoxin β receptor (LTβR) signaling for maintenance, are thought to be the primary sites of prion accumulation in SLOs. However, prion titers in RML-infected TNFR1−/− lymph nodes and rates of neuroinvasion in TNFR1−/− mice remain high despite the absence of mature FDCs. Recently, we discovered that TNFR1-independent prion accumulation in lymph nodes relies on LTβR signaling. Loss of LTβR signaling in TNFR1−/− lymph nodes coincided with the de-differentiation of high endothelial venules (HEVs)—the primary sites of lymphocyte entry into lymph nodes. These findings suggest that HEVs are the sites through which prions initially invade lymph nodes from the bloodstream. Identification of HEVs as entry portals for prions clarifies a number of previous observations concerning peripheral prion pathogenesis. However, a number of questions still remain: What is the mechanism by which prions are taken up by HEVs? Which cells are responsible for delivering prions to lymph nodes? Are HEVs the main entry site for prions into lymph nodes or do alternative routes also exist? These questions and others are considered in this article. PMID:23357827

Quinacrine promotes replication and conformational mutation of chronic wasting disease prions

PubMed Central

Bian, Jifeng; Kang, Hae-Eun; Telling, Glenn C.

2014-01-01

Quinacrine’s ability to reduce levels of pathogenic prion protein (PrPSc) in mouse cells infected with experimentally adapted prions led to several unsuccessful clinical studies in patients with prion diseases, a 10-y investment to understand its mechanism of action, and the production of related compounds with expectations of greater efficacy. We show here, in stark contrast to this reported inhibitory effect, that quinacrine enhances deer and elk PrPSc accumulation and promotes propagation of prions causing chronic wasting disease (CWD), a fatal, transmissible, neurodegenerative disorder of cervids of uncertain zoonotic potential. Surprisingly, despite increased prion titers in quinacrine-treated cells, transmission of the resulting prions produced prolonged incubation times and altered PrPSc deposition patterns in the brains of diseased transgenic mice. This unexpected outcome is consistent with quinacrine affecting the intrinsic properties of the CWD prion. Accordingly, quinacrine-treated CWD prions were comprised of an altered PrPSc conformation. Our findings provide convincing evidence for drug-induced conformational mutation of prions without the prerequisite of generating drug-resistant variants of the original strain. More specifically, they show that a drug capable of restraining prions in one species/strain setting, and consequently used to treat human prion diseases, improves replicative ability in another and therefore force reconsideration of current strategies to screen antiprion compounds. PMID:24711410

Prions and lymphoid organs: solved and remaining mysteries.

PubMed

O'Connor, Tracy; Aguzzi, Adriano

2013-01-01

Prion colonization of secondary lymphoid organs (SLOs) is a critical step preceding neuroinvasion in prion pathogenesis. Follicular dendritic cells (FDCs), which depend on both tumor necrosis factor receptor 1 (TNFR1) and lymphotoxin β receptor (LTβR) signaling for maintenance, are thought to be the primary sites of prion accumulation in SLOs. However, prion titers in RML-infected TNFR1 (-/-) lymph nodes and rates of neuroinvasion in TNFR1 (-/-) mice remain high despite the absence of mature FDCs. Recently, we discovered that TNFR1-independent prion accumulation in lymph nodes relies on LTβR signaling. Loss of LTβR signaling in TNFR1 (-/-) lymph nodes coincided with the de-differentiation of high endothelial venules (HEVs)-the primary sites of lymphocyte entry into lymph nodes. These findings suggest that HEVs are the sites through which prions initially invade lymph nodes from the bloodstream. Identification of HEVs as entry portals for prions clarifies a number of previous observations concerning peripheral prion pathogenesis. However, a number of questions still remain: What is the mechanism by which prions are taken up by HEVs? Which cells are responsible for delivering prions to lymph nodes? Are HEVs the main entry site for prions into lymph nodes or do alternative routes also exist? These questions and others are considered in this article.

Gene knockout of tau expression does not contribute to the pathogenesis of prion disease.

PubMed

Lawson, Victoria A; Klemm, Helen M; Welton, Jeremy M; Masters, Colin L; Crouch, Peter; Cappai, Roberto; Ciccotosto, Giuseppe D

2011-11-01

Prion diseases or transmissible spongiform encephalopathies are a group of fatal and transmissible disorders affecting the central nervous system of humans and animals. The principal agent of prion disease transmission and pathogenesis is proposed to be an abnormal protease-resistant isoform of the normal cellular prion protein. The microtubule-associated protein tau is elevated in patients with Creutzfeldt-Jakob disease. To determine whether tau expression contributes to prion disease pathogenesis, tau knockout and control wild-type mice were infected with the M1000 strain of mouse-adapted human prions. Immunohistochemical analysis for total tau expression in prion-infected wild-type mice indicated tau aggregation in the cytoplasm of a subpopulation of neurons in regions associated with spongiform change. Western immunoblot analysis of brain homogenates revealed a decrease in total tau immunoreactivity and epitope-specific changes in tau phosphorylation. No significant difference in incubation period or other disease features were observed between tau knockout and wild-type mice with clinical prion disease. These results demonstrate that, in this model of prion disease, tau does not contribute to the pathogenesis of prion disease and that changes in the tau protein profile observed in mice with clinical prion disease occurs as a consequence of the prion-induced pathogenesis.

Evidence for two distinct intracellular pools of inorganic sulfate in Penicillium notatum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hunter, D.R.; Segel, I.H.

1985-06-01

A strain of Penicillium notatum unable to metabolize inorganic sulfate can accumulate sulfate internally to an apparent equilibrium concentration 10/sup 5/ times greater than that remaining in the medium. The apparent K/sub eq/ is near constant at all initial external sulfate concentrations below that which would eventually exceed the internal capacity of the cells. Under equilibrium conditions of zero net flux, external /sup 35/SO/sub 4//sup 2 -/ exchanges with internal, unlabeled SO/sub 4//sup 2 -/ at a rate consistent with the kinetic constants with the sulfate transport system. Efflux experiments demonstrated that sulfate occupies two distinct intracellular pools. Pool 1more » is characterized by the rapid release of /sup 35/SO/sub 4//sup 2 -/ when the suspension of preloaded cells is adjusted to 10 mM azide at pH 8.4 (t/sub 1/2/, 0.38 min). /sup 35/SO/sub 4//sup 2 -/ in pool 1 also rapidly exchanges with unlabeled medium sulfate. Pool 2 is characterized by the slow release of /sup 35/SO/sub 4//sup 2 -/ induced by azide at pH 8.4 or unlabeled sulfate (t/sub 1/2/, 32 to 49 min). Early in the /sup 35/SO/sub 4//sup 2 -/ accumulation process, up to 78% of the total transported substrate is found in pool 1. At equilibrium, pool 1 accounts for only about 2% of the total accumulated /sup 35/SO/sub 4//sup 2 -/. Monensin (33 ..mu..m) accelerates the transfer of /sup 35/SO/sub 4//sup 2 -/ from pool 1 to pool 2. Valinomycin (0.2 ..mu..M) and tetraphynylboron/sup -/ (1 mM) retard the transfer of /sup 35/SO/sub 4//sup 2 -/ from pool 1 to pool 2. Pool 2 may reside in a vacuole or other intracellular organelle. A model for the transfer of sulfate from pool 1 to pool 2 is presented.« less

Human prion diseases: surgical lessons learned from iatrogenic prion transmission.

PubMed

Bonda, David J; Manjila, Sunil; Mehndiratta, Prachi; Khan, Fahd; Miller, Benjamin R; Onwuzulike, Kaine; Puoti, Gianfranco; Cohen, Mark L; Schonberger, Lawrence B; Cali, Ignazio

2016-07-01

The human prion diseases, or transmissible spongiform encephalopathies, have captivated our imaginations since their discovery in the Fore linguistic group in Papua New Guinea in the 1950s. The mysterious and poorly understood "infectious protein" has become somewhat of a household name in many regions across the globe. From bovine spongiform encephalopathy (BSE), commonly identified as mad cow disease, to endocannibalism, media outlets have capitalized on these devastatingly fatal neurological conditions. Interestingly, since their discovery, there have been more than 492 incidents of iatrogenic transmission of prion diseases, largely resulting from prion-contaminated growth hormone and dura mater grafts. Although fewer than 9 cases of probable iatrogenic neurosurgical cases of Creutzfeldt-Jakob disease (CJD) have been reported worldwide, the likelihood of some missed cases and the potential for prion transmission by neurosurgery create considerable concern. Laboratory studies indicate that standard decontamination and sterilization procedures may be insufficient to completely remove infectivity from prion-contaminated instruments. In this unfortunate event, the instruments may transmit the prion disease to others. Much caution therefore should be taken in the absence of strong evidence against the presence of a prion disease in a neurosurgical patient. While the Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO) have devised risk assessment and decontamination protocols for the prevention of iatrogenic transmission of the prion diseases, incidents of possible exposure to prions have unfortunately occurred in the United States. In this article, the authors outline the historical discoveries that led from kuru to the identification and isolation of the pathological prion proteins in addition to providing a brief description of human prion diseases and iatrogenic forms of CJD, a brief history of prion disease nosocomial transmission, and a summary of the CDC and WHO guidelines for prevention of prion disease transmission and decontamination of prion-contaminated neurosurgical instruments.

The tip of the iceberg: RNA-binding proteins with prion-like domains in neurodegenerative disease

PubMed Central

King, Oliver D.; Gitler, Aaron D.; Shorter, James

2012-01-01

Prions are self-templating protein conformers that are naturally transmitted between individuals and promote phenotypic change. In yeast, prion-encoded phenotypes can be beneficial, neutral or deleterious depending upon genetic background and environmental conditions. A distinctive and portable ‘prion domain’ enriched in asparagine, glutamine, tyrosine and glycine residues unifies the majority of yeast prion proteins. Deletion of this domain precludes prionogenesis and appending this domain to reporter proteins can confer prionogenicity. An algorithm designed to detect prion domains has successfully identified 19 domains that can confer prion behavior. Scouring the human genome with this algorithm enriches a select group of RNA-binding proteins harboring a canonical RNA recognition motif (RRM) and a putative prion domain. Indeed, of 210 human RRM-bearing proteins, 29 have a putative prion domain, and 12 of these are in the top 60 prion candidates in the entire genome. Startlingly, these RNA-binding prion candidates are inexorably emerging, one by one, in the pathology and genetics of devastating neurodegenerative disorders, including: amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U), Alzheimer’s disease and Huntington’s disease. For example, FUS and TDP-43, which rank 1st and 10th among RRM-bearing prion candidates, form cytoplasmic inclusions in the degenerating motor neurons of ALS patients and mutations in TDP-43 and FUS cause familial ALS. Recently, perturbed RNA-binding proteostasis of TAF15, which is the 2nd ranked RRM-bearing prion candidate, has been connected with ALS and FTLD-U. We strongly suspect that we have now merely reached the tip of the iceberg. We predict that additional RNA-binding prion candidates identified by our algorithm will soon surface as genetic modifiers or causes of diverse neurodegenerative conditions. Indeed, simple prion-like transfer mechanisms involving the prion-like domains of RNA-binding proteins could underlie the classical non-cell-autonomous emanation of neurodegenerative pathology from originating epicenters to neighboring portions of the nervous system. PMID:22445064

Protease resistance of infectious prions is suppressed by removal of a single atom in the cellular prion protein.

PubMed

Leske, Henning; Hornemann, Simone; Herrmann, Uli Simon; Zhu, Caihong; Dametto, Paolo; Li, Bei; Laferriere, Florent; Polymenidou, Magdalini; Pelczar, Pawel; Reimann, Regina Rose; Schwarz, Petra; Rushing, Elisabeth Jane; Wüthrich, Kurt; Aguzzi, Adriano

2017-01-01

Resistance to proteolytic digestion has long been considered a defining trait of prions in tissues of organisms suffering from transmissible spongiform encephalopathies. Detection of proteinase K-resistant prion protein (PrPSc) still represents the diagnostic gold standard for prion diseases in humans, sheep and cattle. However, it has become increasingly apparent that the accumulation of PrPSc does not always accompany prion infections: high titers of prion infectivity can be reached also in the absence of protease resistant PrPSc. Here, we describe a structural basis for the phenomenon of protease-sensitive prion infectivity. We studied the effect on proteinase K (PK) resistance of the amino acid substitution Y169F, which removes a single oxygen atom from the β2-α2 loop of the cellular prion protein (PrPC). When infected with RML or the 263K strain of prions, transgenic mice lacking wild-type (wt) PrPC but expressing MoPrP169F generated prion infectivity at levels comparable to wt mice. The newly generated MoPrP169F prions were biologically indistinguishable from those recovered from prion-infected wt mice, and elicited similar pathologies in vivo. Surprisingly, MoPrP169F prions showed greatly reduced PK resistance and density gradient analyses showed a significant reduction in high-density aggregates. Passage of MoPrP169F prions into mice expressing wt MoPrP led to full recovery of protease resistance, indicating that no strain shift had taken place. We conclude that a subtle structural variation in the β2-α2 loop of PrPC affects the sensitivity of PrPSc to protease but does not impact prion replication and infectivity. With these findings a specific structural feature of PrPC can be linked to a physicochemical property of the corresponding PrPSc.

Human prion diseases: surgical lessons learned from iatrogenic prion transmission

PubMed Central

Bonda, David J.; Manjila, Sunil; Mehndiratta, Prachi; Khan, Fahd; Miller, Benjamin R.; Onwuzulike, Kaine; Puoti, Gianfranco; Cohen, Mark L.; Schonberger, Lawrence B.; Cali, Ignazio

2016-01-01

The human prion diseases, or transmissible spongiform encephalopathies, have captivated our imaginations since their discovery in the Fore linguistic group in Papua New Guinea in the 1950s. The mysterious and poorly understood “infectious protein” has become somewhat of a household name in many regions across the globe. From bovine spongiform encephalopathy (BSE), commonly identified as mad cow disease, to endocannibalism, media outlets have capitalized on these devastatingly fatal neurological conditions. Interestingly, since their discovery, there have been more than 492 incidents of iatrogenic transmission of prion diseases, largely resulting from prion-contaminated growth hormone and dura mater grafts. Although fewer than 9 cases of probable iatrogenic neurosurgical cases of Creutzfeldt-Jakob disease (CJD) have been reported worldwide, the likelihood of some missed cases and the potential for prion transmission by neurosurgery create considerable concern. Laboratory studies indicate that standard decontamination and sterilization procedures may be insufficient to completely remove infectivity from prion-contaminated instruments. In this unfortunate event, the instruments may transmit the prion disease to others. Much caution therefore should be taken in the absence of strong evidence against the presence of a prion disease in a neurosurgical patient. While the Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO) have devised risk assessment and decontamination protocols for the prevention of iatrogenic transmission of the prion diseases, incidents of possible exposure to prions have unfortunately occurred in the United States. In this article, the authors outline the historical discoveries that led from kuru to the identification and isolation of the pathological prion proteins in addition to providing a brief description of human prion diseases and iatrogenic forms of CJD, a brief history of prion disease nosocomial transmission, and a summary of the CDC and WHO guidelines for prevention of prion disease transmission and decontamination of prion-contaminated neurosurgical instruments. PMID:27364252

The Gut-Associated Lymphoid Tissues in the Small Intestine, Not the Large Intestine, Play a Major Role in Oral Prion Disease Pathogenesis

PubMed Central

Donaldson, David S.; Else, Kathryn J.

2015-01-01

ABSTRACT Prion diseases are infectious neurodegenerative disorders characterized by accumulations of abnormally folded cellular prion protein in affected tissues. Many natural prion diseases are acquired orally, and following exposure, the early replication of some prion isolates upon follicular dendritic cells (FDC) within gut-associated lymphoid tissues (GALT) is important for the efficient spread of disease to the brain (neuroinvasion). Prion detection within large intestinal GALT biopsy specimens has been used to estimate human and animal disease prevalence. However, the relative contributions of the small and large intestinal GALT to oral prion pathogenesis were unknown. To address this issue, we created mice that specifically lacked FDC-containing GALT only in the small intestine. Our data show that oral prion disease susceptibility was dramatically reduced in mice lacking small intestinal GALT. Although these mice had FDC-containing GALT throughout their large intestines, these tissues were not early sites of prion accumulation or neuroinvasion. We also determined whether pathology specifically within the large intestine might influence prion pathogenesis. Congruent infection with the nematode parasite Trichuris muris in the large intestine around the time of oral prion exposure did not affect disease pathogenesis. Together, these data demonstrate that the small intestinal GALT are the major early sites of prion accumulation and neuroinvasion after oral exposure. This has important implications for our understanding of the factors that influence the risk of infection and the preclinical diagnosis of disease. IMPORTANCE Many natural prion diseases are acquired orally. After exposure, the accumulation of some prion diseases in the gut-associated lymphoid tissues (GALT) is important for efficient spread of disease to the brain. However, the relative contributions of GALT in the small and large intestines to oral prion pathogenesis were unknown. We show that the small intestinal GALT are the essential early sites of prion accumulation. Furthermore, congruent infection with a large intestinal helminth (worm) around the time of oral prion exposure did not affect disease pathogenesis. This is important for our understanding of the factors that influence the risk of prion infection and the preclinical diagnosis of disease. The detection of prions within large intestinal GALT biopsy specimens has been used to estimate human and animal disease prevalence. However, our data suggest that using these biopsy specimens may miss individuals in the early stages of oral prion infection and significantly underestimate the disease prevalence. PMID:26157121

Microbial communities and greenhouse gas emissions associated with the biodegradation of specified risk material in compost

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Shanwei; Agriculture and Agri-Food Canada, Lethbridge Research Centre, P.O. Box 3000, Lethbridge, Alberta, Canada T1J 4B1; Reuter, Tim

Highlights: ► Addition of feathers altered bacterial and fungal communities in compost. ► Microbial communities degrading SRM and compost matrix were distinct. ► Addition of feathers may enrich for microbial communities that degrade SRM. ► Inclusion of feather in compost increased both CH{sub 4} and N{sub 2}O emissions from compost. ► Density of methanogens and methanotrophs were weakly associated with CH{sub 4} emissions. - Abstract: Provided that infectious prions (PrP{sup Sc}) are inactivated, composting of specified risk material (SRM) may be a viable alternative to rendering and landfilling. In this study, bacterial and fungal communities as well as greenhouse gasmore » emissions associated with the degradation of SRM were examined in laboratory composters over two 14 day composting cycles. Chicken feathers were mixed into compost to enrich for microbial communities involved in the degradation of keratin and other recalcitrant proteins such as prions. Feathers altered the composition of bacterial and fungal communities primarily during the first cycle. The bacterial genera Saccharomonospora, Thermobifida, Thermoactinomycetaceae, Thiohalospira, Pseudomonas, Actinomadura, and Enterobacter, and the fungal genera Dothideomycetes, Cladosporium, Chaetomium, and Trichaptum were identified as candidates involved in SRM degradation. Feathers increased (P < 0.05) headspace concentrations of CH{sub 4} primarily during the early stages of the first cycle and N{sub 2}O during the second. Although inclusion of feathers in compost increases greenhouse gas emissions, it may promote the establishment of microbial communities that are more adept at degrading SRM and recalcitrant proteins such as keratin and PrP{sup Sc}.« less

Perturbation of the Secondary Structure of the Scrapie Prion Protein Under Conditions that Alter Infectivity

NASA Astrophysics Data System (ADS)

Gasset, Maria; Baldwin, Michael A.; Fletterick, Robert J.; Prusiner, Stanley B.

1993-01-01

Limited proteolysis of the scrapie prion protein (PrPSc) generates PrP 27-30, which polymerizes into amyloid. By attenuated total reflection-Fourier transform infrared spectroscopy, PrP 27-30 polymers contained 54% β-sheet, 25% α-helix, 10% turns, and 11% random coil; dispersion into detergent-lipid-protein-complexes preserved infectivity and secondary structure. Almost 60% of the β-sheet was low-frequency infrared-absorbing, reflecting intermolecular aggregation. Decreased low-frequency β-sheet and increased turn content were found after SDS/PAGE, which disassembled the amyloid polymers, denatured PrP 27-30, and diminished scrapie infectivity. Acid-induced transitions were reversible, whereas alkali produced an irreversible transition centered at pH 10 under conditions that diminished infectivity. Whether PrPSc synthesis involves a transition in the secondary structure of one or more domains of the cellular prion protein from α-helical, random coil, or turn into β-sheet remains to be established.

Classical Bovine Spongiform Encephalopathy by Transmission of H-Type Prion in Homologous Prion Protein Context

PubMed Central

Andréoletti, Olivier; Lacroux, Caroline; Prieto, Irene; Lorenzo, Patricia; Larska, Magdalena; Baron, Thierry; Espinosa, Juan-Carlos

2011-01-01

Bovine spongiform encephalopathy (BSE) and BSE-related disorders have been associated with a single major prion strain. Recently, 2 atypical, presumably sporadic forms of BSE have been associated with 2 distinct prion strains that are characterized mainly by distinct Western blot profiles of abnormal protease-resistant prion protein (PrPres), named high-type (BSE-H) and low-type (BSE-L), that also differed from classical BSE. We characterized 5 atypical BSE-H isolates by analyzing their molecular and neuropathologic properties during transmission in transgenic mice expressing homologous bovine prion protein. Unexpectedly, in several inoculated animals, strain features emerged that were highly similar to those of classical BSE agent. These findings demonstrate the capability of an atypical bovine prion to acquire classical BSE–like properties during propagation in a homologous bovine prion protein context and support the view that the epidemic BSE agent could have originated from such a cattle prion. PMID:21888788

Kuru prions and sporadic Creutzfeldt–Jakob disease prions have equivalent transmission properties in transgenic and wild-type mice

PubMed Central

Wadsworth, Jonathan D. F.; Joiner, Susan; Linehan, Jacqueline M.; Desbruslais, Melanie; Fox, Katie; Cooper, Sharon; Cronier, Sabrina; Asante, Emmanuel A.; Mead, Simon; Brandner, Sebastian; Hill, Andrew F.; Collinge, John

2008-01-01

Kuru provides our principal experience of an epidemic human prion disease and primarily affected the Fore linguistic group of the Eastern Highlands of Papua New Guinea. Kuru was transmitted by the practice of consuming dead relatives as a mark of respect and mourning (transumption). To date, detailed information of the prion strain type propagated in kuru has been lacking. Here, we directly compare the transmission properties of kuru prions with sporadic, iatrogenic, and variant Creutzfeldt–Jakob disease (CJD) prions in Prnp-null transgenic mice expressing human prion protein and in wild-type mice. Molecular and neuropathological data from these transmissions show that kuru prions are distinct from variant CJD and have transmission properties equivalent to those of classical (sporadic) CJD prions. These findings are consistent with the hypothesis that kuru originated from chance consumption of an individual with sporadic CJD. PMID:18316717

Prion-based memory of heat stress in yeast

PubMed Central

Chernova, Tatiana A.; Wilkinson, Keith D.

2017-01-01

ABSTRACT Amyloids and amyloid-based prions are self-perpetuating protein aggregates which can spread by converting a normal protein of the same sequence into a prion form. They are associated with diseases in humans and mammals, and control heritable traits in yeast and other fungi. Some amyloids are implicated in biologically beneficial processes. As prion formation generates reproducible memory of a conformational change, prions can be considered as molecular memory devices.  We have demonstrated that in yeast, stress-inducible cytoskeleton-associated protein Lsb2 forms a metastable prion in response to high temperature. This prion promotes conversion of other proteins into prions and can persist in a fraction of cells for a significant number of cell generations after stress, thus maintaining the memory of stress in a population of surviving cells. Acquisition of an amino acid substitution required for Lsb2 to form a prion coincides with acquisition of increased thermotolerance in the evolution of Saccharomyces yeast. Thus the ability to form an Lsb2 prion in response to stress coincides with yeast adaptation to growth at higher temperatures. These findings intimately connect prion formation to the cellular response to environmental stresses. PMID:28521568

RML prions act through Mahogunin and Attractin-independent pathways.

PubMed

Gunn, Teresa M; Carlson, George A

2013-01-01

While the conversion of the normal form of prion protein to a conformationally distinct pathogenic form is recognized to be the primary cause of prion disease, it is not clear how this leads to spongiform change, neuronal dysfunction and death. Mahogunin ring finger-1 (Mgrn1) and Attractin (Atrn) null mutant mice accumulate vacuoles throughout the brain that appear very similar to those associated with prion disease, but they do not accumulate the protease-resistant scrapie form of the prion protein or become sick. A study demonstrating an interaction between cytosolically-exposed prion protein and MGRN1 suggested that disruption of MGRN1 function may contribute to prion disease pathogenesis, but we recently showed that neither loss of MGRN1 nor MGRN1 overexpression influences the onset or progression of prion disease following intracerebral inoculation with Rocky Mountain Laboratory prions. Here, we show that loss of ATRN also has no effect on prion disease onset or progression and discuss possible mechanisms that could cause vacuolation of the central nervous system in Mgrn1 and Atrn null mutant mice and whether the same pathways might contribute to this intriguing phenotype in prion disease.

Prion-based memory of heat stress in yeast.

PubMed

Chernova, Tatiana A; Chernoff, Yury O; Wilkinson, Keith D

2017-05-04

Amyloids and amyloid-based prions are self-perpetuating protein aggregates which can spread by converting a normal protein of the same sequence into a prion form. They are associated with diseases in humans and mammals, and control heritable traits in yeast and other fungi. Some amyloids are implicated in biologically beneficial processes. As prion formation generates reproducible memory of a conformational change, prions can be considered as molecular memory devices.  We have demonstrated that in yeast, stress-inducible cytoskeleton-associated protein Lsb2 forms a metastable prion in response to high temperature. This prion promotes conversion of other proteins into prions and can persist in a fraction of cells for a significant number of cell generations after stress, thus maintaining the memory of stress in a population of surviving cells. Acquisition of an amino acid substitution required for Lsb2 to form a prion coincides with acquisition of increased thermotolerance in the evolution of Saccharomyces yeast. Thus the ability to form an Lsb2 prion in response to stress coincides with yeast adaptation to growth at higher temperatures. These findings intimately connect prion formation to the cellular response to environmental stresses.

Enhanced detection of infectious prions by direct ELISA from the brains of asymptomatic animals using DRM2-118 monoclonal antibody and Gdn-HCl.

PubMed

Hnasko, Robert; Lin, Alice; McGarvey, Jeffery; Stanker, Larry

2018-05-01

In this report we describe the use of a novel anti-prion monoclonal antibody (DRM2-118) for the direct detection of infectious prions by ELISA. Epitope mapping using overlapping hamster (SHa) prion peptides indicates DRM2-118 binding occurs between residues 93-100 and at the 3 10 -helix (residues 163-170) between alpha helix-A and -B. This antibody shows broad species binding to endogenous prions from brain homogenates and corresponding recombinant prion proteins. To evaluate the performance of this MAb for the detection of prion proteins we performed an animal time course and evaluated prion detection from both crude brain homogenates and lipid raft fractions (DRM) by direct ELISA. Prion detection was significantly enhanced by the addition of the chaotropic guanidine-HCl (Gdn-HCl) during protein immobilization with detection of PK-resistant prion from asymptomatic animal brains at (45-DPI) and from lipid rafts at (24-DPI). Our data demonstrates enhanced prion detection from brain lipid rafts of asymptomatic animals by a simple direct ELISA using the DRM2-118 MAb combined with Gdn-HCl. Published by Elsevier B.V.

Stimulating the Release of Exosomes Increases the Intercellular Transfer of Prions.

PubMed

Guo, Belinda B; Bellingham, Shayne A; Hill, Andrew F

2016-03-04

Exosomes are small extracellular vesicles released by cells and play important roles in intercellular communication and pathogen transfer. Exosomes have been implicated in several neurodegenerative diseases, including prion disease and Alzheimer disease. Prion disease arises upon misfolding of the normal cellular prion protein, PrP(C), into the disease-associated isoform, PrP(Sc). The disease has a unique transmissible etiology, and exosomes represent a novel and efficient method for prion transmission. The precise mechanism by which prions are transmitted from cell to cell remains to be fully elucidated, although three hypotheses have been proposed: direct cell-cell contact, tunneling nanotubes, and exosomes. Given the reported presence of exosomes in biological fluids and in the lipid and nucleic acid contents of exosomes, these vesicles represent an ideal mechanism for encapsulating prions and potential cofactors to facilitate prion transmission. This study investigates the relationship between exosome release and intercellular prion dissemination. Stimulation of exosome release through treatment with an ionophore, monensin, revealed a corresponding increase in intercellular transfer of prion infectivity. Conversely, inhibition of exosome release using GW4869 to target the neutral sphingomyelinase pathway induced a decrease in intercellular prion transmission. Further examination of the effect of monensin on PrP conversion revealed that monensin also alters the conformational stability of PrP(C), leading to increased generation of proteinase K-resistant prion protein. The findings presented here provide support for a positive relationship between exosome release and intercellular transfer of prion infectivity, highlighting an integral role for exosomes in facilitating the unique transmissible nature of prions. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Patterns of [PSI+] aggregation allow insights into cellular organization of yeast prion aggregates

PubMed Central

Tyedmers, Jens

2012-01-01

The yeast prion phenomenon is very widespread and mounting evidence suggests that it has an impact on cellular regulatory mechanisms related to phenotypic responses to changing environments. Studying the aggregation patterns of prion amyloids during different stages of the prion life cycle is a first key step to understand major principles of how and where cells generate, organize and turn-over prion aggregates. The induction of the [PSI+] state involves the actin cytoskeleton and quality control compartments such as the Insoluble Protein Deposit (IPOD). An initially unstable transitional induction state can be visualized by overexpression of the prion determinant and displays characteristic large ring- and ribbon-shaped aggregates consisting of poorly fragmented bundles of very long prion fibrils. In the mature prion state, the aggregation pattern is characterized by highly fragmented, shorter prion fibrils that form aggregates, which can be visualized through tagging with fluorescent proteins. The number of aggregates formed varies, ranging from a single large aggregate at the IPOD to multiple smaller ones, depending on several parameters discussed. Aggregate units below the resolution of light microscopy that are detectable by fluorescence correlation spectroscopy are in equilibrium with larger aggregates in this stage and can mediate faithful inheritance of the prion state. Loss of the prion state is often characterized by reduced fragmentation of prion fibrils and fewer, larger aggregates. PMID:22449721

Selective propagation of mouse-passaged scrapie prions with long incubation period from a mixed prion population using GT1-7 cells

PubMed Central

Masujin, Kentaro; Okada, Hiroyuki; Ushiki-Kaku, Yuko; Matsuura, Yuichi; Yokoyama, Takashi

2017-01-01

In our previous study, we demonstrated the propagation of mouse-passaged scrapie isolates with long incubation periods (L-type) derived from natural Japanese sheep scrapie cases in murine hypothalamic GT1-7 cells, along with disease-associated prion protein (PrPSc) accumulation. We here analyzed the susceptibility of GT1-7 cells to scrapie prions by exposure to infected mouse brains at different passages, following interspecies transmission. Wild-type mice challenged with a natural sheep scrapie case (Kanagawa) exhibited heterogeneity of transmitted scrapie prions in early passages, and this mixed population converged upon one with a short incubation period (S-type) following subsequent passages. However, when GT1-7 cells were challenged with these heterologous samples, L-type prions became dominant. This study demonstrated that the susceptibility of GT1-7 cells to L-type prions was at least 105 times higher than that to S-type prions and that L-type prion-specific biological characteristics remained unchanged after serial passages in GT1-7 cells. This suggests that a GT1-7 cell culture model would be more useful for the economical and stable amplification of L-type prions at the laboratory level. Furthermore, this cell culture model might be used to selectively propagate L-type scrapie prions from a mixed prion population. PMID:28636656

Bank Vole Prion Protein As an Apparently Universal Substrate for RT-QuIC-Based Detection and Discrimination of Prion Strains.

PubMed

Orrú, Christina D; Groveman, Bradley R; Raymond, Lynne D; Hughson, Andrew G; Nonno, Romolo; Zou, Wenquan; Ghetti, Bernardino; Gambetti, Pierluigi; Caughey, Byron

2015-06-01

Prions propagate as multiple strains in a wide variety of mammalian species. The detection of all such strains by a single ultrasensitive assay such as Real Time Quaking-induced Conversion (RT-QuIC) would facilitate prion disease diagnosis, surveillance and research. Previous studies have shown that bank voles, and transgenic mice expressing bank vole prion protein, are susceptible to most, if not all, types of prions. Here we show that bacterially expressed recombinant bank vole prion protein (residues 23-230) is an effective substrate for the sensitive RT-QuIC detection of all of the different prion types that we have tested so far--a total of 28 from humans, cattle, sheep, cervids and rodents, including several that have previously been undetectable by RT-QuIC or Protein Misfolding Cyclic Amplification. Furthermore, comparison of the relative abilities of different prions to seed positive RT-QuIC reactions with bank vole and not other recombinant prion proteins allowed discrimination of prion strains such as classical and atypical L-type bovine spongiform encephalopathy, classical and atypical Nor98 scrapie in sheep, and sporadic and variant Creutzfeldt-Jakob disease in humans. Comparison of protease-resistant RT-QuIC conversion products also aided strain discrimination and suggested the existence of several distinct classes of prion templates among the many strains tested.

Selective propagation of mouse-passaged scrapie prions with long incubation period from a mixed prion population using GT1-7 cells.

PubMed

Miyazawa, Kohtaro; Masujin, Kentaro; Okada, Hiroyuki; Ushiki-Kaku, Yuko; Matsuura, Yuichi; Yokoyama, Takashi

2017-01-01

In our previous study, we demonstrated the propagation of mouse-passaged scrapie isolates with long incubation periods (L-type) derived from natural Japanese sheep scrapie cases in murine hypothalamic GT1-7 cells, along with disease-associated prion protein (PrPSc) accumulation. We here analyzed the susceptibility of GT1-7 cells to scrapie prions by exposure to infected mouse brains at different passages, following interspecies transmission. Wild-type mice challenged with a natural sheep scrapie case (Kanagawa) exhibited heterogeneity of transmitted scrapie prions in early passages, and this mixed population converged upon one with a short incubation period (S-type) following subsequent passages. However, when GT1-7 cells were challenged with these heterologous samples, L-type prions became dominant. This study demonstrated that the susceptibility of GT1-7 cells to L-type prions was at least 105 times higher than that to S-type prions and that L-type prion-specific biological characteristics remained unchanged after serial passages in GT1-7 cells. This suggests that a GT1-7 cell culture model would be more useful for the economical and stable amplification of L-type prions at the laboratory level. Furthermore, this cell culture model might be used to selectively propagate L-type scrapie prions from a mixed prion population.

A direct assessment of human prion adhered to steel wire using real-time quaking-induced conversion

PubMed Central

Mori, Tsuyoshi; Atarashi, Ryuichiro; Furukawa, Kana; Takatsuki, Hanae; Satoh, Katsuya; Sano, Kazunori; Nakagaki, Takehiro; Ishibashi, Daisuke; Ichimiya, Kazuko; Hamada, Masahisa; Nakayama, Takehisa; Nishida, Noriyuki

2016-01-01

Accidental transmission of prions during neurosurgery has been reported as a consequence of re-using contaminated surgical instruments. Several decontamination methods have been studied using the 263K-hamster prion; however, no studies have directly evaluated human prions. A newly developed in vitro amplification system, designated real-time quaking-induced conversion (RT-QuIC), has allowed the activity of abnormal prion proteins to be assessed within a few days. RT-QuIC using human recombinant prion protein (PrP) showed high sensitivity for prions as the detection limit of our assay was estimated as 0.12 fg of active prions. We applied this method to detect human prion activity on stainless steel wire. When we put wires contaminated with human Creutzfeldt–Jakob disease brain tissue directly into the test tube, typical PrP-amyloid formation was observed within 48 hours, and we could detect the activity of prions at 50% seeding dose on the wire from 102.8 to 105.8 SD50. Using this method, we also confirmed that the seeding activities on the wire were removed following treatment with NaOH. As seeding activity closely correlated with the infectivity of prions using the bioassay, this wire-QuIC assay will be useful for the direct evaluation of decontamination methods for human prions. PMID:27112110

Quantifying the relative amounts of PrP polymorphisms present in prions isolated from heterozygous prion-infected animals

USDA-ARS?s Scientific Manuscript database

Prions cause protein misfolding diseases, such as transmissible spongiform encephalopathy. They propagate infections by converting a normal cellular prion protein into a prion (PrPSc). PrPC and PrPSc are isosequential and differ only in their respective conformations. PrPC is monomeric and sensit...

The [RNQ+] prion

PubMed Central

Stein, Kevin C

2011-01-01

The formation of fibrillar amyloid is most often associated with protein conformational disorders such as prion diseases, Alzheimer disease and Huntington disease. Interestingly, however, an increasing number of studies suggest that amyloid structures can sometimes play a functional role in normal biology. Several proteins form self-propagating amyloids called prions in the budding yeast Saccharomyces cerevisiae. These unique elements operate by creating a reversible, epigenetic change in phenotype. While the function of the non-prion conformation of the Rnq1 protein is unclear, the prion form, [RNQ+], acts to facilitate the de novo formation of other prions to influence cellular phenotypes. The [RNQ+] prion itself does not adversely affect the growth of yeast, but the overexpression of Rnq1p can form toxic aggregated structures that are not necessarily prions. The [RNQ+] prion is also involved in dictating the aggregation and toxicity of polyglutamine proteins ectopically expressed in yeast. Thus, the [RNQ+] prion provides a tractable model that has the potential to reveal significant insight into the factors that dictate how amyloid structures are initiated and propagated in both physiological and pathological contexts. PMID:22052347

Prion protein immunocytochemistry helps to establish the true incidence of prion diseases.

PubMed

Lantos, P L; McGill, I S; Janota, I; Doey, L J; Collinge, J; Bruce, M T; Whatley, S A; Anderton, B H; Clinton, J; Roberts, G W

1992-11-23

Creutzfeldt-Jakob disease (CJD) and Gerstmann-Strüssler-Scheinker disease (GSSD) are transmissible spongiform encephalopathies or prion diseases affecting man. It has been reported that prion diseases may occur without the histological hallmarks of spongiform encephalopathies: vacuolation of the cerebral grey matter, neuronal loss and astrocytosis. These cases without characteristic neuropathology may go undiagnosed and consequently the true incidence of transmissible dementias is likely to have been under-estimated. Immunocytochemistry using antibodies to prion protein gives positive staining of these cases, albeit the pattern of immunostaining differs from that seen in typical forms. Accumulation of prion protein is a molecular hallmark of prion diseases, and thus a reproducible, speedy and cost-efficient immunocytochemical screening of unusual dementias may help to establish the true incidence of prion diseases.

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

PubMed

Workman, Rob G; Maddison, Ben C; Gough, Kevin C

2017-07-04

Prion 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.

Species-barrier-independent prion replication in apparently resistant species

NASA Astrophysics Data System (ADS)

Hill, Andrew F.; Joiner, Susan; Linehan, Jackie; Desbruslais, Melanie; Lantos, Peter L.; Collinge, John

2000-08-01

Transmission of prions between mammalian species is thought to be limited by a "species barrier," which depends on differences in the primary structure of prion proteins in the infecting inoculum and the host. Here we demonstrate that a strain of hamster prions thought to be nonpathogenic for conventional mice leads to prion replication to high levels in such mice but without causing clinical disease. Prions pathogenic in both mice and hamsters are produced. These results demonstrate the existence of subclinical forms of prion infection with important public health implications, both with respect to iatrogenic transmission from apparently healthy humans and dietary exposure to cattle and other species exposed to bovine spongiform encephalopathy prions. Current definitions of the species barrier, which have been based on clinical end-points, need to be fundamentally reassessed.

Synthesis, x-ray, and low-temperature neutron diffraction study of a rhodium (V) complex: dihydridobis(triethylsilyl)-pentamethylcyclopentadienylrhodium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fernandez, M.J.; Bailey, P.M.; Bentz, P.O.

1984-09-19

Reaction of (C/sub 5/ qentamethyl Rh)/sub 2/Cl/sub 4/) (1) with triethylsilane leads to the novel rhodium(V) complex (eta/sup 5/-C/sub 5/Me/sub 5/Rh(H)/sub 2/(SiE-triethyl/sub 3/)/sub 2/) (2) characterized by NMR spectra (/sup 1/H, /sup 13/C, /sup 29/Si, and /sup 103/Rh), X-ray diffraction, and neutron diffraction at 20 K. The complex shows a four-legged piano stool geometry with the pentamethylcyclopentadienyl eta/sub 5/-bonded to the rhodium (average Rh-C, 2.283 (9) A) on top and the two triethylsilyl ligands trans in the basal plane (Rh-Si, 2.379 (2) A). The neutron diffraction analysis located the two hydrides, which are trans to each other and cis tomore » the triethylsilyls in the basal plane. The mean Rh-H distance is 1.581 (3) A, and the H-Rh-H angle is 94.8 (2)/sup 0/. Complex 2 is rather stable, but it reacts under forcing conditions with neutral ligands (triphenylphosphine, CO, or maleic anhydride) to give (C/sub 5/Me/sub 5/Rh(PPh/sub 3/)H(SiEt/sub 3/)), (C/sub 5/Me/sub 5/Rh(CO)/sub 2/), or (C/sub 5/M3/5Rh(maleic anhydride)/sup 2/). It reacts more easily with electrophiles such as HBF/sub 4/ to give (C/sub 5/Me/sub 5/Rh)/sub 4/H/sub 4/)/sup 2 +/, with HCl to give 1, with AgBF/sub 4/ in MeCN to give (C/sub 5/Me/sub 5/Rh(MeCN)/sub 3/)/sup 2 +/, and with I/sub 2/ to give ((C/sub 5/Me/sub 5/Rh)/sub 2/I/sub 4/). The predominant mode of reaction involves reductive elimination of Et/sub 3/Si-H, which can be strongly promoted by an electrophile. 43 references, 3 figures, 3 tables.« less

Relationship between disease-specific structures of amyloid fibrils and their mechanical properties

NASA Astrophysics Data System (ADS)

Yoon, Gwonchan; Kab Kim, Young; Eom, Kilho; Na, Sungsoo

2013-01-01

It has recently been reported that the mechanical behavior of prion nanofibrils may play a critical role in expression of neurodegenerative diseases. In this work, we have studied the mechanical behavior of HET-s prion nanofibrils using an elastic network model. We have shown that the mechanical properties of prion nanofibrils formed as left-handed β-helices are different from those of non-prion nanofibrils formed as right-handed β-helices. In particular, the bending behavior of prion nanofibrils depends on the length of the nanofibril and that the bending rigidity of the prion nanofibril is larger than that of the non-prion nanofibril.

Lesion of the Olfactory Epithelium Accelerates Prion Neuroinvasion and Disease Onset when Prion Replication Is Restricted to Neurons

PubMed Central

Crowell, Jenna; Wiley, James A.; Bessen, Richard A.

2015-01-01

Natural prion diseases of ruminants are moderately contagious and while the gastrointestinal tract is the primary site of prion agent entry, other mucosae may be entry sites in a subset of infections. In the current study we examined prion neuroinvasion and disease induction following disruption of the olfactory epithelium in the nasal mucosa since this site contains environmentally exposed olfactory sensory neurons that project directly into the central nervous system. Here we provide evidence for accelerated prion neuroinvasion and clinical onset from the olfactory mucosa after disruption and regeneration of the olfactory epithelium and when prion replication is restricted to neurons. In transgenic mice with neuron restricted replication of prions, there was a reduction in survival when the olfactory epithelium was disrupted prior to intranasal inoculation and there was >25% decrease in the prion incubation period. In a second model, the neurotropic DY strain of transmissible mink encephalopathy was not pathogenic in hamsters by the nasal route, but 50% of animals exhibited brain infection and/or disease when the olfactory epithelium was disrupted prior to intranasal inoculation. A time course analysis of prion deposition in the brain following loss of the olfactory epithelium in models of neuron-restricted prion replication suggests that neuroinvasion from the olfactory mucosa is via the olfactory nerve or brain stem associated cranial nerves. We propose that induction of neurogenesis after damage to the olfactory epithelium can lead to prion infection of immature olfactory sensory neurons and accelerate prion spread to the brain. PMID:25822718

Region-specific protein misfolding cyclic amplification reproduces brain tropism of prion strains.

PubMed

Privat, Nicolas; Levavasseur, Etienne; Yildirim, Serfildan; Hannaoui, Samia; Brandel, Jean-Philippe; Laplanche, Jean-Louis; Béringue, Vincent; Seilhean, Danielle; Haïk, Stéphane

2017-10-06

Human prion diseases such as Creutzfeldt-Jakob disease are transmissible brain proteinopathies, characterized by the accumulation of a misfolded isoform of the host cellular prion protein (PrP) in the brain. According to the prion model, prions are defined as proteinaceous infectious particles composed solely of this abnormal isoform of PrP (PrP Sc ). Even in the absence of genetic material, various prion strains can be propagated in experimental models. They can be distinguished by the pattern of disease they produce and especially by the localization of PrP Sc deposits within the brain and the spongiform lesions they induce. The mechanisms involved in this strain-specific targeting of distinct brain regions still are a fundamental, unresolved question in prion research. To address this question, we exploited a prion conversion in vitro assay, protein misfolding cyclic amplification (PMCA), by using experimental scrapie and human prion strains as seeds and specific brain regions from mice and humans as substrates. We show here that region-specific PMCA in part reproduces the specific brain targeting observed in experimental, acquired, and sporadic Creutzfeldt-Jakob diseases. Furthermore, we provide evidence that, in addition to cellular prion protein, other region- and species-specific molecular factors influence the strain-dependent prion conversion process. This important step toward understanding prion strain propagation in the human brain may impact research on the molecular factors involved in protein misfolding and the development of ultrasensitive methods for diagnosing prion disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Cell Biology Approaches to Studying Prion Diseases.

PubMed

Priola, Suzette A

2017-01-01

During the course of prion infection, the normally soluble and protease-sensitive mammalian prion protein (PrP C ) is refolded into an insoluble, partially protease-resistant, and infectious form called PrP Sc . The conformational conversion of PrP C to PrP Sc is a critical event during prion infection and is essential for the production of prion infectivity. This chapter briefly summarizes the ways in which cell biological approaches have enhanced our understanding of how PrP contributes to different aspects of prion pathogenesis.

Matter radii of {sup 32-35}Mg

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kanungo, R.; Perro, C.; Prochazka, A.

The interaction cross sections of {sup 32-35}Mg at 900A MeV have been measured using the fragment separator at GSI. The deviation from the r{sub 0}A{sup 1/3} trend is slightly larger for {sup 35}Mg, signaling the possible formation of a longer tail in the neutron distribution for {sup 35}Mg. The radii extracted from a Glauber model analysis with Fermi densities are consistent with models predicting the development of neutron skins.

Soluble Aβ aggregates can inhibit prion propagation.

PubMed

Sarell, Claire J; Quarterman, Emma; Yip, Daniel C-M; Terry, Cassandra; Nicoll, Andrew J; Wadsworth, Jonathan D F; Farrow, Mark A; Walsh, Dominic M; Collinge, John

2017-11-01

Mammalian prions cause lethal neurodegenerative diseases such as Creutzfeldt-Jakob disease (CJD) and consist of multi-chain assemblies of misfolded cellular prion protein (PrP C ). Ligands that bind to PrP C can inhibit prion propagation and neurotoxicity. Extensive prior work established that certain soluble assemblies of the Alzheimer's disease (AD)-associated amyloid β-protein (Aβ) can tightly bind to PrP C , and that this interaction may be relevant to their toxicity in AD. Here, we investigated whether such soluble Aβ assemblies might, conversely, have an inhibitory effect on prion propagation. Using cellular models of prion infection and propagation and distinct Aβ preparations, we found that the form of Aβ assemblies which most avidly bound to PrP in vitro also inhibited prion infection and propagation. By contrast, forms of Aβ which exhibit little or no binding to PrP were unable to attenuate prion propagation. These data suggest that soluble aggregates of Aβ can compete with prions for binding to PrP C and emphasize the bidirectional nature of the interplay between Aβ and PrP C in Alzheimer's and prion diseases. Such inhibitory effects of Aβ on prion propagation may contribute to the apparent fall-off in the incidence of sporadic CJD at advanced age where cerebral Aβ deposition is common. © 2017 The Authors.

Strain conformation controls the specificity of cross-species prion transmission in the yeast model.

PubMed

Grizel, Anastasia V; Rubel, Aleksandr A; Chernoff, Yury O

2016-07-03

Transmissible self-assembled fibrous cross-β polymer infectious proteins (prions) cause neurodegenerative diseases in mammals and control non-Mendelian heritable traits in yeast. Cross-species prion transmission is frequently impaired, due to sequence differences in prion-forming proteins. Recent studies of prion species barrier on the model of closely related yeast species show that colocalization of divergent proteins is not sufficient for the cross-species prion transmission, and that an identity of specific amino acid sequences and a type of prion conformational variant (strain) play a major role in the control of transmission specificity. In contrast, chemical compounds primarily influence transmission specificity via favoring certain strain conformations, while the species origin of the host cell has only a relatively minor input. Strain alterations may occur during cross-species prion conversion in some combinations. The model is discussed which suggests that different recipient proteins can acquire different spectra of prion strain conformations, which could be either compatible or incompatible with a particular donor strain.

Solvent extraction of thorium(IV), uranium(VI), and europium(III) with lipophilic alkyl-substituted pyridinium salts. Final report for subcontract 9-XZ2-1123E-1, June 1, 1992--December 1, 1995

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ensor, D.D.

1997-01-01

In the treatment of high level nuclear wastes, aromatic pyridinium salts which are radiation-resistant are desired for the extraction of actinides and lanthanides. The solvent extraction of Th{sup +4}, UO{sub 2}{sup +2}, and Eu{sup +3} by three aromatic extractants, 3,5-didodecylpyridinium nitrate (35PY), 2,6-didodecylpyridinium nitrate (26PY), and 1-methyl-3,5-didodecyl-pyridinium iodide (1M35PY) has been studied in nitric acid media. The general order of extractability of the three extractants in toluene was 1M35PY>> 26PY > 35PY. The overall extraction efficiency of the metal ions was Th{sup +4} >UO{sub 2}{sup +2} > Eu{sup +3}. The extraction of HNO{sub 3}, which was competitive with the extractionmore » of metal ions, was quantitatively investigated by NaOH titration and UV spectrometry. The loading capacity suggested that the extracted species in the organic phase for thorium was (R{sub 4}N{sup +}){sub 2}Th(NO{sub 3}{sup -}){sub 6}, where R{sub 4}N{sup +} denotes 1M35PY. A comparison of 1M35PY to the well-characterized extractant, Aliquat-336, an aliphatic ammonium salt was made. At the same extractant concentration, 1M35PY extracted thorium more efficiently than Aliquat-336 at high acidity. Thorium could be readily stripped with dilute nitric acid from 1M35PY. After irradiation of 0.1M 1M35PY with {sup 60}Co at 40R/min for 48 hours, no change in the extraction efficiency of thorium was observed.« less

Molecular Barriers to Zoonotic Transmission of Prions

PubMed Central

Barria, Marcelo A.; Balachandran, Aru; Morita, Masanori; Kitamoto, Tetsuyuki; Barron, Rona; Manson, Jean; Knight, Richard; Ironside, James W.

2014-01-01

The risks posed to human health by individual animal prion diseases cannot be determined a priori and are difficult to address empirically. The fundamental event in prion disease pathogenesis is thought to be the seeded conversion of normal prion protein to its pathologic isoform. We used a rapid molecular conversion assay (protein misfolding cyclic amplification) to test whether brain homogenates from specimens of classical bovine spongiform encephalopathy (BSE), atypical BSE (H-type BSE and L-type BSE), classical scrapie, atypical scrapie, and chronic wasting disease can convert normal human prion protein to the abnormal disease-associated form. None of the tested prion isolates from diseased animals were as efficient as classical BSE in converting human prion protein. However, in the case of chronic wasting disease, there was no absolute barrier to conversion of the human prion protein. PMID:24377702

High-resolution structure of infectious prion protein: the final frontier

PubMed Central

Diaz-Espinoza, Rodrigo; Soto, Claudio

2014-01-01

Prions are the proteinaceous infectious agents responsible for the transmission of prion diseases. The main or sole component of prions is the misfolded prion protein (PrPSc), which is able to template the conversion of the host’s natively folded form of the protein (PrPC). The detailed mechanism of prion replication and the high-resolution structure of PrPSc are unknown. The currently available information on PrPSc structure comes mostly from low-resolution biophysical techniques, which have resulted in quite divergent models. Recent advances in the production of infectious prions, using very pure recombinant protein, offer new hope for PrPSc structural studies. This review highlights the importance of, challenges for and recent progress toward elucidating the elusive structure of PrPSc, arguably the major pending milestone to reach in understanding prions. PMID:22472622

A systematic investigation of production of synthetic prions from recombinant prion protein.

PubMed

Schmidt, Christian; Fizet, Jeremie; Properzi, Francesca; Batchelor, Mark; Sandberg, Malin K; Edgeworth, Julie A; Afran, Louise; Ho, Sammy; Badhan, Anjna; Klier, Steffi; Linehan, Jacqueline M; Brandner, Sebastian; Hosszu, Laszlo L P; Tattum, M Howard; Jat, Parmjit; Clarke, Anthony R; Klöhn, Peter C; Wadsworth, Jonathan D F; Jackson, Graham S; Collinge, John

2015-12-01

According to the protein-only hypothesis, infectious mammalian prions, which exist as distinct strains with discrete biological properties, consist of multichain assemblies of misfolded cellular prion protein (PrP). A critical test would be to produce prion strains synthetically from defined components. Crucially, high-titre 'synthetic' prions could then be used to determine the structural basis of infectivity and strain diversity at the atomic level. While there have been multiple reports of production of prions from bacterially expressed recombinant PrP using various methods, systematic production of high-titre material in a form suitable for structural analysis remains a key goal. Here, we report a novel high-throughput strategy for exploring a matrix of conditions, additives and potential cofactors that might generate high-titre prions from recombinant mouse PrP, with screening for infectivity using a sensitive automated cell-based bioassay. Overall, approximately 20,000 unique conditions were examined. While some resulted in apparently infected cell cultures, this was transient and not reproducible. We also adapted published methods that reported production of synthetic prions from recombinant hamster PrP, but again did not find evidence of significant infectious titre when using recombinant mouse PrP as substrate. Collectively, our findings are consistent with the formation of prion infectivity from recombinant mouse PrP being a rare stochastic event and we conclude that systematic generation of prions from recombinant PrP may only become possible once the detailed structure of authentic ex vivo prions is solved. © 2015 The Authors.

A systematic investigation of production of synthetic prions from recombinant prion protein

PubMed Central

Schmidt, Christian; Fizet, Jeremie; Properzi, Francesca; Batchelor, Mark; Sandberg, Malin K.; Edgeworth, Julie A.; Afran, Louise; Ho, Sammy; Badhan, Anjna; Klier, Steffi; Linehan, Jacqueline M.; Brandner, Sebastian; Hosszu, Laszlo L. P.; Tattum, M. Howard; Jat, Parmjit; Clarke, Anthony R.; Klöhn, Peter C.; Wadsworth, Jonathan D. F.; Jackson, Graham S.; Collinge, John

2015-01-01

According to the protein-only hypothesis, infectious mammalian prions, which exist as distinct strains with discrete biological properties, consist of multichain assemblies of misfolded cellular prion protein (PrP). A critical test would be to produce prion strains synthetically from defined components. Crucially, high-titre ‘synthetic' prions could then be used to determine the structural basis of infectivity and strain diversity at the atomic level. While there have been multiple reports of production of prions from bacterially expressed recombinant PrP using various methods, systematic production of high-titre material in a form suitable for structural analysis remains a key goal. Here, we report a novel high-throughput strategy for exploring a matrix of conditions, additives and potential cofactors that might generate high-titre prions from recombinant mouse PrP, with screening for infectivity using a sensitive automated cell-based bioassay. Overall, approximately 20 000 unique conditions were examined. While some resulted in apparently infected cell cultures, this was transient and not reproducible. We also adapted published methods that reported production of synthetic prions from recombinant hamster PrP, but again did not find evidence of significant infectious titre when using recombinant mouse PrP as substrate. Collectively, our findings are consistent with the formation of prion infectivity from recombinant mouse PrP being a rare stochastic event and we conclude that systematic generation of prions from recombinant PrP may only become possible once the detailed structure of authentic ex vivo prions is solved. PMID:26631378

Increased infectivity of anchorless mouse scrapie prions in transgenic mice overexpressing human prion protein.

PubMed

Race, Brent; Phillips, Katie; Meade-White, Kimberly; Striebel, James; Chesebro, Bruce

2015-06-01

Prion protein (PrP) is found in all mammals, mostly as a glycoprotein anchored to the plasma membrane by a C-terminal glycosylphosphatidylinositol (GPI) linkage. Following prion infection, host protease-sensitive prion protein (PrPsen or PrPC) is converted into an abnormal, disease-associated, protease-resistant form (PrPres). Biochemical characteristics, such as the PrP amino acid sequence, and posttranslational modifications, such as glycosylation and GPI anchoring, can affect the transmissibility of prions as well as the biochemical properties of the PrPres generated. Previous in vivo studies on the effects of GPI anchoring on prion infectivity have not examined cross-species transmission. In this study, we tested the effect of lack of GPI anchoring on a species barrier model using mice expressing human PrP. In this model, anchorless 22L prions derived from tg44 mice were more infectious than 22L prions derived from C57BL/10 mice when tested in tg66 transgenic mice, which expressed wild-type anchored human PrP at 8- to 16-fold above normal. Thus, the lack of the GPI anchor on the PrPres from tg44 mice appeared to reduce the effect of the mouse-human PrP species barrier. In contrast, neither source of prions induced disease in tgRM transgenic mice, which expressed human PrP at 2- to 4-fold above normal. Prion protein (PrP) is found in all mammals, usually attached to cells by an anchor molecule called GPI. Following prion infection, PrP is converted into a disease-associated form (PrPres). While most prion diseases are species specific, this finding is not consistent, and species barriers differ in strength. The amino acid sequence of PrP varies among species, and this variability affects prion species barriers. However, other PrP modifications, including glycosylation and GPI anchoring, may also influence cross-species infectivity. We studied the effect of PrP GPI anchoring using a mouse-to-human species barrier model. Experiments showed that prions produced by mice expressing only anchorless PrP were more infectious than prions produced in mice expressing anchored PrP. Thus, the lack of the GPI anchor on prions reduced the effect of the mouse-human species barrier. Our results suggest that prion diseases that produce higher levels of anchorless PrP may pose an increased risk for cross-species infection. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Meat and bone meal and mineral feed additives may increase the risk of oral prion disease transmission

USGS Publications Warehouse

Johnson, C.J.; McKenzie, D.; Pedersen, J.A.; Aiken, Judd M.

2011-01-01

Ingestion of prion-contaminated materials is postulated to be a primary route of prion disease transmission. Binding of prions to soil (micro)particles dramatically enhances peroral disease transmission relative to unbound prions, and it was hypothesized that micrometer-sized particles present in other consumed materials may affect prion disease transmission via the oral route of exposure. Small, insoluble particles are present in many substances, including soil, human foods, pharmaceuticals, and animal feeds. It is known that meat and bone meal (MBM), a feed additive believed responsible for the spread of bovine spongiform encephalopathy (BSE), contains particles smaller than 20 ??m and that the pathogenic prion protein binds to MBM. The potentiation of disease transmission via the oral route by exposure to MBM or three micrometer-sized mineral feed additives was determined. Data showed that when the disease agent was bound to any of the tested materials, the penetrance of disease was increased compared to unbound prions. Our data suggest that in feed or other prion-contaminated substances consumed by animals or, potentially, humans, the addition of MBM or the presence of microparticles could heighten risks of prion disease acquisition. Copyright ?? 2011 Taylor & Francis Group, LLC.

Meat and bone meal and mineral feed additives may increase the risk of oral prion disease transmission

USGS Publications Warehouse

Johnson, Christopher J.; McKenzie, Debbie; Pedersen, Joel A.; Aiken, Judd M.

2011-01-01

Ingestion of prion-contaminated materials is postulated to be a primary route of prion disease transmission. Binding of prions to soil (micro)particles dramatically enhances peroral disease transmission relative to unbound prions, and it was hypothesized that micrometer-sized particles present in other consumed materials may affect prion disease transmission via the oral route of exposure. Small, insoluble particles are present in many substances, including soil, human foods, pharmaceuticals, and animal feeds. It is known that meat and bone meal (MBM), a feed additive believed responsible for the spread of bovine spongiform encephalopathy (BSE), contains particles smaller than 20 μm and that the pathogenic prion protein binds to MBM. The potentiation of disease transmission via the oral route by exposure to MBM or three micrometer-sized mineral feed additives was determined. Data showed that when the disease agent was bound to any of the tested materials, the penetrance of disease was increased compared to unbound prions. Our data suggest that in feed or other prion-contaminated substances consumed by animals or, potentially, humans, the addition of MBM or the presence of microparticles could heighten risks of prion disease acquisition.

Prion pathogenesis and secondary lymphoid organs (SLO): tracking the SLO spread of prions to the brain.

PubMed

Mabbott, Neil A

2012-01-01

Prion diseases are subacute neurodegenerative diseases that affect humans and a range of domestic and free-ranging animal species. These diseases are characterized by the accumulation of PrP (Sc), an abnormally folded isoform of the cellular prion protein (PrP (C)), in affected tissues. The pathology during prion disease appears to occur almost exclusively within the central nervous system. The extensive neurodegeneration which occurs ultimately leads to the death of the host. An intriguing feature of the prion diseases, when compared with other protein-misfolding diseases, is their transmissibility. Following peripheral exposure, some prion diseases accumulate to high levels within lymphoid tissues. The replication of prions within lymphoid tissue has been shown to be important for the efficient spread of disease to the brain. This article describes recent progress in our understanding of the cellular mechanisms that influence the propagation of prions from peripheral sites of exposure (such as the lumen of the intestine) to the brain. A thorough understanding of these events will lead to the identification of important targets for therapeutic intervention, or alternatively, reveal additional processes that influence disease susceptibility to peripherally-acquired prion diseases.

A Sequence-Dependent DNA Condensation Induced by Prion Protein

PubMed Central

2018-01-01

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. PMID:29657864

A Sequence-Dependent DNA Condensation Induced by Prion Protein.

PubMed

Bera, Alakesh; Biring, Sajal

2018-01-01

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.

Sulfate-chloride exchange by lobster hepatopancreas is regulated by pH-sensitive modifier sites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cattey, M.A.; Ahearn, G.A.; Gerencser, G.A.

1991-03-15

{sup 35}SO{sub 4}{sup 2{minus}} uptake by Atlantic lobster (Homarus americanus) hepatopancreatic epithelial brush border membrane vesicles (BBMV) was stimulated by internal Cl{sup {minus}}, but not internal HCO{sub 3}{sup {minus}}, or external Na{sup +}. Sulfate-chloride exchange was stimulated by inside positive, and inhibited by inside negative, trans-membrane K diffusion potentials. {sup 35}SO{sub 4}{sup 2{minus}}-Cl{sup {minus}} exchange was strongly inhibited by 4,4{prime} diisothiocyanostilbene-2,2{prime}-disulfonic acid (DIDS), 4-acetamido-4{prime}-isotheocynaostilbene-2,2{prime}-disulfonic acid, (SITS), and thiosulfate. Chloride, bicarbonate, furosamide, and bumetanide slightly, yet significantly, cis-inhibited {sup 35}SO{sub 4}{sup 2{minus}}-Cl{sup {minus}} exchange. Altering bilateral pH from 8.0 to 5.4 stimulated {sup 35}SO{sub 4}{sup 2{minus}}-Cl{sup {minus}} exchange when vesicles weremore » loaded with Cl{sup {minus}}, but reduced bilateral pH alone or the presence of pH gradients did not affect {sup 35}SO{sub 4}{sup 2{minus}} transport in the absence of internal Cl{sup {minus}}. {sup 36}Cl uptake into SO{sub 4}{sup 2{minus}}-loaded BBMV was stimulated by an internal negative membrane potential and inhibited when the interior was electrically positive. A model is proposed which suggests that SO{sub 4}{sup 2{minus}}-Cl{sup {minus}} exchange is regulated by internal and external pH-sensitive modifier sites on the anion antiporter and by coupling to the electrogenic 2 Na{sup +}/1 H{sup +} antiporter and by coupling to the electrogenic 2 Na{sup +}/1 H{sup +} antiporter on the same membrane.« less

Identification of novel putative-binding proteins for cellular prion protein and a specific interaction with the STIP1 homology and U-Box-containing protein 1

PubMed Central

Gimenez, Ana Paula Lappas; Richter, Larissa Morato Luciani; Atherino, Mariana Campos; Beirão, Breno Castello Branco; Fávaro, Celso; Costa, Michele Dietrich Moura; Zanata, Silvio Marques; Malnic, Bettina; Mercadante, Adriana Frohlich

2015-01-01

ABSTRACT Prion diseases involve the conversion of the endogenous cellular prion protein, PrPC, into a misfolded infectious isoform, PrPSc. Several functions have been attributed to PrPC, and its role has also been investigated in the olfactory system. PrPC is expressed in both the olfactory bulb (OB) and olfactory epithelium (OE) and the nasal cavity is an important route of transmission of diseases caused by prions. Moreover, Prnp−/− mice showed impaired behavior in olfactory tests. Given the high PrPC expression in OE and its putative role in olfaction, we screened a mouse OE cDNA library to identify novel PrPC-binding partners. Ten different putative PrPC ligands were identified, which were involved in functions such as cellular proliferation and apoptosis, cytoskeleton and vesicle transport, ubiquitination of proteins, stress response, and other physiological processes. In vitro binding assays confirmed the interaction of PrPC with STIP1 homology and U-Box containing protein 1 (Stub1) and are reported here for the first time. Stub1 is a co-chaperone with ubiquitin E3-ligase activity, which is associated with neurodegenerative diseases characterized by protein misfolding and aggregation. Physiological and pathological implications of PrPC-Stub1 interaction are under investigation. The PrPC-binding proteins identified here are not exclusive to the OE, suggesting that these interactions may occur in other tissues and play general biological roles. These data corroborate the proposal that PrPC is part of a multiprotein complex that modulates several cellular functions and provide a platform for further studies on the physiological and pathological roles of prion protein. PMID:26237451

Inherited prion disease A117V is not simply a proteinopathy but produces prions transmissible to transgenic mice expressing homologous prion protein.

PubMed

Asante, Emmanuel A; Linehan, Jacqueline M; Smidak, Michelle; Tomlinson, Andrew; Grimshaw, Andrew; Jeelani, Asif; Jakubcova, Tatiana; Hamdan, Shyma; Powell, Caroline; Brandner, Sebastian; Wadsworth, Jonathan D F; Collinge, John

2013-01-01

Prions are infectious agents causing fatal neurodegenerative diseases of humans and animals. In humans, these have sporadic, acquired and inherited aetiologies. The inherited prion diseases are caused by one of over 30 coding mutations in the human prion protein (PrP) gene (PRNP) and many of these generate infectious prions as evidenced by their experimental transmissibility by inoculation to laboratory animals. However, some, and in particular an extensively studied type of Gerstmann-Sträussler-Scheinker syndrome (GSS) caused by a PRNP A117V mutation, are thought not to generate infectious prions and instead constitute prion proteinopathies with a quite distinct pathogenetic mechanism. Multiple attempts to transmit A117V GSS have been unsuccessful and typical protease-resistant PrP (PrP(Sc)), pathognomonic of prion disease, is not detected in brain. Pathogenesis is instead attributed to production of an aberrant topological form of PrP, C-terminal transmembrane PrP ((Ctm)PrP). Barriers to transmission of prion strains from one species to another appear to relate to structural compatibility of PrP in host and inoculum and we have therefore produced transgenic mice expressing human 117V PrP. We found that brain tissue from GSS A117V patients did transmit disease to these mice and both the neuropathological features of prion disease and presence of PrP(Sc) was demonstrated in the brains of recipient transgenic mice. This PrP(Sc) rapidly degraded during laboratory analysis, suggesting that the difficulty in its detection in patients with GSS A117V could relate to post-mortem proteolysis. We conclude that GSS A117V is indeed a prion disease although the relative contributions of (Ctm)PrP and prion propagation in neurodegeneration and their pathogenetic interaction remains to be established.

De novo generation of infectious prions with bacterially expressed recombinant prion protein.

PubMed

Zhang, Zhihong; Zhang, Yi; Wang, Fei; Wang, Xinhe; Xu, Yuanyuan; Yang, Huaiyi; Yu, Guohua; Yuan, Chonggang; Ma, Jiyan

2013-12-01

The prion hypothesis is strongly supported by the fact that prion infectivity and the pathogenic conformer of prion protein (PrP) are simultaneously propagated in vitro by the serial protein misfolding cyclic amplification (sPMCA). However, due to sPMCA's enormous amplification power, whether an infectious prion can be formed de novo with bacterially expressed recombinant PrP (rPrP) remains to be satisfactorily resolved. To address this question, we performed unseeded sPMCA with rPrP in a laboratory that has never been exposed to any native prions. Two types of proteinase K (PK)-resistant and self-perpetuating recombinant PrP conformers (rPrP-res) with PK-resistant cores of 17 or 14 kDa were generated. A bioassay revealed that rPrP-res(17kDa) was highly infectious, causing prion disease in wild-type mice with an average survival time of about 172 d. In contrast, rPrP-res(14kDa) completely failed to induce any disease. Our findings reveal that sPMCA is sufficient to initiate various self-perpetuating PK-resistant rPrP conformers, but not all of them possess in vivo infectivity. Moreover, generating an infectious prion in a prion-free environment establishes that an infectious prion can be formed de novo with bacterially expressed rPrP.

Porcine prion protein amyloid.

PubMed

Hammarström, Per; Nyström, Sofie

2015-01-01

Mammalian prions are composed of misfolded aggregated prion protein (PrP) with amyloid-like features. Prions are zoonotic disease agents that infect a wide variety of mammalian species including humans. Mammals and by-products thereof which are frequently encountered in daily life are most important for human health. It is established that bovine prions (BSE) can infect humans while there is no such evidence for any other prion susceptible species in the human food chain (sheep, goat, elk, deer) and largely prion resistant species (pig) or susceptible and resistant pets (cat and dogs, respectively). PrPs from these species have been characterized using biochemistry, biophysics and neurobiology. Recently we studied PrPs from several mammals in vitro and found evidence for generic amyloidogenicity as well as cross-seeding fibril formation activity of all PrPs on the human PrP sequence regardless if the original species was resistant or susceptible to prion disease. Porcine PrP amyloidogenicity was among the studied. Experimentally inoculated pigs as well as transgenic mouse lines overexpressing porcine PrP have, in the past, been used to investigate the possibility of prion transmission in pigs. The pig is a species with extraordinarily wide use within human daily life with over a billion pigs harvested for human consumption each year. Here we discuss the possibility that the largely prion disease resistant pig can be a clinically silent carrier of replicating prions.

The celecoxib derivatives AR-12 and AR-14 induce autophagy and clear prion-infected cells from prions.

PubMed

Abdulrahman, Basant A; Abdelaziz, Dalia; Thapa, Simrika; Lu, Li; Jain, Shubha; Gilch, Sabine; Proniuk, Stefan; Zukiwski, Alexander; Schatzl, Hermann M

2017-12-14

Prion diseases are fatal infectious neurodegenerative disorders that affect both humans and animals. The autocatalytic conversion of the cellular prion protein (PrP C ) into the pathologic isoform PrP Sc is a key feature in prion pathogenesis. AR-12 is an IND-approved derivative of celecoxib that demonstrated preclinical activity against several microbial diseases. Recently, AR-12 has been shown to facilitate clearance of misfolded proteins. The latter proposes AR-12 to be a potential therapeutic agent for neurodegenerative disorders. In this study, we investigated the role of AR-12 and its derivatives in controlling prion infection. We tested AR-12 in prion infected neuronal and non-neuronal cell lines. Immunoblotting and confocal microscopy results showed that AR-12 and its analogue AR-14 reduced PrP Sc levels after only 72 hours of treatment. Furthermore, infected cells were cured of PrP Sc after exposure of AR-12 or AR-14 for only two weeks. We partially attribute the influence of the AR compounds on prion propagation to autophagy stimulation, in line with our previous findings that drug-induced stimulation of autophagy has anti-prion effects in vitro and in vivo. Taken together, this study demonstrates that AR-12 and the AR-14 analogue are potential new therapeutic agents for prion diseases and possibly protein misfolding disorders involving prion-like mechanisms.

Crystal Structure of Human Liver [delta][superscript 4]-3-Ketosteroid 5[beta]-Reductase (AKR1D1) and Implications for Substrate Binding and Catalysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Di Costanzo, Luigi; Drury, Jason E.; Penning, Trevor M.

2008-07-15

AKR1D1 (steroid 5{beta}-reductase) reduces all {Delta}{sup 4}-3-ketosteroids to form 5{beta}-dihydrosteroids, a first step in the clearance of steroid hormones and an essential step in the synthesis of all bile acids. The reduction of the carbon-carbon double bond in an {alpha}{beta}-unsaturated ketone by 5{beta}-reductase is a unique reaction in steroid enzymology because hydride transfer from NADPH to the {beta}-face of a {Delta}{sup 4}-3-ketosteroid yields a cis-A/B-ring configuration with an {approx}90{sup o} bend in steroid structure. Here, we report the first x-ray crystal structure of a mammalian steroid hormone carbon-carbon double bond reductase, human {Delta}{sup 4}-3-ketosteroid 5{beta}-reductase (AKR1D1), and its complexes withmore » intact substrates. We have determined the structures of AKR1D1 complexes with NADP{sup +} at 1.79- and 1.35-{angstrom} resolution (HEPES bound in the active site), NADP{sup +} and cortisone at 1.90-{angstrom} resolution, NADP{sup +} and progesterone at 2.03-{angstrom} resolution, and NADP{sup +} and testosterone at 1.62-{angstrom} resolution. Complexes with cortisone and progesterone reveal productive substrate binding orientations based on the proximity of each steroid carbon-carbon double bond to the re-face of the nicotinamide ring of NADP{sup +}. This orientation would permit 4-pro-(R)-hydride transfer from NADPH. Each steroid carbonyl accepts hydrogen bonds from catalytic residues Tyr{sup 58} and Glu{sup 120}. The Y58F and E120A mutants are devoid of activity, supporting a role for this dyad in the catalytic mechanism. Intriguingly, testosterone binds nonproductively, thereby rationalizing the substrate inhibition observed with this particular steroid. The locations of disease-linked mutations thought to be responsible for bile acid deficiency are also revealed.« less

Production of cattle lacking prion protein

PubMed Central

Richt, Jürgen A; Kasinathan, Poothappillai; Hamir, Amir N; Castilla, Joaquin; Sathiyaseelan, Thillai; Vargas, Francisco; Sathiyaseelan, Janaki; Wu, Hua; Matsushita, Hiroaki; Koster, Julie; Kato, Shinichiro; Ishida, Isao; Soto, Claudio; Robl, James M; Kuroiwa, Yoshimi

2010-01-01

Prion diseases are caused by propagation of misfolded forms of the normal cellular prion protein PrPC, such as PrPBSE in bovine spongiform encephalopathy (BSE) in cattle and PrPCJD in Creutzfeldt-Jakob disease (CJD) in humans1. Disruption of PrPC expression in mice, a species that does not naturally contract prion diseases, results in no apparent developmental abnormalities2–5. However, the impact of ablating PrPC function in natural host species of prion diseases is unknown. Here we report the generation and characterization of PrPC-deficient cattle produced by a sequential gene-targeting system6. At over 20 months of age, the cattle are clinically, physiologically, histopathologically, immunologically and reproductively normal. Brain tissue homogenates are resistant to prion propagation in vitro as assessed by protein misfolding cyclic amplification7. PrPC-deficient cattle may be a useful model for prion research and could provide industrial bovine products free of prion proteins. PMID:17195841

Comparative analysis of the prion protein gene sequences in African lion.

PubMed

Wu, Chang-De; Pang, Wan-Yong; Zhao, De-Ming

2006-10-01

The prion protein gene of African lion (Panthera Leo) was first cloned and polymorphisms screened. The results suggest that the prion protein gene of eight African lions is highly homogenous. The amino acid sequences of the prion protein (PrP) of all samples tested were identical. Four single nucleotide polymorphisms (C42T, C81A, C420T, T600C) in the prion protein gene (Prnp) of African lion were found, but no amino acid substitutions. Sequence analysis showed that the higher homology is observed to felis catus AF003087 (96.7%) and to sheep number M31313.1 (96.2%) Genbank accessed. With respect to all the mammalian prion protein sequences compared, the African lion prion protein sequence has three amino acid substitutions. The homology might in turn affect the potential intermolecular interactions critical for cross species transmission of prion disease.

Metal Dyshomeostasis and Their Pathological Role in Prion and Prion-Like Diseases: The Basis for a Nutritional Approach

PubMed Central

Toni, Mattia; Massimino, Maria L.; De Mario, Agnese; Angiulli, Elisa; Spisni, Enzo

2017-01-01

Metal ions are key elements in organisms' life acting like cofactors of many enzymes but they can also be potentially dangerous for the cell participating in redox reactions that lead to the formation of reactive oxygen species (ROS). Any factor inducing or limiting a metal dyshomeostasis, ROS production and cell injury may contribute to the onset of neurodegenerative diseases or play a neuroprotective action. Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, are a group of fatal neurodegenerative disorders affecting the central nervous system (CNS) of human and other mammalian species. The causative agent of TSEs is believed to be the scrapie prion protein PrPSc, the β sheet-rich pathogenic isoform produced by the conformational conversion of the α-helix-rich physiological isoform PrPC. The peculiarity of PrPSc is its ability to self-propagate in exponential fashion in cells and its tendency to precipitate in insoluble and protease-resistance amyloid aggregates leading to neuronal cell death. The expression “prion-like diseases” refers to a group of neurodegenerative diseases that share some neuropathological features with prion diseases such as the involvement of proteins (α-synuclein, amyloid β, and tau) able to precipitate producing amyloid deposits following conformational change. High social impact diseases such as Alzheimer's and Parkinson's belong to prion-like diseases. Accumulating evidence suggests that the exposure to environmental metals is a risk factor for the development of prion and prion-like diseases and that metal ions can directly bind to prion and prion-like proteins affecting the amount of amyloid aggregates. The diet, source of metal ions but also of natural antioxidant and chelating agents such as polyphenols, is an aspect to take into account in addressing the issue of neurodegeneration. Epidemiological data suggest that the Mediterranean diet, based on the abundant consumption of fresh vegetables and on low intake of meat, could play a preventive or delaying role in prion and prion-like neurodegenerative diseases. In this review, metal role in the onset of prion and prion-like diseases is dealt with from a nutritional, cellular, and molecular point of view. PMID:28154522

A naturally occurring variant of the human prion protein completely prevents prion disease.

PubMed

Asante, Emmanuel A; Smidak, Michelle; Grimshaw, Andrew; Houghton, Richard; Tomlinson, Andrew; Jeelani, Asif; Jakubcova, Tatiana; Hamdan, Shyma; Richard-Londt, Angela; Linehan, Jacqueline M; Brandner, Sebastian; Alpers, Michael; Whitfield, Jerome; Mead, Simon; Wadsworth, Jonathan D F; Collinge, John

2015-06-25

Mammalian prions, transmissible agents causing lethal neurodegenerative diseases, are composed of assemblies of misfolded cellular prion protein (PrP). A novel PrP variant, G127V, was under positive evolutionary selection during the epidemic of kuru--an acquired prion disease epidemic of the Fore population in Papua New Guinea--and appeared to provide strong protection against disease in the heterozygous state. Here we have investigated the protective role of this variant and its interaction with the common, worldwide M129V PrP polymorphism. V127 was seen exclusively on a M129 PRNP allele. We demonstrate that transgenic mice expressing both variant and wild-type human PrP are completely resistant to both kuru and classical Creutzfeldt-Jakob disease (CJD) prions (which are closely similar) but can be infected with variant CJD prions, a human prion strain resulting from exposure to bovine spongiform encephalopathy prions to which the Fore were not exposed. Notably, mice expressing only PrP V127 were completely resistant to all prion strains, demonstrating a different molecular mechanism to M129V, which provides its relative protection against classical CJD and kuru in the heterozygous state. Indeed, this single amino acid substitution (G→V) at a residue invariant in vertebrate evolution is as protective as deletion of the protein. Further study in transgenic mice expressing different ratios of variant and wild-type PrP indicates that not only is PrP V127 completely refractory to prion conversion but acts as a potent dose-dependent inhibitor of wild-type prion propagation.

In vitro replication highlights the mutability of prions.

PubMed

Vanni, Ilaria; Di Bari, Michele Angelo; Pirisinu, Laura; D'Agostino, Claudia; Agrimi, Umberto; Nonno, Romolo

2014-01-01

Prions exist as strains, which are thought to reflect PrP(Sc) conformational variants. Prion strains can mutate and it has been proposed that prion mutability depends on an intrinsic heterogeneity of prion populations that would behave as quasispecies. We investigated in vitro prion mutability of 2 strains, by following PrP(Sc) variations of populations serially propagated in PMCA under constant environmental pressure. Each strain was propagated either at low dilution of the seed, i.e., by large population passages, or at limiting dilution, mimicking bottleneck events. In both strains, PrP(Sc) conformational variants were identified only after large population passages, while repeated bottleneck events caused a rapid decline in amplification rates. These findings support the view that mutability is an intrinsic property of prions.

Modelos estereoquimicos na quimica de coordenacao e organometalica de lantanideos e actinideos: aplicacoes a complexos de torio (iv) com boratos de polipirazolilo (Stereochemical models in lanthanide and actinide coordination and organometallic chemistry: Applications to thorium (IV) complexes with polypyrazolylborates). Doctoral thesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

de Almeida, J.C.M.

1990-01-01

A detailed analysis is made of two stereochemical models commonly used in lanthanide and actinide coordination and organometallic chemistry. Li Xing-fu's Cone Packing Model and K. N. Raymond's Ionic Model. Corrections are introduced in the first model as a basis to discuss the stability and structure of known complexes. A Steric Coordination Number is defined for the second model, based on the solid angle to correlate metal-ligand distances in complexes with the ionic radii of the elements and to assign effective radii to the ligands, related to the donating power of the coordinating atoms. As an application of the models,more » the syntheses and characterizations of thorium(IV) complexes with polypyrazolylborates. (HBPz3) {sup -1} and (HB(3.5-Me2Pz)3) {sup -1}, and alkoxides, aryloxides, carboxylates, amides, thiolates, alkyls and cyclopentadienyl are described and their stabilities discussed. The geometries of the complexes in the solid and in solution are discussed and a mechanism is proposed to explain the fluxionality in solution of the complexes with (HBPz3) {sup -1}.« less

Evidence That the [beta] Subunit of Chlamydia trachomatis Ribonucleotide Reductase Is Active with the Manganese Ion of Its Manganese(IV)/Iron(III) Cofactor in Site 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dassama, Laura M.K.; Boal, Amie K.; Krebs, Carsten

2014-10-02

The reaction of a class I ribonucleotide reductase (RNR) begins when a cofactor in the {beta} subunit oxidizes a cysteine residue {approx}35 {angstrom} away in the {alpha} subunit, generating a thiyl radical. In the class Ic enzyme from Chlamydia trachomatis (Ct), the cysteine oxidant is the Mn{sup IV} ion of a Mn{sup IV}/Fe{sup III} cluster, which assembles in a reaction between O{sub 2} and the Mn{sup II}/Fe{sup II} complex of {beta}. The heterodinuclear nature of the cofactor raises the question of which site, 1 or 2, contains the Mn{sup IV} ion. Because site 1 is closer to the conserved locationmore » of the cysteine-oxidizing tyrosyl radical of class Ia and Ib RNRs, we suggested that the Mn{sup IV} ion most likely resides in this site (i.e., {sup 1}Mn{sup IV}/{sup 2}Fe{sup III}), but a subsequent computational study favored its occupation of site 2 ({sup 1}Fe{sup III}/{sup 2}Mn{sup IV}). In this work, we have sought to resolve the location of the Mn{sup IV} ion in Ct RNR-{beta} by correlating X-ray crystallographic anomalous scattering intensities with catalytic activity for samples of the protein reconstituted in vitro by two different procedures. In samples containing primarily Mn{sup IV}/Fe{sup III} clusters, Mn preferentially occupies site 1, but some anomalous scattering from site 2 is observed, implying that both {sup 1}Mn{sup II}/{sup 2}Fe{sup II} and {sup 1}Fe{sup II}/{sup 2}Mn{sup II} complexes are competent to react with O{sub 2} to produce the corresponding oxidized states. However, with diminished Mn{sup II} loading in the reconstitution, there is no evidence for Mn occupancy of site 2, and the greater activity of these 'low-Mn' samples on a per-Mn basis implies that the {sup 1}Mn{sup IV}/{sup 2}Fe{sup III}-{beta} is at least the more active of the two oxidized forms and may be the only active form.« less

Pathways of Prion Spread during Early Chronic Wasting Disease in Deer

PubMed Central

Hoover, Clare E.; Davenport, Kristen A.; Henderson, Davin M.; Denkers, Nathaniel D.; Mathiason, Candace K.; Soto, Claudio; Zabel, Mark D.

2017-01-01

ABSTRACT Among prion infections, two scenarios of prion spread are generally observed: (i) early lymphoid tissue replication or (ii) direct neuroinvasion without substantial antecedent lymphoid amplification. In nature, cervids are infected with chronic wasting disease (CWD) prions by oral and nasal mucosal exposure, and studies of early CWD pathogenesis have implicated pharyngeal lymphoid tissue as the earliest sites of prion accumulation. However, knowledge of chronological events in prion spread during early infection remains incomplete. To investigate this knowledge gap in early CWD pathogenesis, we exposed white-tailed deer to CWD prions by mucosal routes and performed serial necropsies to assess PrPCWD tissue distribution by real-time quaking-induced conversion (RT-QuIC) and tyramide signal amplification immunohistochemistry (TSA-IHC). Although PrPCWD was not detected by either method in the initial days (1 and 3) postexposure, we observed PrPCWD seeding activity and follicular immunoreactivity in oropharyngeal lymphoid tissues at 1 and 2 months postexposure (MPE). At 3 MPE, PrPCWD replication had expanded to all systemic lymphoid tissues. By 4 MPE, the PrPCWD burden in all lymphoid tissues had increased and approached levels observed in terminal disease, yet there was no evidence of nervous system invasion. These results indicate the first site of CWD prion entry is in the oropharynx, and the initial phase of prion amplification occurs in the oropharyngeal lymphoid tissues followed by rapid dissemination to systemic lymphoid tissues. This lymphoid replication phase appears to precede neuroinvasion. IMPORTANCE Chronic wasting disease (CWD) is a universally fatal transmissible spongiform encephalopathy affecting cervids, and natural infection occurs through oral and nasal mucosal exposure to infectious prions. Terminal disease is characterized by PrPCWD accumulation in the brain and lymphoid tissues of affected animals. However, the initial sites of prion accumulation and pathways of prion spread during early CWD infection remain unknown. To investigate the chronological events of early prion pathogenesis, we exposed deer to CWD prions and monitored the tissue distribution of PrPCWD over the first 4 months of infection. We show CWD uptake occurs in the oropharynx with initial prion replication in the draining oropharyngeal lymphoid tissues, rapidly followed by dissemination to systemic lymphoid tissues without evidence of neuroinvasion. These data highlight the two phases of CWD infection: a robust prion amplification in systemic lymphoid tissues prior to neuroinvasion and establishment of a carrier state. PMID:28250130

Creutzfeldt-Jakob disease and mad cows: lessons learnt from yeast cells.

PubMed

Hofmann, J; Wolf, H; Grassmann, A; Arndt, V; Graham, J; Vorberg, I

2012-01-24

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases that affect mammals including humans. The proteinaceous nature of the infectious agent, the prion, and its propagation, challenge established dogmas in biology. It is now widely accepted that prion diseases are caused by unconventional agents principally composed of a misfolded host-encoded protein, PrP. Surprisingly, major break-throughs in prion research came from studies on functionally unrelated proteins in yeast and filamentous fungi. Aggregates composed of these proteins act as epigenetic elements of inheritance that can propagate their alternative states by a conformational switch into an ordered ß-sheet rich polymer just like mammalian prions. Since their discovery prions of lower eukaryotes have provided invaluable insights into all aspects of prion biogenesis. Importantly, yeast prions provide proof-of-principle that distinct protein conformers can be infectious and can serve as genetic elements that have the capacity to encipher strain specific information. As a powerful and tractable model system, yeast prions will continue to increase our understanding of prion-host cell interaction and potential mechanisms of protein-based epigenetic inheritance.

A stretch of residues within the protease-resistant core is not necessary for prion structure and infectivity

PubMed Central

Munoz-Montesino, Carola; Sizun, Christina; Moudjou, Mohammed; Herzog, Laetitia; Reine, Fabienne; Igel-Egalon, Angelique; Barbereau, Clément; Chapuis, Jérôme; Ciric, Danica; Laude, Hubert; Béringue, Vincent; Rezaei, Human; Dron, Michel

2017-01-01

ABSTRACT Mapping out regions of PrP influencing prion conversion remains a challenging issue complicated by the lack of prion structure. The portion of PrP associated with infectivity contains the α-helical domain of the correctly folded protein and turns into a β-sheet-rich insoluble core in prions. Deletions performed so far inside this segment essentially prevented the conversion. Recently we found that deletion of the last C-terminal residues of the helix H2 was fully compatible with prion conversion in the RK13-ovPrP cell culture model, using 3 different infecting strains. This was in agreement with preservation of the overall PrPC structure even after removal of up to one-third of this helix. Prions with internal deletion were infectious for cells and mice expressing the wild-type PrP and they retained prion strain-specific characteristics. We thus identified a piece of the prion domain that is neither necessary for the conformational transition of PrPC nor for the formation of a stable prion structure. PMID:28281924

A stretch of residues within the protease-resistant core is not necessary for prion structure and infectivity.

PubMed

Munoz-Montesino, Carola; Sizun, Christina; Moudjou, Mohammed; Herzog, Laetitia; Reine, Fabienne; Igel-Egalon, Angelique; Barbereau, Clément; Chapuis, Jérôme; Ciric, Danica; Laude, Hubert; Béringue, Vincent; Rezaei, Human; Dron, Michel

2017-01-02

Mapping out regions of PrP influencing prion conversion remains a challenging issue complicated by the lack of prion structure. The portion of PrP associated with infectivity contains the α-helical domain of the correctly folded protein and turns into a β-sheet-rich insoluble core in prions. Deletions performed so far inside this segment essentially prevented the conversion. Recently we found that deletion of the last C-terminal residues of the helix H2 was fully compatible with prion conversion in the RK13-ovPrP cell culture model, using 3 different infecting strains. This was in agreement with preservation of the overall PrP C structure even after removal of up to one-third of this helix. Prions with internal deletion were infectious for cells and mice expressing the wild-type PrP and they retained prion strain-specific characteristics. We thus identified a piece of the prion domain that is neither necessary for the conformational transition of PrP C nor for the formation of a stable prion structure.

Resistance of Soil-Bound Prions to Rumen Digestion

PubMed Central

Saunders, Samuel E.; Bartelt-Hunt, Shannon L.; Bartz, Jason C.

2012-01-01

Before prion uptake and infection can occur in the lower gastrointestinal system, ingested prions are subjected to anaerobic digestion in the rumen of cervids and bovids. The susceptibility of soil-bound prions to rumen digestion has not been evaluated previously. In this study, prions from infectious brain homogenates as well as prions bound to a range of soils and soil minerals were subjected to in vitro rumen digestion, and changes in PrP levels were measured via western blot. Binding to clay appeared to protect noninfectious hamster PrPc from complete digestion, while both unbound and soil-bound infectious PrPSc proved highly resistant to rumen digestion. In addition, no change in intracerebral incubation period was observed following active rumen digestion of unbound hamster HY TME prions and HY TME prions bound to a silty clay loam soil. These results demonstrate that both unbound and soil-bound prions readily survive rumen digestion without a reduction in infectivity, further supporting the potential for soil-mediated transmission of chronic wasting disease (CWD) and scrapie in the environment. PMID:22937149

Immunology of Prion Protein and Prions.

PubMed

Mabbott, Neil A

2017-01-01

Many natural prion diseases are acquired peripherally, such as following the oral consumption of contaminated food or pasture. After peripheral exposure many prion isolates initially accumulate to high levels within the host's secondary lymphoid tissues. The replication of prions within these tissues is essential for their efficient spread to the brain where they ultimately cause neurodegeneration. This chapter describes our current understanding of the critical tissues, cells, and molecules which the prions exploit to mediate their efficient propagation from the site of exposure (such as the intestine) to the brain. Interactions between the immune system and prions are not only restricted to the secondary lymphoid tissues. Therefore, an account of how the activation status of the microglial in the brain can also influence progression of prion disease pathogenesis is provided. Prion disease susceptibility may also be influenced by additional factors such as chronic inflammation, coinfection with other pathogens, and aging. Finally, the potential for immunotherapy to provide a means of safe and effective prophylactic or therapeutic intervention in these currently untreatable diseases is considered. © 2017 Elsevier Inc. All rights reserved.

Prion pathogenesis and secondary lymphoid organs (SLO)

PubMed Central

Mabbott, Neil A.

2012-01-01

Prion diseases are subacute neurodegenerative diseases that affect humans and a range of domestic and free-ranging animal species. These diseases are characterized by the accumulation of PrPSc, an abnormally folded isoform of the cellular prion protein (PrPC), in affected tissues. The pathology during prion disease appears to occur almost exclusively within the central nervous system. The extensive neurodegeneration which occurs ultimately leads to the death of the host. An intriguing feature of the prion diseases, when compared with other protein-misfolding diseases, is their transmissibility. Following peripheral exposure, some prion diseases accumulate to high levels within lymphoid tissues. The replication of prions within lymphoid tissue has been shown to be important for the efficient spread of disease to the brain. This article describes recent progress in our understanding of the cellular mechanisms that influence the propagation of prions from peripheral sites of exposure (such as the lumen of the intestine) to the brain. A thorough understanding of these events will lead to the identification of important targets for therapeutic intervention, or alternatively, reveal additional processes that influence disease susceptibility to peripherally-acquired prion diseases. PMID:22895090

Lack of prion transmission by sexual or parental routes in experimentally infected hamsters.

PubMed

Morales, Rodrigo; Pritzkow, Sandra; Hu, Ping Ping; Duran-Aniotz, Claudia; Soto, Claudio

2013-01-01

Prion diseases are a group of neurodegenerative disorders affecting humans as well as captive and wild animals. The mechanisms and routes governing the natural spread of prions are not completely understood and several hypotheses have been proposed. In this study, we analyzed the effect of gender in prion incubation period, as well as the possibility of prion transmission by sexual and parental contact using 263K infected hamsters as a model. Our results show that males have significantly longer incubation periods compared with females when exposed to the same quantity of infectious material. Importantly, no evidence of sexual or parental prion transmission was found, even 500 d after sexual contact or birth, respectively. Western blotting and PMCA were unable to detect sub-clinical levels of PrP(Sc) in experimental subjects, suggesting a complete absence of prion transmission by these routes. Our results show that sexual and parental transmission of prions does not occur in this model. It remains to be studied whether this conclusion is valid also for other prion strains and species.

Mitigation of Prion Infectivity and Conversion Capacity by a Simulated Natural Process—Repeated Cycles of Drying and Wetting

PubMed Central

Yuan, Qi; Eckland, Thomas; Telling, Glenn; Bartz, Jason; Bartelt-Hunt, Shannon

2015-01-01

Prions enter the environment from infected hosts, bind to a wide range of soil and soil minerals, and remain highly infectious. Environmental sources of prions almost certainly contribute to the transmission of chronic wasting disease in cervids and scrapie in sheep and goats. While much is known about the introduction of prions into the environment and their interaction with soil, relatively little is known about prion degradation and inactivation by natural environmental processes. In this study, we examined the effect of repeated cycles of drying and wetting on prion fitness and determined that 10 cycles of repeated drying and wetting could reduce PrPSc abundance, PMCA amplification efficiency and extend the incubation period of disease. Importantly, prions bound to soil were more susceptible to inactivation by repeated cycles of drying and wetting compared to unbound prions, a result which may be due to conformational changes in soil-bound PrPSc or consolidation of the bonding between PrPSc and soil. This novel finding demonstrates that naturally-occurring environmental process can degrade prions. PMID:25665187

Time resolved infrared studies of C-H bond activation by organometallics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Asplund, M.C.

This work describes how step-scan Fourier Transform Infrared spectroscopy and visible and near infrared ultrafast lasers have been applied to the study of the photochemical activation of C-H bonds in organometallic systems, which allow for the selective breaking of C-H bonds in alkanes. The author has established the photochemical mechanism of C-H activation by Tp{sup *}Rh(CO){sub 2}(Tp{sup *} = HB-Pz{sup *}{sub 3}, Pz = 3,5-dimethylpyrazolyl) in alkane solution. The initially formed monocarbonyl forms a weak solvent complex, which undergoes a change in Tp{sup *} ligand connectivity. The final C-H bond breaking step occurs at different time scales depending on themore » structure of the alkane. In linear solvents, the time scale is <50 ns and cyclic alkanes is {approximately}200 ps. The reactivity of the Tp{sup *}Rh(CO){sub 2} system has also been studied in aromatic solvents. Here the reaction proceeds through two different pathways, with very different time scales. The first proceeds in a manner analogous to alkanes and takes <50 ns. The second proceeds through a Rh-C-C complex, and takes place on a time scale of 1.8 {micro}s.« less

Lichens: Unexpected anti-prion agents?

USGS Publications Warehouse

Rodriguez, Cynthia M.; Bennett, James P.; Johnson, Christopher J.

2012-01-01

The prion diseases sheep scrapie and cervid chronic wasting disease are transmitted, in part, via an environmental reservoir of infectivity; prions released from infected animals persist in the environment and can cause disease years later. Central to controlling disease transmission is the identification of methods capable of inactivating these agents on the landscape. We have found that certain lichens, common, ubiquitous, symbiotic organisms, possess a serine protease capable of degrading prion protein (PrP) from prion-infected animals. The protease functions against a range of prion strains from various hosts and reduces levels of abnormal PrP by at least two logs. We have now tested more than 20 lichen species from several geographical locations and from various taxa and found that approximately half of these species degrade PrP. Critical next steps include examining the effect of lichens on prion infectivity and cloning the protease responsible for PrP degradation. The impact of lichens on prions in the environment remains unknown. We speculate that lichens could have the potential to degrade prions when they are shed from infected animals onto lichens or into environments where lichens are abundant. In addition, lichens are frequently consumed by cervids and many other animals and the effect of dietary lichens on prion disease transmission should also be considered.

Generating Bona Fide Mammalian Prions with Internal Deletions.

PubMed

Munoz-Montesino, Carola; Sizun, Christina; Moudjou, Mohammed; Herzog, Laetitia; Reine, Fabienne; Chapuis, Jérôme; Ciric, Danica; Igel-Egalon, Angelique; Laude, Hubert; Béringue, Vincent; Rezaei, Human; Dron, Michel

2016-08-01

Mammalian prions are PrP proteins with altered structures causing transmissible fatal neurodegenerative diseases. They are self-perpetuating through formation of beta-sheet-rich assemblies that seed conformational change of cellular PrP. Pathological PrP usually forms an insoluble protease-resistant core exhibiting beta-sheet structures but no more alpha-helical content, loosing the three alpha-helices contained in the correctly folded PrP. The lack of a high-resolution prion structure makes it difficult to understand the dynamics of conversion and to identify elements of the protein involved in this process. To determine whether completeness of residues within the protease-resistant domain is required for prions, we performed serial deletions in the helix H2 C terminus of ovine PrP, since this region has previously shown some tolerance to sequence changes without preventing prion replication. Deletions of either four or five residues essentially preserved the overall PrP structure and mutant PrP expressed in RK13 cells were efficiently converted into bona fide prions upon challenge by three different prion strains. Remarkably, deletions in PrP facilitated the replication of two strains that otherwise do not replicate in this cellular context. Prions with internal deletion were self-propagating and de novo infectious for naive homologous and wild-type PrP-expressing cells. Moreover, they caused transmissible spongiform encephalopathies in mice, with similar biochemical signatures and neuropathologies other than the original strains. Prion convertibility and transfer of strain-specific information are thus preserved despite shortening of an alpha-helix in PrP and removal of residues within prions. These findings provide new insights into sequence/structure/infectivity relationship for prions. Prions are misfolded PrP proteins that convert the normal protein into a replicate of their own abnormal form. They are responsible for invariably fatal neurodegenerative disorders. Other aggregation-prone proteins appear to have a prion-like mode of expansion in brains, such as in Alzheimer's or Parkinson's diseases. To date, the resolution of prion structure remains elusive. Thus, to genetically define the landscape of regions critical for prion conversion, we tested the effect of short deletions. We found that, surprisingly, removal of a portion of PrP, the C terminus of alpha-helix H2, did not hamper prion formation but generated infectious agents with an internal deletion that showed characteristics essentially similar to those of original infecting strains. Thus, we demonstrate that completeness of the residues inside prions is not necessary for maintaining infectivity and the main strain-specific information, while reporting one of the few if not the only bona fide prions with an internal deletion. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Generating Bona Fide Mammalian Prions with Internal Deletions

PubMed Central

Munoz-Montesino, Carola; Sizun, Christina; Moudjou, Mohammed; Herzog, Laetitia; Reine, Fabienne; Chapuis, Jérôme; Ciric, Danica; Igel-Egalon, Angelique; Laude, Hubert; Béringue, Vincent; Rezaei, Human

2016-01-01

ABSTRACT Mammalian prions are PrP proteins with altered structures causing transmissible fatal neurodegenerative diseases. They are self-perpetuating through formation of beta-sheet-rich assemblies that seed conformational change of cellular PrP. Pathological PrP usually forms an insoluble protease-resistant core exhibiting beta-sheet structures but no more alpha-helical content, loosing the three alpha-helices contained in the correctly folded PrP. The lack of a high-resolution prion structure makes it difficult to understand the dynamics of conversion and to identify elements of the protein involved in this process. To determine whether completeness of residues within the protease-resistant domain is required for prions, we performed serial deletions in the helix H2 C terminus of ovine PrP, since this region has previously shown some tolerance to sequence changes without preventing prion replication. Deletions of either four or five residues essentially preserved the overall PrP structure and mutant PrP expressed in RK13 cells were efficiently converted into bona fide prions upon challenge by three different prion strains. Remarkably, deletions in PrP facilitated the replication of two strains that otherwise do not replicate in this cellular context. Prions with internal deletion were self-propagating and de novo infectious for naive homologous and wild-type PrP-expressing cells. Moreover, they caused transmissible spongiform encephalopathies in mice, with similar biochemical signatures and neuropathologies other than the original strains. Prion convertibility and transfer of strain-specific information are thus preserved despite shortening of an alpha-helix in PrP and removal of residues within prions. These findings provide new insights into sequence/structure/infectivity relationship for prions. IMPORTANCE Prions are misfolded PrP proteins that convert the normal protein into a replicate of their own abnormal form. They are responsible for invariably fatal neurodegenerative disorders. Other aggregation-prone proteins appear to have a prion-like mode of expansion in brains, such as in Alzheimer's or Parkinson's diseases. To date, the resolution of prion structure remains elusive. Thus, to genetically define the landscape of regions critical for prion conversion, we tested the effect of short deletions. We found that, surprisingly, removal of a portion of PrP, the C terminus of alpha-helix H2, did not hamper prion formation but generated infectious agents with an internal deletion that showed characteristics essentially similar to those of original infecting strains. Thus, we demonstrate that completeness of the residues inside prions is not necessary for maintaining infectivity and the main strain-specific information, while reporting one of the few if not the only bona fide prions with an internal deletion. PMID:27226369

Prion Protein Devoid of the Octapeptide Repeat Region Delays Bovine Spongiform Encephalopathy Pathogenesis in Mice.

PubMed

Hara, Hideyuki; Miyata, Hironori; Das, Nandita Rani; Chida, Junji; Yoshimochi, Tatenobu; Uchiyama, Keiji; Watanabe, Hitomi; Kondoh, Gen; Yokoyama, Takashi; Sakaguchi, Suehiro

2018-01-01

Conformational conversion of the cellular isoform of prion protein, PrP C , into the abnormally folded, amyloidogenic isoform, PrP Sc , is a key pathogenic event in prion diseases, including Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy (BSE) in animals. We previously reported that the octapeptide repeat (OR) region could be dispensable for converting PrP C into PrP Sc after infection with RML prions. We demonstrated that mice transgenically expressing mouse PrP with deletion of the OR region on the PrP knockout background, designated Tg(PrPΔOR)/ Prnp 0 / 0 mice, did not show reduced susceptibility to RML scrapie prions, with abundant accumulation of PrP Sc ΔOR in their brains. We show here that Tg(PrPΔOR)/ Prnp 0 / 0 mice were highly resistant to BSE prions, developing the disease with markedly elongated incubation times after infection with BSE prions. The conversion of PrPΔOR into PrP Sc ΔOR was markedly delayed in their brains. These results suggest that the OR region may have a crucial role in the conversion of PrP C into PrP Sc after infection with BSE prions. However, Tg(PrPΔOR)/ Prnp 0 / 0 mice remained susceptible to RML and 22L scrapie prions, developing the disease without elongated incubation times after infection with RML and 22L prions. PrP Sc ΔOR accumulated only slightly less in the brains of RML- or 22L-infected Tg(PrPΔOR)/ Prnp 0 / 0 mice than PrP Sc in control wild-type mice. Taken together, these results indicate that the OR region of PrP C could play a differential role in the pathogenesis of BSE prions and RML or 22L scrapie prions. IMPORTANCE Structure-function relationship studies of PrP C conformational conversion into PrP Sc are worthwhile to understand the mechanism of the conversion of PrP C into PrP Sc We show here that, by inoculating Tg(PrPΔOR)/ Prnp 0 / 0 mice with the three different strains of RML, 22L, and BSE prions, the OR region could play a differential role in the conversion of PrP C into PrP Sc after infection with RML or 22L scrapie prions and BSE prions. PrPΔOR was efficiently converted into PrP Sc ΔOR after infection with RML and 22L prions. However, the conversion of PrPΔOR into PrP Sc ΔOR was markedly delayed after infection with BSE prions. Further investigation into the role of the OR region in the conversion of PrP C into PrP Sc after infection with BSE prions might be helpful for understanding the pathogenesis of BSE prions. Copyright © 2017 American Society for Microbiology.

Pathogenic prions deviate PrPC signaling in neuronal cells and impair A-beta clearance

PubMed Central

Pradines, E; Hernandez-Rapp, J; Villa-Diaz, A; Dakowski, C; Ardila-Osorio, H; Haik, S; Schneider, B; Launay, J-M; Kellermann, O; Torres, J-M; Mouillet-Richard, S

2013-01-01

The subversion of the normal function exerted by the cellular prion protein (PrPC) in neurons by pathogenic prions is assumed to have a central role in the pathogenesis of transmissible spongiform encephalopathies. Using two murine models of prion infection, the 1C11 neuronal cell line and neurospheres, we document that prion infection is associated with the constitutive activation of signaling targets normally coupled with PrPC, including the Fyn kinase, the mitogen-associated protein kinases ERK1/2 and the CREB transcription factor. PrPC-dependent signaling overactivation in infected cells is associated with the recruitment of p38 and JNK stress-associated kinases. Downstream from CREB, prion-infected cells exhibit reduced activity of the matrix metalloprotease (MMP)-9. As MMP-9 catalyzes the degradation of the amyloid A-beta peptide, the decrease in MMP-9 activity in prion-infected cells causes a significant impairment of the clearance of A-beta, leading to its accumulation. By exploiting two 1C11-infected clones accumulating high or moderate levels of prions, we show that the prion-induced changes are correlated with the level of infectivity. Of note, a dose-dependent increase in A-beta levels was also found in the cerebrospinal fluid of mice inoculated with these infected clones. By demonstrating that pathogenic prions trigger increases in A-beta levels through the deviation of PrPC signaling, our data argue that A-beta may exacerbate prion-induced toxicity. PMID:23303130

Detecting and quantifying prions: Mass spectrometry-based approaches

USDA-ARS?s Scientific Manuscript database

Prions are novel pathogens that cause a set of rare fatal neurological diseases know as transmissible spongiform encephalopathies. Examples of these diseases include Creutzfeldt-Jakob disease, scrapie and chronic wasting disease. Prions are able to recruit a normal cellular prion protein and convert...

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.

PubMed

Hwang, Soyoun; Greenlee, Justin J; Nicholson, Eric M

2017-01-01

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.

Evidence for room-temperature in-diffusion of nickel into silicon

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yarykin, Nikolai, E-mail: nay@iptm.ru; Weber, Jörg

2016-09-05

Interstitial nickel in crystalline Si is shown to be a fast diffuser at room temperature. In this study, Ni is incorporated in Si by wet chemical etching in nickel-contaminated alkaline solutions. Nickel in-diffusion is observed by means of detecting the electrically active NiVO defect, which is formed due to Ni capture to the vacancy–oxygen complex in electron-irradiated Si. The depth profiles of the NiVO concentration measured by the deep-level transient spectroscopy technique extend to ∼15 μm in the samples doped with Ni at 35 °C for 30 min. This allows us to get a lower estimate for the nickel diffusivity at this temperaturemore » as 10{sup −9} cm{sup 2}/s. The activation energy for electron emission from the NiVO level and the apparent capture cross section are equal to 371 meV and 3 × 10{sup −15} cm{sup 2}, respectively. The NiVO complex dissociates at 300 °C reestablishing the initial concentration of the VO centers.« less

Increased Abundance of M Cells in the Gut Epithelium Dramatically Enhances Oral Prion Disease Susceptibility

PubMed Central

Sehgal, Anuj; Rios, Daniel

2016-01-01

Many natural prion diseases of humans and animals are considered to be acquired through oral consumption of contaminated food or pasture. Determining the route by which prions establish host infection will identify the important factors that influence oral prion disease susceptibility and to which intervention strategies can be developed. After exposure, the early accumulation and replication of prions within small intestinal Peyer’s patches is essential for the efficient spread of disease to the brain. To replicate within Peyer’s patches, the prions must first cross the gut epithelium. M cells are specialised epithelial cells within the epithelia covering Peyer’s patches that transcytose particulate antigens and microorganisms. M cell-development is dependent upon RANKL-RANK-signalling, and mice in which RANK is deleted only in the gut epithelium completely lack M cells. In the specific absence of M cells in these mice, the accumulation of prions within Peyer’s patches and the spread of disease to the brain was blocked, demonstrating a critical role for M cells in the initial transfer of prions across the gut epithelium in order to establish host infection. Since pathogens, inflammatory stimuli and aging can modify M cell-density in the gut, these factors may also influence oral prion disease susceptibility. Mice were therefore treated with RANKL to enhance M cell density in the gut. We show that prion uptake from the gut lumen was enhanced in RANKL-treated mice, resulting in shortened survival times and increased disease susceptibility, equivalent to a 10-fold higher infectious titre of prions. Together these data demonstrate that M cells are the critical gatekeepers of oral prion infection, whose density in the gut epithelium directly limits or enhances disease susceptibility. Our data suggest that factors which alter M cell-density in the gut epithelium may be important risk factors which influence host susceptibility to orally acquired prion diseases. PMID:27973593

High hydrophobic amino acid exposure is responsible of the neurotoxic effects induced by E200K or D202N disease-related mutations of the human prion protein.

PubMed

Corsaro, Alessandro; Thellung, Stefano; Bucciarelli, Tonino; Scotti, Luca; Chiovitti, Katia; Villa, Valentina; D'Arrigo, Cristina; Aceto, Antonio; Florio, Tullio

2011-03-01

Mutations in prion protein are thought to be causative of inherited prion diseases favoring the spontaneous conversion of the normal prion protein into the scrapie-like pathological prion protein. We previously reported that, by controlled thermal denaturation, human prion protein fragment 90-231 acquires neurotoxic properties when transformed in a β-rich conformation, resembling the scrapie-like conformation. In this study we generated prion protein fragment 90-231 bearing mutations identified in familial prion diseases (D202N and E200K), to analyze their role in the induction of a neurotoxic conformation. Prion protein fragment 90-231(wild type) and the D202N mutant were not toxic in native conformation but induced cell death only after thermal denaturation. Conversely, prion protein fragment 90-231(E200K) was highly toxic in its native structure, suggesting that E200K mutation per se favors the acquisition of a peptide neurotoxic conformation. To identify the structural determinants of prion protein fragment 90-231 toxicity, we show that while the wild type peptide is structured in α-helix, hPrP90-231 E200K is spontaneously refolded in a β-structured conformer characterized by increased proteinase K resistance and propensity to generate fibrils. However, the most significant difference induced by E200K mutation in prion protein fragment 90-231 structure in native conformation we observed, was an increase in the exposure of hydrophobic amino-acids on protein surface that was detected in wild type and D202N proteins only after thermal denaturation. In conclusion, we propose that increased hydrophobicity is one of the main determinants of toxicity induced by different mutations in prion protein-derived peptides. Copyright © 2010 Elsevier Ltd. All rights reserved.

Acquisition of Drug Resistance and Dependence by Prions

PubMed Central

Oelschlegel, Anja M.; Weissmann, Charles

2013-01-01

We have reported that properties of prion strains may change when propagated in different environments. For example, when swainsonine-sensitive 22L prions were propagated in PK1 cells in the presence of swainsonine, drug-resistant variants emerged. We proposed that prions constitute quasi- populations comprising a range of variants with different properties, from which the fittest are selected in a particular environment. Prion populations developed heterogeneity even after biological cloning, indicating that during propagation mutation-like processes occur at the conformational level. Because brain-derived 22L prions are naturally swainsonine resistant, it was not too surprising that prions which had become swa sensitive after propagation in cells could revert to drug resistance. Because RML prions, both after propagation in brain or in PK1 cells, are swainsonine sensitive, we investigated whether it was nonetheless possible to select swainsonine-resistant variants by propagation in the presence of the drug. Interestingly, this was not possible with the standard line of PK1 cells, but in certain PK1 sublines not only swainsonine-resistant, but even swainsonine-dependent populations (i.e. that propagated more rapidly in the presence of the drug) could be isolated. Once established, they could be passaged indefinitely in PK1 cells, even in the absence of the drug, without losing swainsonine dependence. The misfolded prion protein (PrPSc) associated with a swainsonine-dependent variant was less rapidly cleared in PK1 cells than that associated with its drug-sensitive counterpart, indicating that likely structural differences of the misfolded PrP underlie the properties of the prions. In summary, propagation of prions in the presence of an inhibitory drug may not only cause the selection of drug-resistant prions but even of stable variants that propagate more efficiently in the presence of the drug. These adaptations are most likely due to conformational changes of the abnormal prion protein. PMID:23408888

Increased Abundance of M Cells in the Gut Epithelium Dramatically Enhances Oral Prion Disease Susceptibility.

PubMed

Donaldson, David S; Sehgal, Anuj; Rios, Daniel; Williams, Ifor R; Mabbott, Neil A

2016-12-01

Many natural prion diseases of humans and animals are considered to be acquired through oral consumption of contaminated food or pasture. Determining the route by which prions establish host infection will identify the important factors that influence oral prion disease susceptibility and to which intervention strategies can be developed. After exposure, the early accumulation and replication of prions within small intestinal Peyer's patches is essential for the efficient spread of disease to the brain. To replicate within Peyer's patches, the prions must first cross the gut epithelium. M cells are specialised epithelial cells within the epithelia covering Peyer's patches that transcytose particulate antigens and microorganisms. M cell-development is dependent upon RANKL-RANK-signalling, and mice in which RANK is deleted only in the gut epithelium completely lack M cells. In the specific absence of M cells in these mice, the accumulation of prions within Peyer's patches and the spread of disease to the brain was blocked, demonstrating a critical role for M cells in the initial transfer of prions across the gut epithelium in order to establish host infection. Since pathogens, inflammatory stimuli and aging can modify M cell-density in the gut, these factors may also influence oral prion disease susceptibility. Mice were therefore treated with RANKL to enhance M cell density in the gut. We show that prion uptake from the gut lumen was enhanced in RANKL-treated mice, resulting in shortened survival times and increased disease susceptibility, equivalent to a 10-fold higher infectious titre of prions. Together these data demonstrate that M cells are the critical gatekeepers of oral prion infection, whose density in the gut epithelium directly limits or enhances disease susceptibility. Our data suggest that factors which alter M cell-density in the gut epithelium may be important risk factors which influence host susceptibility to orally acquired prion diseases.

Physical, chemical and kinetic factors affecting prion infectivity

PubMed Central

Properzi, Francesca; Badhan, Anjna; Klier, Steffi; Schmidt, Christian; Klöhn, Peter C.; Wadsworth, Jonathan D. F.; Clarke, Anthony R.; Jackson, Graham S.; Collinge, John

2016-01-01

ABSTRACT The mouse-adapted scrapie prion strain RML is one of the most widely used in prion research. The introduction of a cell culture-based assay of RML prions, the scrapie cell assay (SCA) allows more rapid and precise prion titration. A semi-automated version of this assay (ASCA) was applied to explore a range of conditions that might influence the infectivity and properties of RML prions. These include resistance to freeze-thaw procedures; stability to endogenous proteases in brain homogenate despite prolonged exposure to varying temperatures; distribution of infective material between pellet and supernatant after centrifugation, the effect of reducing agents and the influence of detergent additives on the efficiency of infection. Apparent infectivity is increased significantly by interaction with cationic detergents. Importantly, we have also elucidated the relationship between the duration of exposure of cells to RML prions and the transmission of infection. We established that the infection process following contact of cells with RML prions is rapid and followed an exponential time course, implying a single rate-limiting process. PMID:27282252

Physical, chemical and kinetic factors affecting prion infectivity.

PubMed

Properzi, Francesca; Badhan, Anjna; Klier, Steffi; Schmidt, Christian; Klöhn, Peter C; Wadsworth, Jonathan D F; Clarke, Anthony R; Jackson, Graham S; Collinge, John

2016-05-03

The mouse-adapted scrapie prion strain RML is one of the most widely used in prion research. The introduction of a cell culture-based assay of RML prions, the scrapie cell assay (SCA) allows more rapid and precise prion titration. A semi-automated version of this assay (ASCA) was applied to explore a range of conditions that might influence the infectivity and properties of RML prions. These include resistance to freeze-thaw procedures; stability to endogenous proteases in brain homogenate despite prolonged exposure to varying temperatures; distribution of infective material between pellet and supernatant after centrifugation, the effect of reducing agents and the influence of detergent additives on the efficiency of infection. Apparent infectivity is increased significantly by interaction with cationic detergents. Importantly, we have also elucidated the relationship between the duration of exposure of cells to RML prions and the transmission of infection. We established that the infection process following contact of cells with RML prions is rapid and followed an exponential time course, implying a single rate-limiting process.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maas, Z. G.; Pilachowski, C. A.; Hinkle, K., E-mail: zmaas@indiana.edu, E-mail: cpilacho@indiana.edu, E-mail: hinkle@noao.edu

Chlorine abundances are reported in 15 evolved giants and 1 M dwarf in the solar neighborhood. The Cl abundance was measured using the vibration-rotation 1-0 P8 line of H{sup 35}Cl at 3.69851 μ m. The high-resolution L -band spectra were observed using the Phoenix infrared spectrometer on the Kitt Peak Mayall 4 m telescope. The average [{sup 35}Cl/Fe] abundance in stars with −0.72 < [Fe/H] < 0.20 is [{sup 35}Cl/Fe] = (−0.10 ± 0.15) dex. The mean difference between the [{sup 35}Cl/Fe] ratios measured in our stars and chemical evolution model values is (0.16 ± 0.15) dex. The [{sup 35}Cl/Ca] ratio has an offset of ∼0.35more » dex above model predictions, suggesting that chemical evolution models are underproducing Cl at the high metallicity range. Abundances of C, N, O, Si, and Ca were also measured in our spectral region and are consistent with F and G dwarfs. The Cl versus O abundances from our sample match Cl abundances measured in planetary nebula and H ii regions. In one star where both H{sup 35}Cl and H{sup 37}Cl could be measured, a {sup 35}Cl/{sup 37}Cl isotope ratio of 2.2 ± 0.4 was found, consistent with values found in the Galactic ISM and predicted chemical evolution models.« less

Secretin receptor involvement in prion-infected cells and animals.

PubMed

Kimura, Tomohiro; Nishizawa, Keiko; Oguma, Ayumi; Nishimura, Yuki; Sakasegawa, Yuji; Teruya, Kenta; Nishijima, Ichiko; Doh-ura, Katsumi

2015-07-08

The cellular mechanisms behind prion biosynthesis and metabolism remain unclear. Here we show that secretin signaling via the secretin receptor regulates abnormal prion protein formation in prion-infected cells. Animal studies demonstrate that secretin receptor deficiency slightly, but significantly, prolongs incubation time in female but not male mice. This gender-specificity is consistent with our finding that prion-infected cells are derived from females. Therefore, our results provide initial insights into the reasons why age of disease onset in certain prion diseases is reported to occur slightly earlier in females than males. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Prions and prion-like proteins.

PubMed

Fraser, Paul E

2014-07-18

Prions are self-replicating protein aggregates and are the primary causative factor in a number of neurological diseases in mammals. The prion protein (PrP) undergoes a conformational transformation leading to aggregation into an infectious cellular pathogen. Prion-like protein spreading and transmission of aggregates between cells have also been demonstrated for other proteins associated with Alzheimer disease and Parkinson disease. This protein-only phenomenon may therefore have broader implications in neurodegenerative disorders. The minireviews in this thematic series highlight the recent advances in prion biology and the roles these unique proteins play in disease. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Automated decontamination of surface-adherent prions.

PubMed

Schmitt, A; Westner, I M; Reznicek, L; Michels, W; Mitteregger, G; Kretzschmar, H A

2010-09-01

At present there is no routinely available decontamination procedure in washer-disinfectors to allow the reliable inactivation and/or elimination of prions present on reusable surgical instruments. This means that is not possible to provide assurance for preventing iatrogenic transmission of prion diseases. We need effective procedures in prion decontamination that can be integrated into the usual routine of reprocessing surgical instruments. This article reports on the evaluation of an automated process designed to decontaminate prions in washer-disinfectors using a quantitative, highly sensitive in vivo assay for surface-adherent 22L prions. The automated process showed great advantages when compared with conventional alkaline cleaning. In contrast, the new process was as effective as autoclaving at 134 degrees C for 2h and left no detectable prion infectivity, even for heavily contaminated surfaces. This indicates a reduction of surface-adherent prion infectivity of >7 log units. Due to its compatibility with even delicate surgical instruments, the process can be integrated into the large scale reprocessing of instruments in a central sterile supply department. The system could potentially make an important contribution to the prevention of iatrogenic transmission of prions. Copyright 2010 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.

Disturbed vesicular trafficking of membrane proteins in prion disease.

PubMed

Uchiyama, Keiji; Miyata, Hironori; Sakaguchi, Suehiro

2013-01-01

The pathogenic mechanism of prion diseases remains unknown. We recently reported that prion infection disturbs post-Golgi trafficking of certain types of membrane proteins to the cell surface, resulting in reduced surface expression of membrane proteins and abrogating the signal from the proteins. The surface expression of the membrane proteins was reduced in the brains of mice inoculated with prions, well before abnormal symptoms became evident. Prions or pathogenic prion proteins were mainly detected in endosomal compartments, being particularly abundant in recycling endosomes. Some newly synthesized membrane proteins are delivered to the surface from the Golgi apparatus through recycling endosomes, and some endocytosed membrane proteins are delivered back to the surface through recycling endosomes. These results suggest that prions might cause neuronal dysfunctions and cell loss by disturbing post-Golgi trafficking of membrane proteins via accumulation in recycling endosomes. Interestingly, it was recently shown that delivery of a calcium channel protein to the cell surface was impaired and its function was abrogated in a mouse model of hereditary prion disease. Taken together, these results suggest that impaired delivery of membrane proteins to the cell surface is a common pathogenic event in acquired and hereditary prion diseases.

Selfish prion of Rnq1 mutant in yeast.

PubMed

Kurahashi, Hiroshi; Shibata, Shoichiro; Ishiwata, Masao; Nakamura, Yoshikazu

2009-05-01

[PIN(+)] is a prion form of Rnq1 in Saccharomyces cerevisiae and is necessary for the de novo induction of a second prion, [PSI(+)]. We previously isolated a truncated form of Rnq1, named Rnq1Delta100, as a [PSI(+)]-eliminating factor in S. cerevisiae. Rnq1Delta100 deletes the N-terminal non-prion domain of Rnq1, and eliminates [PSI(+)] in [PIN(+)] yeast. Here we found that [PIN(+)] is transmissible to Rnq1Delta100 in the absence of full-length Rnq1, forming a novel prion variant [RNQ1Delta100(+)]. [RNQ1Delta100(+)] has similar [PIN(+)] properties as it stimulates the de novo induction of [PSI(+)] and is eliminated by the null hsp104Delta mutation, but not by Hsp104 overproduction. In contrast, [RNQ1Delta100(+)] inherits the inhibitory activity and hampers the maintenance of [PSI(+)] though less efficiently than [PIN(+)] made of Rnq1-Rnq1Delta100 co-aggregates. Interestingly, [RNQ1Delta100(+)] prion was eliminated by de novo [PSI(+)] induction. Thus, the [RNQ1Delta100(+)] prion demonstrates selfish activity to eliminate a heterologous prion in S. cerevisiae, showing the first instance of a selfish prion variant in living organisms.

Neuronal death induced by misfolded prion protein is due to NAD+ depletion and can be relieved in vitro and in vivo by NAD+ replenishment

PubMed Central

Zhou, Minghai; Ottenberg, Gregory; Sferrazza, Gian Franco; Hubbs, Christopher; Fallahi, Mohammad; Rumbaugh, Gavin; Brantley, Alicia F.

2015-01-01

The mechanisms of neuronal death in protein misfolding neurodegenerative diseases such as Alzheimer’s, Parkinson’s and prion diseases are poorly understood. We used a highly toxic misfolded prion protein (TPrP) model to understand neurotoxicity induced by prion protein misfolding. We show that abnormal autophagy activation and neuronal demise is due to severe, neuron-specific, nicotinamide adenine dinucleotide (NAD+) depletion. Toxic prion protein-exposed neuronal cells exhibit dramatic reductions of intracellular NAD+ followed by decreased ATP production, and are completely rescued by treatment with NAD+ or its precursor nicotinamide because of restoration of physiological NAD+ levels. Toxic prion protein-induced NAD+ depletion results from PARP1-independent excessive protein ADP-ribosylations. In vivo, toxic prion protein-induced degeneration of hippocampal neurons is prevented dose-dependently by intracerebral injection of NAD+. Intranasal NAD+ treatment of prion-infected sick mice significantly improves activity and delays motor impairment. Our study reveals NAD+ starvation as a novel mechanism of autophagy activation and neurodegeneration induced by a misfolded amyloidogenic protein. We propose the development of NAD+ replenishment strategies for neuroprotection in prion diseases and possibly other protein misfolding neurodegenerative diseases. PMID:25678560

MEAT AND BONE MEAL AND MINERAL FEED ADDITIVES MAY INCREASE THE RISK OF ORAL PRION DISEASE TRANSMISSION

PubMed Central

Johnson, Christopher J.; McKenzie, Debbie; Pedersen, Joel A.; Aiken, Judd M.

2011-01-01

Ingestion of prion-contaminated materials is postulated to be a primary route of prion disease transmission. Binding of prions to soil (micro)particles dramatically enhances peroral disease transmission relative to unbound prions, and it was hypothesized that micrometer–sized particles present in other consumed materials may affect prion disease transmission via the oral route of exposure. Small, insoluble particles are present in many substances, including soil, human foods, pharmaceuticals, and animal feeds. It is known that meat and bone meal (MBM), a feed additive believed responsible for the spread of bovine spongiform encephalopathy (BSE), contains particles smaller than 20 μm and that the pathogenic prion protein binds to MBM. The potentiation of disease transmission via the oral route by exposure to MBM or three micrometer-sized mineral feed additives was determined. Data showed that when the disease agent was bound to any of the tested materials, the penetrance of disease was increased compared to unbound prions. Our data suggest that in feed or other prion–contaminated substances consumed by animals or, potentially, humans, the addition of MBM or the presence of microparticles could heighten risks of prion disease acquisition. PMID:21218345

Using small molecule reagents to help distinguish among prion structural models

USDA-ARS?s Scientific Manuscript database

The only demonstrated difference between infectious prions (PrPSc) and the isosequential normal cellular prion protein (PrPC) is conformation. The structure of PrPC has been determined by a variety of instrumental techniques. The structure of prions remains uncertain. Recent instrumental analysis h...

Pathways of Prion Spread during Early Chronic Wasting Disease in Deer.

PubMed

Hoover, Clare E; Davenport, Kristen A; Henderson, Davin M; Denkers, Nathaniel D; Mathiason, Candace K; Soto, Claudio; Zabel, Mark D; Hoover, Edward A

2017-05-15

Among prion infections, two scenarios of prion spread are generally observed: (i) early lymphoid tissue replication or (ii) direct neuroinvasion without substantial antecedent lymphoid amplification. In nature, cervids are infected with chronic wasting disease (CWD) prions by oral and nasal mucosal exposure, and studies of early CWD pathogenesis have implicated pharyngeal lymphoid tissue as the earliest sites of prion accumulation. However, knowledge of chronological events in prion spread during early infection remains incomplete. To investigate this knowledge gap in early CWD pathogenesis, we exposed white-tailed deer to CWD prions by mucosal routes and performed serial necropsies to assess PrP CWD tissue distribution by real-time quaking-induced conversion (RT-QuIC) and tyramide signal amplification immunohistochemistry (TSA-IHC). Although PrP CWD was not detected by either method in the initial days (1 and 3) postexposure, we observed PrP CWD seeding activity and follicular immunoreactivity in oropharyngeal lymphoid tissues at 1 and 2 months postexposure (MPE). At 3 MPE, PrP CWD replication had expanded to all systemic lymphoid tissues. By 4 MPE, the PrP CWD burden in all lymphoid tissues had increased and approached levels observed in terminal disease, yet there was no evidence of nervous system invasion. These results indicate the first site of CWD prion entry is in the oropharynx, and the initial phase of prion amplification occurs in the oropharyngeal lymphoid tissues followed by rapid dissemination to systemic lymphoid tissues. This lymphoid replication phase appears to precede neuroinvasion. IMPORTANCE Chronic wasting disease (CWD) is a universally fatal transmissible spongiform encephalopathy affecting cervids, and natural infection occurs through oral and nasal mucosal exposure to infectious prions. Terminal disease is characterized by PrP CWD accumulation in the brain and lymphoid tissues of affected animals. However, the initial sites of prion accumulation and pathways of prion spread during early CWD infection remain unknown. To investigate the chronological events of early prion pathogenesis, we exposed deer to CWD prions and monitored the tissue distribution of PrP CWD over the first 4 months of infection. We show CWD uptake occurs in the oropharynx with initial prion replication in the draining oropharyngeal lymphoid tissues, rapidly followed by dissemination to systemic lymphoid tissues without evidence of neuroinvasion. These data highlight the two phases of CWD infection: a robust prion amplification in systemic lymphoid tissues prior to neuroinvasion and establishment of a carrier state. Copyright © 2017 American Society for Microbiology.

Oral Prion Disease Pathogenesis Is Impeded in the Specific Absence of CXCR5-Expressing Dendritic Cells

PubMed Central

Bradford, Barry M.; Reizis, Boris

2017-01-01

ABSTRACT After oral exposure, the early replication of certain prion strains upon stromal cell-derived follicular dendritic cells (FDC) in the Peyer's patches in the small intestine is essential for the efficient spread of disease to the brain. However, little is known of how prions are initially conveyed from the gut lumen to establish infection on FDC. Our previous data suggest that mononuclear phagocytes such as CD11c+ conventional dendritic cells play an important role in the initial propagation of prions from the gut lumen into Peyer's patches. However, whether these cells conveyed orally acquired prions toward FDC within Peyer's patches was not known. The chemokine CXCL13 is expressed by FDC and follicular stromal cells and modulates the homing of CXCR5-expressing cells toward the FDC-containing B cell follicles. Here, novel compound transgenic mice were created in which a CXCR5 deficiency was specifically restricted to CD11c+ cells. These mice were used to determine whether CXCR5-expressing conventional dendritic cells propagate prions toward FDC after oral exposure. Our data show that in the specific absence of CXCR5-expressing conventional dendritic cells the early accumulation of prions upon FDC in Peyer's patches and the spleen was impaired, and disease susceptibility significantly reduced. These data suggest that CXCR5-expressing conventional dendritic cells play an important role in the efficient propagation of orally administered prions toward FDC within Peyer's patches in order to establish host infection. IMPORTANCE Many natural prion diseases are acquired by oral consumption of contaminated food or pasture. Once the prions reach the brain they cause extensive neurodegeneration, which ultimately leads to death. In order for the prions to efficiently spread from the gut to the brain, they first replicate upon follicular dendritic cells within intestinal Peyer's patches. How the prions are first delivered to follicular dendritic cells to establish infection was unknown. Understanding this process is important since treatments which prevent prions from infecting follicular dendritic cells can block their spread to the brain. We created mice in which mobile conventional dendritic cells were unable to migrate toward follicular dendritic cells. In these mice the early accumulation of prions on follicular dendritic cells was impaired and oral prion disease susceptibility was reduced. This suggests that prions exploit conventional dendritic cells to facilitate their initial delivery toward follicular dendritic cells to establish host infection. PMID:28275192

Attachment of Pathogenic Prion Protein to Model Oxide Surfaces

PubMed Central

Jacobson, Kurt H.; Kuech, Thomas R.; Pedersen, Joel A.

2014-01-01

Prions are the infectious agents in the class of fatal neurodegenerative diseases known as transmissible spongiform encephalopathies, which affect humans, deer, sheep, and cattle. Prion diseases of deer and sheep can be transmitted via environmental routes, and soil is has been implicated in the transmission of these diseases. Interaction with soil particles is expected to govern the transport, bioavailability and persistence of prions in soil environments. A mechanistic understanding of prion interaction with soil components is critical for understanding the behavior of these proteins in the environment. Here, we report results of a study to investigate the interactions of prions with model oxide surfaces (Al2O3, SiO2) using quartz crystal microbalance with dissipation monitoring and optical waveguide light mode spectroscopy. The efficiency of prion attachment to Al2O3 and SiO2 depended strongly on pH and ionic strength in a manner consistent with electrostatic forces dominating interaction with these oxides. The N-terminal portion of the protein appeared to facilitate attachment to Al2O3 under globally electrostatically repulsive conditions. We evaluated the utility of recombinant prion protein as a surrogate for prions in attachment experiments and found that its behavior differed markedly from that of the infectious agent. Our findings suggest that prions preferentially associate with positively charged mineral surfaces in soils (e.g., iron and aluminum oxides). PMID:23611152

Indexing amyloid peptide diffraction from serial femtosecond crystallography: new algorithms for sparse patterns

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brewster, Aaron S.; Sawaya, Michael R.; University of California, Los Angeles, CA 90095-1570

2015-02-01

Special methods are required to interpret sparse diffraction patterns collected from peptide crystals at X-ray free-electron lasers. Bragg spots can be indexed from composite-image powder rings, with crystal orientations then deduced from a very limited number of spot positions. Still diffraction patterns from peptide nanocrystals with small unit cells are challenging to index using conventional methods owing to the limited number of spots and the lack of crystal orientation information for individual images. New indexing algorithms have been developed as part of the Computational Crystallography Toolbox (cctbx) to overcome these challenges. Accurate unit-cell information derived from an aggregate data setmore » from thousands of diffraction patterns can be used to determine a crystal orientation matrix for individual images with as few as five reflections. These algorithms are potentially applicable not only to amyloid peptides but also to any set of diffraction patterns with sparse properties, such as low-resolution virus structures or high-throughput screening of still images captured by raster-scanning at synchrotron sources. As a proof of concept for this technique, successful integration of X-ray free-electron laser (XFEL) data to 2.5 Å resolution for the amyloid segment GNNQQNY from the Sup35 yeast prion is presented.« less

A Multiscale Approach to Characterize the Early Aggregation Steps of the Amyloid-Forming Peptide GNNQQNY from the Yeast Prion Sup-35

PubMed Central

Nasica-Labouze, Jessica; Meli, Massimiliano; Derreumaux, Philippe; Colombo, Giorgio; Mousseau, Normand

2011-01-01

The self-organization of peptides into amyloidogenic oligomers is one of the key events for a wide range of molecular and degenerative diseases. Atomic-resolution characterization of the mechanisms responsible for the aggregation process and the resulting structures is thus a necessary step to improve our understanding of the determinants of these pathologies. To address this issue, we combine the accelerated sampling properties of replica exchange molecular dynamics simulations based on the OPEP coarse-grained potential with the atomic resolution description of interactions provided by all-atom MD simulations, and investigate the oligomerization process of the GNNQQNY for three system sizes: 3-mers, 12-mers and 20-mers. Results for our integrated simulations show a rich variety of structural arrangements for aggregates of all sizes. Elongated fibril-like structures can form transiently in the 20-mer case, but they are not stable and easily interconvert in more globular and disordered forms. Our extensive characterization of the intermediate structures and their physico-chemical determinants points to a high degree of polymorphism for the GNNQQNY sequence that can be reflected at the macroscopic scale. Detailed mechanisms and structures that underlie amyloid aggregation are also provided. PMID:21625573

Prion Replication Elicits Cytopathic Changes in Differentiated Neurosphere Cultures

PubMed Central

Iwamaru, Yoshifumi; Takenouchi, Takato; Imamura, Morikazu; Shimizu, Yoshihisa; Miyazawa, Kohtaro; Mohri, Shirou; Yokoyama, Takashi

2013-01-01

The molecular mechanisms of prion-induced cytotoxicity remain largely obscure. Currently, only a few cell culture models have exhibited the cytopathic changes associated with prion infection. In this study, we introduced a cell culture model based on differentiated neurosphere cultures isolated from the brains of neonatal prion protein (PrP)-null mice and transgenic mice expressing murine PrP (dNP0 and dNP20 cultures). Upon exposure to mouse Chandler prions, dNP20 cultures supported the de novo formation of abnormal PrP and the resulting infectivity, as assessed by bioassays. Furthermore, this culture was susceptible to various prion strains, including mouse-adapted scrapie, bovine spongiform encephalopathy, and Gerstmann-Sträussler-Scheinker syndrome prions. Importantly, a subset of the cells in the infected culture that was mainly composed of astrocyte lineage cells consistently displayed late-occurring, progressive signs of cytotoxicity as evidenced by morphological alterations, decreased cell viability, and increased lactate dehydrogenase release. These signs of cytotoxicity were not observed in infected dNP0 cultures, suggesting the requirement of endogenous PrP expression for prion-induced cytotoxicity. Degenerated cells positive for glial fibrillary acidic protein accumulated abnormal PrP and exhibited features of apoptotic death as assessed by active caspase-3 and terminal deoxynucleotidyltransferase nick-end staining. Furthermore, caspase inhibition provided partial protection from prion-mediated cell death. These results suggest that differentiated neurosphere cultures can provide an in vitro bioassay for mouse prions and permit the study of the molecular basis for prion-induced cytotoxicity at the cellular level. PMID:23740992

Evidence that bank vole PrP is a universal acceptor for prions.

PubMed

Watts, Joel C; Giles, Kurt; Patel, Smita; Oehler, Abby; DeArmond, Stephen J; Prusiner, Stanley B

2014-04-01

Bank voles are uniquely susceptible to a wide range of prion strains isolated from many different species. To determine if this enhanced susceptibility to interspecies prion transmission is encoded within the sequence of the bank vole prion protein (BVPrP), we inoculated Tg(M109) and Tg(I109) mice, which express BVPrP containing either methionine or isoleucine at polymorphic codon 109, with 16 prion isolates from 8 different species: humans, cattle, elk, sheep, guinea pigs, hamsters, mice, and meadow voles. Efficient disease transmission was observed in both Tg(M109) and Tg(I109) mice. For instance, inoculation of the most common human prion strain, sporadic Creutzfeldt-Jakob disease (sCJD) subtype MM1, into Tg(M109) mice gave incubation periods of ∼200 days that were shortened slightly on second passage. Chronic wasting disease prions exhibited an incubation time of ∼250 days, which shortened to ∼150 days upon second passage in Tg(M109) mice. Unexpectedly, bovine spongiform encephalopathy and variant CJD prions caused rapid neurological dysfunction in Tg(M109) mice upon second passage, with incubation periods of 64 and 40 days, respectively. Despite the rapid incubation periods, other strain-specified properties of many prion isolates--including the size of proteinase K-resistant PrPSc, the pattern of cerebral PrPSc deposition, and the conformational stability--were remarkably conserved upon serial passage in Tg(M109) mice. Our results demonstrate that expression of BVPrP is sufficient to engender enhanced susceptibility to a diverse range of prion isolates, suggesting that BVPrP may be a universal acceptor for prions.

Comparative prion disease gene expression profiling using the prion disease mimetic, cuprizone

PubMed Central

Moody, Laura R; Herbst, Allen J; Yoo, Han Sang; Vanderloo, Joshua P

2009-01-01

Identification of genes expressed in response to prion infection may elucidate biomarkers for disease, identify factors involved in agent replication, mechanisms of neuropathology and therapeutic targets. Although several groups have sought to identify gene expression changes specific to prion disease, expression profiles rife with cell population changes have consistently been identified. Cuprizone, a neurotoxicant, qualitatively mimics the cell population changes observed in prion disease, resulting in both spongiform change and astrocytosis. The use of cuprizone-treated animals as an experimental control during comparative expression profiling allows for the identification of transcripts whose expression increases during prion disease and remains unchanged during cuprizone-triggered neuropathology. In this study, expression profiles from the brains of mice preclinically and clinically infected with Rocky Mountain Laboratory (RML) mouse-adapted scrapie agent and age-matched controls were profiled using Affymetrix gene arrays. In total, 164 genes were differentially regulated during prion infection. Eighty-three of these transcripts have been previously undescribed as differentially regulated during prion disease. A 0.4% cuprizone diet was utilized as a control for comparative expression profiling. Cuprizone treatment induced spongiosis and astrocyte proliferation as indicated by glial fibrillary acidic protein (Gfap) transcriptional activation and immunohistochemistry. Gene expression profiles from brain tissue obtained from cuprizone-treated mice identified 307 differentially regulated transcript changes. After comparative analysis, 17 transcripts unaffected by cuprizone treatment but increasing in expression from preclinical to clinical prion infection were identified. Here we describe the novel use of the prion disease mimetic, cuprizone, to control for cell population changes in the brain during prion infection. PMID:19535908

The chemistry of prions: small molecules, protein conformers and mass spectrometry

USDA-ARS?s Scientific Manuscript database

Background/Introduction. Prions propagate by converting a normal cellular isoform (PrPC) into the prion isoform (PrPSc) in a template-driven process. The lysines in PrPC are highly conserved and strongly influence prion propagation, based on studies using natural polymorphisms of PrPC and transg...

Correlation of cellular factors and differential scrapie prion permissiveness in ovine microglia

USDA-ARS?s Scientific Manuscript database

Prion diseases are fatal neurodegenerative disorders by which the native cellular prion protein (PrP-C) is misfolded into an accumulating, disease-associated isoform (PrP-D). To improve the understanding of prion pathogenesis and develop effective treatments, it is essential to elucidate factors con...

DOE Office of Scientific and Technical Information (OSTI.GOV)

Javahery, G.; Petrie, S.; Wang, J.

Results are reported for the reactions of the fullerene ions C[sub 60[center dot]+], C[sub 70[center dot]+], C[sub 60][sup 2+], C[sub 70][sup 2+], and C[sub 60[center dot]3+] with the nitriles HCN, CH[sub 3]CN, CH[sub 2]CHCN, CH[sub 3]CH[sub 2]CN, CH[sub 2]CHCH[sub 2]CN, CH[sub 3]CH[sub 2]CH[sub 2]CN, (CH[sub 3])[sub 2]CHCN, C[sub 2]N[sub 2], and CH[sub 2](CN)[sub 2]. The reactions were studied using a selected-ion flow tube (SIFT) at 294 [+-] 2 K and a helium buffer gas pressure of 0.35 [+-] 0.01 Torr and exhibited a wide range of chemical behavior. For the monocations C[sub 60][sup [center dot]+] and C[sub 70][sup [center dot]+],more » no detectable reaction occurred with any of the nitriles. For the dications C[sub 60][sup 2+], and C[sub 70][sup 2+], the only primary or secondary product channel evident in all instances was addition: tertiary association was seen to be considerably less efficient than the primary and secondary reactions, and the formation of a quadruple adduct C[sub 60](RCN)[sub 4][sup 2+] was only detected in the reaction with butyronitrile, CH[sub 3]CH[sub 2]CH[sub 2]CN. The observed primary rate coefficients show a very clear dependence upon the molecular complexity of the nitrile: association proceeds more efficiently for nitriles with a higher number of hydrogen atoms. This observation is explained in terms of a model which relates the number of C-H bonds - or, alternatively, the number of internal rotational modes - in the collision complex to the lifetime and probability of stabilization of this complex. 42 refs., 5 figs., 3 tabs.« less

A novel copper-hydrogen peroxide formulation for prion decontamination.

PubMed

Solassol, Jerome; Pastore, Manuela; Crozet, Carole; Perrier, Veronique; Lehmann, Sylvain

2006-09-15

With the appearance of variant Creutzfeldt-Jakob disease (CJD) and the detection of infectious prions in the peripheral organs of persons with sporadic CJD, the development of decontamination methods that are compatible with medical equipment has become a major issue. Here, we show that a formulation of copper metal ions in combination with hydrogen peroxide dramatically reduces the level of prion protein (PrP)(Sc) (the scrapie isoform of PrP) present in homogenates of samples from prion-infected brains, including brain samples from humans with CJD. An animal bioassay confirmed the reduction in prion infectivity, indicating that this novel Cu(2+)-H(2)O(2) formulation has great potential for prion decontamination.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hanft, J.M.; Jones, R.J.

This study was designed to compare the uptake and distribution of /sup 14/C among fructose, glucose, sucrose, and starch in the cob, pedicel, and endosperm tissues of maize (Zea mays L.) kernels induced to abort by high temperature with those that develop normally. Kernels cultured in vitro at 309 and 35/sup 0/C were transferred to (/sup 14/C)sucrose media 10 days after pollination. Kernels cultured at 35/sup 0/C aborted prior to the onset of linear dry matter accumulation. Significant uptake into the cob, pedicel, and endosperm of radioactivity associated with the soluble and starch fractions of the tissues was detected aftermore » 24 hours in culture on atlageled media. After 8 days in culture on (/sup 14/C)sucrose media, 48 and 40% of the radioactivity associated with the cob carbohydrates was found in the reducing sugars at 30 and 35/sup 0/C, respectively. Of the total carbohydrates, a higher percentage of label was associated with sucrose and lower percentage with fructose and glucose in pedicel tissue of kernels cultured at 35/sup 0/C compared to kernels cultured at 30/sup 0/C. These results indicate that sucrose was not cleaved to fructose and glucose as rapidly during the unloading process in the pedicel of kernels induced to abort by high temperature. Kernels cultured at 35/sup 0/C had a much lower proportion of label associated with endosperm starch (29%) than did kernels cultured at 30/sup 0/C (89%). Kernels cultured at 35/sup 0/C had a correspondingly higher proportion of /sup 14/C in endosperm fructose, glucose, and sucrose.« less

An overview of animal prion diseases

PubMed Central

2011-01-01

Prion diseases are transmissible neurodegenerative conditions affecting human and a wide range of animal species. The pathogenesis of prion diseases is associated with the accumulation of aggregates of misfolded conformers of host-encoded cellular prion protein (PrPC). Animal prion diseases include scrapie of sheep and goats, bovine spongiform encephalopathy (BSE) or mad cow disease, transmissible mink encephalopathy, feline spongiform encephalopathy, exotic ungulate spongiform encephalopathy, chronic wasting disease of cervids and spongiform encephalopathy of primates. Although some cases of sporadic atypical scrapie and BSE have also been reported, animal prion diseases have basically occurred via the acquisition of infection from contaminated feed or via the exposure to contaminated environment. Scrapie and chronic wasting disease are naturally sustaining epidemics. The transmission of BSE to human has caused more than 200 cases of variant Cruetzfeldt-Jacob disease and has raised serious public health concerns. The present review discusses the epidemiology, clinical neuropathology, transmissibility and genetics of animal prion diseases. PMID:22044871

Prion 2005: Between Fundamentals and Society's Needs.

PubMed

Treiber, Carina

2006-01-25

Prion diseases for the most part affect individuals older than 60 years of age and share features with other diseases characterized by protein deposits in the brain, such as Alzheimer's disease and Parkinson's disease. The international conference "Prion 2005: Between Fundamentals and Society's Needs," organized by the German Transmissible Spongiform Encephalopathies Research Platform, aimed to integrate and coordinate the research efforts of participants to better achieve prevention, treatment, control, and management of prion diseases, including Creutzfeldt-Jakob disease and fatal familial insomnia in humans. Several main topics were discussed, such as the molecular characteristics of prion strains, the cell biology of cellular and pathogenic forms of the prion proteins, the pathogenesis of the diseases they cause, emerging problems, and promising approaches for therapy and new diagnostic tools. The presentations at the Prion 2005 conference provided new insights in both basic and applied research, which will have broad implications for society's needs.

Saccharomyces cerevisiae: a sexy yeast with a prion problem.

PubMed

Kelly, Amy C; Wickner, Reed B

2013-01-01

Yeast prions are infectious proteins that spread exclusively by mating. The frequency of prions in the wild therefore largely reflects the rate of spread by mating counterbalanced by prion growth slowing effects in the host. We recently showed that the frequency of outcross mating is about 1% of mitotic doublings with 23-46% of total matings being outcrosses. These findings imply that even the mildest forms of the [PSI+], [URE3] and [PIN+] prions impart > 1% growth/survival detriment on their hosts. Our estimate of outcrossing suggests that Saccharomyces cerevisiae is far more sexual than previously thought and would therefore be more responsive to the adaptive effects of natural selection compared with a strictly asexual yeast. Further, given its large effective population size, a growth/survival detriment of > 1% for yeast prions should strongly select against prion-infected strains in wild populations of Saccharomyces cerevisiae.

Molecular modeling of the conformational dynamics of the cellular prion protein

NASA Astrophysics Data System (ADS)

Nguyen, Charles; Colling, Ian; Bartz, Jason; Soto, Patricia

2014-03-01

Prions are infectious agents responsible for transmissible spongiform encephalopathies (TSEs), a type of fatal neurodegenerative disease in mammals. Prions propagate biological information by conversion of the non-pathological version of the prion protein to the infectious conformation, PrPSc. A wealth of knowledge has shed light on the nature and mechanism of prion protein conversion. In spite of the significance of this problem, we are far from fully understanding the conformational dynamics of the cellular isoform. To remedy this situation we employ multiple biomolecular modeling techniques such as docking and molecular dynamics simulations to map the free energy landscape and determine what specific regions of the prion protein are most conductive to binding. The overall goal is to characterize the conformational dynamics of the cell form of the prion protein, PrPc, to gain insight into inhibition pathways against misfolding. NE EPSCoR FIRST Award to Patricia Soto.

Soil clay content underlies prion infection odds

PubMed Central

David Walter, W.; Walsh, Daniel P.; Farnsworth, Matthew L.; Winkelman, Dana L.; Miller, Michael W.

2011-01-01

Environmental factors—especially soil properties—have been suggested as potentially important in the transmission of infectious prion diseases. Because binding to montmorillonite (an aluminosilicate clay mineral) or clay-enriched soils had been shown to enhance experimental prion transmissibility, we hypothesized that prion transmission among mule deer might also be enhanced in ranges with relatively high soil clay content. In this study, we report apparent influences of soil clay content on the odds of prion infection in free-ranging deer. Analysis of data from prion-infected deer herds in northern Colorado, USA, revealed that a 1% increase in the clay-sized particle content in soils within the approximate home range of an individual deer increased its odds of infection by up to 8.9%. Our findings suggest that soil clay content and related environmental properties deserve greater attention in assessing risks of prion disease outbreaks and prospects for their control in both natural and production settings. PMID:21326232

Soil clay content underlies prion infection odds.

PubMed

David Walter, W; Walsh, Daniel P; Farnsworth, Matthew L; Winkelman, Dana L; Miller, Michael W

2011-02-15

Environmental factors-especially soil properties-have been suggested as potentially important in the transmission of infectious prion diseases. Because binding to montmorillonite (an aluminosilicate clay mineral) or clay-enriched soils had been shown to enhance experimental prion transmissibility, we hypothesized that prion transmission among mule deer might also be enhanced in ranges with relatively high soil clay content. In this study, we report apparent influences of soil clay content on the odds of prion infection in free-ranging deer. Analysis of data from prion-infected deer herds in northern Colorado, USA, revealed that a 1% increase in the clay-sized particle content in soils within the approximate home range of an individual deer increased its odds of infection by up to 8.9%. Our findings suggest that soil clay content and related environmental properties deserve greater attention in assessing risks of prion disease outbreaks and prospects for their control in both natural and production settings.

Clinically Unsuspected Prion Disease Among Patients With Dementia Diagnoses in an Alzheimer's Disease Database.

PubMed

Maddox, Ryan A; Blase, J L; Mercaldo, N D; Harvey, A R; Schonberger, L B; Kukull, W A; Belay, E D

2015-12-01

Brain tissue analysis is necessary to confirm prion diseases. Clinically unsuspected cases may be identified through neuropathologic testing. National Alzheimer's Coordinating Center (NACC) Minimum and Neuropathologic Data Set for 1984 to 2005 were reviewed. Eligible patients had dementia, underwent autopsy, had available neuropathologic data, belonged to a currently funded Alzheimer's Disease Center (ADC), and were coded as having an Alzheimer's disease clinical diagnosis or a nonprion disease etiology. For the eligible patients with neuropathology indicating prion disease, further clinical information, collected from the reporting ADC, determined whether prion disease was considered before autopsy. Of 6000 eligible patients in the NACC database, 7 (0.12%) were clinically unsuspected but autopsy-confirmed prion disease cases. The proportion of patients with dementia with clinically unrecognized but autopsy-confirmed prion disease was small. Besides confirming clinically suspected cases, neuropathology is useful to identify unsuspected clinically atypical cases of prion disease. © The Author(s) 2015.

Increased expression of p62/SQSTM1 in prion diseases and its association with pathogenic prion protein.

PubMed

Homma, Takujiro; Ishibashi, Daisuke; Nakagaki, Takehiro; Satoh, Katsuya; Sano, Kazunori; Atarashi, Ryuichiro; Nishida, Noriyuki

2014-03-28

Prion diseases are neurodegenerative disorders characterized by the aggregation of abnormally folded prion protein (PrP(Sc)). In this study, we focused on the mechanism of clearance of PrP(Sc), which remains unclear. p62 is a cytosolic protein known to mediate both the formation and degradation of aggregates of abnormal proteins. The levels of p62 protein increased in prion-infected brains and persistently infected cell cultures. Upon proteasome inhibition, p62 co-localized with PrP(Sc), forming a large aggregate in the perinuclear region, hereafter referred to as PrP(Sc)-aggresome. These aggregates were surrounded with autophagosome marker LC3 and lysosomes in prion-infected cells. Moreover, transient expression of the phosphomimic form of p62, which has enhanced ubiquitin-binding activity, reduced the amount of PrP(Sc) in prion-infected cells, indicating that the activation of p62 could accelerate the clearance of PrP(Sc). Our findings would thus suggest that p62 could be a target for the therapeutic control of prion diseases.

Infectivity-associated PrP(Sc) and disease duration-associated PrP(Sc) of mouse BSE prions.

PubMed

Miyazawa, Kohtaro; Okada, Hiroyuki; Masujin, Kentaro; Iwamaru, Yoshifumi; Yokoyama, Takashi

2015-01-01

Disease-related prion protein (PrP(Sc)), which is a structural isoform of the host-encoded cellular prion protein, is thought to be a causative agent of transmissible spongiform encephalopathies. However, the specific role of PrP(Sc) in prion pathogenesis and its relationship to infectivity remain controversial. A time-course study of prion-affected mice was conducted, which showed that the prion infectivity was not simply proportional to the amount of PrP(Sc) in the brain. Centrifugation (20,000 ×g) of the brain homogenate showed that most of the PrP(Sc) was precipitated into the pellet, and the supernatant contained only a slight amount of PrP(Sc). Interestingly, mice inoculated with the obtained supernatant showed incubation periods that were approximately 15 d longer than those of mice inoculated with the crude homogenate even though both inocula contained almost the same infectivity. Our results suggest that a small population of fine PrP(Sc) may be responsible for prion infectivity and that large, aggregated PrP(Sc) may contribute to determining prion disease duration.

Infectivity-associated PrPSc and disease duration-associated PrPSc of mouse BSE prions

PubMed Central

Miyazawa, Kohtaro; Okada, Hiroyuki; Masujin, Kentaro; Iwamaru, Yoshifumi; Yokoyama, Takashi

2015-01-01

ABSTRACT Disease-related prion protein (PrPSc), which is a structural isoform of the host-encoded cellular prion protein, is thought to be a causative agent of transmissible spongiform encephalopathies. However, the specific role of PrPSc in prion pathogenesis and its relationship to infectivity remain controversial. A time-course study of prion-affected mice was conducted, which showed that the prion infectivity was not simply proportional to the amount of PrPSc in the brain. Centrifugation (20,000 ×g) of the brain homogenate showed that most of the PrPSc was precipitated into the pellet, and the supernatant contained only a slight amount of PrPSc. Interestingly, mice inoculated with the obtained supernatant showed incubation periods that were approximately 15 d longer than those of mice inoculated with the crude homogenate even though both inocula contained almost the same infectivity. Our results suggest that a small population of fine PrPSc may be responsible for prion infectivity and that large, aggregated PrPSc may contribute to determining prion disease duration. PMID:26555211

Yeast prions are useful for studying protein chaperones and protein quality control.

PubMed

Masison, Daniel C; Reidy, Michael

2015-01-01

Protein chaperones help proteins adopt and maintain native conformations and play vital roles in cellular processes where proteins are partially folded. They comprise a major part of the cellular protein quality control system that protects the integrity of the proteome. Many disorders are caused when proteins misfold despite this protection. Yeast prions are fibrous amyloid aggregates of misfolded proteins. The normal action of chaperones on yeast prions breaks the fibers into pieces, which results in prion replication. Because this process is necessary for propagation of yeast prions, even small differences in activity of many chaperones noticeably affect prion phenotypes. Several other factors involved in protein processing also influence formation, propagation or elimination of prions in yeast. Thus, in much the same way that the dependency of viruses on cellular functions has allowed us to learn much about cell biology, the dependency of yeast prions on chaperones presents a unique and sensitive way to monitor the functions and interactions of many components of the cell's protein quality control system. Our recent work illustrates the utility of this system for identifying and defining chaperone machinery interactions.

Rebels with a cause: molecular features and physiological consequences of yeast prions.

PubMed

Garcia, David M; Jarosz, Daniel F

2014-02-01

Prions are proteins that convert between structurally and functionally distinct states, at least one of which is self-perpetuating. The prion fold templates the conversion of native protein, altering its structure and function, and thus serves as a protein-based element of inheritance. Molecular chaperones ensure that these prion aggregates are divided and faithfully passed from mother cells to their daughters. Prions were originally identified as the cause of several rare neurodegenerative diseases in mammals, but the last decade has brought great progress in understanding their broad importance in biology and evolution. Most prion proteins regulate information flow in signaling networks, or otherwise affect gene expression. Consequently, switching into and out of prion states creates diverse new traits – heritable changes based on protein structure rather than nucleic acid. Despite intense study of the molecular mechanisms of this paradigm-shifting, epigenetic mode of inheritance, many key questions remain. Recent studies in yeast that support the view that prions are common, often beneficial elements of inheritance that link environmental stress to the appearance of new traits.

Correlation analysis for the incubation period of prion disease.

PubMed

Bae, Se-Eun; Jung, Sunghoon; Kim, Ha-Yeon; Son, Hyeon S

2012-07-01

Previous studies have shown that genetic quantitative trait loci (QTL), strain barriers, inoculation dose and inoculation method modulate the incubation period of prion diseases. We examined the relationship between a diverse set of physical, genetic and immunological characteristics and the incubation period of prion disease using correlation analyses. We found that incubation period was highly correlated with brain weight. In addition, mean corpuscular volume and cell size were strongly correlated with incubation period, indicating that the physical magnitude of prion-infected organs or individual cells may be important in determining the incubation period. Given the same prion inoculation dose, animals with a lower brain weight, mean corpuscular volume or cell size may experience more virulent disease, as the effective concentration of abnormal prion, which might regulate the attachment rate of prions to aggregates, is increased with smaller capacity of brains and cells. This is partly consistent with previous theoretical modeling. The strong correlations between incubation period and physical properties of the brain and cells in this study suggest that the mechanism underlying prion disease pathology may be physical, indicating that the incubation process is governed by simple chemical stoichiometry.

Biochemical Characterization of Prion Strains in Bank Voles

PubMed Central

Pirisinu, Laura; Marcon, Stefano; Di Bari, Michele Angelo; D’Agostino, Claudia; Agrimi, Umberto; Nonno, Romolo

2013-01-01

Prions exist as different strains exhibiting distinct disease phenotypes. Currently, the identification of prion strains is still based on biological strain typing in rodents. However, it has been shown that prion strains may be associated with distinct PrPSc biochemical types. Taking advantage of the availability of several prion strains adapted to a novel rodent model, the bank vole, we investigated if any prion strain was actually associated with distinctive PrPSc biochemical characteristics and if it was possible to univocally identify strains through PrPSc biochemical phenotypes. We selected six different vole-adapted strains (three human-derived and three animal-derived) and analyzed PrPSc from individual voles by epitope mapping of protease resistant core of PrPSc (PrPres) and by conformational stability and solubility assay. Overall, we discriminated five out of six prion strains, while two different scrapie strains showed identical PrPSc types. Our results suggest that the biochemical strain typing approach here proposed was highly discriminative, although by itself it did not allow us to identify all prion strains analyzed. PMID:25437201

Unraveling the key to the resistance of canids to prion diseases

PubMed Central

Fernández-Borges, Natalia; Sánchez-Martín, Manuel A.; Pumarola, Martí

2017-01-01

One of the characteristics of prions is their ability to infect some species but not others and prion resistant species have been of special interest because of their potential in deciphering the determinants for susceptibility. Previously, we developed different in vitro and in vivo models to assess the susceptibility of species that were erroneously considered resistant to prion infection, such as members of the Leporidae and Equidae families. Here we undertake in vitro and in vivo approaches to understand the unresolved low prion susceptibility of canids. Studies based on the amino acid sequence of the canine prion protein (PrP), together with a structural analysis in silico, identified unique key amino acids whose characteristics could orchestrate its high resistance to prion disease. Cell- and brain-based PMCA studies were performed highlighting the relevance of the D163 amino acid in proneness to protein misfolding. This was also investigated by the generation of a novel transgenic mouse model carrying this substitution and these mice showed complete resistance to disease despite intracerebral challenge with three different mouse prion strains (RML, 22L and 301C) known to cause disease in wild-type mice. These findings suggest that dog D163 amino acid is primarily, if not totally, responsible for the prion resistance of canids. PMID:29131852

Prion disease tempo determined by host-dependent substrate reduction

PubMed Central

Mays, Charles E.; Kim, Chae; Haldiman, Tracy; van der Merwe, Jacques; Lau, Agnes; Yang, Jing; Grams, Jennifer; Di Bari, Michele A.; Nonno, Romolo; Telling, Glenn C.; Kong, Qingzhong; Langeveld, Jan; McKenzie, Debbie; Westaway, David; Safar, Jiri G.

2014-01-01

The symptoms of prion infection can take years or decades to manifest following the initial exposure. Molecular markers of prion disease include accumulation of the misfolded prion protein (PrPSc), which is derived from its cellular precursor (PrPC), as well as downregulation of the PrP-like Shadoo (Sho) glycoprotein. Given the overlapping cellular environments for PrPC and Sho, we inferred that PrPC levels might also be altered as part of a host response during prion infection. Using rodent models, we found that, in addition to changes in PrPC glycosylation and proteolytic processing, net reductions in PrPC occur in a wide range of prion diseases, including sheep scrapie, human Creutzfeldt-Jakob disease, and cervid chronic wasting disease. The reduction in PrPC results in decreased prion replication, as measured by the protein misfolding cyclic amplification technique for generating PrPSc in vitro. While PrPC downregulation is not discernible in animals with unusually short incubation periods and high PrPC expression, slowly evolving prion infections exhibit downregulation of the PrPC substrate required for new PrPSc synthesis and as a receptor for pathogenic signaling. Our data reveal PrPC downregulation as a previously unappreciated element of disease pathogenesis that defines the extensive, presymptomatic period for many prion strains. PMID:24430187

Prions and prion diseases: fundamentals and mechanistic details.

PubMed

Ryou, Chongsuk

2007-07-01

Prion diseases, often called transmissible spongiform encephalopathies (TSEs), are infectious diseases that accompany neurological dysfunctions in many mammalian hosts. Prion diseases include Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE, "mad cow disease") in cattle, scrapie in sheep, and chronic wasting disease (CWD) in deer and elks. The cause of these fatal diseases is a proteinaceous pathogen termed prion that lacks functional nucleic acids. As demonstrated in the BSE outbreak and its transmission to humans, the onset of disease is not limited to a certain species but can be transmissible from one host species to another. Such a striking nature ofprions has generated huge concerns in public health and attracted serious attention in the scientific communities. To date, the potential transmission ofprions to humans via foodbome infectiorn and iatrogenic routes has not been alleviated. Rather, the possible transmission of human to human or cervids to human aggravates the terrifying situation across the globe. In this review, basic features about prion diseases including clinical and pathological characteristics, etiology, and transmission of diseases are described. Based on recently accumulated evidences, the molecular and biochemical aspects of prions, with an emphasis on the molecular interactions involved in prion conversion that is critical during prion replication and pathogenesis, are also addressed.

Prion disease susceptibility is affected by β-structure folding propensity and local side-chain interactions in PrP

PubMed Central

Khan, M. Qasim; Sweeting, Braden; Mulligan, Vikram Khipple; Arslan, Pharhad Eli; Cashman, Neil R.; Pai, Emil F.; Chakrabartty, Avijit

2010-01-01

Prion diseases occur when the normally α-helical prion protein (PrP) converts to a pathological β-structured state with prion infectivity (PrPSc). Exposure to PrPSc from other mammals can catalyze this conversion. Evidence from experimental and accidental transmission of prions suggests that mammals vary in their prion disease susceptibility: Hamsters and mice show relatively high susceptibility, whereas rabbits, horses, and dogs show low susceptibility. Using a novel approach to quantify conformational states of PrP by circular dichroism (CD), we find that prion susceptibility tracks with the intrinsic propensity of mammalian PrP to convert from the native, α-helical state to a cytotoxic β-structured state, which exists in a monomer–octamer equilibrium. It has been controversial whether β-structured monomers exist at acidic pH; sedimentation equilibrium and dual-wavelength CD evidence is presented for an equilibrium between a β-structured monomer and octamer in some acidic pH conditions. Our X-ray crystallographic structure of rabbit PrP has identified a key helix-capping motif implicated in the low prion disease susceptibility of rabbits. Removal of this capping motif increases the β-structure folding propensity of rabbit PrP to match that of PrP from mouse, a species more susceptible to prion disease. PMID:21041683

Prion disease susceptibility is affected by beta-structure folding propensity and local side-chain interactions in PrP.

PubMed

Khan, M Qasim; Sweeting, Braden; Mulligan, Vikram Khipple; Arslan, Pharhad Eli; Cashman, Neil R; Pai, Emil F; Chakrabartty, Avijit

2010-11-16

Prion diseases occur when the normally α-helical prion protein (PrP) converts to a pathological β-structured state with prion infectivity (PrP(Sc)). Exposure to PrP(Sc) from other mammals can catalyze this conversion. Evidence from experimental and accidental transmission of prions suggests that mammals vary in their prion disease susceptibility: Hamsters and mice show relatively high susceptibility, whereas rabbits, horses, and dogs show low susceptibility. Using a novel approach to quantify conformational states of PrP by circular dichroism (CD), we find that prion susceptibility tracks with the intrinsic propensity of mammalian PrP to convert from the native, α-helical state to a cytotoxic β-structured state, which exists in a monomer-octamer equilibrium. It has been controversial whether β-structured monomers exist at acidic pH; sedimentation equilibrium and dual-wavelength CD evidence is presented for an equilibrium between a β-structured monomer and octamer in some acidic pH conditions. Our X-ray crystallographic structure of rabbit PrP has identified a key helix-capping motif implicated in the low prion disease susceptibility of rabbits. Removal of this capping motif increases the β-structure folding propensity of rabbit PrP to match that of PrP from mouse, a species more susceptible to prion disease.

Direct Detection of Soil-Bound Prions

PubMed Central

Genovesi, Sacha; Leita, Liviana; Sequi, Paolo; Andrighetto, Igino; Sorgato, M. Catia; Bertoli, Alessandro

2007-01-01

Scrapie and chronic wasting disease are contagious prion diseases affecting sheep and cervids, respectively. Studies have indicated that horizontal transmission is important in sustaining these epidemics, and that environmental contamination plays an important role in this. In the perspective of detecting prions in soil samples from the field by more direct methods than animal-based bioassays, we have developed a novel immuno-based approach that visualises in situ the major component (PrPSc) of prions sorbed onto agricultural soil particles. Importantly, the protocol needs no extraction of the protein from soil. Using a cell-based assay of infectivity, we also report that samples of agricultural soil, or quartz sand, acquire prion infectivity after exposure to whole brain homogenates from prion-infected mice. Our data provide further support to the notion that prion-exposed soils retain infectivity, as recently determined in Syrian hamsters intracerebrally or orally challanged with contaminated soils. The cell approach of the potential infectivity of contaminated soil is faster and cheaper than classical animal-based bioassays. Although it suffers from limitations, e.g. it can currently test only a few mouse prion strains, the cell model can nevertheless be applied in its present form to understand how soil composition influences infectivity, and to test prion-inactivating procedures. PMID:17957252

Ultraviolet-ozone treatment reduces levels of disease-associated prion protein and prion infectivity

USGS Publications Warehouse

Johnson, C.J.; Gilbert, P.; McKenzie, D.; Pedersen, J.A.; Aiken, Judd M.

2009-01-01

Background. Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases caused by novel infectious agents referred to as prions. Prions appear to be composed primarily, if not exclusively, of a misfolded isoform of the cellular prion protein. TSE infectivity is remarkably stable and can resist many aggressive decontamination procedures, increasing human, livestock and wildlife exposure to TSEs. Findings. We tested the hypothesis that UV-ozone treatment reduces levels of the pathogenic prion protein and inactivates the infectious agent. We found that UV-ozone treatment decreased the carbon and prion protein content in infected brain homogenate to levels undetectable by dry-ashing carbon analysis or immunoblotting, respectively. After 8 weeks of ashing, UV-ozone treatment reduced the infectious titer of treated material by a factor of at least 105. A small amount of infectivity, however, persisted despite UV-ozone treatment. When bound to either montmorillonite clay or quartz surfaces, PrPTSE was still susceptible to degradation by UV-ozone. Conclusion. Our findings strongly suggest that UV-ozone treatment can degrade pathogenic prion protein and inactivate prions, even when the agent is associated with surfaces. Using larger UV-ozone doses or combining UV-ozone treatment with other decontaminant methods may allow the sterilization of TSE-contaminated materials. ?? 2009 Aiken et al; licensee BioMed Central Ltd.

Optimization of Aryl Amides that Extend Survival in Prion-Infected Mice.

PubMed

Giles, Kurt; Berry, David B; Condello, Carlo; Dugger, Brittany N; Li, Zhe; Oehler, Abby; Bhardwaj, Sumita; Elepano, Manuel; Guan, Shenheng; Silber, B Michael; Olson, Steven H; Prusiner, Stanley B

2016-09-01

Developing therapeutics for neurodegenerative diseases (NDs) prevalent in the aging population remains a daunting challenge. With the growing understanding that many NDs progress by conformational self-templating of specific proteins, the prototypical prion diseases offer a platform for ND drug discovery. We evaluated high-throughput screening hits with the aryl amide scaffold and explored the structure-activity relationships around three series differing in their N-aryl core: benzoxazole, benzothiazole, and cyano. Potent anti-prion compounds were advanced to pharmacokinetic studies, and the resulting brain-penetrant leads from each series, together with a related N-aryl piperazine lead, were escalated to long-term dosing and efficacy studies. Compounds from each of the four series doubled the survival of mice infected with a mouse-passaged prion strain. Treatment with aryl amides altered prion strain properties, as evidenced by the distinct patterns of neuropathological deposition of prion protein and associated astrocytic gliosis in the brain; however, none of the aryl amide compounds resulted in drug-resistant prion strains, in contrast to previous studies on compounds with the 2-aminothiazole (2-AMT) scaffold. As seen with 2-AMTs and other effective anti-prion compounds reported to date, the novel aryl amides reported here were ineffective in prolonging the survival of transgenic mice infected with human prions. Most encouraging is our discovery that aryl amides show that the development of drug resistance is not an inevitable consequence of efficacious anti-prion therapeutics. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Optimization of Aryl Amides that Extend Survival in Prion-Infected Mice

PubMed Central

Giles, Kurt; Berry, David B.; Condello, Carlo; Dugger, Brittany N.; Li, Zhe; Oehler, Abby; Bhardwaj, Sumita; Elepano, Manuel; Guan, Shenheng; Silber, B. Michael; Olson, Steven H.

2016-01-01

Developing therapeutics for neurodegenerative diseases (NDs) prevalent in the aging population remains a daunting challenge. With the growing understanding that many NDs progress by conformational self-templating of specific proteins, the prototypical prion diseases offer a platform for ND drug discovery. We evaluated high-throughput screening hits with the aryl amide scaffold and explored the structure–activity relationships around three series differing in their N-aryl core: benzoxazole, benzothiazole, and cyano. Potent anti-prion compounds were advanced to pharmacokinetic studies, and the resulting brain-penetrant leads from each series, together with a related N-aryl piperazine lead, were escalated to long-term dosing and efficacy studies. Compounds from each of the four series doubled the survival of mice infected with a mouse-passaged prion strain. Treatment with aryl amides altered prion strain properties, as evidenced by the distinct patterns of neuropathological deposition of prion protein and associated astrocytic gliosis in the brain; however, none of the aryl amide compounds resulted in drug-resistant prion strains, in contrast to previous studies on compounds with the 2-aminothiazole (2-AMT) scaffold. As seen with 2-AMTs and other effective anti-prion compounds reported to date, the novel aryl amides reported here were ineffective in prolonging the survival of transgenic mice infected with human prions. Most encouraging is our discovery that aryl amides show that the development of drug resistance is not an inevitable consequence of efficacious anti-prion therapeutics. PMID:27317802

Biological and biochemical characterization of mice expressing prion protein devoid of the octapeptide repeat region after infection with prions.

PubMed

Yamaguchi, Yoshitaka; Miyata, Hironori; Uchiyama, Keiji; Ootsuyama, Akira; Inubushi, Sachiko; Mori, Tsuyoshi; Muramatsu, Naomi; Katamine, Shigeru; Sakaguchi, Suehiro

2012-01-01

Accumulating lines of evidence indicate that the N-terminal domain of prion protein (PrP) is involved in prion susceptibility in mice. In this study, to investigate the role of the octapeptide repeat (OR) region alone in the N-terminal domain for the susceptibility and pathogenesis of prion disease, we intracerebrally inoculated RML scrapie prions into tg(PrPΔOR)/Prnp(0/0) mice, which express mouse PrP missing only the OR region on the PrP-null background. Incubation times of these mice were not extended. Protease-resistant PrPΔOR, or PrP(Sc)ΔOR, was easily detectable but lower in the brains of these mice, compared to that in control wild-type mice. Consistently, prion titers were slightly lower and astrogliosis was milder in their brains. However, in their spinal cords, PrP(Sc)ΔOR and prion titers were abundant and astrogliosis was as strong as in control wild-type mice. These results indicate that the role of the OR region in prion susceptibility and pathogenesis of the disease is limited. We also found that the PrP(Sc)ΔOR, including the pre-OR residues 23-50, was unusually protease-resistant, indicating that deletion of the OR region could cause structural changes to the pre-OR region upon prion infection, leading to formation of a protease-resistant structure for the pre-OR region.

Homogenous photocatalytic decontamination of prion infected stainless steel and titanium surfaces.

PubMed

Berberidou, Chrysanthi; Xanthopoulos, Konstantinos; Paspaltsis, Ioannis; Lourbopoulos, Athanasios; Polyzoidou, Eleni; Sklaviadis, Theodoros; Poulios, Ioannis

2013-01-01

Prions are notorious for their extraordinary resistance to traditional methods of decontamination, rendering their transmission a public health risk. Iatrogenic Creutzfeldt-Jakob disease (iCJD) via contaminated surgical instruments and medical devices has been verified both experimentally and clinically. Standard methods for prion inactivation by sodium hydroxide or sodium hypochlorite have failed, in some cases, to fully remove prion infectivity, while they are often impractical for routine applications. Prion accumulation in peripheral tissues and indications of human-to-human bloodborne prion transmission, highlight the need for novel, efficient, yet user-friendly methods of prion inactivation. Here we show both in vitro and in vivo that homogenous photocatalytic oxidation, mediated by the photo-Fenton reagent, has the potential to inactivate the pathological prion isoform adsorbed on metal substrates. Photocatalytic oxidation with 224 μg mL(-1) Fe (3+), 500 μg mL(-1) h(-1) H 2O 2, UV-A for 480 min lead to 100% survival in golden Syrian hamsters after intracranial implantation of stainless steel wires infected with the 263K prion strain. Interestingly, photocatalytic treatment of 263K infected titanium wires, under the same experimental conditions, prolonged the survival interval significantly, but failed to eliminate infectivity, a result that we correlate with the increased adsorption of PrP(Sc) on titanium, in comparison to stainless steel. Our findings strongly indicate that our, user--and environmentally--friendly protocol can be safely applied to the decontamination of prion infected stainless steel surfaces.

Experimental sheep BSE prions generate the vCJD phenotype when serially passaged in transgenic mice expressing human prion protein.

PubMed

Joiner, Susan; Asante, Emmanuel A; Linehan, Jacqueline M; Brock, Lara; Brandner, Sebastian; Bellworthy, Susan J; Simmons, Marion M; Hope, James; Collinge, John; Wadsworth, Jonathan D F

2018-03-15

The epizootic prion disease of cattle, bovine spongiform encephalopathy (BSE), causes variant Creutzfeldt-Jakob disease (vCJD) in humans following dietary exposure. While it is assumed that all cases of vCJD attributed to a dietary aetiology are related to cattle BSE, sheep and goats are susceptible to experimental oral challenge with cattle BSE prions and farmed animals in the UK were undoubtedly exposed to BSE-contaminated meat and bone meal during the late 1980s and early 1990s. Although no natural field cases of sheep BSE have been identified, it cannot be excluded that some BSE-infected sheep might have entered the European human food chain. Evaluation of the zoonotic potential of sheep BSE prions has been addressed by examining the transmission properties of experimental brain isolates in transgenic mice that express human prion protein, however to-date there have been relatively few studies. Here we report that serial passage of experimental sheep BSE prions in transgenic mice expressing human prion protein with methionine at residue 129 produces the vCJD phenotype that mirrors that seen when the same mice are challenged with vCJD prions from patient brain. These findings are congruent with those reported previously by another laboratory, and thereby strongly reinforce the view that sheep BSE prions could have acted as a causal agent of vCJD within Europe. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Fiber Diffraction of the Prion-Forming Domain HET-s(218-289) Shows Dehydration-Induced Deformation of a Complex Amyloid Structure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wan, William; Stubbs, Gerald

2014-05-01

Amyloids are filamentous protein aggregates that can be formed by many different proteins and are associated with both disease and biological functions. The pathogenicities or biological functions of amyloids are determined by their particular molecular structures, making accurate structural models a requirement for understanding their biological effects. One potential factor that can affect amyloid structures is hydration. Previous studies of simple stacked β-sheet amyloids have suggested that dehydration does not impact structure, but other studies indicated dehydration-related structural changes of a putative water-filled nanotube. Our results show that dehydration significantly affects the molecular structure of the fungal prion-forming domain HET-s(218–289),more » which forms a β-solenoid with no internal solvent-accessible regions. The dehydration-related structural deformation of HET-s(218–289) indicates that water can play a significant role in complex amyloid structures, even when no obvious water-accessible cavities are present.« less

Diagnostic and prognostic value of human prion detection in cerebrospinal fluid.

PubMed

Foutz, Aaron; Appleby, Brian S; Hamlin, Clive; Liu, Xiaoqin; Yang, Sheng; Cohen, Yvonne; Chen, Wei; Blevins, Janis; Fausett, Cameron; Wang, Han; Gambetti, Pierluigi; Zhang, Shulin; Hughson, Andrew; Tatsuoka, Curtis; Schonberger, Lawrence B; Cohen, Mark L; Caughey, Byron; Safar, Jiri G

2017-01-01

Several prion amplification systems have been proposed for detection of prions in cerebrospinal fluid (CSF), most recently, the measurements of prion seeding activity with second-generation real-time quaking-induced conversion (RT-QuIC). The objective of this study was to investigate the diagnostic performance of the RT-QuIC prion test in the broad phenotypic spectrum of prion diseases. We performed CSF RT-QuIC testing in 2,141 patients who had rapidly progressive neurological disorders, determined diagnostic sensitivity and specificity in 272 cases that were autopsied, and evaluated the impact of mutations and polymorphisms in the PRNP gene, and type 1 or type 2 human prions on diagnostic performance. The 98.5% diagnostic specificity and 92% sensitivity of CSF RT-QuIC in a blinded retrospective analysis matched the 100% specificity and 95% sensitivity of a blind prospective study. The CSF RT-QuIC differentiated 94% of cases of sporadic Creutzfeldt-Jakob disease (sCJD) MM1 from the sCJD MM2 phenotype, and 80% of sCJD VV2 from sCJD VV1. The mixed prion type 1-2 and cases heterozygous for codon 129 generated intermediate CSF RT-QuIC patterns, whereas genetic prion diseases revealed distinct profiles for each PRNP gene mutation. The diagnostic performance of the improved CSF RT-QuIC is superior to surrogate marker tests for prion diseases such as 14-3-3 and tau proteins, and together with PRNP gene sequencing the test allows the major prion subtypes to be differentiated in vivo. This differentiation facilitates prediction of the clinicopathological phenotype and duration of the disease-two important considerations for envisioned therapeutic interventions. ANN NEUROL 2017;81:79-92. © 2016 American Neurological Association.

Diagnostic and Prognostic Value of Human Prion Detection in Cerebrospinal Fluid

PubMed Central

Foutz, Aaron; Appleby, Brian S.; Hamlin, Clive; Liu, Xiaoqin; Yang, Sheng; Cohen, Yvonne; Chen, Wei; Blevins, Janis; Fausett, Cameron; Wang, Han; Gambetti, Pierluigi; Zhang, Shulin; Hughson, Andrew; Tatsuoka, Curtis; Schonberger, Lawrence B.; Cohen, Mark L.; Caughey, Byron; Safar, Jiri G.

2016-01-01

Objective Several prion amplification systems have been proposed for detection of prions in cerebrospinal fluid (CSF), most recently, the measurements of prion seeding activity with second-generation real-time quaking-induced conversion (RT-QuIC). The objective of this study was to investigate the diagnostic performance of the RT-QuIC prion test in the broad phenotypic spectrum of prion diseases. Methods We performed CSF RT-QuIC testing in 2,141 patients who had rapidly progressive neurological disorders, determined diagnostic sensitivity and specificity in 272 cases which were autopsied, and evaluated the impact of mutations and polymorphisms in the PRNP gene, and Type 1 or Type 2 of human prions on diagnostic performance. Results The 98.5% diagnostic specificity and 92% sensitivity of CSF RT-QuIC in a blinded retrospective analysis matched the 100% specificity and 95% sensitivity of a blind prospective study. The CSF RT-QuIC differentiated 94% of cases of sporadic Creutzfeldt-Jakob disease (sCJD) MM1 from the sCJD MM2 phenotype, and 80% of sCJD VV2 from sCJD VV1. The mixed prion type 1–2 and cases heterozygous for codon 129 generated intermediate CSF RT-QuIC patterns, while genetic prion diseases revealed distinct profiles for each PRNP gene mutation. Interpretation The diagnostic performance of the improved CSF RT-QuIC is superior to surrogate marker tests for prion diseases such as 14-3-3 and Tau proteins and together with PRNP gene sequencing, the test allows the major prion subtypes to be differentiated in vivo. This differentiation facilitates prediction of the clinicopathological phenotype and duration of the disease—two important considerations for envisioned therapeutic interventions. PMID:27893164

Prion Strain Characterization of a Novel Subtype of Creutzfeldt-Jakob Disease.

PubMed

Galeno, Roberta; Di Bari, Michele Angelo; Nonno, Romolo; Cardone, Franco; Sbriccoli, Marco; Graziano, Silvia; Ingrosso, Loredana; Fiorini, Michele; Valanzano, Angelina; Pasini, Giulia; Poleggi, Anna; Vinci, Ramona; Ladogana, Anna; Puopolo, Maria; Monaco, Salvatore; Agrimi, Umberto; Zanusso, Gianluigi; Pocchiari, Maurizio

2017-06-01

In 2007, we reported a patient with an atypical form of Creutzfeldt-Jakob disease (CJD) heterozygous for methionine-valine (MV) at codon 129 who showed a novel pathological prion protein (PrP TSE ) conformation with an atypical glycoform (AG) profile and intraneuronal PrP deposition. In the present study, we further characterize the conformational properties of this pathological prion protein (PrP TSE MV AG ), showing that PrP TSE MV AG is composed of multiple conformers with biochemical properties distinct from those of PrP TSE type 1 and type 2 of MV sporadic CJD (sCJD). Experimental transmission of CJD-MV AG to bank voles and gene-targeted transgenic mice carrying the human prion protein gene (TgHu mice) showed unique transmission rates, survival times, neuropathological changes, PrP TSE deposition patterns, and PrP TSE glycotypes that are distinct from those of sCJD-MV1 and sCJD-MV2. These biochemical and experimental data suggest the presence of a novel prion strain in CJD-MV AG IMPORTANCE Sporadic Creutzfeldt-Jakob disease is caused by the misfolding of the cellular prion protein, which assumes two different major conformations (type 1 and type 2) and, together with the methionine/valine polymorphic codon 129 of the prion protein gene, contribute to the occurrence of distinct clinical-pathological phenotypes. Inoculation in laboratory rodents of brain tissues from the six possible combinations of pathological prion protein types with codon 129 genotypes results in the identification of 3 or 4 strains of prions. We report on the identification of a novel strain of Creutzfeldt-Jakob disease isolated from a patient who carried an abnormally glycosylated pathological prion protein. This novel strain has unique biochemical characteristics, does not transmit to humanized transgenic mice, and shows exclusive transmission properties in bank voles. The identification of a novel human prion strain improves our understanding of the pathogenesis of the disease and of possible mechanisms of prion transmission. Copyright © 2017 American Society for Microbiology.

Implications of prion adaptation and evolution paradigm for human neurodegenerative diseases.

PubMed

Kabir, M Enamul; Safar, Jiri G

2014-01-01

There is a growing body of evidence indicating that number of human neurodegenerative diseases, including Alzheimer disease, Parkinson disease, fronto-temporal dementias, and amyotrophic lateral sclerosis, propagate in the brain via prion-like intercellular induction of protein misfolding. Prions cause lethal neurodegenerative diseases in humans, the most prevalent being sporadic Creutzfeldt-Jakob disease (sCJD); they self-replicate and spread by converting the cellular form of prion protein (PrP(C)) to a misfolded pathogenic conformer (PrP(Sc)). The extensive phenotypic heterogeneity of human prion diseases is determined by polymorphisms in the prion protein gene, and by prion strain-specific conformation of PrP(Sc). Remarkably, even though informative nucleic acid is absent, prions may undergo rapid adaptation and evolution in cloned cells and upon crossing the species barrier. In the course of our investigation of this process, we isolated distinct populations of PrP(Sc) particles that frequently co-exist in sCJD. The human prion particles replicate independently and undergo competitive selection of those with lower initial conformational stability. Exposed to mutant substrate, the winning PrP(Sc) conformers are subject to further evolution by natural selection of the subpopulation with the highest replication rate due to the lowest stability. Thus, the evolution and adaptation of human prions is enabled by a dynamic collection of distinct populations of particles, whose evolution is governed by the selection of progressively less stable, faster replicating PrP(Sc) conformers. This fundamental biological mechanism may explain the drug resistance that some prions gained after exposure to compounds targeting PrP(Sc). Whether the phenotypic heterogeneity of other neurodegenerative diseases caused by protein misfolding is determined by the spectrum of misfolded conformers (strains) remains to be established. However, the prospect that these conformers may evolve and adapt by a prion-like mechanism calls for the reevaluation of therapeutic strategies that target aggregates of misfolded proteins, and argues for new therapeutic approaches that will focus on prior pathogenetic steps.

Chimeric elk/mouse prion proteins in transgenic mice.

PubMed

Tamgüney, Gültekin; Giles, Kurt; Oehler, Abby; Johnson, Natrina L; DeArmond, Stephen J; Prusiner, Stanley B

2013-02-01

Chronic wasting disease (CWD) of deer and elk is a highly communicable neurodegenerative disorder caused by prions. Investigations of CWD are hampered by slow bioassays in transgenic (Tg) mice. Towards the development of Tg mice that will be more susceptible to CWD prions, we created a series of chimeric elk/mouse transgenes that encode the N terminus of elk PrP (ElkPrP) up to residue Y168 and the C terminus of mouse PrP (MoPrP) beyond residue 169 (mouse numbering), designated Elk3M(SNIVVK). Between codons 169 and 219, six residues distinguish ElkPrP from MoPrP: N169S, T173N, V183I, I202V, I214V and R219K. Using chimeric elk/mouse PrP constructs, we generated 12 Tg mouse lines and determined incubation times after intracerebral inoculation with the mouse-passaged RML scrapie or Elk1P CWD prions. Unexpectedly, one Tg mouse line expressing Elk3M(SNIVVK) exhibited incubation times of <70 days when inoculated with RML prions; a second line had incubation times of <90 days. In contrast, mice expressing full-length ElkPrP had incubation periods of >250 days for RML prions. Tg(Elk3M,SNIVVK) mice were less susceptible to CWD prions than Tg(ElkPrP) mice. Changing three C-terminal mouse residues (202, 214 and 219) to those of elk doubled the incubation time for mouse RML prions and rendered the mice resistant to Elk1P CWD prions. Mutating an additional two residues from mouse to elk at codons 169 and 173 increased the incubation times for mouse prions to >300 days, but made the mice susceptible to CWD prions. Our findings highlight the role of C-terminal residues in PrP that control the susceptibility and replication of prions.

Action of tremorgenic mycotoxins on GABA/sub A/ receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gant, D.B.; Cole, R.J.; Valdes, J.J.

1987-11-09

The effects of four tremorgenic and one nontremorgenic mycotoxins were studied on ..gamma..-aminobutyric acid (GABA/sub A/) receptor binding and function in rat brain and on binding of a voltage-operated Cl/sup -/ channel in Torpedo electric organ. None of the mycotoxins had significant effect on (/sup 3/H)muscimol or (/sup 3/H)flunitrazepam binding to the GAMA/sup A/ receptor. However, only the four tremorgenic mycotoxins inhibited GABA-induced /sup 36/Cl/sup -/ influx and (/sup 35/S)t-butylbicyclophosphorothionate ((/sup 35/S)TBPS) binding in rate brain membranes, while the nontremorgenic verruculotoxin had no effect. Inhibition of (/sup 35/S)TBPS binding by paspalinine was non-competitive. This suggests that tremorgenic mycotoxins inhibit GABA/submore » A/ receptor function by binding close to the receptor's Cl/sup -/ channel. On the voltage-operated Cl/sup -/ channel, only high concentrations of verruculogen and verruculotoxin caused significant inhibition of the channel's binding of (/sup 35/S)TBPS. The data suggest that the tremorgenic action of these mycotoxins may be due in part to their inhibition of GABA/sub A/ receptor function. 21 references, 4 figures, 2 tables.« less

Melanin or a Melanin-Like Substance Interacts with the N-Terminal Portion of Prion Protein and Inhibits Abnormal Prion Protein Formation in Prion-Infected Cells.

PubMed

Hamanaka, Taichi; Nishizawa, Keiko; Sakasegawa, Yuji; Oguma, Ayumi; Teruya, Kenta; Kurahashi, Hiroshi; Hara, Hideyuki; Sakaguchi, Suehiro; Doh-Ura, Katsumi

2017-03-15

Prion diseases are progressive fatal neurodegenerative illnesses caused by the accumulation of transmissible abnormal prion protein (PrP). To find treatments for prion diseases, we searched for substances from natural resources that inhibit abnormal PrP formation in prion-infected cells. We found that high-molecular-weight components from insect cuticle extracts reduced abnormal PrP levels. The chemical nature of these components was consistent with that of melanin. In fact, synthetic melanin produced from tyrosine or 3-hydroxy-l-tyrosine inhibited abnormal PrP formation. Melanin did not modify cellular or cell surface PrP levels, nor did it modify lipid raft or cellular cholesterol levels. Neither did it enhance autophagy or lysosomal function. Melanin was capable of interacting with PrP at two N-terminal domains. Specifically, it strongly interacted with the PrP region of amino acids 23 to 50 including a positively charged amino acid cluster and weakly interacted with the PrP octarepeat peptide region of residues 51 to 90. However, the in vitro and in vivo data were inconsistent with those of prion-infected cells. Abnormal PrP formation in protein misfolding cyclic amplification was not inhibited by melanin. Survival after prion infection was not significantly altered in albino mice or exogenously melanin-injected mice compared with that of control mice. These data suggest that melanin, a main determinant of skin color, is not likely to modify prion disease pathogenesis, even though racial differences in the incidence of human prion diseases have been reported. Thus, the findings identify an interaction between melanin and the N terminus of PrP, but the pathophysiological roles of the PrP-melanin interaction remain unclear. IMPORTANCE The N-terminal region of PrP is reportedly important for neuroprotection, neurotoxicity, and abnormal PrP formation, as this region is bound by many factors, such as metal ions, lipids, nucleic acids, antiprion compounds, and several proteins, including abnormal PrP in prion disease and the Aβ oligomer in Alzheimer's disease. In the present study, melanin, a main determinant of skin color, was newly found to interact with this N-terminal region and inhibits abnormal PrP formation in prion-infected cells. However, the data for prion infection in mice lacking melanin production suggest that melanin is not associated with the prion disease mechanism, although the incidence of prion disease is reportedly much higher in white people than in black people. Thus, the roles of the PrP-melanin interaction remain to be further elucidated, but melanin might be a useful competitive tool for evaluating the functions of other ligands at the N-terminal region. Copyright © 2017 American Society for Microbiology.

Melanin or a Melanin-Like Substance Interacts with the N-Terminal Portion of Prion Protein and Inhibits Abnormal Prion Protein Formation in Prion-Infected Cells

PubMed Central

Hamanaka, Taichi; Nishizawa, Keiko; Sakasegawa, Yuji; Oguma, Ayumi; Teruya, Kenta; Kurahashi, Hiroshi; Hara, Hideyuki; Sakaguchi, Suehiro

2017-01-01

ABSTRACT Prion diseases are progressive fatal neurodegenerative illnesses caused by the accumulation of transmissible abnormal prion protein (PrP). To find treatments for prion diseases, we searched for substances from natural resources that inhibit abnormal PrP formation in prion-infected cells. We found that high-molecular-weight components from insect cuticle extracts reduced abnormal PrP levels. The chemical nature of these components was consistent with that of melanin. In fact, synthetic melanin produced from tyrosine or 3-hydroxy-l-tyrosine inhibited abnormal PrP formation. Melanin did not modify cellular or cell surface PrP levels, nor did it modify lipid raft or cellular cholesterol levels. Neither did it enhance autophagy or lysosomal function. Melanin was capable of interacting with PrP at two N-terminal domains. Specifically, it strongly interacted with the PrP region of amino acids 23 to 50 including a positively charged amino acid cluster and weakly interacted with the PrP octarepeat peptide region of residues 51 to 90. However, the in vitro and in vivo data were inconsistent with those of prion-infected cells. Abnormal PrP formation in protein misfolding cyclic amplification was not inhibited by melanin. Survival after prion infection was not significantly altered in albino mice or exogenously melanin-injected mice compared with that of control mice. These data suggest that melanin, a main determinant of skin color, is not likely to modify prion disease pathogenesis, even though racial differences in the incidence of human prion diseases have been reported. Thus, the findings identify an interaction between melanin and the N terminus of PrP, but the pathophysiological roles of the PrP-melanin interaction remain unclear. IMPORTANCE The N-terminal region of PrP is reportedly important for neuroprotection, neurotoxicity, and abnormal PrP formation, as this region is bound by many factors, such as metal ions, lipids, nucleic acids, antiprion compounds, and several proteins, including abnormal PrP in prion disease and the Aβ oligomer in Alzheimer's disease. In the present study, melanin, a main determinant of skin color, was newly found to interact with this N-terminal region and inhibits abnormal PrP formation in prion-infected cells. However, the data for prion infection in mice lacking melanin production suggest that melanin is not associated with the prion disease mechanism, although the incidence of prion disease is reportedly much higher in white people than in black people. Thus, the roles of the PrP-melanin interaction remain to be further elucidated, but melanin might be a useful competitive tool for evaluating the functions of other ligands at the N-terminal region. PMID:28077650

Photodissociation spectroscopy of the dysprosium monochloride molecular ion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dunning, Alexander, E-mail: alexander.dunning@gmail.com; Schowalter, Steven J.; Puri, Prateek

2015-09-28

We have performed a combined experimental and theoretical study of the photodissociation cross section of the molecular ion DyCl{sup +}. The photodissociation cross section for the photon energy range 35 500 cm{sup −1} to 47 500 cm{sup −1} is measured using an integrated ion trap and time-of-flight mass spectrometer; we observe a broad, asymmetric profile that is peaked near 43 000 cm{sup −1}. The theoretical cross section is determined from electronic potentials and transition dipole moments calculated using the relativistic configuration-interaction valence-bond and coupled-cluster methods. The electronic structure of DyCl{sup +} is extremely complex due to the presence of multiple open electronic shells,more » including the 4f{sup 10} configuration. The molecule has nine attractive potentials with ionically bonded electrons and 99 repulsive potentials dissociating to a ground state Dy{sup +} ion and Cl atom. We explain the lack of symmetry in the cross section as due to multiple contributions from one-electron-dominated transitions between the vibrational ground state and several resolved repulsive excited states.« less

Detecting and discriminating among pathogenic protein conformers(prions), using mass spectrometry-based and antibody-based approaches(Abstract)

USDA-ARS?s Scientific Manuscript database

A set of fatal neurological diseases that includes scrapie and chronic wasting disease (CWD) are caused by a pathological protein referred to as a prion (PrPSc). A prion propagates an infection by converting a normal cellular protein (PrPC) into a prion. Unlike viral, bacterial, or fungal pathogens,...

The Transcription Terminator Rho: A First Bacterial Prion.

PubMed

Pallarès, Irantzu; Ventura, Salvador

2017-06-01

Traditionally associated with neurodegenerative diseases, prions are increasingly recognized for their potential to confer beneficial traits on eukaryotic organisms. The discovery of the first bacterial prion suggests that the sustained mechanism of prion assembly is an ancient molecular tool aimed at providing fast and persistent adaptation to changing environments. Copyright © 2017 Elsevier Ltd. All rights reserved.

Ophthalmic Surgery in Prion Diseases

PubMed Central

Hamaguchi, Tsuyoshi; Noguchi-Shinohara, Moeko; Nakamura, Yosikazu; Sato, Takeshi; Kitamoto, Tetsuyuki; Mizusawa, Hidehiro

2007-01-01

Eleven (1.8%) of 597 patients underwent ophthalmic surgery within 1 month before the onset of prion disease or after the onset. All ophthalmologists reused surgical instruments that had been incompletely sterilized to eliminate infectious prion protein. Ophthalmologists should be aware of prion diseases as a possible cause of visual symptoms and use disposable instruments whenever possible. PMID:17370537

Ovine leukocyte profiles do not associate with variation in the prion gene, but are breed-dependent

USDA-ARS?s Scientific Manuscript database

Prion genotype in sheep confer resistance to scrapie. In cattle, lymphocyte profile has been found to be associated with prion genotype. Therefore, the aim of this study was to determine if variations in the sheep prion gene were associated with leukocyte populations as measured by complete blood ce...

[A review of the current research on prions. The evidence suggests the possibility of transmission of the mad cow disease to humans].

PubMed

Grandien, M; Wahren, B

1998-11-25

Further evidence of the transmissibility of bovine spongiform encephalopathy (BSE) across the species barrier from cow to man has been derived from epidemiological analysis and the characterisation of prion strains. Recent research has shown the persistence of prions after experimental transmission to resistant murine species, and subclinical persistence in cows. The accumulation of pathological prion proteins in tonsils and appendix has been demonstrated prior to clinical confirmation of the presence of the new variant of Creutzfeldt-Jakob disease. Current prion research is focused on the involvement of B lymphocytes as carriers, on the species barrier and cellular receptors, and on macromolecules involved in the conformational change from normal to pathological prion proteins.

Chronic wasting disease prion infection of differentiated neurospheres.

PubMed

Iwamaru, Yoshifumi; Mathiason, Candace K; Telling, Glenn C; Hoover, Edward A

2017-07-04

A possible strategy to develop more diverse cell culture systems permissive to infection with naturally occurring prions is to exploit culture of neurospheres from transgenic mice expressing the normal prion protein (PrP) of the native host species. Accordingly, we developed differentiated neurosphere cultures from the cervid PrP-expressing mice to investigate whether this in vitro system would support replication of non-adapted cervid-origin chronic wasting disease (CWD) prions. Here we report the successful amplification of disease-associated PrP in differentiated neurosphere cultures within 3 weeks after exposure to CWD prions from both white-tailed deer or elk. This neurosphere culture system provides a new in vitro tool that can be used to assess non-adapted CWD prion propagation and transmission.

Practical auxiliary basis implementation of Rung 3.5 functionals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Janesko, Benjamin G., E-mail: b.janesko@tcu.edu; Scalmani, Giovanni; Frisch, Michael J.

2014-07-21

Approximate exchange-correlation functionals for Kohn-Sham density functional theory often benefit from incorporating exact exchange. Exact exchange is constructed from the noninteracting reference system's nonlocal one-particle density matrix γ(r{sup -vector},r{sup -vector}′). Rung 3.5 functionals attempt to balance the strengths and limitations of exact exchange using a new ingredient, a projection of γ(r{sup -vector},r{sup -vector} ′) onto a semilocal model density matrix γ{sub SL}(ρ(r{sup -vector}),∇ρ(r{sup -vector}),r{sup -vector}−r{sup -vector} ′). γ{sub SL} depends on the electron density ρ(r{sup -vector}) at reference point r{sup -vector}, and is closely related to semilocal model exchange holes. We present a practical implementation of Rung 3.5 functionals, expandingmore » the r{sup -vector}−r{sup -vector} ′ dependence of γ{sub SL} in an auxiliary basis set. Energies and energy derivatives are obtained from 3D numerical integration as in standard semilocal functionals. We also present numerical tests of a range of properties, including molecular thermochemistry and kinetics, geometries and vibrational frequencies, and bandgaps and excitation energies. Rung 3.5 functionals typically provide accuracy intermediate between semilocal and hybrid approximations. Nonlocal potential contributions from γ{sub SL} yield interesting successes and failures for band structures and excitation energies. The results enable and motivate continued exploration of Rung 3.5 functional forms.« less

Signal Transduction by a Fungal NOD-Like Receptor Based on Propagation of a Prion Amyloid Fold

PubMed Central

Daskalov, Asen; Habenstein, Birgit; Martinez, Denis; Debets, Alfons J. M.; Sabaté, Raimon; Loquet, Antoine; Saupe, Sven J.

2015-01-01

In the fungus Podospora anserina, the [Het-s] prion induces programmed cell death by activating the HET-S pore-forming protein. The HET-s β-solenoid prion fold serves as a template for converting the HET-S prion-forming domain into the same fold. This conversion, in turn, activates the HET-S pore-forming domain. The gene immediately adjacent to het-S encodes NWD2, a Nod-like receptor (NLR) with an N-terminal motif similar to the elementary repeat unit of the β-solenoid fold. NLRs are immune receptors controlling cell death and host defense processes in animals, plants and fungi. We have proposed that, analogously to [Het-s], NWD2 can activate the HET-S pore-forming protein by converting its prion-forming region into the β-solenoid fold. Here, we analyze the ability of NWD2 to induce formation of the β-solenoid prion fold. We show that artificial NWD2 variants induce formation of the [Het-s] prion, specifically in presence of their cognate ligands. The N-terminal motif is responsible for this prion induction, and mutations predicted to affect the β-solenoid fold abolish templating activity. In vitro, the N-terminal motif assembles into infectious prion amyloids that display a structure resembling the β-solenoid fold. In vivo, the assembled form of the NWD2 N-terminal region activates the HET-S pore-forming protein. This study documenting the role of the β-solenoid fold in fungal NLR function further highlights the general importance of amyloid and prion-like signaling in immunity-related cell fate pathways. PMID:25671553

Prion Disease Induces Alzheimer Disease-Like Neuropathologic Changes

PubMed Central

Tousseyn, Thomas; Bajsarowicz, Krystyna; Sánchez, Henry; Gheyara, Ania; Oehler, Abby; Geschwind, Michael; DeArmond, Bernadette; DeArmond, Stephen J.

2016-01-01

We examined the brains of 266 patients with prion diseases (PrionD) and found that 46 (17%) had Alzheimer disease (AD)-like changes. To explore potential mechanistic links between PrionD and AD, we exposed human brain aggregates (Hu BrnAggs) to brain homogenate from a patient with sporadic Creutzfeldt-Jakob disease (CJD) and found that the neurons in the Hu BrnAggs produced many β-amyloid (β42) inclusions, whereas uninfected, control-exposed Hu BrnAggs did not. Western blots of 20-pooled CJD-infected BrnAggs verified higher Aβ42 levels than controls. We next examined the CA1 region of the hippocampus from 14 patients with PrionD and found that 5 patients had low levels of scrapie-associated prion protein (PrPSc), many Aβ42 intraneuronal inclusions, low APOE-4, and no significant nerve cell loss. Seven patients had high levels of PrPSc, low Aβ42, high APOE-4 and 40% nerve cell loss, suggesting that APOE-4 and PrPSc together cause neuron loss in PrionD. There were also increased levels of hyperphosphorylated tau protein (Hτ) and Hτ-positive neuropil threads and neuron bodies in both PrionD and AD groups. The brains of 6 age-matched control patients without dementia did not contain Aβ42 deposits; however, there were rare Hτ-positive threads in 5 controls and 2 controls had a few Hτ-positive nerve cell bodies. We conclude that PrionD may trigger biochemical changes similar to AD and suggest that PrionD are diseases of PrPSc, Aβ42, APOE-4 and abnormal tau. PMID:26226132

Adsorption mechanisms of selenium oxyanions at the aluminum oxide/water interface

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peak, Derek

2008-06-09

Sorption processes at the mineral/water interface typically control the mobility and bioaccessibility of many inorganic contaminants such as oxyanions. Selenium is an important micronutrient for human and animal health, but at elevated concentrations selenium toxicity is a concern. The objective of this study was to determine the bonding mechanisms of selenate (SeO{sub 4}{sup 2-}) and selenite (SeO{sub 3}{sup 2-}) on hydrous aluminum oxide (HAO) over a wide range of reaction pH using extended X-ray absorption fine structure (EXAFS) spectroscopy. Additionally, selenate adsorption on corundum ({alpha}-Al{sub 2}O{sub 3}) was studied to determine if adsorption mechanisms change as the aluminum oxide surfacemore » structure changes. The overall findings were that selenite forms a mixture of outer-sphere and inner-sphere bidentate-binuclear (corner-sharing) surface complexes on HAO, selenate forms primarily outer-sphere surface complexes on HAO, and on corundum selenate forms outer-sphere surface complexes at pH 3.5 but inner-sphere monodentate surface complexes at pH 4.5 and above. It is possible that the lack of inner-sphere complex formation at pH 3.5 is caused by changes in the corundum surface at low pH or secondary precipitate formation. The results are consistent with a structure-based reactivity for metal oxides, wherein hydrous metal oxides form outer-sphere complexes with sulfate and selenate, but inner-sphere monodentate surface complexes are formed between sulfate and selenate and {alpha}-Me{sub 2}O{sub 3}.« less

Production of Prnp-/- goats by gene targeting in adult fibroblasts.

PubMed

Zhu, Caihong; Li, Bei; Yu, Guohua; Chen, Jianquan; Yu, Huiqing; Chen, Juan; Xu, Xujun; Wu, Youbing; Zhang, Aimin; Cheng, Guoxiang

2009-04-01

Homozygous mice devoid of functional Prnp are resistant to scrapie and prion propagation, but heterozygous mice for Prnp disruption still suffer from prion disease and prion deposition. We have previously generated heterozygous cloned goats with one allele of Prnp functional disruption. To obtain goats with both alleles of Prnp be disrupted which would be resistant to scrapie completely, a second-round gene targeting was applied to disrupt the wild type allele of Prnp in the heterozygous goats. By second-round gene targeting, we successfully disrupted the wild type allele of Prnp in primary Prnp (+/-) goat skin fibroblasts and obtained a Prnp (-/-) cell line without Prnp expression. This is the first report on successful targeting modification in primary adult somatic cells of animals. These cells were used as nuclear donors for somatic cell cloning to produce Prnp (-/-) goats. A total of 57 morulae or blastocytes developed from the reconstructed embryos were transferred to 31 recipients, which produced 7 pregnancies at day 35. At 73 days of gestation, we obtained one cloned fetus with Prnp (-/-) genotype. Our research not only indicated that multiple genetic modifications could be accomplished by multi-round gene targeting in primary somatic cells, but also provided strong evidence that gene targeting in adult cells other than fetal cells could be applied to introduce precise genetic modifications in animals without destroying the embryos.

Human Tonsil-Derived Follicular Dendritic-Like Cells are Refractory to Human Prion Infection in Vitro and Traffic Disease-Associated Prion Protein to Lysosomes

PubMed Central

Krejciova, Zuzana; De Sousa, Paul; Manson, Jean; Ironside, James W.; Head, Mark W.

2014-01-01

The molecular mechanisms involved in human cellular susceptibility to prion infection remain poorly defined. This is due, in part, to the absence of any well characterized and relevant cultured human cells susceptible to infection with human prions, such as those involved in Creutzfeldt-Jakob disease. In variant Creutzfeldt-Jakob disease, prion replication is thought to occur first in the lymphoreticular system and then spread into the brain. We have, therefore, examined the susceptibility of a human tonsil-derived follicular dendritic cell-like cell line (HK) to prion infection. HK cells were found to display a readily detectable, time-dependent increase in cell-associated abnormal prion protein (PrPTSE) when exposed to medium spiked with Creutzfeldt-Jakob disease brain homogenate, resulting in a coarse granular perinuclear PrPTSE staining pattern. Despite their high level of cellular prion protein expression, HK cells failed to support infection, as judged by longer term maintenance of PrPTSE accumulation. Colocalization studies revealed that exposure of HK cells to brain homogenate resulted in increased numbers of detectable lysosomes and that these structures immunostained intensely for PrPTSE after exposure to Creutzfeldt-Jakob disease brain homogenate. Our data suggest that human follicular dendritic-like cells and perhaps other human cell types are able to avoid prion infection by efficient lysosomal degradation of PrPTSE. PMID:24183781

Tau prions from Alzheimer’s disease and chronic traumatic encephalopathy patients propagate in cultured cells

PubMed Central

Woerman, Amanda L.; Aoyagi, Atsushi; Patel, Smita; Kazmi, Sabeen A.; Lobach, Iryna; Grinberg, Lea T.; McKee, Ann C.; Seeley, William W.; Olson, Steven H.; Prusiner, Stanley B.

2016-01-01

Tau prions are thought to aggregate in the central nervous system, resulting in neurodegeneration. Among the tauopathies, Alzheimer’s disease (AD) is the most common, whereas argyrophilic grain disease (AGD), corticobasal degeneration (CBD), chronic traumatic encephalopathy (CTE), Pick’s disease (PiD), and progressive supranuclear palsy (PSP) are less prevalent. Brain extracts from deceased individuals with PiD, a neurodegenerative disorder characterized by three-repeat (3R) tau prions, were used to infect HEK293T cells expressing 3R tau fused to yellow fluorescent protein (YFP). Extracts from AGD, CBD, and PSP patient samples, which contain four-repeat (4R) tau prions, were transmitted to HEK293 cells expressing 4R tau fused to YFP. These studies demonstrated that prion propagation in HEK cells requires isoform pairing between the infecting prion and the recipient substrate. Interestingly, tau aggregates in AD and CTE, containing both 3R and 4R isoforms, were unable to robustly infect either 3R- or 4R-expressing cells. However, AD and CTE prions were able to replicate in HEK293T cells expressing both 3R and 4R tau. Unexpectedly, increasing the level of 4R isoform expression alone supported the propagation of both AD and CTE prions. These results allowed us to determine the levels of tau prions in AD and CTE brain extracts. PMID:27911827

Generic amyloidogenicity of mammalian prion proteins from species susceptible and resistant to prions.

PubMed

Nyström, Sofie; Hammarström, Per

2015-05-11

Prion diseases are lethal, infectious diseases associated with prion protein (PrP) misfolding. A large number of mammals are susceptible to both sporadic and acquired prion diseases. Although PrP is highly conserved and ubiquitously expressed in all mammals, not all species exhibit prion disease. By employing full length recombinant PrP from five known prion susceptible species (human, cattle, cat, mouse and hamster) and two species considered to be prion resistant (pig and dog) the amyloidogenicity of these PrPs has been delineated. All the mammalian PrPs, even from resistant species, were swiftly converted from the native state to amyloid-like structure when subjected to a native condition conversion assay. The PrPs displayed amyloidotypic tinctorial and ultrastructural hallmarks. Self-seeded conversion of the PrPs displayed significantly decreased lag phases demonstrating that nucleation dependent polymerization is a dominating mechanism in the fibrillation process. Fibrils from Aβ1-40, Aβ1-42, Lysozyme, Insulin and Transthyretin did not accelerate conversion of HuPrP whereas fibrils from HuPrP90-231 and HuPrP121-231 as well as full length PrPs of all PrPs efficiently seeded conversion showing specificity of the assay requiring the C-terminal PrP sequence. Our findings have implications for PrP misfolding and could have ramifications in the context of prion resistant species and silent carriers.

The non-octarepeat copper binding site of the prion protein is a key regulator of prion conversion

NASA Astrophysics Data System (ADS)

Giachin, Gabriele; Mai, Phuong Thao; Tran, Thanh Hoa; Salzano, Giulia; Benetti, Federico; Migliorati, Valentina; Arcovito, Alessandro; Longa, Stefano Della; Mancini, Giordano; D'Angelo, Paola; Legname, Giuseppe

2015-10-01

The conversion of the prion protein (PrPC) into prions plays a key role in transmissible spongiform encephalopathies. Despite the importance for pathogenesis, the mechanism of prion formation has escaped detailed characterization due to the insoluble nature of prions. PrPC interacts with copper through octarepeat and non-octarepeat binding sites. Copper coordination to the non-octarepeat region has garnered interest due to the possibility that this interaction may impact prion conversion. We used X-ray absorption spectroscopy to study copper coordination at pH 5.5 and 7.0 in human PrPC constructs, either wild-type (WT) or carrying pathological mutations. We show that mutations and pH cause modifications of copper coordination in the non-octarepeat region. In the WT at pH 5.5, copper is anchored to His96 and His111, while at pH 7 it is coordinated by His111. Pathological point mutations alter the copper coordination at acidic conditions where the metal is anchored to His111. By using in vitro approaches, cell-based and computational techniques, we propose a model whereby PrPC coordinating copper with one His in the non-octarepeat region converts to prions at acidic condition. Thus, the non-octarepeat region may act as the long-sought-after prion switch, critical for disease onset and propagation.

Transmissibility of Gerstmann-Sträussler-Scheinker syndrome in rodent models: New insights into the molecular underpinnings of prion infectivity.

PubMed

Nonno, Romolo; Angelo Di Bari, Michele; Agrimi, Umberto; Pirisinu, Laura

2016-11-01

Prion diseases, or transmissible spongiform encephalopathies, have revealed the bewildering phenomenon of transmissibility in neurodegenerative diseases. Hence, the experimental transmissibility of prion-like neurodegenerative diseases via template directed misfolding has become the focus of intense research. Gerstmann-Sträussler-Scheinker disease (GSS) is an inherited prion disease associated with mutations in the prion protein gene. However, with the exception of a few GSS cases with P102L mutation characterized by co-accumulation of protease-resistant PrP core (PrP res ) of ∼21 kDa, attempts to transmit to rodents GSS associated to atypical misfolded prion protein with ∼8 kDa PrP res have been unsuccessful. As a result, these GSS subtypes have often been considered as non-transmissible proteinopathies rather than true prion diseases. In a recent study we inoculated bank voles with GSS cases associated with P102L, A117V and F198S mutations and found that they transmitted efficiently and produced distinct pathological phenotypes, irrespective of the presence of 21 kDa PrP res in the inoculum. This study demonstrates that GSS is a genuine prion disease characterized by both transmissibility and strain variation. We discuss the implications of these findings for the understanding of the heterogeneous clinic-pathological phenotypes of GSS and of the molecular underpinnings of prion infectivity.

Regulation of MicroRNAs-Mediated Autophagic Flux: A New Regulatory Avenue for Neurodegenerative Diseases With Focus on Prion Diseases

PubMed Central

Shah, Syed Zahid Ali; Zhao, Deming; Hussain, Tariq; Sabir, Naveed; Yang, Lifeng

2018-01-01

Prion diseases are fatal neurological disorders affecting various mammalian species including humans. Lack of proper diagnostic tools and non-availability of therapeutic remedies are hindering the control strategies for prion diseases. MicroRNAs (miRNAs) are abundant endogenous short non-coding essential RNA molecules that negatively regulate the target genes after transcription. Several biological processes depend on miRNAs, and altered profiles of these miRNAs are potential biomarkers for various neurodegenerative diseases, including prion diseases. Autophagic flux degrades the misfolded prion proteins to reduce chronic endoplasmic reticulum stress and enhance cell survival. Recent evidence suggests that specific miRNAs target and regulate the autophagic mechanism, which is critical for alleviating cellular stress. miRNAs-mediated regulation of these specific proteins involved in the autophagy represents a new target with highly significant therapeutic prospects. Here, we will briefly describe the biology of miRNAs, the use of miRNAs as potential biomarkers with their credibility, the regulatory mechanism of miRNAs in major neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and prion diseases, degradation pathways for aggregated prion proteins, the role of autophagy in prion diseases. Finally, we will discuss the miRNAs-modulated autophagic flux in neurodegenerative diseases and employ them as potential therapeutic intervention strategy in prion diseases. PMID:29867448

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

USDA-ARS?s Scientific Manuscript database

Prions are amyloid-forming proteins that cause transmissible spongiform encephalopathies through a process involving conversion from normal cellular prion protein to pathogenic misfolded conformation. This conversion has been used for in vitro assays including serial protein misfolding amplification...

Sensitive and specific detection of classical scrapie prions in the brain of goats by real-time quaking-induced conversion

USDA-ARS?s Scientific Manuscript database

The real-time quaking-induced conversion (RT-QuIC) is a rapid, specific, and sensitive prion seeding activity detection assay that uses recombinant prion protein (rPrPSen) to detect sub-infectious levels of the abnormal isoforms of the prion protein (PrPSc). Although RT-QuIC has been successfully us...

Mammalian prions

PubMed Central

Salamat, Muhammad Khalid; Munoz-Montesino, Carola; Moudjou, Mohammed; Rezaei, Human; Laude, Hubert; Béringue, Vincent; Dron, Michel

2013-01-01

Upon prion infection, abnormal prion protein (PrPSc) self-perpetuate by conformational conversion of α-helix-rich PrPC into β sheet enriched form, leading to formation and deposition of PrPSc aggregates in affected brains. However the process remains poorly understood at the molecular level and the regions of PrP critical for conversion are still debated. Minimal amino acid substitutions can impair prion replication at many places in PrP. Conversely, we recently showed that bona fide prions could be generated after introduction of eight and up to 16 additional amino acids in the H2-H3 inter-helix loop of PrP. Prion replication also accommodated the insertions of an octapeptide at different places in the last turns of H2. This reverse genetic approach reveals an unexpected tolerance of prions to substantial sequence changes in the protease-resistant part which is associated with infectivity. It also demonstrates that conversion does not require the presence of a specific sequence in the middle of the H2-H3 area. We discuss the implications of our findings according to different structural models proposed for PrPSc and questioned the postulated existence of an N- or C-terminal prion domain in the protease-resistant region. PMID:23232499

Sex effects in mouse prion disease incubation time.

PubMed

Akhtar, Shaheen; Wenborn, Adam; Brandner, Sebastian; Collinge, John; Lloyd, Sarah E

2011-01-01

Prion disease incubation time in mice is determined by many factors including PrP expression level, Prnp alleles, genetic background, prion strain and route of inoculation. Sex differences have been described in age of onset for vCJD and in disease duration for both vCJD and sporadic CJD and have also been shown in experimental models. The sex effects reported for mouse incubation times are often contradictory and detail only one strain of mice or prions, resulting in broad generalisations and a confusing picture. To clarify the effect of sex on prion disease incubation time in mice we have compared male and female transmission data from twelve different inbred lines of mice inoculated with at least two prion strains, representing both mouse-adapted scrapie and BSE. Our data show that sex can have a highly significant difference on incubation time. However, this is limited to particular mouse and prion strain combinations. No sex differences were seen in endogenous PrP(C) levels nor in the neuropathological markers of prion disease: PrP(Sc) distribution, spongiosis, neuronal loss and gliosis. These data suggest that when comparing incubation times between experimental groups, such as testing the effects of modifier genes or therapeutics, single sex groups should be used.

Prion diseases and adult neurogenesis: how do prions counteract the brain's endogenous repair machinery?

PubMed

Relaño-Ginés, Aroa; Lehmann, Sylvain; Crozet, Carole

2014-01-01

Scientific advances in stem cell biology and adult neurogenesis have raised the hope that neurodegenerative disorders could benefit from stem cell-based therapy. Adult neurogenesis might be part of the physiological regenerative process, however it might become impaired by the disease's mechanism and therefore contribute to neurodegeneration. In prion disorders this endogenous repair system has rarely been studied. Whether adult neurogenesis plays a role or not in brain repair or in the propagation of prion pathology remains unclear. We have recently investigated the status of adult neural stem cells isolated from prion-infected mice. We were able to show that neural stem cells accumulate and replicate prions thus resulting in an alteration of their neuronal destiny. We also reproduced these results in adult neural stem cells, which were infected in vitro. The fact that endogenous adult neurogenesis could be altered by the accumulation of misfolded prion protein represents another great challenge. Inhibiting prion propagation in these cells would thus help the endogenous neurogenesis to compensate for the injured neuronal system. Moreover, understanding the endogenous modulation of the neurogenesis system would help develop effective neural stem cell-based therapies.

Discovery of small molecules binding to the normal conformation of prion by combining virtual screening and multiple biological activity evaluation methods

NASA Astrophysics Data System (ADS)

Li, Lanlan; Wei, Wei; Jia, Wen-Juan; Zhu, Yongchang; Zhang, Yan; Chen, Jiang-Huai; Tian, Jiaqi; Liu, Huanxiang; He, Yong-Xing; Yao, Xiaojun

2017-12-01

Conformational conversion of the normal cellular prion protein, PrPC, into the misfolded isoform, PrPSc, is considered to be a central event in the development of fatal neurodegenerative diseases. Stabilization of prion protein at the normal cellular form (PrPC) with small molecules is a rational and efficient strategy for treatment of prion related diseases. However, few compounds have been identified as potent prion inhibitors by binding to the normal conformation of prion. In this work, to rational screening of inhibitors capable of stabilizing cellular form of prion protein, multiple approaches combining docking-based virtual screening, steady-state fluorescence quenching, surface plasmon resonance and thioflavin T fluorescence assay were used to discover new compounds interrupting PrPC to PrPSc conversion. Compound 3253-0207 that can bind to PrPC with micromolar affinity and inhibit prion fibrillation was identified from small molecule databases. Molecular dynamics simulation indicated that compound 3253-0207 can bind to the hotspot residues in the binding pocket composed by β1, β2 and α2, which are significant structure moieties in conversion from PrPC to PrPSc.

Decontamination of prions in a plasma product manufacturing environment.

PubMed

Bellon, Anne; Comoy, Emmanuel; Simoneau, Steve; Mornac, Sandrine; Dehen, Capucine; Perrin, Audrey; Arzel, Aude; Arrabal, Samuel; Baron, Henry; Laude, Hubert; You, Bruno; Deslys, Jean-Philippe; Flan, Benoit

2014-04-01

The high resistance of prions to inactivating treatments requires the proper management of decontaminating procedures of equipment in contact with materials of human or animal origin destined for medical purposes. Sodium hydroxide (NaOH) is widely used today for this purpose as it inactivates a wide variety of pathogens including prions. Several NaOH treatments were tested on prions bound to either stainless steel or chromatographic resins in industrial conditions with multiple prion strains. Data show a strong correlation between inactivation results obtained by immunochemical detection of the prion protein and those obtained with infectivity assays and establish effective inactivation treatments for prions bound to stainless steel or chromatographic resins (ion exchange and affinity), including treatments with lower NaOH concentrations. Furthermore, no obvious strain-specific behavior difference was observed between experimental models. The results generated by these investigations show that industrial NaOH decontamination regimens (in combination with the NaCl elution in the case of the chromatography process) attain substantial prion inactivation and/or removal between batches, thus providing added assurance to the biologic safety of the final plasma-derived medicinal products. © 2013 American Association of Blood Banks.

Correlation analysis for the incubation period of prion disease

PubMed Central

Bae, Se-Eun; Jung, Sunghoon; Kim, Ha-Yeon; Son, Hyeon S.

2012-01-01

Previous studies have shown that genetic quantitative trait loci (QTL), strain barriers, inoculation dose and inoculation method modulate the incubation period of prion diseases. We examined the relationship between a diverse set of physical, genetic and immunological characteristics and the incubation period of prion disease using correlation analyses. We found that incubation period was highly correlated with brain weight. In addition, mean corpuscular volume and cell size were strongly correlated with incubation period, indicating that the physical magnitude of prion-infected organs or individual cells may be important in determining the incubation period. Given the same prion inoculation dose, animals with a lower brain weight, mean corpuscular volume or cell size may experience more virulent disease, as the effective concentration of abnormal prion, which might regulate the attachment rate of prions to aggregates, is increased with smaller capacity of brains and cells. This is partly consistent with previous theoretical modeling. The strong correlations between incubation period and physical properties of the brain and cells in this study suggest that the mechanism underlying prion disease pathology may be physical, indicating that the incubation process is governed by simple chemical stoichiometry. PMID:22561168

Alteration of the endocannabinoid system in mouse brain during prion disease.

PubMed

Petrosino, S; Ménard, B; Zsürger, N; Di Marzo, V; Chabry, J

2011-03-17

Prion diseases are neurodegenerative disorders characterized by deposition of the pathological prion protein (PrPsc) within the brain of affected humans and animals. Microglial cell activation is a common feature of prion diseases; alterations of various neurotransmitter systems and neurotransmission have been also reported. Owing to its ability to modulate both neuroimmune responses and neurotransmission, it was of interest to study the brain endocannabinoid system in a prion-infected mouse model. The production of the endocannabinoid, 2-arachidonoyglycerol (2-AG), was enhanced 10 weeks post-infection, without alteration of the other endocannabinoid, anandamide. The CB2 receptor expression was up-regulated in brains of prion-infected mice as early as 10 weeks and up to 32 weeks post-infection whereas the mRNAs of other cannabinoid receptors (CBRs) remain unchanged. The observed alterations of the endocannabinoid system were specific for prion infection since no significant changes were observed in the brain of prion-resistant mice, that is, mice devoid of the Prnp gene. Our study highlights important alterations of the endocannabinoid system during early stages of the disease long before the clinical signs of the disease. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Disease Transmission by Misfolded Prion-Protein Isoforms, Prion-Like Amyloids, Functional Amyloids and the Central Dogma.

PubMed

Daus, Martin L

2016-01-04

In 1982, the term "prions" (proteinaceous infectious particles) was coined to specify a new principle of infection. A misfolded isoform of a cellular protein has been described as the causative agent of a fatal neurodegenerative disease. At the beginning of prion research scientists assumed that the infectious agent causing transmissible spongiform encephalopathy (TSE) was a virus, but some unconventional properties of these pathogens were difficult to bring in line with the prevailing viral model. The discovery that prions (obviously devoid of any coding nucleic acid) can store and transmit information similarly to DNA was initially even denoted as being "heretical" but is nowadays mainly accepted by the scientific community. This review describes, from a historical point of view, how the "protein-only hypothesis" expands the Central Dogma. Definition of both, the prion principle and the Central Dogma, have been essential steps to understand information storage and transfer within and among cells and organisms. Furthermore, the current understanding of the infectivity of prion-proteins after misfolding is summarized succinctly. Finally, prion-like amyloids and functional amyloids, as found in yeast and bacteria, will be discussed.

Divergent prion strain evolution driven by PrPC expression level in transgenic mice

PubMed Central

Le Dur, Annick; Laï, Thanh Lan; Stinnakre, Marie-George; Laisné, Aude; Chenais, Nathalie; Rakotobe, Sabine; Passet, Bruno; Reine, Fabienne; Soulier, Solange; Herzog, Laetitia; Tilly, Gaëlle; Rézaei, Human; Béringue, Vincent; Vilotte, Jean-Luc; Laude, Hubert

2017-01-01

Prions induce a fatal neurodegenerative disease in infected host brain based on the refolding and aggregation of the host-encoded prion protein PrPC into PrPSc. Structurally distinct PrPSc conformers can give rise to multiple prion strains. Constrained interactions between PrPC and different PrPSc strains can in turn lead to certain PrPSc (sub)populations being selected for cross-species transmission, or even produce mutation-like events. By contrast, prion strains are generally conserved when transmitted within the same species, or to transgenic mice expressing homologous PrPC. Here, we compare the strain properties of a representative sheep scrapie isolate transmitted to a panel of transgenic mouse lines expressing varying levels of homologous PrPC. While breeding true in mice expressing PrPC at near physiological levels, scrapie prions evolve consistently towards different strain components in mice beyond a certain threshold of PrPC overexpression. Our results support the view that PrPC gene dosage can influence prion evolution on homotypic transmission. PMID:28112164

Potential approaches for heterologous prion protein treatment of prion diseases

PubMed Central

Seelig, Davis M.; Goodman, Patricia A.; Skinner, Pamela J.

2016-01-01

ABSTRACT Prion diseases, or transmissible spongiform encephalopathies (TSEs) are progressive, fatal neurodegenerative diseases with no effective treatment. The pathology of these diseases involves the conversion of a protease sensitive form of the cellular prion protein (PrPC) into a protease resistant infectious form (PrPres). The efficiency of this conversion is predicated upon a number of factors, most notably a strong homology between cellular PrPC and PrPres. In our recently published study, we infected mice with the RML-Chandler strain of scrapie and treated them with heterologous hamster prion proteins. This treatment was seen to reduce clinical signs of prion disease, to delay the onset of clinical symptoms and to prolong survival. In this current article we discuss potential mechanisms of action of treatment with heterologous prion proteins. We also discuss potential extensions of these studies using a heterologous rabbit PrP-based treatment strategy or a peptide based strategy, and improvement of treatment delivery including a lentiviral-based system. PMID:26636482

Discovery of Novel Anti-prion Compounds Using In Silico and In Vitro Approaches

PubMed Central

Hyeon, Jae Wook; Choi, Jiwon; Kim, Su Yeon; Govindaraj, Rajiv Gandhi; Jam Hwang, Kyu; Lee, Yeong Seon; An, Seong Soo A.; Lee, Myung Koo; Joung, Jong Young; No, Kyoung Tai; Lee, Jeongmin

2015-01-01

Prion diseases are associated with the conformational conversion of the physiological form of cellular prion protein (PrPC) to the pathogenic form, PrPSc. Compounds that inhibit this process by blocking conversion to the PrPSc could provide useful anti-prion therapies. However, no suitable drugs have been identified to date. To identify novel anti-prion compounds, we developed a combined structure- and ligand-based virtual screening system in silico. Virtual screening of a 700,000-compound database, followed by cluster analysis, identified 37 compounds with strong interactions with essential hotspot PrP residues identified in a previous study of PrPC interaction with a known anti-prion compound (GN8). These compounds were tested in vitro using a multimer detection system, cell-based assays, and surface plasmon resonance. Some compounds effectively reduced PrPSc levels and one of these compounds also showed a high binding affinity for PrPC. These results provide a promising starting point for the development of anti-prion compounds. PMID:26449325

Soil clay content underlies prion infection odds

USGS Publications Warehouse

David, Walter W.; Walsh, D.P.; Farnsworth, Matthew L.; Winkelman, D.L.; Miller, M.W.

2011-01-01

Environmental factors-especially soil properties-have been suggested as potentially important in the transmission of infectious prion diseases. Because binding to montmorillonite (an aluminosilicate clay mineral) or clay-enriched soils had been shown to enhance experimental prion transmissibility, we hypothesized that prion transmission among mule deer might also be enhanced in ranges with relatively high soil clay content. In this study, we report apparent influences of soil clay content on the odds of prion infection in free-ranging deer. Analysis of data from prion-infected deer herds in northern Colorado, USA, revealed that a 1% increase in the clay-sized particle content in soils within the approximate home range of an individual deer increased its odds of infection by up to 8.9%. Our findings suggest that soil clay content and related environmental properties deserve greater attention in assessing risks of prion disease outbreaks and prospects for their control in both natural and production settings. ?? 2011 Macmillan Publishers Limited. All rights reserved.

Crystallographic Studies of Prion Protein (PrP) Segments Suggest How Structural Changes Encoded by Polymorphism at Residue 129 Modulate Susceptibility to Human Prion Disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Apostol, Marcin I.; Sawaya, Michael R.; Cascio, Duilio

2010-09-23

A single nucleotide polymorphism (SNP) in codon 129 of the human prion gene, leading to a change from methionine to valine at residue 129 of prion protein (PrP), has been shown to be a determinant in the susceptibility to prion disease. However, the molecular basis of this effect remains unexplained. In the current study, we determined crystal structures of prion segments having either Met or Val at residue 129. These 6-residue segments of PrP centered on residue 129 are 'steric zippers,' pairs of interacting {beta}-sheets. Both structures of these 'homozygous steric zippers' reveal direct intermolecular interactions between Met or Valmore » in one sheet and the identical residue in the mating sheet. These two structures, plus a structure-based model of the heterozygous Met-Val steric zipper, suggest an explanation for the previously observed effects of this locus on prion disease susceptibility and progression.« less

Identification of Two Prion Protein Regions That Modify Scrapie Incubation Time

PubMed Central

Supattapone, Surachai; Muramoto, Tamaki; Legname, Giuseppe; Mehlhorn, Ingrid; Cohen, Fred E.; DeArmond, Stephen J.; Prusiner, Stanley B.; Scott, Michael R.

2001-01-01

A series of prion transmission experiments was performed in transgenic (Tg) mice expressing either wild-type, chimeric, or truncated prion protein (PrP) molecules. Following inoculation with Rocky Mountain Laboratory (RML) murine prions, scrapie incubation times for Tg(MoPrP)4053, Tg(MHM2)294/Prnp0/0, and Tg(MoPrP,Δ23–88)9949/Prnp0/0 mice were ∼50, 120, and 160 days, respectively. Similar scrapie incubation times were obtained after inoculation of these lines of Tg mice with either MHM2(MHM2(RML)) or MoPrP(Δ23–88)(RML) prions, excluding the possibility that sequence-dependent transmission barriers could account for the observed differences. Tg(MHM2)294/Prnp0/0 mice displayed prolonged scrapie incubation times with four different strains of murine prions. These data provide evidence that the N terminus of MoPrP and the chimeric region of MHM2 PrP (residues 108 through 111) both influence the inherent efficiency of prion propagation. PMID:11152514

Inhibition of GNNQQNY prion peptide aggregation by trehalose: a mechanistic view.

PubMed

Katyal, Nidhi; Deep, Shashank

2017-07-26

Deposition of amyloid fibrils is the seminal event in the pathogenesis of numerous neurodegenerative diseases. The formation of this amyloid assembly is the manifestation of a cascade of structural transitions including toxic oligomer formation in the early stages of aggregation. Thus a viable therapeutic strategy involves the use of small molecular ligands to interfere with this assembly. In this perspective, we have explored the kinetics of aggregate formation of the fibril forming GNNQQNY peptide fragment from the yeast prion protein SUP35 using multiple all atom MD simulations with explicit solvent and provided mechanistic insights into the way trehalose, an experimentally known aggregation inhibitor, modulates the aggregation pathway. The results suggest that the assimilation process is impeded by different barriers at smaller and larger oligomeric sizes: the initial one being easily surpassed at higher temperatures and peptide concentrations. The kinetic profile demonstrates that trehalose delays the aggregation process by increasing both these activation barriers, specifically the latter one. It increases the sampling of small-sized aggregates that lack the beta sheet conformation. Analysis reveals that the barrier in the growth of larger stable oligomers causes the formation of multiple stable small oligomers which then fuse together bimolecularly. The PCA of 26 properties was carried out to deconvolute the events within the temporary lag phases, which suggested dynamism in lags involving an increase in interchain contacts and burial of SASA. The predominant growth route is monomer addition, which changes to condensation on account of a large number of depolymerisation events in the presence of trehalose. The favourable interaction of trehalose specifically with the sidechain of the peptide promotes crowding of trehalose molecules in its vicinity - the combination of both these factors imparts the observed behaviour. Furthermore, increasing trehalose concentration leads to faster expulsion of water molecules than interpeptide interactions. These expelled water molecules have larger translational movement, suggesting an entropy factor to favor the assembly process. Different conformations observed under this condition suggest the role of water molecules in guiding the morphology of the aggregates as well. A similar scenario exists on increasing peptide concentration.

Infectious Prions in the Saliva and Blood of Deer with Chronic Wasting Disease

NASA Astrophysics Data System (ADS)

Mathiason, Candace K.; Powers, Jenny G.; Dahmes, Sallie J.; Osborn, David A.; Miller, Karl V.; Warren, Robert J.; Mason, Gary L.; Hays, Sheila A.; Hayes-Klug, Jeanette; Seelig, Davis M.; Wild, Margaret A.; Wolfe, Lisa L.; Spraker, Terry R.; Miller, Michael W.; Sigurdson, Christina J.; Telling, Glenn C.; Hoover, Edward A.

2006-10-01

A critical concern in the transmission of prion diseases, including chronic wasting disease (CWD) of cervids, is the potential presence of prions in body fluids. To address this issue directly, we exposed cohorts of CWD-naïve deer to saliva, blood, or urine and feces from CWD-positive deer. We found infectious prions capable of transmitting CWD in saliva (by the oral route) and in blood (by transfusion). The results help to explain the facile transmission of CWD among cervids and prompt caution concerning contact with body fluids in prion infections.

Modelling charge interactions in the prion protein: predictions for pathogenesis.

PubMed

Warwicker, J

1999-04-30

Calculations are presented for the pH-dependence of stability and membrane charge complementarity of prion protein fragments. The theoretical results are compared with reported characterisations of prion protein folding in vitro. Discussion of models for conformational change and pathogenesis in vivo leads to the prediction of amino acids that could mediate sensitivity to the endosomal pH and to a design strategy for recombinant prion proteins with an increased susceptibility to prion proteinSc-like properties in vitro. In this model, the protective effect of certain basic polymorphisms can be interpreted in terms of oligomerisation on a negatively-charged surface.

Preliminary results of human PrPC protein studied by spectroscopic techniques

NASA Astrophysics Data System (ADS)

Nowakowski, Michał; Czapla-Masztafiak, Joanna; Kozak, Maciej; Zhukov, Igor; Zhukova, Lilia; Szlachetko, Jakub; Kwiatek, Wojciech M.

2017-11-01

Neurodegenerative diseases are one of the malfunctions of human nervous system, being a class of complex and prominent pathologies. The human prion Protease Resistant Protein (PrP) is protein regulating copper metabolism in mammalian cells through binding of Cu(II) ions to specific fragments. Nowadays misfolding of this protein is associated with development of prion diseases. Therefore, it is crucial to obtain structural information about coordination of Cu(II) by PrP protein. Herein, we report X-ray absorption spectroscopy (XAS) measurements, carried out on SuperXAS beamline (SLS, PSI Villigen) on PrPC-Cu(II) complexes. Obtained results were compared with theoretical predictions done by FEFF 9.6 software. Complementary to XAS data, Atomic Force Microscopy (AFM) measurements were conducted to obtain low resolution structural information about prepared sample that allow to develop protocol of fixing PrPC molecules on solid substrate used for further experiments. It has been established that folded C-terminal domain of PrPC protein has around 5 nm in diameter. Presented results showed that both XAS and AFM methods are useful tools in detailed examination of complexes of human PrPC either with Cu(II) or with other divalent metal ions.

Using mass spectrometry and small molecule reagents to detect distinctive structural features of different prion conformations (strains)

USDA-ARS?s Scientific Manuscript database

A prion (PrPSc) is a conformer of a normal cellular prion protein (PrPC). Although they are isosequential, PrPSc is an infectious protein able to convert PrPC into the prion conformation and thereby propagate an infection. PrPC is monomeric while PrPSc is a multimer. PrPSc can adopt more than one co...

Syntheses of monomeric (. eta. sup 5 -pentamethylcyclopentadienyl)platinum(IV) methyl and bromo complexes and of (hydrotris(3,5-dimethyl-1-pyrazolyl)borato)trimethylplatinum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roth, S.; Ramamoorthy, V.; Sharp, P.R.

1990-09-05

The reaction of Cp*MgCl{center dot}THF (Cp* = C{sub 5}Me{sub 5}) with 1 equiv of PtMe{sub 3}I and PtMe{sub 2}Br{sub 2} produces Cp*PtMe{sub 3} (1) and Cp*PtMe{sub 2}Br (2), respectively. Reaction of 2 with Br{sub 2} produces Cp*PtMeBr{sub 2} (3) in good yield. The structures of 2 and 3 have been determined by x-ray crystallography, and the crystal structure data are reported. Complex 2 crystallizes in the monoclinic space group, P2{sub 1}/m, and complex 3 crystallizes in the monoclinic space group, P2{sub 1}/m. The molecules reside on mirror planes and are monomeric pseudotetrahedral Pt(IV) complexes with piano stool type geometries andmore » {eta}{sup 5}-Cp* groups. Both molecules have Br atoms on the mirror. This leads to a disorder of the Me and the second Br positions in complex 3. The average Pt-C(Cp*) bond length is 2.25 (7) {angstrom} in 2 and 2.22 (4) {angstrom} in 3. The Pt-C(Me) and Pt-Br bond lengths in 2 are 2.07 (2) and 2.498 (2) {angstrom}, respectively. The ordered Pt-Br bond length in 3 is 2.496 (2) {angstrom}. Treatment of 1 with halogens results in the cleavage of the Pt-Cp* bond. The reaction of PtMe{sub 3}I with KTp* (Tp* = (HB(3,5-dimethylpyrazolyl){sub 3}){sup {minus}}) in thf gives Tp*PtMe{sub 3} (4) in almost quantitative yield. The reaction of 4 with Br{sub 2} brominates the 4-position of the pyrazolyl ring only. 28 refs., 2 figs., 5 tabs.« less

Prions: Introducing a Complex Scientific Controversy to a Biology Classroom

ERIC Educational Resources Information Center

Zaitsev, Igor V.

2009-01-01

Thomas Kuhn, in "The Structure of Scientific Revolutions," posited that science does not progress by the steady accumulation of knowledge, but rather by a system of competition among paradigms. They vie for supremacy through greater parsimony, explanatory power, and popularity among the community of scientists (Kuhn, 1962). The current…

Replica Exchange Molecular Dynamics Study of Dimerization in Prion Protein: Multiple Modes of Interaction and Stabilization.

PubMed

Chamachi, Neharika G; Chakrabarty, Suman

2016-08-04

The pathological forms of prions are known to be a result of misfolding, oligomerization, and aggregation of the cellular prion. While the mechanism of misfolding and aggregation in prions has been widely studied using both experimental and computational tools, the structural and energetic characterization of the dimer form have not garnered as much attention. On one hand dimerization can be the first step toward a nucleation-like pathway to aggregation, whereas on the other hand it may also increase the conformational stability preventing self-aggregation. In this work, we have used extensive all-atom replica exchange molecular dynamics simulations of both monomer and dimer forms of a mouse prion protein to understand the structural, dynamic, and thermodynamic stability of dimeric prion as compared to the monomeric form. We show that prion proteins can dimerize spontaneously being stabilized by hydrophobic interactions as well as intermolecular hydrogen bonding and salt bridge formation. We have computed the conformational free energy landscapes for both monomer and dimer forms to compare the thermodynamic stability and misfolding pathways. We observe large conformational heterogeneity among the various modes of interactions between the monomers and the strong intermolecular interactions may lead to as high as 20% β-content. The hydrophobic regions in helix-2, surrounding coil regions, terminal regions along with the natively present β-sheet region appear to actively participate in prion-prion intermolecular interactions. Dimerization seems to considerably suppress the inherent dynamic instability observed in monomeric prions, particularly because the regions of structural frustration constitute the dimer interface. Further, we demonstrate an interesting reversible coupling between the Q160-G131 interaction (which leads to inhibition of β-sheet extension) and the G131-V161 H-bond formation.

The Oral Secretion of Infectious Scrapie Prions Occurs in Preclinical Sheep with a Range of PRNP Genotypes

PubMed Central

Gough, Kevin C.; Baker, Claire A.; Rees, Helen C.; Terry, Linda A.; Spiropoulos, John; Thorne, Leigh

2012-01-01

Preclinical sheep with the highly scrapie-susceptible VRQ/VRQ PRNP genotype secrete prions from the oral cavity. In order to further understand the significance of orally available prions, buccal swabs were taken from sheep with a range of PRNP genotypes and analyzed by serial protein misfolding cyclic amplification (sPMCA). Prions were detected in buccal swabs from scrapie-exposed sheep of genotypes linked to high (VRQ/VRQ and ARQ/VRQ) and low (ARR/VRQ and AHQ/VRQ) lymphoreticular system involvement in scrapie pathogenesis. For both groups, the level of prion detection was significantly higher than that for scrapie-resistant ARR/ARR sheep which were kept in the same farm environment and acted as sentinel controls for prions derived from the environment which might contaminate the oral cavity. In addition, sheep with no exposure to the scrapie agent did not contain any measurable prions within the oral cavity. Furthermore, prions were detected in sheep over a wide age range representing various stages of preclinical disease. These data demonstrate that orally available scrapie prions may be a common feature in sheep incubating scrapie, regardless of the PRNP genotype and any associated high-level accumulation of PrPSc within lymphoreticular tissues. PrPSc was present in buccal swabs from a large proportion of sheep with PRNP genotypes associated with relatively low disease penetrance, indicating that subclinical scrapie infection is likely to be a common occurrence. The significance of positive sPMCA reactions was confirmed by the transmission of infectivity in buccal swab extracts to Tg338 mice, illustrating the likely importance of orally available prions in the horizontal transmission of scrapie. PMID:22013047

PrPC Governs Susceptibility to Prion Strains in Bank Vole, While Other Host Factors Modulate Strain Features

PubMed Central

Espinosa, J. C.; Nonno, R.; Di Bari, M.; Aguilar-Calvo, P.; Pirisinu, L.; Fernández-Borges, N.; Vanni, I.; Vaccari, G.; Marín-Moreno, A.; Frassanito, P.; Lorenzo, P.; Agrimi, U.

2016-01-01

ABSTRACT Bank vole is a rodent species that shows differential susceptibility to the experimental transmission of different prion strains. In this work, the transmission features of a panel of diverse prions with distinct origins were assayed both in bank vole expressing methionine at codon 109 (Bv109M) and in transgenic mice expressing physiological levels of bank vole PrPC (the BvPrP-Tg407 mouse line). This work is the first systematic comparison of the transmission features of a collection of prion isolates, representing a panel of diverse prion strains, in a transgenic-mouse model and in its natural counterpart. The results showed very similar transmission properties in both the natural species and the transgenic-mouse model, demonstrating the key role of the PrP amino acid sequence in prion transmission susceptibility. However, differences in the PrPSc types propagated by Bv109M and BvPrP-Tg407 suggest that host factors other than PrPC modulate prion strain features. IMPORTANCE The differential susceptibility of bank voles to prion strains can be modeled in transgenic mice, suggesting that this selective susceptibility is controlled by the vole PrP sequence alone rather than by other species-specific factors. Differences in the phenotypes observed after prion transmissions in bank voles and in the transgenic mice suggest that host factors other than the PrPC sequence may affect the selection of the substrain replicating in the animal model. PMID:27654300

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

PubMed

Caughey, Byron; Orru, Christina D; Groveman, Bradley R; Hughson, Andrew G; Manca, Matteo; Raymond, Lynne D; Raymond, Gregory J; Race, Brent; Saijo, Eri; Kraus, Allison

2017-01-01

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. © 2017 Elsevier Inc. All rights reserved.

Effects of a naturally occurring amino acid substitution in bovine PrP: a model for inherited prion disease in a natural host species

USDA-ARS?s Scientific Manuscript database

The most common hereditary prion disease is human Creutzfeldt-Jakob disease (CJD) associated with a mutation in the prion gene (PRNP) resulting in a glutamic acid to lysine substitution at position 200 (E200K) in the prion protein. Models of E200K CJD in transgenic mice have proven interesting but h...

Mammalian prions: tolerance to sequence changes-how far?

PubMed

Salamat, Muhammad Khalid; Munoz-Montesino, Carola; Moudjou, Mohammed; Rezaei, Human; Laude, Hubert; Béringue, Vincent; Dron, Michel

2013-01-01

Upon prion infection, abnormal prion protein (PrP (Sc) ) self-perpetuate by conformational conversion of α-helix-rich PrP (C) into β sheet enriched form, leading to formation and deposition of PrP (Sc) aggregates in affected brains. However the process remains poorly understood at the molecular level and the regions of PrP critical for conversion are still debated. Minimal amino acid substitutions can impair prion replication at many places in PrP. Conversely, we recently showed that bona fide prions could be generated after introduction of eight and up to 16 additional amino acids in the H2-H3 inter-helix loop of PrP. Prion replication also accommodated the insertions of an octapeptide at different places in the last turns of H2. This reverse genetic approach reveals an unexpected tolerance of prions to substantial sequence changes in the protease-resistant part which is associated with infectivity. It also demonstrates that conversion does not require the presence of a specific sequence in the middle of the H2-H3 area. We discuss the implications of our findings according to different structural models proposed for PrP (Sc) and questioned the postulated existence of an N- or C-terminal prion domain in the protease-resistant region.

Role of Prion Replication in the Strain-dependent Brain Regional Distribution of Prions*

PubMed Central

Hu, Ping Ping; Morales, Rodrigo; Duran-Aniotz, Claudia; Moreno-Gonzalez, Ines; Khan, Uffaf; Soto, Claudio

2016-01-01

One intriguing feature of prion diseases is their strain variation. Prion strains are differentiated by the clinical consequences they generate in the host, their biochemical properties, and their potential to infect other animal species. The selective targeting of these agents to specific brain structures have been extensively used to characterize prion strains. However, the molecular basis dictating strain-specific neurotropism are still elusive. In this study, isolated brain structures from animals infected with four hamster prion strains (HY, DY, 139H, and SSLOW) were analyzed for their content of protease-resistant PrPSc. Our data show that these strains have different profiles of PrP deposition along the brain. These patterns of accumulation, which were independent of regional PrPC production, were not reproduced by in vitro replication when different brain regions were used as substrate for the misfolding-amplification reaction. On the contrary, our results show that in vitro replication efficiency depended exclusively on the amount of PrPC present in each part of the brain. Our results suggest that the variable regional distribution of PrPSc in distinct strains is not determined by differences on prion formation, but on other factors or cellular pathways. Our findings may contribute to understand the molecular mechanisms of prion pathogenesis and strain diversity. PMID:27056328

Progress and problems in the biology, diagnostics, and therapeutics of prion diseases

PubMed Central

Aguzzi, Adriano; Heikenwalder, Mathias; Miele, Gino

2004-01-01

The term “prion” was introduced by Stanley Prusiner in 1982 to describe the atypical infectious agent that causes transmissible spongiform encephalopathies, a group of infectious neurodegenerative diseases that include scrapie in sheep, Creutzfeldt-Jakob disease in humans, chronic wasting disease in cervids, and bovine spongiform encephalopathy in cattle. Over the past twenty years, the word “prion” has been taken to signify various subtly different concepts. In this article, we refer to the prion as the transmissible principle underlying prion diseases, without necessarily implying any specific biochemical or structural identity. When Prusiner started his seminal work, the study of transmissible spongiform encephalopathies was undertaken by only a handful of scientists. Since that time, the “mad cow” crisis has put prion diseases on the agenda of both politicians and the media. Significant progress has been made in prion disease research, and many aspects of prion pathogenesis are now understood. And yet the diagnostic procedures available for prion diseases are not nearly as sensitive as they ought to be, and no therapeutic intervention has been shown to reliably affect the course of the diseases. This article reviews recent progress in the areas of pathogenesis of, diagnostics of, and therapy for prion diseases and highlights some conspicuous problems that remain to be addressed in each of these fields. PMID:15254579

Sporadic Fatal Insomnia in an Adolescent

PubMed Central

Blase, Jennifer L.; Cracco, Laura; Schonberger, Lawrence B.; Maddox, Ryan A.; Cohen, Yvonne; Cali, Ignazio

2014-01-01

The occurrence of sporadic prion disease among adolescents is extremely rare. A prion disease was confirmed in an adolescent with disease onset at 13 years of age. Genetic, neuropathologic, and biochemical analyses of the patient’s autopsy brain tissue were consistent with sporadic fatal insomnia, a type of sporadic prion disease. There was no evidence of an environmental source of infection, and this patient represents the youngest documented case of sporadic prion disease. Although rare, a prion disease diagnosis should not be discounted in adolescents exhibiting neurologic signs. Brain tissue testing is necessary for disease confirmation and is particularly beneficial in cases with an unusual clinical presentation. PMID:24488737

Recombinant human prion protein inhibits prion propagation in vitro.

PubMed

Yuan, Jue; Zhan, Yi-An; Abskharon, Romany; Xiao, Xiangzhu; Martinez, Manuel Camacho; Zhou, Xiaochen; Kneale, Geoff; Mikol, Jacqueline; Lehmann, Sylvain; Surewicz, Witold K; Castilla, Joaquín; Steyaert, Jan; Zhang, Shulin; Kong, Qingzhong; Petersen, Robert B; Wohlkonig, Alexandre; Zou, Wen-Quan

2013-10-09

Prion diseases are associated with the conformational conversion of the cellular prion protein (PrP(C)) into the pathological scrapie isoform (PrP(Sc)) in the brain. Both the in vivo and in vitro conversion of PrP(C) into PrP(Sc) is significantly inhibited by differences in amino acid sequence between the two molecules. Using protein misfolding cyclic amplification (PMCA), we now report that the recombinant full-length human PrP (rHuPrP23-231) (that is unglycosylated and lacks the glycophosphatidylinositol anchor) is a strong inhibitor of human prion propagation. Furthermore, rHuPrP23-231 also inhibits mouse prion propagation in a scrapie-infected mouse cell line. Notably, it binds to PrP(Sc), but not PrP(C), suggesting that the inhibitory effect of recombinant PrP results from blocking the interaction of brain PrP(C) with PrP(Sc). Our findings suggest a new avenue for treating prion diseases, in which a patient's own unglycosylated and anchorless PrP is used to inhibit PrP(Sc) propagation without inducing immune response side effects.

Protein conformation determines the sensibility to high pressure treatment of infectious scrapie prions.

PubMed

Heindl, Philipp; García, Avelina Fernández; Butz, Peter; Pfaff, Eberhard; Tauscher, Bernhard

2006-03-01

Application of high pressure can be used for gentle pasteurizing of food, minimizing undesirable alterations such as vitamin losses and changes in taste and color. In addition, pressure has become a useful tool for investigating structural changes in proteins. Treatments of proteins with high pressure can reveal conformations that are not obtainable by other physical variables like temperature, since pressure favors structural transitions accompanied with smaller volumes. Here, we discuss both the potential use of high pressure to inactivate infectious TSE material and the application of this thermodynamic parameter for the investigation of prion folding. This review summarizes our findings on the effects of pressure on the structure of native infectious scrapie prions in hamster brain homogenates and on the structure of infectious prion rods isolated from diseased hamsters brains. Native prions were found to be pressure sensitive, whereas isolated prions revealed an extreme pressure-resistant structure. The discussion will be focused on the different pressure behavior of these prion isoforms, which points out differences in the protein structure that have not been taken into consideration before.

Experimental and Theoretical Insights into the Inhibition Mechanism of Prion Fibrillation by Resveratrol and its Derivatives.

PubMed

Li, Lanlan; Zhu, Yongchang; Zhou, Shuangyan; An, Xiaoli; Zhang, Yan; Bai, Qifeng; He, Yong-Xing; Liu, Huanxiang; Yao, Xiaojun

2017-12-20

Resveratrol and its derivatives have been shown to display beneficial effects to neurodegenerative diseases. However, the molecular mechanism of resveratrol and its derivatives on prion conformational conversion is poorly understood. In this work, the interaction mechanism between prion and resveratrol as well as its derivatives was investigated using steady-state fluorescence quenching, Thioflavin T binding assay, Western blotting, and molecular dynamics simulation. Protein fluorescence quenching method and Thioflavin T assay revealed that resveratrol and its derivatives could interact with prion and interrupt prion fibril formation. Molecular dynamics simulation results indicated that resveratrol can stabilize the PrP 127-147 peptide mainly through π-π stacking interactions between resveratrol and Tyr128. The hydrogen bonds interactions between resveratrol and the PrP 127-147 peptide could further reduce the flexibility and the propensity to aggregate. The results of this study not only can provide useful information about the interaction mechanism between resveratrol and prion, but also can provide useful clues for further design of new inhibitors inhibiting prion aggregation.

NMR shielding and spin–rotation constants of {sup 175}LuX (X = {sup 19}F, {sup 35}Cl, {sup 79}Br, {sup 127}I) molecules

DOE Office of Scientific and Technical Information (OSTI.GOV)

Demissie, Taye B.

2015-12-31

This presentation demonstrates the relativistic effects on the spin-rotation constants, absolute nuclear magnetic resonance (NMR) shielding constants and shielding spans of {sup 175}LuX (X = {sup 19}F, {sup 35}Cl, {sup 79}Br, {sup 127}I) molecules. The results are obtained from calculations performed using density functional theory (non-relativistic and four-component relativistic) and coupled-cluster calculations. The spin-rotation constants are compared with available experimental values. In most of the molecules studied, relativistic effects make an order of magnitude difference on the NMR absolute shielding constants.

Genetics Home Reference: prion disease

MedlinePlus

... from eating beef products containing PrP Sc from cattle with prion disease . In cows, this form of the disease is known as bovine spongiform encephalopathy (BSE) or, more commonly, "mad cow disease." Another example of an acquired human prion ...

MAD COW DISEASE: New Recruits for French Prion Research.

PubMed

Casassus, B

2000-12-01

As panic over "mad cow disease" engulfs France and threatens to spread to other countries in Western Europe, French research minister Roger-Gérard Schwartzenberg last week unveiled detailed plans for spending $27 million the government has earmarked for prion disease research in 2001. Next year's budget for studying prions--infectious, abnormal proteins linked to bovine spongiform encephalopathy and its human form, variant Creutzfeldt-Jakob disease--will triple France's current prion research spending.

A role for astroglia in prion diseases.

PubMed

Aguzzi, Adriano; Liu, Yingjun

2017-12-04

In this issue of JEM, Krejciova et al. (https://doi.org/10.1084/jem.20161547) report that astrocytes derived from human iPSCs can replicate human CJD prions. These observations provide a new, potentially very valuable model for studying human prions in cellula and for identifying antiprion compounds that might serve as clinical candidates. Furthermore, they add to the evidence that astrocytes may not be just innocent bystanders in prion diseases. © 2017 Aguzzi and Liu.

Fate of Prions in Soil: A Review

PubMed Central

Smith, Christen B.; Booth, Clarissa J.; Pedersen, Joel A.

2011-01-01

Prions are the etiological agents of transmissible spongiform encephalopathies (TSEs), a class of fatal neurodegenerative diseases affecting humans and other mammals. The pathogenic prion protein is a misfolded form of the host-encoded prion protein and represents the predominant, if not sole, component of the infectious agent. Environmental routes of TSE transmission are implicated in epizootics of sheep scrapie and chronic wasting disease (CWD) of deer, elk, and moose. Soil represents a plausible environmental reservoir of scrapie and CWD agents, which can persist in the environment for years. Attachment to soil particles likely influences the persistence and infectivity of prions in the environment. Effective methods to inactivate TSE agents in soil are currently lacking, and the effects of natural degradation mechanisms on TSE infectivity are largely unknown. An improved understanding of the processes affecting the mobility, persistence, and bioavailability of prions in soil is needed for the management of TSE-contaminated environments. PMID:21520752

Mineral licks as environmental reservoirs of chronic wasting disease prions

PubMed Central

Plummer, Ian H.; Johnson, Chad J.; Chesney, Alexandra R.; Pedersen, Joel A.

2018-01-01

Chronic wasting disease (CWD) is a fatal neurodegenerative disease of deer, elk, moose, and reindeer (cervids) caused by misfolded prion proteins. The disease has been reported across North America and recently discovered in northern Europe. Transmission of CWD in wild cervid populations can occur through environmental routes, but limited ability to detect prions in environmental samples has prevented the identification of potential transmission “hot spots”. We establish widespread CWD prion contamination of mineral licks used by free-ranging cervids in an enzootic area in Wisconsin, USA. We show mineral licks can serve as reservoirs of CWD prions and thus facilitate disease transmission. Furthermore, mineral licks attract livestock and other wildlife that also obtain mineral nutrients via soil and water consumption. Exposure to CWD prions at mineral licks provides potential for cross-species transmission to wildlife, domestic animals, and humans. Managing deer use of mineral licks warrants further consideration to help control outbreaks of CWD. PMID:29719000

Aβ seeds and prions: How close the fit?

PubMed

Rasmussen, Jay; Jucker, Mathias; Walker, Lary C

2017-07-04

The prion paradigm is increasingly invoked to explain the molecular pathogenesis of neurodegenerative diseases involving the misfolding and aggregation of proteins other than the prion protein (PrP). Extensive evidence from in vitro and in vivo studies indicates that misfolded and aggregated Aβ peptide, which is the probable molecular trigger for Alzheimer's disease, manifests all of the key characteristics of canonical mammalian prions. These features include a β-sheet rich architecture, tendency to polymerize into amyloid, templated corruption of like protein molecules, ability to form structurally and functionally variant strains, systematic spread by neuronal transport, and resistance to inactivation by heat and formaldehyde. In addition to Aβ, a growing body of research supports the view that the prion-like molecular transformation of specific proteins drives the onset and course of a remarkable variety of clinicopathologically diverse diseases. As such, the expanded prion paradigm could conceptually unify fundamental and translational investigations of these disorders.

Allelic variants of hereditary prions: The bimodularity principle.

PubMed

Tikhodeyev, Oleg N; Tarasov, Oleg V; Bondarev, Stanislav A

2017-01-02

Modern biology requires modern genetic concepts equally valid for all discovered mechanisms of inheritance, either "canonical" (mediated by DNA sequences) or epigenetic. Applying basic genetic terms such as "gene" and "allele" to protein hereditary factors is one of the necessary steps toward these concepts. The basic idea that different variants of the same prion protein can be considered as alleles has been previously proposed by Chernoff and Tuite. In this paper, the notion of prion allele is further developed. We propose the idea that any prion allele is a bimodular hereditary system that depends on a certain DNA sequence (DNA determinant) and a certain epigenetic mark (epigenetic determinant). Alteration of any of these 2 determinants may lead to establishment of a new prion allele. The bimodularity principle is valid not only for hereditary prions; it seems to be universal for any epigenetic hereditary factor.

Molecular mechanisms for protein-encoded inheritance

PubMed Central

Wiltzius, Jed J. W.; Landau, Meytal; Nelson, Rebecca; Sawaya, Michael R.; Apostol, Marcin I.; Goldschmidt, Lukasz; Soriaga, Angela B.; Cascio, Duilio; Rajashankar, Kanagalaghatta; Eisenberg, David

2013-01-01

Strains are phenotypic variants, encoded by nucleic acid sequences in chromosomal inheritance and by protein “conformations” in prion inheritance and transmission. But how is a protein “conformation” stable enough to endure transmission between cells or organisms? Here new polymorphic crystal structures of segments of prion and other amyloid proteins offer structural mechanisms for prion strains. In packing polymorphism, prion strains are encoded by alternative packings (polymorphs) of β-sheets formed by the same segment of a protein; in a second mechanism, segmental polymorphism, prion strains are encoded by distinct β-sheets built from different segments of a protein. Both forms of polymorphism can produce enduring “conformations,” capable of encoding strains. These molecular mechanisms for transfer of information into prion strains share features with the familiar mechanism for transfer of information by nucleic acid inheritance, including sequence specificity and recognition by non-covalent bonds. PMID:19684598

Allelic variants of hereditary prions: The bimodularity principle

PubMed Central

Tikhodeyev, Oleg N.; Tarasov, Oleg V.; Bondarev, Stanislav A.

2017-01-01

ABSTRACT Modern biology requires modern genetic concepts equally valid for all discovered mechanisms of inheritance, either “canonical” (mediated by DNA sequences) or epigenetic. Applying basic genetic terms such as “gene” and “allele” to protein hereditary factors is one of the necessary steps toward these concepts. The basic idea that different variants of the same prion protein can be considered as alleles has been previously proposed by Chernoff and Tuite. In this paper, the notion of prion allele is further developed. We propose the idea that any prion allele is a bimodular hereditary system that depends on a certain DNA sequence (DNA determinant) and a certain epigenetic mark (epigenetic determinant). Alteration of any of these 2 determinants may lead to establishment of a new prion allele. The bimodularity principle is valid not only for hereditary prions; it seems to be universal for any epigenetic hereditary factor. PMID:28281926

Bile Acids Reduce Prion Conversion, Reduce Neuronal Loss, and Prolong Male Survival in Models of Prion Disease

PubMed Central

Cortez, Leonardo M.; Campeau, Jody; Norman, Grant; Kalayil, Marian; Van der Merwe, Jacques; McKenzie, Debbie

2015-01-01

ABSTRACT Prion diseases are fatal neurodegenerative disorders associated with the conversion of cellular prion protein (PrPC) into its aberrant infectious form (PrPSc). There is no treatment available for these diseases. The bile acids tauroursodeoxycholic acid (TUDCA) and ursodeoxycholic acid (UDCA) have been recently shown to be neuroprotective in other protein misfolding disease models, including Parkinson's, Huntington's and Alzheimer's diseases, and also in humans with amyotrophic lateral sclerosis. Here, we studied the therapeutic efficacy of these compounds in prion disease. We demonstrated that TUDCA and UDCA substantially reduced PrP conversion in cell-free aggregation assays, as well as in chronically and acutely infected cell cultures. This effect was mediated through reduction of PrPSc seeding ability, rather than an effect on PrPC. We also demonstrated the ability of TUDCA and UDCA to reduce neuronal loss in prion-infected cerebellar slice cultures. UDCA treatment reduced astrocytosis and prolonged survival in RML prion-infected mice. Interestingly, these effects were limited to the males, implying a gender-specific difference in drug metabolism. Beyond effects on PrPSc, we found that levels of phosphorylated eIF2α were increased at early time points, with correlated reductions in postsynaptic density protein 95. As demonstrated for other neurodegenerative diseases, we now show that TUDCA and UDCA may have a therapeutic role in prion diseases, with effects on both prion conversion and neuroprotection. Our findings, together with the fact that these natural compounds are orally bioavailable, permeable to the blood-brain barrier, and U.S. Food and Drug Administration-approved for use in humans, make these compounds promising alternatives for the treatment of prion diseases. IMPORTANCE Prion diseases are fatal neurodegenerative diseases that are transmissible to humans and other mammals. There are no disease-modifying therapies available, despite decades of research. Treatment targets have included inhibition of protein accumulation, clearance of toxic aggregates, and prevention of downstream neurodegeneration. No one target may be sufficient; rather, compounds which have a multimodal mechanism, acting on different targets, would be ideal. TUDCA and UDCA are bile acids that may fulfill this dual role. Previous studies have demonstrated their neuroprotective effects in several neurodegenerative disease models, and we now demonstrate that this effect occurs in prion disease, with an added mechanistic target of upstream prion seeding. Importantly, these are natural compounds which are orally bioavailable, permeable to the blood-brain barrier, and U.S. Food and Drug Administration-approved for use in humans with primary biliary cirrhosis. They have recently been proven efficacious in human amyotrophic lateral sclerosis. Therefore, these compounds are promising options for the treatment of prion diseases. PMID:25972546

$sup 35$S Metabolism in the Physiology of Mammals Reproduction; METABOLISME DU 35S DANS LA PHYSIOLOGIE DE LA REPRODUCTION CHEZ LES MAMMIFERES

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moricard, R.; Gothie, S.; Rodriguez-Galindo, M.

1959-10-31

From ovulation to nidation in the uterus, the spermatozoa, the second order ovocyte, and the fertilized egg are free in the genital secretions whose sulfur amount depends on ovarian hormones. The spermatozoa can fix S/sup 35/ in vitro and they seem to be able to metabolize this element during their ascent through the genital tract. In the uterus and in the mucosa of the cervix, there is an intense and fast metabolism of the S/sup 35/ in the guinea pig. In the fallopian tube, fecondation takes place in the secretions in which S/sup 35/ metabolism is at the highest degreemore » at fertilization. As soon as the second polar body is expelled, the pellucida has a very high concentration of S/sup 35/, whether the latter is injected in the form of Na/sub 2/S/sup 35/O/sub 4/ or cystine. This concentration is not only due to fertilization, it appears also in unfertilized eggs. In vitro cultures of ovarian ovocytes, tubal eggs, and uterine blastocytes show the protective part played by the pellucida and its concentrating power, a function which does not seem to be performed by the pellucida of first-order ovocytes. (auth)« less

Prion propagation and toxicity occur in vitro with two-phase kinetics specific to strain and neuronal type.

PubMed

Hannaoui, Samia; Maatouk, Layal; Privat, Nicolas; Levavasseur, Etienne; Faucheux, Baptiste A; Haïk, Stéphane

2013-03-01

Prion diseases, or transmissible spongiform encephalopathies (TSEs), are fatal neurodegenerative disorders that occur in humans and animals. The neuropathological hallmarks of TSEs are spongiosis, glial proliferation, and neuronal loss. The only known specific molecular marker of TSEs is the abnormal isoform (PrP(Sc)) of the host-encoded prion protein (PrP(C)), which accumulates in the brain of infected subjects and forms infectious prion particles. Although this transmissible agent lacks a specific nucleic acid component, several prion strains have been isolated. Prion strains are characterized by differences in disease outcome, PrP(Sc) distribution patterns, and brain lesion profiles at the terminal stage of the disease. The molecular factors and cellular mechanisms involved in strain-specific neuronal tropism and toxicity remain largely unknown. Currently, no cellular model exists to facilitate in vitro studies of these processes. A few cultured cell lines that maintain persistent scrapie infections have been developed, but only two of them have shown the cytotoxic effects associated with prion propagation. In this study, we have developed primary neuronal cultures to assess in vitro neuronal tropism and toxicity of different prion strains (scrapie strains 139A, ME7, and 22L). We have tested primary neuronal cultures enriched in cerebellar granular, striatal, or cortical neurons. Our results showed that (i) a strain-specific neuronal tropism operated in vitro; (ii) the cytotoxic effect varied among strains and neuronal cell types; (iii) prion propagation and toxicity occurred in two kinetic phases, a replicative phase followed by a toxic phase; and (iv) neurotoxicity peaked when abnormal PrP accumulation reached a plateau.

Prion Propagation and Toxicity Occur In Vitro with Two-Phase Kinetics Specific to Strain and Neuronal Type

PubMed Central

Hannaoui, Samia; Maatouk, Layal; Privat, Nicolas; Levavasseur, Etienne; Faucheux, Baptiste A.

2013-01-01

Prion diseases, or transmissible spongiform encephalopathies (TSEs), are fatal neurodegenerative disorders that occur in humans and animals. The neuropathological hallmarks of TSEs are spongiosis, glial proliferation, and neuronal loss. The only known specific molecular marker of TSEs is the abnormal isoform (PrPSc) of the host-encoded prion protein (PrPC), which accumulates in the brain of infected subjects and forms infectious prion particles. Although this transmissible agent lacks a specific nucleic acid component, several prion strains have been isolated. Prion strains are characterized by differences in disease outcome, PrPSc distribution patterns, and brain lesion profiles at the terminal stage of the disease. The molecular factors and cellular mechanisms involved in strain-specific neuronal tropism and toxicity remain largely unknown. Currently, no cellular model exists to facilitate in vitro studies of these processes. A few cultured cell lines that maintain persistent scrapie infections have been developed, but only two of them have shown the cytotoxic effects associated with prion propagation. In this study, we have developed primary neuronal cultures to assess in vitro neuronal tropism and toxicity of different prion strains (scrapie strains 139A, ME7, and 22L). We have tested primary neuronal cultures enriched in cerebellar granular, striatal, or cortical neurons. Our results showed that (i) a strain-specific neuronal tropism operated in vitro; (ii) the cytotoxic effect varied among strains and neuronal cell types; (iii) prion propagation and toxicity occurred in two kinetic phases, a replicative phase followed by a toxic phase; and (iv) neurotoxicity peaked when abnormal PrP accumulation reached a plateau. PMID:23255799

Rapid and Highly Sensitive Detection of Variant Creutzfeldt - Jakob Disease Abnormal Prion Protein on Steel Surfaces by Protein Misfolding Cyclic Amplification: Application to Prion Decontamination Studies

PubMed Central

Belondrade, Maxime; Nicot, Simon; Béringue, Vincent; Coste, Joliette; Lehmann, Sylvain; Bougard, Daisy

2016-01-01

The prevalence of variant Creutzfeldt-Jakob disease (vCJD) in the population remains uncertain, although it has been estimated that 1 in 2000 people in the United Kingdom are positive for abnormal prion protein (PrPTSE) by a recent survey of archived appendix tissues. The prominent lymphotropism of vCJD prions raises the possibility that some surgical procedures may be at risk of iatrogenic vCJD transmission in healthcare facilities. It is therefore vital that decontamination procedures applied to medical devices before their reprocessing are thoroughly validated. A current limitation is the lack of a rapid model permissive to human prions. Here, we developed a prion detection assay based on protein misfolding cyclic amplification (PMCA) technology combined with stainless-steel wire surfaces as carriers of prions (Surf-PMCA). This assay allowed the specific detection of minute quantities (10−8 brain dilution) of either human vCJD or ovine scrapie PrPTSE adsorbed onto a single steel wire, within a two week timeframe. Using Surf-PMCA we evaluated the performance of several reference and commercially available prion-specific decontamination procedures. Surprisingly, we found the efficiency of several marketed reagents to remove human vCJD PrPTSE was lower than expected. Overall, our results demonstrate that Surf-PMCA can be used as a rapid and ultrasensitive assay for the detection of human vCJD PrPTSE adsorbed onto a metallic surface, therefore facilitating the development and validation of decontamination procedures against human prions. PMID:26800081

PrPC Governs Susceptibility to Prion Strains in Bank Vole, While Other Host Factors Modulate Strain Features.

PubMed

Espinosa, J C; Nonno, R; Di Bari, M; Aguilar-Calvo, P; Pirisinu, L; Fernández-Borges, N; Vanni, I; Vaccari, G; Marín-Moreno, A; Frassanito, P; Lorenzo, P; Agrimi, U; Torres, J M

2016-12-01

Bank vole is a rodent species that shows differential susceptibility to the experimental transmission of different prion strains. In this work, the transmission features of a panel of diverse prions with distinct origins were assayed both in bank vole expressing methionine at codon 109 (Bv109M) and in transgenic mice expressing physiological levels of bank vole PrP C (the BvPrP-Tg407 mouse line). This work is the first systematic comparison of the transmission features of a collection of prion isolates, representing a panel of diverse prion strains, in a transgenic-mouse model and in its natural counterpart. The results showed very similar transmission properties in both the natural species and the transgenic-mouse model, demonstrating the key role of the PrP amino acid sequence in prion transmission susceptibility. However, differences in the PrP Sc types propagated by Bv109M and BvPrP-Tg407 suggest that host factors other than PrP C modulate prion strain features. The differential susceptibility of bank voles to prion strains can be modeled in transgenic mice, suggesting that this selective susceptibility is controlled by the vole PrP sequence alone rather than by other species-specific factors. Differences in the phenotypes observed after prion transmissions in bank voles and in the transgenic mice suggest that host factors other than the PrP C sequence may affect the selection of the substrain replicating in the animal model. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

A transfectant RK13 cell line permissive to classical caprine scrapie prion propagation.

PubMed

Dassanayake, Rohana P; Zhuang, Dongyue; Truscott, Thomas C; Madsen-Bouterse, Sally A; O'Rourke, Katherine I; Schneider, David A

2016-03-03

To assess scrapie infectivity associated with caprine-origin tissues, bioassay can be performed using kids, lambs or transgenic mice expressing caprine or ovine prion (PRNP) alleles, but the incubation periods are fairly long. Although several classical ovine scrapie prion permissive cell lines with the ability to detect brain-derived scrapie prion have been available, no classical caprine scrapie permissive cell line is currently available. Therefore, the aims of this study were to generate a rabbit kidney epithelial cell line (RK13) stably expressing caprine wild-type PRNP (cpRK13) and then to assess permissiveness of cpRK13 cells to classical caprine scrapie prion propagation. The cpRK13 and plasmid control RK13 (pcRK13) cells were incubated with brain-derived classical caprine scrapie inocula prepared from goats or ovinized transgenic mice (Tg338, express ovine VRQ allele) infected with caprine scrapie. Significant PrP(Sc) accumulation, which is indicative of scrapie prion propagation, was detected by TSE ELISA and immunohistochemistry in cpRK13 cells inoculated with classical caprine scrapie inocula. Western blot analysis revealed the typical proteinase K-resistant 3 PrP(res) isoforms in the caprine scrapie prion inoculated cpRK13 cell lysate. Importantly, PrP(Sc) accumulation was not detected in similarly inoculated pcRK13 cells, whether by TSE ELISA, immunohistochemistry, or western blot. These findings suggest that caprine scrapie prions can be propagated in cpRK13 cells, thus this cell line may be a useful tool for the assessment of classical caprine prions in the brain tissues of goats.

Alteration of the chronic wasting disease species barrier by in vitro prion amplification

USGS Publications Warehouse

Kurt, Timothy D.; Seelig, Davis M.; Schneider, Jay R.; Johnson, Christopher J.; Telling, Glenn C.; Heisey, Dennis M.; Hoover, Edward A.

2011-01-01

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) of cervids now detected in 19 states of the United States, three Canadian provinces, and South Korea. Whether noncervid species can be infected by CWD and thereby serve as reservoirs for the infection is not known. To investigate this issue, we previously used serial protein misfolding cyclic amplification (sPMCA) to demonstrate that CWD prions can amplify in brain homogenates from several species sympatric with cervids, including prairie voles (Microtus ochrogaster) and field mice (Peromyscus spp.). Here, we show that prairie voles are susceptible to mule deer CWD prions in vivo and that sPMCA amplification of CWD prions in vole brain enhances the infectivity of CWD for this species. Prairie voles inoculated with sPMCA products developed clinical signs of TSE disease approximately 300 days prior to, and more consistently than, those inoculated with CWD prions from deer brain. Moreover, the deposition patterns and biochemical properties of protease-resistant form of PrP (PrPRES) in the brains of affected voles differed from those in cervidized transgenic (CerPrP) mice infected with CWD. In addition, voles inoculated orally with sPMCA products developed clinical signs of TSE and were positive for PrPRES deposition, whereas those inoculated orally with deer-origin CWD prions did not. These results demonstrate that transspecies sPMCA of CWD prions can enhance the infectivity and adapt the host range of CWD prions and thereby may be useful to assess determinants of prion species barriers.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matzel, J P; Jacobsen, B; Hutcheon, I D

The abundance of short-lived radionuclides (SLRs) in early solar system materials provide key information about their nucleosynthetic origin and can constrain the timing of early solar system events. Excesses of {sup 36}S ({sup 36}S*) correlated with {sup 35}Cl/{sup 34}S ratios provide direct evidence for in situ decay of {sup 36}Cl ({tau}{sub 1/2} {approx} 0.3 Ma) and have been reported in sodalite (Na{sub 8}Al{sub 6}Si{sub 6}O{sub 24}Cl{sub 2}) and wadalite (Ca{sub 6}Al{sub 5}Si{sub 2}O{sub 16}Cl{sub 3}) in CAIs and chondrules from the Allende and Ningqiang CV carbonaceous chondrites. While previous studies demonstrate unequivocally that {sup 36}Cl was extant in the earlymore » solar system, no consensus on the origin or initial abundance of {sup 36}Cl has emerged. Understanding the origin of {sup 36}Cl, as well as the reported variation in the initial {sup 36}Cl/{sup 35}Cl ratio, requires addressing when, where and how chlorine was incorporated into CAIs and chondrules. These factors are key to distinguishing between stellar nucleosynthesis or energetic particle irradiation for the origin of {sup 36}Cl. Wadalite is a chlorine-rich secondary mineral with structural and chemical affinities to grossular. The high chlorine ({approx}12 wt%) and very low sulfur content (<<0.01 wt%) make wadalite ideal for studies of the {sup 36}Cl-{sup 36}S system. Wadalite is present in Allende CAIs exclusively in the interior regions either in veins crosscutting melilite or in zones between melilite and anorthite associated with intergrowths of grossular, monticellite, and wollastonite. Wadalite and sodalite most likely resulted from open-system alteration of primary minerals with a chlorine-rich fluid phase. We recently reported large {sup 36}S* correlated with {sup 35}Cl/{sup 34}S in wadalite in Allende Type B CAI AJEF, yielding a ({sup 36}Cl/{sup 35}Cl){sub 0} ratio of (1.7 {+-} 0.3) x 10{sup -5}. This value is the highest reported {sup 36}Cl/{sup 35}Cl ratio and is {approx}5 times greater than values for sodalite in some CAIs and chondrules. We report here new {sup 36}Cl-{sup 36}S isotope measurements of wadalite in two Type B CAIs (TS34 and Egg-6) and sodalite in a fine-grained CAI (Pink Angel) from Allende.« less

Emergence of two prion subtypes in ovine PrP transgenic mice infected with human MM2-cortical Creutzfeldt-Jakob disease prions.

PubMed

Chapuis, Jérôme; Moudjou, Mohammed; Reine, Fabienne; Herzog, Laetitia; Jaumain, Emilie; Chapuis, Céline; Quadrio, Isabelle; Boulliat, Jacques; Perret-Liaudet, Armand; Dron, Michel; Laude, Hubert; Rezaei, Human; Béringue, Vincent

2016-02-05

Mammalian prions are proteinaceous pathogens responsible for a broad range of fatal neurodegenerative diseases in humans and animals. These diseases can occur spontaneously, such as Creutzfeldt-Jakob disease (CJD) in humans, or be acquired or inherited. Prions are primarily formed of macromolecular assemblies of the disease-associated prion protein PrP(Sc), a misfolded isoform of the host-encoded prion protein PrP(C). Within defined host-species, prions can exist as conformational variants or strains. Based on both the M/V polymorphism at codon 129 of PrP and the electrophoretic signature of PrP(Sc) in the brain, sporadic CJD is classified in different subtypes, which may encode different strains. A transmission barrier, the mechanism of which remains unknown, limits prion cross-species propagation. To adapt to the new host, prions have the capacity to 'mutate' conformationally, leading to the emergence of a variant with new biological properties. Here, we transmitted experimentally one rare subtype of human CJD, designated cortical MM2 (129 MM with type 2 PrP(Sc)), to transgenic mice overexpressing either human or the VRQ allele of ovine PrP(C). In marked contrast with the reported absence of transmission to knock-in mice expressing physiological levels of human PrP, this subtype transmitted faithfully to mice overexpressing human PrP, and exhibited unique strain features. Onto the ovine PrP sequence, the cortical MM2 subtype abruptly evolved on second passage, thereby allowing emergence of a pair of strain variants with distinct PrP(Sc) biochemical characteristics and differing tropism for the central and lymphoid tissues. These two strain components exhibited remarkably distinct replicative properties in cell-free amplification assay, allowing the 'physical' cloning of the minor, lymphotropic component, and subsequent isolation in ovine PrP mice and RK13 cells. Here, we provide in-depth assessment of the transmissibility and evolution of one rare subtype of sporadic CJD upon homologous and heterologous transmission. The notion that the environment or matrix where replication is occurring is key to the selection and preferential amplification of prion substrain components raises new questions on the determinants of prion replication within and between species. These data also further interrogate on the interplay between animal and human prions.

Sulforaphane-induced autophagy flux prevents prion protein-mediated neurotoxicity through AMPK pathway.

PubMed

Lee, J-H; Jeong, J-K; Park, S-Y

2014-10-10

Prion diseases are neurodegenerative and infectious disorders that involve accumulation of misfolded scrapie prion protein, and which are characterized by spongiform degeneration. Autophagy, a major homeostatic process responsible for the degradation of cytoplasmic components, has garnered attention as the potential target for neurodegenerative diseases such as prion disease. We focused on protective effects of sulforaphane found in cruciferous vegetables on prion-mediated neurotoxicity and the mechanism of sulforaphane related to autophagy. In human neuroblastoma cells, sulforaphane protected prion protein (PrP) (106-126)-mediated neurotoxicity and increased autophagy flux marker microtubule-associated protein 1 light chain 3-II protein levels, following a decrease of p62 protein level. Pharmacological and genetical inhibition of autophagy by 3MA, wortmannin and knockdown of autophagy-related 5 (ATG5) led to block the effect of sulforaphane against PrP (106-126)-induced neurotoxicity. Furthermore we demonstrated that both sulforaphane-induced autophagy and protective effect of sulforaphane against PrP (106-126)-induced neurotoxicity are dependent on the AMP-activated protein kinase (AMPK) signaling. The present results indicated that sulforaphane of cruciferous vegetables enhanced autophagy flux led to the protection effects against prion-mediated neurotoxicity, which was regulated by AMPK signaling pathways in human neuron cells. Our data also suggest that sulforaphane has a potential value as a therapeutic tool in neurodegenerative disease including prion diseases. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

Persistence of pathogenic prion protein during simulated wastewater treatment processes

USGS Publications Warehouse

Hinckley, G.T.; Johnson, C.J.; Jacobson, K.H.; Bartholomay, C.; Mcmahon, K.D.; McKenzie, D.; Aiken, Judd M.; Pedersen, J.A.

2008-01-01

Transmissible spongiform encephalopathies (TSEs, prion diseases) are a class of fatal neurodegenerative diseases affecting a variety of mammalian species including humans. A misfolded form of the prion protein (PrP TSE) is the major, if not sole, component of the infectious agent. Prions are highly resistant to degradation and to many disinfection procedures suggesting that, if prions enter wastewater treatment systems through sewers and/or septic systems (e.g., from slaughterhouses, necropsy laboratories, rural meat processors, private game dressing) or through leachate from landfills that have received TSE-contaminated material, prions could survive conventional wastewater treatment Here, we report the results of experiments examining the partitioning and persistence of PrPTSE during simulated wastewater treatment processes including activated and mesophilic anaerobic sludge digestion. Incubation with activated sludge did not result in significant PrPTSE degradation. PrPTSE and prion infectivity partitioned strongly to activated sludge solids and are expected to enter biosolids treatment processes. A large fraction of PrPTSE survived simulated mesophilic anaerobic sludge digestion. The small reduction in recoverable PrPTSE after 20-d anaerobic sludge digestion appeared attributable to a combination of declining extractability with time and microbial degradation. Our results suggest that if prions were to enter municipal wastewater treatment systems, most would partition to activated sludge solids, survive mesophilic anaerobic digestion, and be present in treated biosolids. ?? 2008 American Chemical Society.

Molecular pathogenesis of sporadic prion diseases in man

PubMed Central

Safar, Jiri G.

2012-01-01

The yeast, fungal and mammalian prions determine heritable and infectious traits that are encoded in alternative conformations of proteins. They cause lethal sporadic, familial and infectious neurodegenerative conditions in man, including Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), kuru, sporadic fatal insomnia (SFI) and likely variable protease-sensitive prionopathy (VPSPr). The most prevalent of human prion diseases is sporadic (s)CJD. Recent advances in amplification and detection of prions led to considerable optimism that early and possibly preclinical diagnosis and therapy might become a reality. Although several drugs have already been tested in small numbers of sCJD patients, there is no clear evidence of any agent’s efficacy. Therefore, it remains crucial to determine the full spectrum of sCJD prion strains and the conformational features in the pathogenic human prion protein governing replication of sCJD prions. Research in this direction is essential for the rational development of diagnostic as well as therapeutic strategies. Moreover, there is growing recognition that fundamental processes involved in human prion propagation – intercellular induction of protein misfolding and seeded aggregation of misfolded host proteins – are of far wider significance. This insight leads to new avenues of research in the ever-widening spectrum of age-related human neurodegenerative diseases that are caused by protein misfolding and that pose a major challenge for healthcare. PMID:22421210

Role of Prion Replication in the Strain-dependent Brain Regional Distribution of Prions.

PubMed

Hu, Ping Ping; Morales, Rodrigo; Duran-Aniotz, Claudia; Moreno-Gonzalez, Ines; Khan, Uffaf; Soto, Claudio

2016-06-10

One intriguing feature of prion diseases is their strain variation. Prion strains are differentiated by the clinical consequences they generate in the host, their biochemical properties, and their potential to infect other animal species. The selective targeting of these agents to specific brain structures have been extensively used to characterize prion strains. However, the molecular basis dictating strain-specific neurotropism are still elusive. In this study, isolated brain structures from animals infected with four hamster prion strains (HY, DY, 139H, and SSLOW) were analyzed for their content of protease-resistant PrP(Sc) Our data show that these strains have different profiles of PrP deposition along the brain. These patterns of accumulation, which were independent of regional PrP(C) production, were not reproduced by in vitro replication when different brain regions were used as substrate for the misfolding-amplification reaction. On the contrary, our results show that in vitro replication efficiency depended exclusively on the amount of PrP(C) present in each part of the brain. Our results suggest that the variable regional distribution of PrP(Sc) in distinct strains is not determined by differences on prion formation, but on other factors or cellular pathways. Our findings may contribute to understand the molecular mechanisms of prion pathogenesis and strain diversity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of Alprenolol Hydrochloride as an Anti-prion Compound Using Surface Plasmon Resonance Imaging.

PubMed

Miyazaki, Yukiko; Ishikawa, Takeshi; Kamatari, Yuji O; Nakagaki, Takehiro; Takatsuki, Hanae; Ishibashi, Daisuke; Kuwata, Kazuo; Nishida, Noriyuki; Atarashi, Ryuichiro

2018-04-27

Prion diseases are transmissible neurodegenerative disorders of humans and animals, which are characterized by the aggregation of abnormal prion protein (PrP Sc ) in the central nervous system. Although several small compounds that bind to normal PrP (PrP C ) have been shown to inhibit structural conversion of the protein, an effective therapy for human prion disease remains to be established. In this study, we screened 1200 existing drugs approved by the US Food and Drug Administration (FDA) for anti-prion activity using surface plasmon resonance imaging (SPRi). Of these drugs, 31 showed strong binding activity to recombinant human PrP, and three of these reduced the accumulation of PrP Sc in prion-infected cells. One of the active compounds, alprenolol hydrochloride, which is used clinically as a β-adrenergic blocker for hypertension, also reduced the accumulation of PrP Sc in the brains of prion-infected mice at the middle stage of the disease when the drug was administered orally with their daily water from the day after infection. Docking simulation analysis suggested that alprenolol hydrochloride fitted into the hotspot within mouse PrP C , which is known as the most fragile structure within the protein. These findings provide evidence that SPRi is useful in identifying effective drug candidates for neurodegenerative diseases caused by abnormal protein aggregation, such as prion diseases.

Infrared Microspectroscopy: A Multiple-Screening Platform for Investigating Single-Cell Biochemical Perturbations upon Prion Infection

PubMed Central

2011-01-01

Prion diseases are a group of fatal neurodegenerative disorders characterized by the accumulation of prions in the central nervous system. The pathogenic prion (PrPSc) possesses the capability to convert the host-encoded cellular isoform of the prion protein, PrPC, into nascent PrPSc. The present work aims at providing novel insight into cellular response upon prion infection evidenced by synchrotron radiation infrared microspectroscopy (SR-IRMS). This non-invasive, label-free analytical technique was employed to investigate the biochemical perturbations undergone by prion infected mouse hypothalamic GT1-1 cells at the cellular and subcellular level. A decrement in total cellular protein content upon prion infection was identified by infrared (IR) whole-cell spectra and validated by bicinchoninic acid assay and single-cell volume analysis by atomic force microscopy (AFM). Hierarchical cluster analysis (HCA) of IR data discriminated between infected and uninfected cells and allowed to deduce an increment of lysosomal bodies within the cytoplasm of infected GT1-1 cells, a hypothesis further confirmed by SR-IRMS at subcellular spatial resolution and fluorescent microscopy. The purpose of this work, therefore, consists of proposing IRMS as a powerful multiscreening platform, drawing on the synergy with conventional biological assays and microscopy techniques in order to increase the accuracy of investigations performed at the single-cell level. PMID:22778865

Screening of intact yeasts and cell extracts to reduce Scrapie prions during biotransformation of food waste.

PubMed

Huyben, David; Boqvist, Sofia; Passoth, Volkmar; Renström, Lena; Allard Bengtsson, Ulrika; Andréoletti, Olivier; Kiessling, Anders; Lundh, Torbjörn; Vågsholm, Ivar

2018-02-08

Yeasts can be used to convert organic food wastes to protein-rich animal feed in order to recapture nutrients. However, the reuse of animal-derived waste poses a risk for the transmission of infectious prions that can cause neurodegeneration and fatality in humans and animals. The aim of this study was to investigate the ability of yeasts to reduce prion activity during the biotransformation of waste substrates-thereby becoming a biosafety hurdle in such a circular food system. During pre-screening, 30 yeast isolates were spiked with Classical Scrapie prions and incubated for 72 h in casein substrate, as a waste substitute. Based on reduced Scrapie seeding activity, waste biotransformation and protease activities, intact cells and cell extracts of 10 yeasts were further tested. Prion analysis showed that five yeast species reduced Scrapie seeding activity by approximately 1 log10 or 90%. Cryptococcus laurentii showed the most potential to reduce prion activity since both intact and extracted cells reduced Scrapie by 1 log10 and achieved the highest protease activity. These results show that select forms of yeast can act as a prion hurdle during the biotransformation of waste. However, the limited ability of yeasts to reduce prion activity warrants caution as a sole barrier to transmission as higher log reductions are needed before using waste-cultured yeast in circular food systems.

Phosphorylated human tau associates with mouse prion protein amyloid in scrapie-infected mice but does not increase progression of clinical disease.

PubMed

Race, Brent; Phillips, Katie; Kraus, Allison; Chesebro, Bruce

2016-07-03

Tauopathies are a family of neurodegenerative diseases in which fibrils of human hyperphosphorylated tau (P-tau) are believed to cause neuropathology. In Alzheimer disease, P-tau associates with A-beta amyloid and contributes to disease pathogenesis. In familial human prion diseases and variant CJD, P-tau often co-associates with prion protein amyloid, and might also accelerate disease progression. To test this latter possibility, here we compared progression of amyloid prion disease in vivo after scrapie infection of mice with and without expression of human tau. The mice used expressed both anchorless prion protein (PrP) and membrane-anchored PrP, that generate disease associated amyloid and non-amyloid PrP (PrPSc) after scrapie infection. Human P-tau induced by scrapie infection was only rarely associated with non-amyloid PrPSc, but abundant human P-tau was detected at extracellular, perivascular and axonal deposits associated with amyloid PrPSc. This pathology was quite similar to that seen in familial prion diseases. However, association of human and mouse P-tau with amyloid PrPSc did not diminish survival time following prion infection in these mice. By analogy, human P-tau may not affect prion disease progression in humans. Alternatively, these results might be due to other factors, including rapidity of disease, blocking effects by mouse tau, or low toxicity of human P-tau in this model.

Enzymatic Formulation Capable of Degrading Scrapie Prion under Mild Digestion Conditions

PubMed Central

Okoroma, Emeka A.; Purchase, Diane; Garelick, Hemda; Morris, Roger; Neale, Michael H.; Windl, Otto; Abiola, Oduola O.

2013-01-01

The prion agent is notoriously resistant to common proteases and conventional sterilisation procedures. The current methods known to destroy prion infectivity such as incineration, alkaline and thermal hydrolysis are harsh, destructive, environmentally polluting and potentially hazardous, thus limit their applications for decontamination of delicate medical and laboratory devices, remediation of prion contaminated environment and for processing animal by-products including specified risk materials and carcases. Therefore, an environmentally friendly, non-destructive enzymatic degradation approach is highly desirable. A feather-degrading Bacillus licheniformis N22 keratinase has been isolated which degraded scrapie prion to undetectable level of PrPSc signals as determined by Western Blot analysis. Prion infectivity was verified by ex vivo cell-based assay. An enzymatic formulation combining N22 keratinase and biosurfactant derived from Pseudomonas aeruginosa degraded PrPSc at 65°C in 10 min to undetectable level -. A time-course degradation analysis carried out at 50°C over 2 h revealed the progressive attenuation of PrPSc intensity. Test of residual infectivity by standard cell culture assay confirmed that the enzymatic formulation reduced PrPSc infectivity to undetectable levels as compared to cells challenged with untreated standard scrapie sheep prion (SSBP/1) (p-value = 0.008 at 95% confidence interval). This novel enzymatic formulation has significant potential application for prion decontamination in various environmentally friendly systems under mild treatment conditions. PMID:23874511

Infrared microspectroscopy: a multiple-screening platform for investigating single-cell biochemical perturbations upon prion infection.

PubMed

Didonna, Alessandro; Vaccari, Lisa; Bek, Alpan; Legname, Giuseppe

2011-03-16

Prion diseases are a group of fatal neurodegenerative disorders characterized by the accumulation of prions in the central nervous system. The pathogenic prion (PrP(Sc)) possesses the capability to convert the host-encoded cellular isoform of the prion protein, PrP(C), into nascent PrP(Sc). The present work aims at providing novel insight into cellular response upon prion infection evidenced by synchrotron radiation infrared microspectroscopy (SR-IRMS). This non-invasive, label-free analytical technique was employed to investigate the biochemical perturbations undergone by prion infected mouse hypothalamic GT1-1 cells at the cellular and subcellular level. A decrement in total cellular protein content upon prion infection was identified by infrared (IR) whole-cell spectra and validated by bicinchoninic acid assay and single-cell volume analysis by atomic force microscopy (AFM). Hierarchical cluster analysis (HCA) of IR data discriminated between infected and uninfected cells and allowed to deduce an increment of lysosomal bodies within the cytoplasm of infected GT1-1 cells, a hypothesis further confirmed by SR-IRMS at subcellular spatial resolution and fluorescent microscopy. The purpose of this work, therefore, consists of proposing IRMS as a powerful multiscreening platform, drawing on the synergy with conventional biological assays and microscopy techniques in order to increase the accuracy of investigations performed at the single-cell level.

Intranasal inoculation of white-tailed deer (Odocoileus virginianus) with lyophilized chronic wasting disease prion particulate complexed to montmorillonite clay

USDA-ARS?s Scientific Manuscript database

Chronic wasting disease (CWD) is the transmissible spongiform encephalopathy or TSE of deer and elk, occurring primarily in North America. The TSEs are fatal neurodegenerative disorders associated with conversion of a normal cell protein to a pathogenic and potentially infectious agent by post trans...

Disinfectants and Prions

USDA-ARS?s Scientific Manuscript database

Prions are novel pathogens that are believed to be composed solely of protein. They are capable of converting a normal cellular protein into the infectious isoform and thereby propagating an infection. Prion infections are characterized by a long asymptomatic incubation period followed by a relative...

Synthetic Prions Provide Clues for Understanding Prion Diseases.

PubMed

Imberdis, Thibaut; Harris, David A

2016-04-01

This Commentary highlights the article by Makarava et al that discusses the formation of synthetic prions and the role of substrate levels in their evolution. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

PrPC expression and prion seeding activity in the alimentary tract and lymphoid tissue of deer

PubMed Central

Davenport, Kristen A.; Hoover, Clare E.; Bian, Jifeng; Telling, Glenn C.; Mathiason, Candace K.; Hoover, Edward A.

2017-01-01

The agent responsible for prion diseases is a misfolded form of a normal protein (PrPC). The prion hypothesis stipulates that PrPC must be present for the disease to manifest. Cervid populations across the world are infected with chronic wasting disease, a horizontally-transmissible prion disease that is likely spread via oral exposure to infectious prions (PrPCWD). Though PrPCWD has been identified in many tissues, there has been little effort to characterize the overall PrPC expression in cervids and its relationship to PrPCWD accumulation. We used immunohistochemistry (IHC), western blot and enzyme-linked immunosorbent assay to describe PrPC expression in naïve white-tailed deer. We used real-time, quaking-induced conversion (RT-QuIC) to detect prion seeding activity in CWD-infected deer. We assessed tissues comprising the alimentary tract, alimentary-associated lymphoid tissue and systemic lymphoid tissue from 5 naïve deer. PrPC was expressed in all tissues, though expression was often very low compared to the level in the CNS. IHC identified specific cell types wherein PrPC expression is very high. To compare the distribution of PrPC to PrPCWD, we examined 5 deer with advanced CWD infection. Using RT-QuIC, we detected prion seeding activity in all 21 tissues. In 3 subclinical deer sacrificed 4 months post-inoculation, we detected PrPCWD consistently in alimentary-associated lymphoid tissue, irregularly in alimentary tract tissues, and not at all in the brain. Contrary to our hypothesis that PrPC levels dictate prion accumulation, PrPC expression was higher in the lower gastrointestinal tissues than in the alimentary-associated lymphoid system and was higher in salivary glands than in the oropharyngeal lymphoid tissue. These data suggest that PrPC expression is not the sole driver of prion accumulation and that alimentary tract tissues accumulate prions before centrifugal spread from the brain occurs. PMID:28880938

Prions: Beyond a Single Protein

PubMed Central

Das, Alvin S.

2016-01-01

SUMMARY Since the term protein was first coined in 1838 and protein was discovered to be the essential component of fibrin and albumin, all cellular proteins were presumed to play beneficial roles in plants and mammals. However, in 1967, Griffith proposed that proteins could be infectious pathogens and postulated their involvement in scrapie, a universally fatal transmissible spongiform encephalopathy in goats and sheep. Nevertheless, this novel hypothesis had not been evidenced until 1982, when Prusiner and coworkers purified infectious particles from scrapie-infected hamster brains and demonstrated that they consisted of a specific protein that he called a “prion.” Unprecedentedly, the infectious prion pathogen is actually derived from its endogenous cellular form in the central nervous system. Unlike other infectious agents, such as bacteria, viruses, and fungi, prions do not contain genetic materials such as DNA or RNA. The unique traits and genetic information of prions are believed to be encoded within the conformational structure and posttranslational modifications of the proteins. Remarkably, prion-like behavior has been recently observed in other cellular proteins—not only in pathogenic roles but also serving physiological functions. The significance of these fascinating developments in prion biology is far beyond the scope of a single cellular protein and its related disease. PMID:27226089

Smart protein biogate as a mediator to regulate competitive host-guest interaction for sensitive ratiometric electrochemical assay of prion

NASA Astrophysics Data System (ADS)

Yu, Peng; Zhang, Xiaohua; Zhou, Jiawan; Xiong, Erhu; Li, Xiaoyu; Chen, Jinhua

2015-11-01

A novel competitive host-guest strategy regulated by protein biogate was developed for sensitive and selective analysis of prion protein. The methylene blue (MB)-tagged prion aptamer (MB-Apt) was introduced to the multiwalled carbon nanotubes-β-cyclodextrins (MWCNTs-β-CD) composites-modified glassy carbon (GC) electrode through the host-guest interaction between β-CD and MB. In the absence of prion, MB-Apt could be displaced by ferrocenecarboxylic acid (FCA) due to its stronger binding affinity to β-CD, resulting in a large oxidation peak of FCA. However, in the presence of prion, the specific prion-aptamer interaction drove the formation of protein biogate to seal the cavity of β-CD, which hindered the guest displacement of MB by FCA and resulted in the oxidation peak current of MB (IMB) increased and that of FCA (IFCA) decreased. The developed aptasensor showed good response towards the target (prion protein) with a low detection limit of 160 fM. By changing the specific aptamers, this strategy could be easily extended to detect other proteins, showing promising potential for extensive applications in bioanalysis.

Live imaging of prions reveals nascent PrPSc in cell-surface, raft-associated amyloid strings and webs

PubMed Central

Rouvinski, Alexander; Karniely, Sharon; Kounin, Maria; Moussa, Sanaa; Goldberg, Miri D.; Warburg, Gabriela; Lyakhovetsky, Roman; Papy-Garcia, Dulce; Kutzsche, Janine; Korth, Carsten; Carlson, George A.; Godsave, Susan F.; Peters, Peter J.; Luhr, Katarina; Kristensson, Krister

2014-01-01

Mammalian prions refold host glycosylphosphatidylinositol-anchored PrPC into β-sheet–rich PrPSc. PrPSc is rapidly truncated into a C-terminal PrP27-30 core that is stable for days in endolysosomes. The nature of cell-associated prions, their attachment to membranes and rafts, and their subcellular locations are poorly understood; live prion visualization has not previously been achieved. A key obstacle has been the inaccessibility of PrP27-30 epitopes. We overcame this hurdle by focusing on nascent full-length PrPSc rather than on its truncated PrP27-30 product. We show that N-terminal PrPSc epitopes are exposed in their physiological context and visualize, for the first time, PrPSc in living cells. PrPSc resides for hours in unexpected cell-surface, slow moving strings and webs, sheltered from endocytosis. Prion strings observed by light and scanning electron microscopy were thin, micrometer-long structures. They were firmly cell associated, resisted phosphatidylinositol-specific phospholipase C, aligned with raft markers, fluoresced with thioflavin, and were rapidly abolished by anti-prion glycans. Prion strings and webs are the first demonstration of membrane-anchored PrPSc amyloids. PMID:24493590

Disease Transmission by Misfolded Prion-Protein Isoforms, Prion-Like Amyloids, Functional Amyloids and the Central Dogma

PubMed Central

Daus, Martin L.

2016-01-01

In 1982, the term “prions” (proteinaceous infectious particles) was coined to specify a new principle of infection. A misfolded isoform of a cellular protein has been described as the causative agent of a fatal neurodegenerative disease. At the beginning of prion research scientists assumed that the infectious agent causing transmissible spongiform encephalopathy (TSE) was a virus, but some unconventional properties of these pathogens were difficult to bring in line with the prevailing viral model. The discovery that prions (obviously devoid of any coding nucleic acid) can store and transmit information similarly to DNA was initially even denoted as being “heretical” but is nowadays mainly accepted by the scientific community. This review describes, from a historical point of view, how the “protein-only hypothesis” expands the Central Dogma. Definition of both, the prion principle and the Central Dogma, have been essential steps to understand information storage and transfer within and among cells and organisms. Furthermore, the current understanding of the infectivity of prion-proteins after misfolding is summarized succinctly. Finally, prion-like amyloids and functional amyloids, as found in yeast and bacteria, will be discussed. PMID:26742083

Cell-surface prion protein interacts with glycosaminoglycans.

PubMed Central

Pan, Tao; Wong, Boon-Seng; Liu, Tong; Li, Ruliang; Petersen, Robert B; Sy, Man-Sun

2002-01-01

We used ELISA and flow cytometry to study the binding of prion protein PrP to glycosaminoglycans (GAGs). We found that recombinant human PrP (rPrP) binds GAGs including chondroitin sulphate A, chondroitin sulphate B, hyaluronic acid, and heparin. rPrP binding to GAGs occurs via the N-terminus, a region known to bind divalent cations. Additionally, rPrP binding to GAGs is enhanced in the presence of Cu2+ and Zn2+, but not Ca2+ and Mn2+. rPrP binds heparin strongest, and the binding is inhibited by certain heparin analogues, including heparin disaccharide and sulphate-containing monosaccharides, but not by acetylated heparin. Full-length normal cellular prion protein (PrPC), but not N-terminally truncated PrPC species, from human brain bind GAGs in a similar Cu2+/Zn2+-enhanced fashion. We found that GAGs specifically bind to a synthetic peptide corresponding to amino acid residues 23-35 in the N-terminus of rPrP. We further demonstrated that while both wild-type PrPC and an octapeptide-repeat-deleted mutant PrP produced by transfected cells bound heparin at the cell surface, the PrP N-terminal deletion mutant and non-transfectant control failed to bind heparin. Binding of heparin to wild-type PrPC on the cell surface results in a reduction of the level of cell-surface PrPC. These results provide strong evidence that PrPC is a surface receptor for GAGs. PMID:12186633

Statistical Mechanics of Prion Diseases

NASA Astrophysics Data System (ADS)

Slepoy, A.; Singh, R. R.; Pázmándi, F.; Kulkarni, R. V.; Cox, D. L.

2001-07-01

We present a two-dimensional, lattice based, protein-level statistical mechanical model for prion diseases (e.g., mad cow disease) with concomitant prion protein misfolding and aggregation. Our studies lead us to the hypothesis that the observed broad incubation time distribution in epidemiological data reflect fluctuation dominated growth seeded by a few nanometer scale aggregates, while much narrower incubation time distributions for innoculated lab animals arise from statistical self-averaging. We model ``species barriers'' to prion infection and assess a related treatment protocol.

Theoretical Modeling of Molecular Mechanisms, Time Scales and Strains in Prion Diseases

DTIC Science & Technology

2008-01-01

transmembrane form of the prion protein is localized in the Golgi apparatus of neurons. J. Biol. Chem. 280: 15855-15864. 5 Baskakov, I.V., G. Legname, M.A...research on the prion problem, particularly on a promising new model for the in vitro mammalian prion fibril which involves a new suggestion for a C... new model for in vitro grown fibrils, new domain swapped oligomer models, and models of the influence of proline mutations on the kinetics of

CWDPRNP: A tool for cervid prion sequence analysis in program R

USGS Publications Warehouse

Miller, William L.; Walter, W. David

2017-01-01

Chronic wasting disease is a fatal, neurological disease caused by an infectious prion protein, which affects economically and ecologically important members of the family Cervidae. Single nucleotide polymorphisms within the prion protein gene have been linked to differential susceptibility to the disease in many species. Wildlife managers are seeking to determine the frequencies of disease-associated alleles and genotypes and delineate spatial genetic patterns. The CWDPRNP package, implemented in program R, provides a unified framework for analyzing prion protein gene variability and spatial structure.

Integrity of Helix 2-Helix 3 Domain of the PrP Protein Is Not Mandatory for Prion Replication*

PubMed Central

Salamat, Khalid; Moudjou, Mohammed; Chapuis, Jérôme; Herzog, Laetitia; Jaumain, Emilie; Béringue, Vincent; Rezaei, Human; Pastore, Annalisa; Laude, Hubert; Dron, Michel

2012-01-01

The process of prion conversion is not yet well understood at the molecular level. The regions critical for the conformational change of PrP remain mostly debated and the extent of sequence change acceptable for prion conversion is poorly documented. To achieve progress on these issues, we applied a reverse genetic approach using the Rov cell system. This allowed us to test the susceptibility of a number of insertion mutants to conversion into prion in the absence of wild-type PrP molecules. We were able to propagate several prions with 8 to 16 extra amino acids, including a polyglycine stretch and His or FLAG tags, inserted in the middle of the protease-resistant fragment. These results demonstrate the possibility to increase the length of the loop between helices H2 and H3 up to 4-fold, without preventing prion replication. They also indicate that this loop probably remains unstructured in PrPSc. We also showed that bona fide prions can be produced following insertion of octapeptides in the two C-terminal turns of H2. These insertions do not interfere with the overall fold of the H2-H3 domain indicating that the highly conserved sequence of the terminal part of H2 is not critical for the conversion. Altogether these data showed that the amplitude of modifications acceptable for prion conversion in the core of the globular domain of PrP is much greater than one might have assumed. These observations should help to refine structural models of PrPSc and elucidate the conformational changes underlying prions generation. PMID:22511770

Integrity of helix 2-helix 3 domain of the PrP protein is not mandatory for prion replication.

PubMed

Salamat, Khalid; Moudjou, Mohammed; Chapuis, Jérôme; Herzog, Laetitia; Jaumain, Emilie; Béringue, Vincent; Rezaei, Human; Pastore, Annalisa; Laude, Hubert; Dron, Michel

2012-06-01

The process of prion conversion is not yet well understood at the molecular level. The regions critical for the conformational change of PrP remain mostly debated and the extent of sequence change acceptable for prion conversion is poorly documented. To achieve progress on these issues, we applied a reverse genetic approach using the Rov cell system. This allowed us to test the susceptibility of a number of insertion mutants to conversion into prion in the absence of wild-type PrP molecules. We were able to propagate several prions with 8 to 16 extra amino acids, including a polyglycine stretch and His or FLAG tags, inserted in the middle of the protease-resistant fragment. These results demonstrate the possibility to increase the length of the loop between helices H2 and H3 up to 4-fold, without preventing prion replication. They also indicate that this loop probably remains unstructured in PrP(Sc). We also showed that bona fide prions can be produced following insertion of octapeptides in the two C-terminal turns of H2. These insertions do not interfere with the overall fold of the H2-H3 domain indicating that the highly conserved sequence of the terminal part of H2 is not critical for the conversion. Altogether these data showed that the amplitude of modifications acceptable for prion conversion in the core of the globular domain of PrP is much greater than one might have assumed. These observations should help to refine structural models of PrP(Sc) and elucidate the conformational changes underlying prions generation.

Genetic predictions of prion disease susceptibility in carnivore species based on variability of the prion gene coding region.

PubMed

Stewart, Paula; Campbell, Lauren; Skogtvedt, Susan; Griffin, Karen A; Arnemo, Jon M; Tryland, Morten; Girling, Simon; Miller, Michael W; Tranulis, Michael A; Goldmann, Wilfred

2012-01-01

Mammalian species vary widely in their apparent susceptibility to prion diseases. For example, several felid species developed prion disease (feline spongiform encephalopathy or FSE) during the bovine spongiform encephalopathy (BSE) epidemic in the United Kingdom, whereas no canine BSE cases were detected. Whether either of these or other groups of carnivore species can contract other prion diseases (e.g. chronic wasting disease or CWD) remains an open question. Variation in the host-encoded prion protein (PrP(C)) largely explains observed disease susceptibility patterns within ruminant species, and may explain interspecies differences in susceptibility as well. We sequenced and compared the open reading frame of the PRNP gene encoding PrP(C) protein from 609 animal samples comprising 29 species from 22 genera of the Order Carnivora; amongst these samples were 15 FSE cases. Our analysis revealed that FSE cases did not encode an identifiable disease-associated PrP polymorphism. However, all canid PrPs contained aspartic acid or glutamic acid at codon 163 which we propose provides a genetic basis for observed susceptibility differences between canids and felids. Among other carnivores studied, wolverine (Gulo gulo) and pine marten (Martes martes) were the only non-canid species to also express PrP-Asp163, which may impact on their prion diseases susceptibility. Populations of black bear (Ursus americanus) and mountain lion (Puma concolor) from Colorado showed little genetic variation in the PrP protein and no variants likely to be highly resistant to prions in general, suggesting that strain differences between BSE and CWD prions also may contribute to the limited apparent host range of the latter.

Reduced Abundance and Subverted Functions of Proteins in Prion-Like Diseases: Gained Functions Fascinate but Lost Functions Affect Aetiology.

PubMed

Allison, W Ted; DuVal, Michèle G; Nguyen-Phuoc, Kim; Leighton, Patricia L A

2017-10-24

Prions have served as pathfinders that reveal many aspects of proteostasis in neurons. The recent realization that several prominent neurodegenerative diseases spread via a prion-like mechanism illuminates new possibilities for diagnostics and therapeutics. Thus, key proteins in Alzheimer Disease and Amyotrophic lateral sclerosis (ALS), including amyloid-β precursor protein, Tau and superoxide dismutase 1 (SOD1), spread to adjacent cells in their misfolded aggregated forms and exhibit template-directed misfolding to induce further misfolding, disruptions to proteostasis and toxicity. Here we invert this comparison to ask what these prion-like diseases can teach us about the broad prion disease class, especially regarding the loss of these key proteins' function(s) as they misfold and aggregate. We also consider whether functional amyloids might reveal a role for subverted protein function in neurodegenerative disease. Our synthesis identifies SOD1 as an exemplar of protein functions being lost during prion-like protein misfolding, because SOD1 is inherently unstable and loses function in its misfolded disease-associated form. This has under-appreciated parallels amongst the canonical prion diseases, wherein the normally folded prion protein, PrP C , is reduced in abundance in fatal familial insomnia patients and during the preclinical phase in animal models, apparently via proteostatic mechanisms. Thus while template-directed misfolding and infectious properties represent gain-of-function that fascinates proteostasis researchers and defines (is required for) the prion(-like) diseases, loss and subversion of the functions attributed to hallmark proteins in neurodegenerative disease needs to be integrated into design towards effective therapeutics. We propose experiments to uniquely test these ideas.

Isolation of Novel Synthetic Prion Strains by Amplification in Transgenic Mice Coexpressing Wild-Type and Anchorless Prion Proteins

PubMed Central

Raymond, Gregory J.; Race, Brent; Hollister, Jason R.; Offerdahl, Danielle K.; Moore, Roger A.; Kodali, Ravindra; Raymond, Lynne D.; Hughson, Andrew G.; Rosenke, Rebecca; Long, Dan; Dorward, David W.

2012-01-01

Mammalian prions are thought to consist of misfolded aggregates (protease-resistant isoform of the prion protein [PrPres]) of the cellular prion protein (PrPC). Transmissible spongiform encephalopathy (TSE) can be induced in animals inoculated with recombinant PrP (rPrP) amyloid fibrils lacking mammalian posttranslational modifications, but this induction is inefficient in hamsters or transgenic mice overexpressing glycosylphosphatidylinositol (GPI)-anchored PrPC. Here we show that TSE can be initiated by inoculation of misfolded rPrP into mice that express wild-type (wt) levels of PrPC and that synthetic prion strain propagation and selection can be affected by GPI anchoring of the host's PrPC. To create prions de novo, we fibrillized mouse rPrP in the absence of molecular cofactors, generating fibrils with a PrPres-like protease-resistant banding profile. These fibrils induced the formation of PrPres deposits in transgenic mice coexpressing wt and GPI-anchorless PrPC (wt/GPI−) at a combined level comparable to that of PrPC expression in wt mice. Secondary passage into mice expressing wt, GPI−, or wt plus GPI− PrPC induced TSE disease with novel clinical, histopathological, and biochemical phenotypes. Contrary to laboratory-adapted mouse scrapie strains, the synthetic prion agents exhibited a preference for conversion of GPI− PrPC and, in one case, caused disease only in GPI− mice. Our data show that novel TSE agents can be generated de novo solely from purified mouse rPrP after amplification in mice coexpressing normal levels of wt and anchorless PrPC. These observations provide insight into the minimal elements required to create prions in vitro and suggest that the PrPC GPI anchor can modulate the propagation of synthetic TSE strains. PMID:22915801

Genetic Predictions of Prion Disease Susceptibility in Carnivore Species Based on Variability of the Prion Gene Coding Region

PubMed Central

Stewart, Paula; Campbell, Lauren; Skogtvedt, Susan; Griffin, Karen A.; Arnemo, Jon M.; Tryland, Morten; Girling, Simon; Miller, Michael W.; Tranulis, Michael A.; Goldmann, Wilfred

2012-01-01

Mammalian species vary widely in their apparent susceptibility to prion diseases. For example, several felid species developed prion disease (feline spongiform encephalopathy or FSE) during the bovine spongiform encephalopathy (BSE) epidemic in the United Kingdom, whereas no canine BSE cases were detected. Whether either of these or other groups of carnivore species can contract other prion diseases (e.g. chronic wasting disease or CWD) remains an open question. Variation in the host-encoded prion protein (PrPC) largely explains observed disease susceptibility patterns within ruminant species, and may explain interspecies differences in susceptibility as well. We sequenced and compared the open reading frame of the PRNP gene encoding PrPC protein from 609 animal samples comprising 29 species from 22 genera of the Order Carnivora; amongst these samples were 15 FSE cases. Our analysis revealed that FSE cases did not encode an identifiable disease-associated PrP polymorphism. However, all canid PrPs contained aspartic acid or glutamic acid at codon 163 which we propose provides a genetic basis for observed susceptibility differences between canids and felids. Among other carnivores studied, wolverine (Gulo gulo) and pine marten (Martes martes) were the only non-canid species to also express PrP-Asp163, which may impact on their prion diseases susceptibility. Populations of black bear (Ursus americanus) and mountain lion (Puma concolor) from Colorado showed little genetic variation in the PrP protein and no variants likely to be highly resistant to prions in general, suggesting that strain differences between BSE and CWD prions also may contribute to the limited apparent host range of the latter. PMID:23236380

hTERT-immortalized ovine microglia propagate natural scrapie isolates

USDA-ARS?s Scientific Manuscript database

Scrapie is a transmissible fatal neurodegenerative disease of sheep that is caused by a novel infectious agent called a prion. The study of prions under controlled culture conditions is crucial to advancing knowledge regarding prion processes of replication and accumulation within cells. Unfortunate...

PrPc Does Not Mediate Internalization of PrPSc but Is Required at an Early Stage for De Novo Prion Infection of Rov Cells▿

PubMed Central

Paquet, Sophie; Daude, Nathalie; Courageot, Marie-Pierre; Chapuis, Jérôme; Laude, Hubert; Vilette, Didier

2007-01-01

We have studied the interactions of exogenous prions with an epithelial cell line inducibly expressing PrPc protein and permissive to infection by a sheep scrapie agent. We demonstrate that abnormal PrP (PrPSc) and prion infectivity are efficiently internalized in Rov cells, whether or not PrPc is expressed. At odds with earlier studies implicating cellular heparan sulfates in PrPSc internalization, we failed to find any involvement of such molecules in Rov cells, indicating that prions can enter target cells by several routes. We further show that PrPSc taken up in the absence of PrPc was unable to promote efficient prion multiplication once PrPc expression was restored in the cells. This observation argues that interaction of PrPSc with PrPc has to occur early, in a specific subcellular compartment(s), and is consistent with the view that the first prion multiplication events may occur at the cell surface. PMID:17626095

Pathogenic prion protein is degraded by a manganese oxide mineral found in soils

USGS Publications Warehouse

Russo, F.; Johnson, C.J.; McKenzie, D.; Aiken, Judd M.; Pedersen, J.A.

2009-01-01

Prions, the aetiological agents of transmissible spongiform encephalopathies, exhibit extreme resistance to degradation. Soil can retain prion infectivity in the environment for years. Reactive soil components may, however, contribute to the inactivation of prions in soil. Members of the birnessite family of manganese oxides (MnO2) rank among the strongest natural oxidants in soils. Here, we report the abiotic degradation of pathogenic prion protein (PrPTSE) by a synthetic analogue of naturally occurring birnessite minerals. Aqueous MnO2 suspensions degraded the PrPTSE as evidenced by decreased immunoreactivity and diminished ability to seed protein misfolding cyclic amplification reactions. Birnessite-mediated PrPTSE degradation increased as a solution's pH decreased, consistent with the pH-dependence of the redox potential of MnO2. Exposure to 5.6 mg MnO2 ml-1 (PrPTSE:MnO2=1 : 110) decreased PrPTSE levels by ???4 orders of magnitude. Manganese oxides may contribute to prion degradation in soil environments rich in these minerals. ?? 2009 SGM.

Molecular modelling indicates that the pathological conformations of prion proteins might be beta-helical.

PubMed Central

Downing, D T; Lazo, N D

1999-01-01

Creutzfeldt-Jakob disease, kuru, scrapie and bovine spongiform encephalopathy are diseases of the mammalian central nervous system that involve the conversion of a cellular protein into an insoluble extracellular isoform. Spectroscopic studies have shown that the precursor protein contains mainly alpha-helical and random-coil conformations, whereas the prion isoform is largely in the beta conformation. The pathogenic prion is resistant to denaturation and protease digestion and can promote the conversion of the precursor protein to the pathogenic form. These properties have yet to be explained in terms of the structural conformations of the proteins. In the present study, molecular modelling showed that prion proteins could adopt the beta-helical conformation, which has been established for a number of fibrous proteins and has been suggested previously as the basis of amyloid fibrils. The beta-helical conformation provides explanations for the biophysical and biochemical stability of prions, their ability to form templates for the transmission of pathological conformation, and the existence of phenotypical strains of the prion diseases. PMID:10510313

A novel copper(II) coordination at His186 in full-length murine prion protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watanabe, Yasuko; Hiraoka, Wakako; Igarashi, Manabu

2010-04-09

To explore Cu(II) ion coordination by His{sup 186} in the C-terminal domain of full-length prion protein (moPrP), we utilized the magnetic dipolar interaction between a paramagnetic metal, Cu(II) ion, and a spin probe introduced in the neighborhood of the postulated binding site by the spin labeling technique (SDSL technique). Six moPrP mutants, moPrP(D143C), moPrP(Y148C), moPrP(E151C), moPrP(Y156C), moPrP(T189C), and moPrP(Y156C,H186A), were reacted with a methane thiosulfonate spin probe and a nitroxide residue (R1) was created in the binding site of each one. Line broadening of the ESR spectra was induced in the presence of Cu(II) ions in moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), andmore » moPrP(T189R1) but not moPrP(D143R1). This line broadening indicated the presence of electron-electron dipolar interaction between Cu(II) and the nitroxide spin probe, suggesting that each interspin distance was within 20 A. The interspin distance ranges between Cu(II) and the spin probes of moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) were estimated to be 12.1 A, 18.1 A, 10.7 A, and 8.4 A, respectively. In moPrP(Y156R1,H186A), line broadening between Cu(II) and the spin probe was not observed. These results suggest that a novel Cu(II) binding site is involved in His186 in the Helix2 region of the C-terminal domain of moPrP{sup C}.« less

Cleaning, disinfection and sterilization of surface prion contamination.

PubMed

McDonnell, G; Dehen, C; Perrin, A; Thomas, V; Igel-Egalon, A; Burke, P A; Deslys, J P; Comoy, E

2013-12-01

Prion contamination is a risk during device reprocessing, being difficult to remove and inactivate. Little is known of the combined effects of cleaning, disinfection and sterilization during a typical reprocessing cycle in clinical practice. To investigate the combination of cleaning, disinfection and/or sterilization on reducing the risk of surface prion contamination. In vivo test methods were used to study the impact of cleaning alone and cleaning combined with thermal disinfection and high- or low-temperature sterilization processes. A standardized test method, based on contamination of stainless steel wires with high titres of scrapie-infected brain homogenates, was used to determine infectivity reduction. Traditional chemical methods of surface decontamination against prions were confirmed to be effective, but extended steam sterilization was more variable. Steam sterilization alone reduced the risk of prion contamination under normal or extended exposure conditions, but did show significant variation. Thermal disinfection had no impact in these studies. Cleaning with certain defined formulations in combination with steam sterilization can be an effective prion decontamination process, in particular with alkaline formulations. Low-temperature, gaseous hydrogen peroxide sterilization was also confirmed to reduce infectivity in the presence and absence of cleaning. Prion decontamination is affected by the full reprocessing cycle used on contaminated surfaces. The correct use of defined cleaning, disinfection and sterilization methods as tested in this report in the scrapie infectivity assay can provide a standard precaution against prion contamination. Copyright © 2013 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Mechanism of Scrapie Prion Precipitation with Phosphotungstate Anions

PubMed Central

2015-01-01

The phosphotungstate anion (PTA) is widely used to facilitate the precipitation of disease-causing prion protein (PrPSc) from infected tissue for applications in structural studies and diagnostic approaches. However, the mechanism of this precipitation is not understood. In order to elucidate the nature of the PTA interaction with PrPSc under physiological conditions, solutions of PTA were characterized by NMR spectroscopy at varying pH. At neutral pH, the parent [PW12O40]3– ion decomposes to give a lacunary [PW11O39]7– (PW11) complex and a single orthotungstate anion [WO4]2– (WO4). To measure the efficacy of each component of PTA, increasing concentrations of PW11, WO4, and mixtures thereof were used to precipitate PrPSc from brain homogenates of scrapie prion-infected mice. The amount of PrPSc isolated, quantified by ELISA and immunoblotting, revealed that both PW11 and WO4 contribute to PrPSc precipitation. Incubation with sarkosyl, PTA, or individual components of PTA resulted in separation of higher-density PrP aggregates from the neuronal lipid monosialotetrahexosylganglioside (GM1), as observed by sucrose gradient centrifugation. These experiments revealed that yield and purity of PrPSc were greater with polyoxometalates (POMs), which substantially supported the separation of lipids from PrPSc in the samples. Interaction of POMs and sarkosyl with brain homogenates promoted the formation of fibrillar PrPSc aggregates prior to centrifugation, likely through the separation of lipids like GM1 from PrPSc. We propose that this separation of lipids from PrP is a major factor governing the facile precipitation of PrPSc by PTA from tissue and might be optimized further for the detection of prions. PMID:25695325

ERK1/2 and p38 MAP kinases control prion protein fragment 90-231-induced astrocyte proliferation and microglia activation.

PubMed

Thellung, Stefano; Villa, Valentina; Corsaro, Alessandro; Pellistri, Francesca; Venezia, Valentina; Russo, Claudio; Aceto, Antonio; Robello, Mauro; Florio, Tullio

2007-11-01

Astrogliosis and microglial activation are a common feature during prion diseases, causing the release of chemoattractant and proinflammatory factors as well as reactive free radicals, involved in neuronal degeneration. The recombinant protease-resistant domain of the prion protein (PrP90-231) displays in vitro neurotoxic properties when refolded in a beta-sheet-rich conformer. Here, we report that PrP90-231 induces the secretion of several cytokines, chemokines, and nitric oxide (NO) release, in both type I astrocytes and microglial cells. PrP90-231 elicited in both cell types the activation of ERK1/2 MAP kinase that displays, in astrocytes, a rapid kinetics and a proliferative response. Conversely, in microglia, PrP90-231-dependent MAP kinase activation was delayed and long lasting, inducing functional activation and growth arrest. In microglial cells, NO release, dependent on the expression of the inducible NO synthase (iNOS), and the secretion of the chemokine CCL5 were Ca(2+) dependent and under the control of the MAP kinases ERK1/2 and p38: ERK1/2 inhibition, using PD98059, reduced iNOS expression, while p38 blockade by PD169316 inhibited CCL5 release. In summary, we demonstrate that glial cells are activated by extracellular misfolded PrP90-231 resulting in a proliferative/secretive response of astrocytes and functional activation of microglia, both dependent on MAP kinase activation. In particular, in microglia, PrP90-231 activated a complex signalling cascade involved in the regulation of NO and chemokine release. These data argue in favor of a causal role for misfolded prion protein in sustaining glial activation and, possibly, glia-mediated neuronal death.

Dependence of intramolecular electron-transfer rates on driving force, pH, and temperature in ammineruthenium-modified ferrocytochromes c

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wishart, J.F.; Sun, J.; Su, C.

1997-01-23

Several ruthenium ammine complexes were used to modify horse-heart cytochrome c at histidine-33, creating a series of (NH{sub 3}){sub 4}(L)Ru-Cyt c derivatives (L = H{sub 2}O/OH{sup -}, ammonia, 4-ethylpyridine, 3,5-lutidine, pyridine, isonicotinamide, N-methylpyrazinium) with a wide range of driving forces for Fe-to-Ru electron transfer (-{Delta}G{degree} = -0.125 to +0.46 eV). Electron-transfer rates and activation parameters were measured by pulse radiolysis using azide or carbonate radicals. The driving-force dependence of electron-transfer rates between redox centers of the same charge types obeys Marcus-Hush theory. The activationless rate limit for all of the ruthenium derivatives except the N-methylpyrazinium complex is 3.9x10{sup 5} s{supmore » -1}. Thermodynamic parameters obtained from nonisothermal differential pulse voltammetry show that the electron-transfer reactions are entropy-driven. The thermodynamic and kinetic effects of phosphate ion binding to the ruthenium center are examined. The rate of intramolecular electron transfer in (NH{sub 3}){sub 4}(isn)Ru{sup III}-Cyt c{sup II} decreases at high pH, with a midpoint at pH 9.1. 28 refs., 4 figs., 3 tabs.« less

Prion peripheralization is a host-driven trait of prion infection, independent of strain

USDA-ARS?s Scientific Manuscript database

Chronic wasting disease (CWD), like scrapie of sheep, is a horizontally transmissible spongiform encephalopathy. Proposed natural routes of transmission for both agents include saliva, urine, and feces, and are likely related to an accumulation of misfolded prion proteins in peripheral excretory tis...

Limited amplification of chronic wasting disease prions in the peripheral tissues of intracerebrally inoculated cattle

USDA-ARS?s Scientific Manuscript database

Chronic wasting disease (CWD) is a fatal neurodegenerative disease, classified as a prion disease or transmissible spongiform encephalopathy (TSE) similar to bovine spongiform encephalopathy (BSE). Cervids affected by CWD accumulate an abnormal protease resistant prion protein throughout the central...

Determining the relative susceptibility of four prion protein genotypes to atypical scrapie

USDA-ARS?s Scientific Manuscript database

Atypical scrapie is a sheep prion (PrPSc) disease whose epidemiology is consistent with a sporadic origin and is associated with specific polymorphisms of the normal cellular prion protein (PrPC). We describe a mass spectrometry-based method of detecting and quantifying the polymorphisms of sheep P...

Disparate modes of evolution shaped modern prion (PRNP) and prion-related doppel (PRND) variation in domestic cattle

USDA-ARS?s Scientific Manuscript database

Previous investigations aimed at determining whether the mammalian prion protein actually facilitates tangible molecular aspects of either a discrete or pleiotropic functional niche have been debated, especially given the apparent absence of overt behavioral or physiological phenotypes associated wi...

Cross-species transmission of CWD prions.

PubMed

Kurt, Timothy D; Sigurdson, Christina J

2016-01-01

Prions cause fatal neurodegenerative diseases in humans and animals and can be transmitted zoonotically. Chronic wasting disease (CWD) is a highly transmissible prion disease of wild deer and elk that affects cervids over extensive regions of the United States and Canada. The risk of cross-species CWD transmission has been experimentally evaluated in a wide array of mammals, including non-human primates and mouse models expressing human cellular prion protein. Here we review the determinants of cross-species CWD transmission, and propose a model that may explain a structural barrier for CWD transmission to humans.

Biosafety of Prions.

PubMed

Bistaffa, Edoardo; Rossi, Martina; De Luca, Chiara M G; Moda, Fabio

2017-01-01

Prions are the infectious agents that cause devastating and untreatable disorders known as Transmissible Spongiform Encephalopathies (TSEs). The pathologic events and the infectious nature of these transmissible agents are not completely understood yet. Due to the difficulties in inactivating prions, working with them requires specific recommendations and precautions. Moreover, with the advent of innovative technologies, such as the Protein Misfolding Cyclic Amplification (PMCA) and the Real Time Quaking-Induced Conversion (RT-QuIC), prions could be amplified in vitro and the infectious features of the amplified products need to be carefully assessed. © 2017 Elsevier Inc. All rights reserved.

Prion Diseases as Transmissible Zoonotic Diseases

PubMed Central

Lee, Jeongmin; Kim, Su Yeon; Hwang, Kyu Jam; Ju, Young Ran; Woo, Hee-Jong

2013-01-01

Prion diseases, also called transmissible spongiform encephalopathies (TSEs), lead to neurological dysfunction in animals and are fatal. Infectious prion proteins are causative agents of many mammalian TSEs, including scrapie (in sheep), chronic wasting disease (in deer and elk), bovine spongiform encephalopathy (BSE; in cattle), and Creutzfeldt–Jakob disease (CJD; in humans). BSE, better known as mad cow disease, is among the many recently discovered zoonotic diseases. BSE cases were first reported in the United Kingdom in 1986. Variant CJD (vCJD) is a disease that was first detected in 1996, which affects humans and is linked to the BSE epidemic in cattle. vCJD is presumed to be caused by consumption of contaminated meat and other food products derived from affected cattle. The BSE epidemic peaked in 1992 and decreased thereafter; this decline is continuing sharply owing to intensive surveillance and screening programs in the Western world. However, there are still new outbreaks and/or progression of prion diseases, including atypical BSE, and iatrogenic CJD and vCJD via organ transplantation and blood transfusion. This paper summarizes studies on prions, particularly on prion molecular mechanisms, BSE, vCJD, and diagnostic procedures. Risk perception and communication policies of the European Union for the prevention of prion diseases are also addressed to provide recommendations for appropriate government policies in Korea. PMID:24159531

The Endoplasmic Reticulum Chaperone GRP78/BiP Modulates Prion Propagation in vitro and in vivo.

PubMed

Park, Kyung-Won; Eun Kim, Gyoung; Morales, Rodrigo; Moda, Fabio; Moreno-Gonzalez, Ines; Concha-Marambio, Luis; Lee, Amy S; Hetz, Claudio; Soto, Claudio

2017-03-23

Prion diseases are fatal neurodegenerative disorders affecting several mammalian species, characterized by the accumulation of the misfolded form of the prion protein, which is followed by the induction of endoplasmic reticulum (ER) stress and the activation of the unfolded protein response (UPR). GRP78, also called BiP, is a master regulator of the UPR, reducing ER stress levels and apoptosis due to an enhancement of the cellular folding capacity. Here, we studied the role of GRP78 in prion diseases using several in vivo and in vitro approaches. Our results show that a reduction in the expression of this molecular chaperone accelerates prion pathogenesis in vivo. In addition, we observed that prion replication in cell culture was inversely related to the levels of expression of GRP78 and that both proteins interact in the cellular context. Finally, incubation of PrP Sc with recombinant GRP78 led to the dose-dependent reduction of protease-resistant PrP Sc in vitro. Our results uncover a novel role of GRP78 in reducing prion pathogenesis, suggesting that modulating its levels/activity may offer a novel opportunity for designing therapeutic approaches for these diseases. These findings may also have implications for other diseases involving the accumulation of misfolded proteins.

Detection and Control of Prion Diseases in Food Animals

PubMed Central

Hedlin, Peter; Taschuk, Ryan; Potter, Andrew; Griebel, Philip; Napper, Scott

2012-01-01

Transmissible spongiform encephalopathies (TSEs), or prion diseases, represent a unique form of infectious disease based on misfolding of a self-protein (PrPC) into a pathological, infectious conformation (PrPSc). Prion diseases of food animals gained notoriety during the bovine spongiform encephalopathy (BSE) outbreak of the 1980s. In particular, disease transmission to humans, to the generation of a fatal, untreatable disease, elevated the perspective on livestock prion diseases from food production to food safety. While the immediate threat posed by BSE has been successfully addressed through surveillance and improved management practices, another prion disease is rapidly spreading. Chronic wasting disease (CWD), a prion disease of cervids, has been confirmed in wild and captive populations with devastating impact on the farmed cervid industries. Furthermore, the unabated spread of this disease through wild populations threatens a natural resource that is a source of considerable economic benefit and national pride. In a worst-case scenario, CWD may represent a zoonotic threat either through direct transmission via consumption of infected cervids or through a secondary food animal, such as cattle. This has energized efforts to understand prion diseases as well as to develop tools for disease detection, prevention, and management. Progress in each of these areas is discussed. PMID:23738120

Distinct prion-like strains of amyloid beta implicated in phenotypic diversity of Alzheimer's disease.

PubMed

Cohen, Mark; Appleby, Brian; Safar, Jiri G

2016-01-01

Vast evidence on human prions demonstrates that variable disease phenotypes, rates of propagation, and targeting of distinct brain structures are determined by unique conformers (strains) of pathogenic prion protein (PrP(Sc)). Recent progress in the development of advanced biophysical tools that inventory structural characteristics of amyloid beta (Aβ) in the brain cortex of phenotypically diverse Alzheimer's disease (AD) patients, revealed unique spectrum of oligomeric particles in the cortex of rapidly progressive cases, implicating these structures in variable rates of propagation in the brain, and in distict disease manifestation. Since only ∼30% of phenotypic diversity of AD can be explained by polymorphisms in risk genes, these and transgenic bioassay data argue that structurally distinct Aβ particles play a major role in the diverse pathogenesis of AD, and may behave as distinct prion-like strains encoding diverse phenotypes. From these observations and our growing understanding of prions, there is a critical need for new strain-specific diagnostic strategies for misfolded proteins causing these elusive disorders. Since targeted drug therapy can induce mutation and evolution of prions into new strains, effective treatments of AD will require drugs that enhance clearance of pathogenic conformers, reduce the precursor protein, or inhibit the conversion of precursors into prion-like states.

Dissociation of recombinant prion autocatalysis from infectivity.

PubMed

Noble, Geoffrey P; Supattapone, Surachai

2015-01-01

Within the mammalian prion field, the existence of recombinant prion protein (PrP) conformers with self-replicating (ie. autocatalytic) activity in vitro but little to no infectious activity in vivo challenges a key prediction of the protein-only hypothesis of prion replication--that autocatalytic PrP conformers should be infectious. To understand this dissociation of autocatalysis from infectivity, we recently performed a structural and functional comparison between a highly infectious and non-infectious pair of autocatalytic recombinant PrP conformers derived from the same initial prion strain. (1) We identified restricted, C-terminal structural differences between these 2 conformers and provided evidence that these relatively subtle differences prevent the non-infectious conformer from templating the conversion of native PrP(C) substrates containing a glycosylphosphatidylinositol (GPI) anchor. (1) In this article we discuss a model, consistent with these findings, in which recombinant PrP, lacking post-translational modifications and associated folding constraints, is capable of adopting a wide variety of autocatalytic conformations. Only a subset of these recombinant conformers can be adopted by post-translationally modified native PrP(C), and this subset represents the recombinant conformers with high specific infectivity. We examine this model's implications for the generation of highly infectious recombinant prions and the protein-only hypothesis of prion replication.

Nanomedicine for prion disease treatment: new insights into the role of dendrimers.

PubMed

McCarthy, James M; Appelhans, Dietmar; Tatzelt, Jörg; Rogers, Mark S

2013-01-01

Despite their devastating impact, no effective therapeutic yet exists for prion diseases at the symptomatic stage in humans or animals. Progress is hampered by the difficulty in identifying compounds that affect PrP (Sc) and the necessity of any potential therapeutic to gain access to the CNS. Synthetic polymers known as dendrimers are a particularly promising candidate in this area. Studies with cell culture models of prion disease and prion infected brain homogenate have demonstrated that numerous species of dendrimers eliminate PrP (Sc) in a dose and time dependent fashion and specific glycodendrimers are capable of crossing the CNS. However, despite their potential a number of important questions remained unanswered such as what makes an effective dendrimer and how dendrimers eliminate prions intracellularly. In a number of recent studies we have tackled these questions and revealed for the first time that a specific dendrimer can inhibit the intracellular conversion of PrP (C) to PrP (Sc) and that a high density of surface reactive groups is a necessity for dendrimers in vitro anti-prion activity. Understanding how a therapeutic works is a vital component in maximising its activity and these studies therefore represent a significant development in the race to find effective treatments for prion diseases.

Structural variants of yeast prions show conformer-specific requirements for chaperone activity

PubMed Central

Stein, Kevin C.; True, Heather L.

2016-01-01

Summary Molecular chaperones monitor protein homeostasis and defend against the misfolding and aggregation of proteins that is associated with protein conformational disorders. In these diseases, a variety of different aggregate structures can form. These are called prion strains, or variants, in prion diseases, and cause variation in disease pathogenesis. Here, we use variants of the yeast prions [RNQ+] and [PSI+] to explore the interactions of chaperones with distinct aggregate structures. We found that prion variants show striking variation in their relationship with Hsp40s. Specifically, the yeast Hsp40 Sis1, and its human ortholog Hdj1, had differential capacities to process prion variants, suggesting that Hsp40 selectivity has likely changed through evolution. We further show that such selectivity involves different domains of Sis1, with some prion conformers having a greater dependence on particular Hsp40 domains. Moreover, [PSI+] variants were more sensitive to certain alterations in Hsp70 activity as compared to [RNQ+] variants. Collectively, our data indicate that distinct chaperone machinery is required, or has differential capacity, to process different aggregate structures. Elucidating the intricacies of chaperone-client interactions, and how these are altered by particular client structures, will be crucial to understanding how this system can go awry in disease and contribute to pathological variation. PMID:25060529

Propagation of prion strains through specific conformers of the prion protein.

PubMed Central

Scott, M R; Groth, D; Tatzelt, J; Torchia, M; Tremblay, P; DeArmond, S J; Prusiner, S B

1997-01-01

Two prion strains with identical incubation periods in mice exhibited distinct incubation periods and different neuropathological profiles upon serial transmission to transgenic mice expressing chimeric Syrian hamster/mouse (MH2M) prion protein (PrP) genes [Tg(MH2M) mice] and subsequent transmission to Syrian hamsters. After transmission to Syrian hamsters, the Me7 strain was indistinguishable from the previously established Syrian hamster strain Sc237, despite having been derived from an independent ancestral source. This apparent convergence suggests that prion diversity may be limited. The Me7 mouse strain could also be transmitted directly to Syrian hamsters, but when derived in this way, its properties were distinct from those of Me7 passaged through Tg(MH2M) mice. The Me7 strain did not appear permanently altered in either case, since the original incubation period could be restored by effectively reversing the series of passages. Prion diversity enciphered in the conformation of the scrapie isoform of PrP (PrP(Sc)) (G. C. Telling et al., Science 274:2079-2082, 1996) seems to be limited by the sequence of the PrP substrates serially converted into PrP(Sc), while prions are propagated through interactions between the cellular and scrapie isoforms of PrP. PMID:9371560

Structure Prediction of Protein Complexes

NASA Astrophysics Data System (ADS)

Pierce, Brian; Weng, Zhiping

Protein-protein interactions are critical for biological function. They directly and indirectly influence the biological systems of which they are a part. Antibodies bind with antigens to detect and stop viruses and other infectious agents. Cell signaling is performed in many cases through the interactions between proteins. Many diseases involve protein-protein interactions on some level, including cancer and prion diseases.

Two new Co(II)-MOFs based on polymeric chain building units: Crystal structures, and magnetic properties

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Min-Le; Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of the Ministry of Education, College of Chemistry & Materials Science, Northwest University, Xi’an 710069; Wu, Ya-Pan

2015-10-15

Two new Co(II) metal-organic frameworks, namely [Co{sub 2}(bpm)(H{sub 2}O){sub 3}(L)]{sub n} (1) and ([Co(bpe)(H{sub 2}O)(H{sub 2}L)]·(bpe){sub 0.5}·(H{sub 2}O)){sub n} (2), (H{sub 4}L=1,1′:2′,1″-terphenyl-3,3″,4′,5′-tetracarboxylic acid, bpm=bis(4-pyridyl)amine, bpe=1,2-bis(4-pyridyl)ethene), have been obtained under hydrothermal conditions. Both complexes 1 and 2 have been characterized by elemental analysis, IR spectra, single-crystal X-ray diffraction, powder X-ray diffraction (PXRD), and thermogravimetric analysis (TGA). Complexes 1 and 2 consist of 1D Co(II) chains bridging by carboxylate groups in syn-anti fashion. 1 shows a novel 3D tri-nodal (4,6,10)-connected net with a (3.4{sup 3}.5{sup 2}){sub 2}(3{sup 2}.4{sup 14}.5{sup 12}.6{sup 12}.7{sup 4}.8)(3{sup 2}.4{sup 2}.5{sup 5}.6{sup 4}.7{sup 2}) topology. While 2 exhibits amore » 2D sql layer. Magnetic susceptibility measurements indicate that both 1 and 2 show weak antiferromagnetic interactions between the adjacent Co(II) ions in 300–8 K for 1 and 300–16 K for 2, respectively, and then 2 also displays ferromagnetic coupling at lower temperatures. - Graphical abstract: Two Co(II) coordination polymers with similar metal chain bridging by carboxylates in syn-anti fashion have been synthesized. Both 1 and 2 show weak antiferromagnetic interactions in high temperature, and then 2 also displays ferromagnetic coupling at lower temperatures. - Highlights: • Two Co(II) coordination polymers with similar metal chain bridging by carboxylates. • A novel 3D (4,6,10)-connected net and a 2D sql layers. • A antiferromagnetic coupling at high temperature for 1 and 2. • A ferromagnetic coupling at lower temperature for 2.« less

Orally administered indomethacin acutely reduces cellular prion protein in the small intestine and modestly increases survival of mice exposed to infectious prions.

PubMed

Martin, Gary R; Sharkey, Keith A; Jirik, Frank R

2015-05-01

The oral uptake of infectious prions represents a common way to acquire a prion disease; thus, host factors, such as gut inflammation and intestinal "leakiness", have the potential to influence infectivity. For example, the ingestion of nonsteroidal anti-inflammatory drugs (NSAIDs) is known to induce intestinal inflammation and increase intestinal permeability. Previously, we reported that normal cellular prion protein (PrP(C)) expression was increased in experimental colitis, and since the level of PrP(C) expressed is a determinant of prion disease propagation, we hypothesized that NSAID administration prior to the oral inoculation of mice with infectious prions would increase intestinal PrP(C) expression and accelerate the onset of neurological disease. In the long-term experiments, one group of mice was gavaged with indomethacin, followed by a second gavage with brain homogenate containing mouse-adapted scrapie (ME7). Control mice received ME7 brain homogenate alone. Brain and splenic tissues were harvested at several time points for immunoblotting, including at the onset of clinical signs of disease. In a second series of experiments, mice were gavaged with indomethacin to assess the acute effects of this treatment on intestinal PrP(C) expression. Acutely, NSAID treatment reduced intestinal PrP(C) expression, and chronically, there was a modest delay in the onset of neurological disease. In contrast to our hypothesis, brief exposure to an NSAID decreased intestinal PrP(C) expression and led to a modest survival advantage following oral ingestion of infectious prions.

Transmission of chronic wasting disease identifies a prion strain causing cachexia and heart infection in hamsters.

PubMed

Bessen, Richard A; Robinson, Cameron J; Seelig, Davis M; Watschke, Christopher P; Lowe, Diana; Shearin, Harold; Martinka, Scott; Babcock, Alex M

2011-01-01

Chronic wasting disease (CWD) is an emerging prion disease of free-ranging and captive cervids in North America. In this study we established a rodent model for CWD in Syrian golden hamsters that resemble key features of the disease in cervids including cachexia and infection of cardiac muscle. Following one to three serial passages of CWD from white-tailed deer into transgenic mice expressing the hamster prion protein gene, CWD was subsequently passaged into Syrian golden hamsters. In one passage line there were preclinical changes in locomotor activity and a loss of body mass prior to onset of subtle neurological symptoms around 340 days. The clinical symptoms included a prominent wasting disease, similar to cachexia, with a prolonged duration. Other features of CWD in hamsters that were similar to cervid CWD included the brain distribution of the disease-specific isoform of the prion protein, PrP(Sc), prion infection of the central and peripheral neuroendocrine system, and PrP(Sc) deposition in cardiac muscle. There was also prominent PrP(Sc) deposition in the nasal mucosa on the edge of the olfactory sensory epithelium with the lumen of the nasal airway that could have implications for CWD shedding into nasal secretions and disease transmission. Since the mechanism of wasting disease in prion diseases is unknown this hamster CWD model could provide a means to investigate the physiological basis of cachexia, which we propose is due to a prion-induced endocrinopathy. This prion disease phenotype has not been described in hamsters and we designate it as the 'wasting' or WST strain of hamster CWD.

PRION-1 scales analysis supports use of functional outcome measures in prion disease

PubMed Central

Mead, S.; Ranopa, M.; Gopalakrishnan, G.S.; Thompson, A.G.B.; Rudge, P.; Wroe, S.; Kennedy, A.; Hudson, F.; MacKay, A.; Darbyshire, J.H.; Walker, A.S.

2011-01-01

Objectives: Human prion diseases are heterogeneous but invariably fatal neurodegenerative disorders with no known effective therapy. PRION-1, the largest clinical trial in prion disease to date, showed no effect of the potential therapeutic quinacrine on survival. Although there are several limitations to the usefulness of survival as an outcome measure, there have been no comprehensive studies of alternatives. Methods: To address this we did comparative analyses of neurocognitive, psychiatric, global, clinician-rated, and functional scales, focusing on validity, variability, and impact on statistical power over 77 person-years follow-up in 101 symptomatic patients in PRION-1. Results: Quinacrine had no demonstrable benefit on any of the 8 scales (p > 0.4). All scales had substantial numbers of patients with the worst possible score at enrollment (Glasgow Coma Scale score being least affected) and were impacted by missing data due to disease progression. These effects were more significant for cognitive/psychiatric scales than global, clinician-rated, or functional scales. The Barthel and Clinical Dementia Rating scales were the most valid and powerful in simulated clinical trials of an effective therapeutic. A combination of selected subcomponents from these 2 scales gave somewhat increased power, compared to use of survival, to detect clinically relevant effects in future clinical trials of feasible size. Conclusions: Our findings have implications for the choice of primary outcome measure in prion disease clinical trials. Prion disease presents the unusual opportunity to follow patients with a neurodegenerative disease through their entire clinical course, and this provides insights relevant to designing outcome measures in related conditions. PMID:22013183

Molecular approaches to detecting and discriminating among prions, a class of pathogenic molecules(Abstract)

USDA-ARS?s Scientific Manuscript database

Prions (PrPSc)are the pathogens that cause a set of fatal neurological diseases that include scrapie and chronic wasting disease (CWD). They are composed solely of protein and unlike viral, bacterial, or fungal pathogens, the information necessary to convert the normal cellular prion protein (PrPC) ...

Genetic variation of the prion protein gene (PRNP) in alpaca (Vicugna pacos)

USDA-ARS?s Scientific Manuscript database

Transmissible spongiform encephalopathies (TSE) are caused by accumulation of a misfolded form of the prion protein (PrP). The normal cellular isoform of PrP is produced by the prion gene (PRNP) and is highly expressed in the central nervous system. Currently, there is an absence of information rega...

Does methionine oxidation influence the progression of classical or atypical scrapie

USDA-ARS?s Scientific Manuscript database

Introduction Prions are pathological proteins that propagate by converting a normal cellular prion protein (PrPC) into a prion (PrPSc). PrPC and PrPSc possess identical covalent structures and only differ in their conformations. The conversion of the PrPC conformation to the PrPSc one is template ...

Transmission of chronic wasting disease of white-tailed deer to Suffolk sheep following intracranial inoculation

USDA-ARS?s Scientific Manuscript database

Background: Interspecies transmission studies are an opportunity to better understand the potential host ranges of prion diseases. Chronic wasting disease (CWD) of cervids and scrapie of sheep and goats have a similar tissue distribution of abnormal prion protein (PrPSc) and prion disease exposure a...

Resistance of fallow deer (dama dama) to chronic wasting disease under natural exposure in a heavily contaminated environment

USDA-ARS?s Scientific Manuscript database

Chronic wasting disease or CWD is a transmissible spongiform encephalopathy or prion disorder of cervid ruminants in several regions of the US and Canada. The prion disorders are characterized by misfolding of the host cellular prion protein into a relatively protease resistant and potentially neur...

Disease-associated prion protein in neural and lymphoid tissues of mink (Mustela vison) inoculated with transmissible mink encephalopathy

USDA-ARS?s Scientific Manuscript database

Transmissible mink encephalopathy (TME) is a prion disorder of farmed raised mink. As with the other transmissible spongiform encephalopathies, the disorder is associated with accumulation of the misfolded prion protein in the brain and an invariably fatal outcome. TME outbreaks have been rare but...

Detection of BSE prions by RT-QuIC in cattle with subclinical BSE

USDA-ARS?s Scientific Manuscript database

Bovine spongiform encephalopathy (BSE) belongs to a group of fatal prion diseases that result from the misfolding of the cellular prion protein (PrPC) into a pathogenic form (PrPSc) that accumulates in the brain and some lymphoid tissues. In vitro assays such as serial protein misfolding amplificati...

The first report of prion-related protein gene (PRNT) polymorphisms in goat.

PubMed

Kim, Yong-Chan; Jeong, Byung-Hoon

2017-06-01

Prion protein is encoded by the prion protein gene (PRNP). Polymorphisms of several members of the prion gene family have shown association with prion diseases in several species. Recent studies on a novel member of the prion gene family in rams have shown that prion-related protein gene (PRNT) has a linkage with codon 26 of prion-like protein (PRND). In a previous study, codon 26 polymorphism of PRND has shown connection with PRNP haplotype which is strongly associated with scrapie vulnerability. In addition, the genotype of a single nucleotide polymorphism (SNP) at codon 26 of PRND is related to fertilisation capacity. These findings necessitate studies on the SNP of PRNT gene which is connected with PRND. In goat, several polymorphism studies have been performed for PRNP, PRND, and shadow of prion protein gene (SPRN). However, polymorphism on PRNT has not been reported. Hence, the objective of this study was to determine the genotype and allelic distribution of SNPs of PRNT in 238 Korean native goats and compare PRNT DNA sequences between Korean native goats and several ruminant species. A total of five SNPs, including PRNT c.-114G > T, PRNT c.-58A > G in the upstream of PRNT gene, PRNT c.71C > T (p.Ala24Val) and PRNT c.102G > A in the open reading frame (ORF) and c.321C > T in the downstream of PRNT gene, were found in this study. All five SNPs of caprine PRNT gene in Korean native goat are in complete linkage disequilibrium (LD) with a D' value of 1.0. Interestingly, comparative sequence analysis of the PRNT gene revealed five mismatches between DNA sequences of Korean native goats and those of goats deposited in the GenBank. Korean native black goats also showed 5 mismatches in PRNT ORF with cattle. To the best of our knowledge, this is the first genetic research of the PRNT gene in goat.

Primary transmission of chronic wasting disease versus scrapie prions from small ruminants to transgenic mice expressing ovine or cervid prion protein.

PubMed

Madsen-Bouterse, Sally A; Schneider, David A; Zhuang, Dongyue; Dassanayake, Rohana P; Balachandran, Aru; Mitchell, Gordon B; O'Rourke, Katherine I

2016-09-01

Development of mice expressing either ovine (Tg338) or cervid (TgElk) prion protein (PrP) have aided in characterization of scrapie and chronic wasting disease (CWD), respectively. Experimental inoculation of sheep with CWD prions has demonstrated the potential for interspecies transmission but, infection with CWD versus classical scrapie prions may be difficult to differentiate using validated diagnostic platforms. In this study, mouse bioassay in Tg338 and TgElk was utilized to evaluate transmission of CWD versus scrapie prions from small ruminants. Mice (≥5 per homogenate) were inoculated with brain homogenates from clinically affected sheep or goats with naturally acquired classical scrapie, white-tailed deer with naturally acquired CWD (WTD-CWD) or sheep with experimentally acquired CWD derived from elk (sheep-passaged-CWD). Survival time (time to clinical disease) and attack rates (brain accumulation of protease resistant PrP, PrPres) were determined. Inoculation with classical scrapie prions resulted in clinical disease and 100 % attack rates in Tg338, but no clinical disease at endpoint (>300 days post-inoculation, p.i.) and low attack rates (6.8 %) in TgElk. Inoculation with WTD-CWD prions yielded no clinical disease or brain PrPres accumulation in Tg338 at endpoint (>500 days p.i.), but rapid onset of clinical disease (~121 days p.i.) and 100 % attack rate in TgElk. Sheep-passaged-CWD resulted in transmission to both mouse lines with 100 % attack rates at endpoint in Tg338 and an attack rate of ~73 % in TgElk with some culled due to clinical disease. These primary transmission observations demonstrate the potential of bioassay in Tg338 and TgElk to help differentiate possible infection with CWD versus classical scrapie prions in sheep and goats.

Biochemical Characterization of Prions.

PubMed

Fiorini, Michele; Bongianni, Matilde; Monaco, Salvatore; Zanusso, Gianluigi

2017-01-01

Prion disease or transmissible spongiform encephalopathies are characterized by the presence of the abnormal form of the prion protein (PrP Sc ). The pathological and transmissible properties of PrP Sc are enciphered in its secondary and tertiary structures. Since it's well established that different strains of prions are linked to different conformations of PrP Sc , biochemical characterization of prions seems a preliminary but reliable approach to detect, analyze, and compare prion strains. Experimental biochemical procedures might be helpful in distinguishing PrP Sc physicochemical properties and include resistance to proteinase K (PK) digestion, insolubility in nonionic detergents, PK-resistance under denaturing conditions and sedimentation properties in sucrose gradients. This biochemical approach has been extensively applied in human prion disorders and subsequently expanded for PrP Sc characterization in animals. In particular, in sporadic Creutzfedlt-Jakob disease (sCJD) PrP Sc is characterized by two main glycotypes conventionally named Type 1 and Type 2, based on the apparent gel migration at 21 and 19kDa of the PrP Sc PK-resistant fragment. An additional PrP Sc type was identified in sCJD characterized by an unglycosylated dominant glycoform pattern and in 2010 a variably protease-sensitive prionopathy (VPSPr) was reported showing a PrP Sc with an electrophoretic ladder like pattern. Additionally, the presence of PrP Sc truncated fragments completes the electrophoretic characterization of different prion strains. By two-dimensional (2D) electrophoretic analysis additional PrP Sc pattern was identified, since this procedure provides information about the isoelectric point and the different peptides length related to PK cleavage, as well as to glycosylation extent or GPI anchor presence. We here provide and extensive review on PrP Sc biochemical analysis in human and animal prion disorders. Further, we show that PrP Sc glycotypes observed in CJD share similarities with PrP Sc in bovine spongiform encephalopathy forms (BSE). © 2017 Elsevier Inc. All rights reserved.

Inactivation of Prions and Amyloid Seeds with Hypochlorous Acid

PubMed Central

Kraus, Allison; Phillips, Katie; Contreras, Luis; Zanusso, Gianluigi; Caughey, Byron

2016-01-01

Hypochlorous acid (HOCl) is produced naturally by neutrophils and other cells to kill conventional microbes in vivo. Synthetic preparations containing HOCl can also be effective as microbial disinfectants. Here we have tested whether HOCl can also inactivate prions and other self-propagating protein amyloid seeds. Prions are deadly pathogens that are notoriously difficult to inactivate, and standard microbial disinfection protocols are often inadequate. Recommended treatments for prion decontamination include strongly basic (pH ≥~12) sodium hypochlorite bleach, ≥1 N sodium hydroxide, and/or prolonged autoclaving. These treatments are damaging and/or unsuitable for many clinical, agricultural and environmental applications. We have tested the anti-prion activity of a weakly acidic aqueous formulation of HOCl (BrioHOCl) that poses no apparent hazard to either users or many surfaces. For example, BrioHOCl can be applied directly to skin and mucous membranes and has been aerosolized to treat entire rooms without apparent deleterious effects. Here, we demonstrate that immersion in BrioHOCl can inactivate not only a range of target microbes, including spores of Bacillus subtilis, but also prions in tissue suspensions and on stainless steel. Real-time quaking-induced conversion (RT-QuIC) assays showed that BrioHOCl treatments eliminated all detectable prion seeding activity of human Creutzfeldt-Jakob disease, bovine spongiform encephalopathy, cervine chronic wasting disease, sheep scrapie and hamster scrapie; these findings indicated reductions of ≥103- to 106-fold. Transgenic mouse bioassays showed that all detectable hamster-adapted scrapie infectivity in brain homogenates or on steel wires was eliminated, representing reductions of ≥~105.75-fold and >104-fold, respectively. Inactivation of RT-QuIC seeding activity correlated with free chlorine concentration and higher order aggregation or destruction of proteins generally, including prion protein. BrioHOCl treatments had similar effects on amyloids composed of human α-synuclein and a fragment of human tau. These results indicate that HOCl can block the self-propagating activity of prions and other amyloids. PMID:27685252

Binding of (/sup 3/H)forskolin to platelet membranes and solubilized proteins from bovine brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nelson, C.A.; Seamon, K.B.

1986-05-01

(/sup 3/H)Forskolin ((/sup 3/H)FSK) bound to platelet membranes with a Kd of 20 nM and a Bmax of 125 fmol/mg protein. The Bmax was increased to 400 fmol/mg protein in the presence of GppNHp (or NaF) and MgCl/sub 2/ with no change in Kd. PGE/sub 1/ decreased the EC50 of GppNHp to increase the Bmax for (/sup 3/H)FSK binding from 600 nM to 35 nM. In contrast, PGE/sub 1/ had no effect on the EC50 of NaF to increase (/sup 3/H)FSK binding. (/sup 3/H)FSK binding increased slowly over 60 min when forskolin and GppNHp were added to membranes simultaneously atmore » 20/sup 0/C. Preincubation of membranes with GppNHp at 20/sup 5/C also caused a linear increase in adenylate cyclase specific activity over 60 minutes. (/sup 3/H)FSK bound to solubilized protein from bovine brain membrane with a Kd of 22 nM. GppNHp increased the number of binding sites in solubilized proteins only if membranes were not preincubated with GppNHp prior to solubilization. In conclusion the number of binding sites for (/sup 3/H)FSK is increased by agents that activate adenylate cyclase through the Ns protein. These sites appear to be associated with an activated complex of the Ns protein and adenylate cyclase.« less

Sod1 deficiency reduces incubation time in mouse models of prion disease.

PubMed

Akhtar, Shaheen; Grizenkova, Julia; Wenborn, Adam; Hummerich, Holger; Fernandez de Marco, Mar; Brandner, Sebastian; Collinge, John; Lloyd, Sarah E

2013-01-01

Prion infections, causing neurodegenerative conditions such as Creutzfeldt-Jakob disease and kuru in humans, scrapie in sheep and BSE in cattle are characterised by prolonged and variable incubation periods that are faithfully reproduced in mouse models. Incubation time is partly determined by genetic factors including polymorphisms in the prion protein gene. Quantitative trait loci studies in mice and human genome-wide association studies have confirmed that multiple genes are involved. Candidate gene approaches have also been used and identified App, Il1-r1 and Sod1 as affecting incubation times. In this study we looked for an association between App, Il1-r1 and Sod1 representative SNPs and prion disease incubation time in the Northport heterogeneous stock of mice inoculated with the Chandler/RML prion strain. No association was seen with App, however, significant associations were seen with Il1-r1 (P = 0.02) and Sod1 (P<0.0001) suggesting that polymorphisms at these loci contribute to the natural variation observed in incubation time. Furthermore, following challenge with Chandler/RML, ME7 and MRC2 prion strains, Sod1 deficient mice showed highly significant reductions in incubation time of 20, 13 and 24%, respectively. No differences were detected in Sod1 expression or activity. Our data confirm the protective role of endogenous Sod1 in prion disease.

Enzymatic Digestion of Chronic Wasting Disease Prions Bound to Soil

PubMed Central

SAUNDERS, SAMUEL E.; BARTZ, JASON C.; VERCAUTEREN, KURT C.; BARTELT-HUNT, SHANNON L.

2010-01-01

Chronic wasting disease (CWD) and sheep scrapie can be transmitted via indirect environmental routes, and it is known that soil can serve as a reservoir of prion infectivity. Given the strong interaction between the prion protein (PrP) and soil, we hypothesized that binding to soil enhances prion resistance to enzymatic digestion, thereby facilitating prion longevity in the environment and providing protection from host degradation. We characterized the performance of a commercially available subtilisin enzyme, the Prionzyme, to degrade soil-bound and unbound CWD and HY TME PrP as a function of pH, temperature, and treatment time. The subtilisin enzyme effectively degraded PrP adsorbed to a wide range of soils and soil minerals below the limits of detection. Signal loss occurred rapidly at high pH (12.5) and within 7 d under conditions representative of the natural environment (pH 7.4, 22°C). We observed no apparent difference in enzyme effectiveness between bound and unbound CWD PrP. Our results show that although adsorbed prions do retain relative resistance to enzymatic digestion compared with other brain homogenate proteins, they can be effectively degraded when bound to soil. Our results also suggest a topical application of a subtilisin enzyme solution may be an effective decontamination method to limit disease transmission via environmental ‘hot spots’ of prion infectivity. PMID:20450190

Semi-purification procedures of prions from a prion-infected brain using sucrose has no influence on the nonenzymatic glycation of the disease-associated prion isoform.

PubMed

Choi, Yeong-Gon; Kim, Jae-Il; Choi, Eun-Kyoung; Carp, Richard I; Kim, Yong-Sun

2016-01-01

Previous studies have shown that the Nε-carboxymethyl group is linked to not only one or more N-terminal Lys residues but also to one or more Lys residues of the protease-resistant core region of the pathogenic prion isoform (PrPSc) in prion-infected brains. Using an anti-advanced glycation end product (AGE) antibody, we detected nonenzymatically glycated PrPSc (AGE-PrPSc) in prion-infected brains following concentration by a series of ultracentrifugation steps with a sucrose cushion. In the present study, the levels of in vitro nonenzymatic glycation of PrPSc using sucrose were investigated to determine whether sucrose cushion can artificially and nonenzymatically induce in vitro glycation during ultracentrifugation. The first insoluble pellet fraction following the first ultracentrifugation (PU1st) collected from 263K scrapie-infected brains was incubated with sucrose, glucose or colloidal silica coated with polyvinylpyrrolidone (percoll). None of the compounds in vitro resulted in AGE-PrPSc. Nonetheless, glucose and percoll produced AGEs in vitro from other proteins within PU1st of the infected brains. This reaction could lead to the AGE-modified polymer(s) of nonenzymatic glycation-prone protein(s). This study showed that PrPSc is not nonenzymatically glycated in vitro with sucrose, glucose or percoll and that AGE-modified PrPSc can be isolated and enriched from prion-infected brains.

The Structural Architecture of an Infectious Mammalian Prion Using Electron Cryomicroscopy.

PubMed

Vázquez-Fernández, Ester; Vos, Matthijn R; Afanasyev, Pavel; Cebey, Lino; Sevillano, Alejandro M; Vidal, Enric; Rosa, Isaac; Renault, Ludovic; Ramos, Adriana; Peters, Peter J; Fernández, José Jesús; van Heel, Marin; Young, Howard S; Requena, Jesús R; Wille, Holger

2016-09-01

The structure of the infectious prion protein (PrPSc), which is responsible for Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy, has escaped all attempts at elucidation due to its insolubility and propensity to aggregate. PrPSc replicates by converting the non-infectious, cellular prion protein (PrPC) into the misfolded, infectious conformer through an unknown mechanism. PrPSc and its N-terminally truncated variant, PrP 27-30, aggregate into amorphous aggregates, 2D crystals, and amyloid fibrils. The structure of these infectious conformers is essential to understanding prion replication and the development of structure-based therapeutic interventions. Here we used the repetitive organization inherent to GPI-anchorless PrP 27-30 amyloid fibrils to analyze their structure via electron cryomicroscopy. Fourier-transform analyses of averaged fibril segments indicate a repeating unit of 19.1 Å. 3D reconstructions of these fibrils revealed two distinct protofilaments, and, together with a molecular volume of 18,990 Å3, predicted the height of each PrP 27-30 molecule as ~17.7 Å. Together, the data indicate a four-rung β-solenoid structure as a key feature for the architecture of infectious mammalian prions. Furthermore, they allow to formulate a molecular mechanism for the replication of prions. Knowledge of the prion structure will provide important insights into the self-propagation mechanisms of protein misfolding.

Assessment of prion reduction filters in decreasing infectivity of ultracentrifuged 263K scrapie-infected brain homogenates in "spiked" human blood and red blood cells.

PubMed

Cardone, Franco; Sowemimo-Coker, Samuel; Abdel-Haq, Hanin; Sbriccoli, Marco; Graziano, Silvia; Valanzano, Angelina; Berardi, Vito Angelo; Galeno, Roberta; Puopolo, Maria; Pocchiari, Maurizio

2014-04-01

The safety of red blood cells (RBCs) is of concern because of the occurrence of four transfusion-transmitted variant Creutzfeldt-Jakob disease (vCJD) cases in the United Kingdom. The absence of validated screening tests requires the use of procedures to remove prions from blood to minimize the risk of transmission. These procedures must be validated using infectious prions in a form that is as close as possible to one in blood. Units of human whole blood (WB) and RBCs were spiked with high-speed supernatants of 263K scrapie-infected hamster brain homogenates. Spiked samples were leukoreduced and then passed through prion-removing filters (Pall Corporation). In another experiment, RBCs from 263K scrapie-infected hamsters were treated as above, and residual infectivity was measured by bioassay. The overall removal of infectivity by the filters from prion-spiked WB and RBCs was approximately two orders of magnitude. No infectivity was detected in filtered hamster RBCs endogenously infected with scrapie. The use of prion-removing filters may help to reduce the risk of transfusion-transmitted vCJD. To avoid overestimation of prion removal efficiency in validation studies, it may be more appropriate to use supernates from ultracentrifugation of scrapie-infected hamster brain homogenate rather than the current standard brain homogenates. © 2013 American Association of Blood Banks.

Evolutionary Descent of Prion Genes from the ZIP Family of Metal Ion Transporters

PubMed Central

Schmitt-Ulms, Gerold; Ehsani, Sepehr; Watts, Joel C.; Westaway, David; Wille, Holger

2009-01-01

In the more than twenty years since its discovery, both the phylogenetic origin and cellular function of the prion protein (PrP) have remained enigmatic. Insights into a possible function of PrP may be obtained through the characterization of its molecular neighborhood in cells. Quantitative interactome data demonstrated the spatial proximity of two metal ion transporters of the ZIP family, ZIP6 and ZIP10, to mammalian prion proteins in vivo. A subsequent bioinformatic analysis revealed the unexpected presence of a PrP-like amino acid sequence within the N-terminal, extracellular domain of a distinct sub-branch of the ZIP protein family that includes ZIP5, ZIP6 and ZIP10. Additional structural threading and orthologous sequence alignment analyses argued that the prion gene family is phylogenetically derived from a ZIP-like ancestral molecule. The level of sequence homology and the presence of prion protein genes in most chordate species place the split from the ZIP-like ancestor gene at the base of the chordate lineage. This relationship explains structural and functional features found within mammalian prion proteins as elements of an ancient involvement in the transmembrane transport of divalent cations. The phylogenetic and spatial connection to ZIP proteins is expected to open new avenues of research to elucidate the biology of the prion protein in health and disease. PMID:19784368

Low copper and high manganese levels in prion protein plaques

USGS Publications Warehouse

Johnson, Christopher J.; Gilbert, P.U.P.A.; Abrecth, Mike; Baldwin, Katherine L.; Russell, Robin E.; Pedersen, Joel A.; McKenzie, Debbie

2013-01-01

Accumulation of aggregates rich in an abnormally folded form of the prion protein characterize the neurodegeneration caused by transmissible spongiform encephalopathies (TSEs). The molecular triggers of plaque formation and neurodegeneration remain unknown, but analyses of TSE-infected brain homogenates and preparations enriched for abnormal prion protein suggest that reduced levels of copper and increased levels of manganese are associated with disease. The objectives of this study were to: (1) assess copper and manganese levels in healthy and TSE-infected Syrian hamster brain homogenates; (2) determine if the distribution of these metals can be mapped in TSE-infected brain tissue using X-ray photoelectron emission microscopy (X-PEEM) with synchrotron radiation; and (3) use X-PEEM to assess the relative amounts of copper and manganese in prion plaques in situ. In agreement with studies of other TSEs and species, we found reduced brain levels of copper and increased levels of manganese associated with disease in our hamster model. We also found that the in situ levels of these metals in brainstem were sufficient to image by X-PEEM. Using immunolabeled prion plaques in directly adjacent tissue sections to identify regions to image by X-PEEM, we found a statistically significant relationship of copper-manganese dysregulation in prion plaques: copper was depleted whereas manganese was enriched. These data provide evidence for prion plaques altering local transition metal distribution in the TSE-infected central nervous system.

Nuclear Data Sheets for A = 34

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nica N.; Nica,N.; Singh,B.

2012-07-01

Nuclear spectroscopic information for experimentally investigated nuclides of mass 34 (Ne, Na, Mg, Al, Si, P, S, Cl, Ar, K, Ca) has been evaluated. The principal sources of the Adopted Levels presented for nuclides close to the stability line are Endt's evaluations (1990En08, 1978En02). The data sets for reactions and decays, including all available gamma-ray data, are based mostly on the original literature. There are no data available for the excited states in {sup 34}Ne and {sup 34}Na. The existence of the {sup 34}K and {sup 34}Ca nuclides has been searched and reported in a secondary publication but no evidencemore » was found for their detection. Both nuclides are possible candidates for one or two-proton emission. Only upper limits of half-lives have been proposed based on expected cross sections. Decay schemes for the {beta} decays of {sup 34}Ne, {sup 34}Na, {sup 34}Mg, and {sup 34}Si are not known. Very little information is available for {beta}-delayed neutron decays of {sup 35}Na, {sup 35}Mg and {sup 35}Si which lead to population of levels in A = 34 nuclides. Neutron-rich nuclides in this mass region are relevant to 'island of inversion'.« less

Primary transmission of chronic wasting disease versus scrapie prions from small ruminants to transgenic mice expressing ovine and cervid prion protein

USDA-ARS?s Scientific Manuscript database

Identifying transmissible spongiform encephalopathy (TSE) reservoirs that could lead to disease re-emergence is imperative to U.S. scrapie eradication efforts. Transgenic mice expressing the cervid (TgElk) or ovine (Tg338) prion protein have aided characterization of chronic wasting disease (CWD) an...

Molecular Model of Prion Transmission to Humans

PubMed Central

Wight, Darren; Barron, Rona; Jeffrey, Martin; Manson, Jean; Prowse, Christopher; Ironside, James W.; Head, Mark W.

2009-01-01

To assess interspecies barriers to transmission of transmissible spongiform encephalopathies, we investigated the ability of disease-associated prion proteins (PrPd) to initiate conversion of the human normal cellular form of prion protein of the 3 major PRNP polymorphic variants in vitro. Protein misfolding cyclic amplification showed that conformation of PrPd partly determines host susceptibility. PMID:19961689

Quantitating PrP polymorphisms present in prions from heterozygous scrapie-infected sheep

USDA-ARS?s Scientific Manuscript database

Scrapie is a prion (PrPSc) disease of sheep. The incubation period of sheep scrapie is strongly influenced by polymorphisms at positions 136, 154, and 171 of a sheep’s normal cellular prion protein (PrPC). Chymotrypsin was used to digest sheep recombinant PrP to identify a set of characteristic pept...

Clinical features in prion protein-deficient and wild-type cattle inoculated with transmissible mink encephalopathy (TME)

USDA-ARS?s Scientific Manuscript database

Background: Transmissible spongiform encephalopathies (TSEs) or prion diseases are caused by the propagation of a misfolded form (PrP**d) of the normal cellular prion protein, PrP**c. Recently, we have reported the generation and characterization of PrP**C-deficient cattle (PrP-/-) produced by a seq...

76 FR 71294 - Prions; Proposed Amendment To Clarify Product Performance Data for Products With Prion-Related...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-11-17

... control pest microorganisms that pose a threat to human health and whose presence cannot readily be... control pest microorganisms that pose a threat to human health and whose presence cannot readily be... of prions (that poses a threat to human health), an applicant or registrant would be required by...

Classical scrapie prions in ovine blood are associated with B lymphocytes and platelets-rich plasma

USDA-ARS?s Scientific Manuscript database

Classical scrapie is a naturally occurring fatal brain disease of sheep and goats which is caused by prions, a novel class of infectious agent, and is accompanied by the accumulation of abnormal isoforms of prion protein (PrP-Sc) in certain neural and lymphoid tissues. Although collection of a blood...

Lack of Prion Accumulation in Lymphoid Tissues of Scrapie-affected Sheep with the AA136, QR171 Prion Protein Genotype

USDA-ARS?s Scientific Manuscript database

Background: Sheep scrapie is a transmissible spongiform encephalopathy which can be transmitted horizontally through the shedding of an infectious conformer (PrP**Sc) of the normal cellular prion protein (PrP**c). Genetics profoundly influence the susceptibility of sheep to scrapie. PrP**c amino-aci...

Recombinant Prion Protein Refolded with Lipid and RNA Has the Biochemical Hallmarks of a Prion but Lacks In Vivo Infectivity

PubMed Central

Timmes, Andrew G.; Moore, Roger A.; Fischer, Elizabeth R.; Priola, Suzette A.

2013-01-01

During prion infection, the normal, protease-sensitive conformation of prion protein (PrPC) is converted via seeded polymerization to an abnormal, infectious conformation with greatly increased protease-resistance (PrPSc). In vitro, protein misfolding cyclic amplification (PMCA) uses PrPSc in prion-infected brain homogenates as an initiating seed to convert PrPC and trigger the self-propagation of PrPSc over many cycles of amplification. While PMCA reactions produce high levels of protease-resistant PrP, the infectious titer is often lower than that of brain-derived PrPSc. More recently, PMCA techniques using bacterially derived recombinant PrP (rPrP) in the presence of lipid and RNA but in the absence of any starting PrPSc seed have been used to generate infectious prions that cause disease in wild-type mice with relatively short incubation times. These data suggest that lipid and/or RNA act as cofactors to facilitate the de novo formation of high levels of prion infectivity. Using rPrP purified by two different techniques, we generated a self-propagating protease-resistant rPrP molecule that, regardless of the amount of RNA and lipid used, had a molecular mass, protease resistance and insolubility similar to that of PrPSc. However, we were unable to detect prion infectivity in any of our reactions using either cell-culture or animal bioassays. These results demonstrate that the ability to self-propagate into a protease-resistant insoluble conformer is not unique to infectious PrP molecules. They suggest that the presence of RNA and lipid cofactors may facilitate the spontaneous refolding of PrP into an infectious form while also allowing the de novo formation of self-propagating, but non-infectious, rPrP-res. PMID:23936256

Detection of Chronic Wasting Disease Prions in Salivary, Urinary, and Intestinal Tissues of Deer: Potential Mechanisms of Prion Shedding and Transmission▿

PubMed Central

Haley, Nicholas J.; Mathiason, Candace K.; Carver, Scott; Zabel, Mark; Telling, Glenn C.; Hoover, Edward A.

2011-01-01

Efficient horizontal transmission is a signature trait of chronic wasting disease (CWD) in cervids. Infectious prions shed into excreta appear to play a key role in this facile transmission, as has been demonstrated by bioassays of cervid and transgenic species and serial protein misfolding cyclic amplification (sPMCA). However, the source(s) of infectious prions in these body fluids has yet to be identified. In the present study, we analyzed tissues proximate to saliva, urine, and fecal production by sPMCA in an attempt to elucidate this unique aspect of CWD pathogenesis. Oropharyngeal, urogenital, and gastrointestinal tissues along with blood and obex from CWD-exposed cervids (comprising 27 animals and >350 individual samples) were analyzed and scored based on the apparent relative CWD burden. PrPCWD-generating activity was detected in a range of tissues and was highest in the salivary gland, urinary bladder, and distal intestinal tract. In the same assays, blood from the same animals and unseeded normal brain homogenate controls (n = 116 of 117) remained negative. The PrP-converting activity in peripheral tissues varied from 10−11- to 100-fold of that found in brain of the same animal. Deer with highest levels of PrPCWD amplification in the brain had higher and more widely disseminated prion amplification in excretory tissues. Interestingly, PrPCWD was not demonstrable in these excretory tissues by conventional Western blotting, suggesting a low prion burden or the presence of protease-sensitive infectious prions destroyed by harsh proteolytic treatments. These findings offer unique insights into the transmission of CWD in particular and prion infection and trafficking overall. PMID:21525361

Ozone inactivation of infectious prions in rendering plant and municipal wastewaters.

PubMed

Ding, Ning; Neumann, Norman F; Price, Luke M; Braithwaite, Shannon L; Balachandran, Aru; Belosevic, Miodrag; Gamal El-Din, Mohamed

2014-02-01

Disposal of tissues and organs associated with prion accumulation and infectivity in infected animals (designated as Specified Risk Materials [SRM]) is strictly regulated by the Canadian Food Inspection Agency (CFIA); however, the contamination of wastewater from slaughterhouses that handle SRM still poses public concern. In this study, we examined for the first time the partitioning of infectious prions in rendering plant wastewater and found that a large proportion of infectious prions were partitioned into the scum layer formed at the top after gravity separation, while quite a few infectious prions still remained in the wastewater. Subsequently, we assessed the ozone inactivation of infectious prions in the raw, natural gravity-separated and dissolved air flotation (DAF)-treated (i.e., primary-treated) rendering plant wastewater, and in a municipal final effluent (i.e., secondary-treated municipal wastewater). At applied ozone doses of 43.4-44.6 mg/L, ozone was instantaneously depleted in the raw rendering plant wastewater, while a greater than 4-log10 inactivation was achieved at a 5 min exposure in the DAF-treated rendering plant wastewater. Prion inactivation in the municipal final effluent was conducted with two levels of applied ozone doses of 13.4 and 22.5mg/L, and a greater than 4-log10 inactivation was achieved at a 5 min exposure with the higher ozone dose. Efficiency factor Hom (EFH) models were used to model (i.e., fit) the experimental data. The CT (disinfectant concentration multiplied by contact time) values were determined for 2- and 3-log10 inactivation in the municipal final effluent treated with an ozone dose of 13.4 mg/L. Our results indicate that ozone could serve as a final barrier for prion inactivation in primary- and/or secondary-treated wastewaters. © 2013.

Photoelectron spectroscopy and electronic structure of ScO{sub n}{sup {minus}}(n = 1--4) and YO{sub n}{sup {minus}}(n = 1--5): Strong electron correlation effects in ScO{sup {minus}} and YO{sup {minus}}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, H.; Wang, L.S.

A photoelectron spectroscopic study of ScO{sub n}{sup {minus}} (n = 1--4) and YO{sub n}{sup {minus}} (n = 1--5) was carried out at three photon energies: 532, 355, and 266 nm. Vibrationally resolved photoelectron spectra were obtained for ScO{sup {minus}} and YO{sup {minus}}. The electron affinities of both ScO and YO were measured to be identical (1.35 eV) within the experimental accuracy ({+-}0.02 eV). Three low-lying excited states were observed for the monoxides, {Alpha}{prime}{sup 2}{Delta}, {Alpha}{sup 2}{Pi}, and {Beta}{sup 2}{Sigma}{sup +}. The latter two excited states resulted from two-electron detachment, suggesting unusually strong electron correlation (configuration interaction) effects in the groundmore » state of the anions. The excitation energies of the low-lying states were also found to be similar for the two monoxides except that YO has a smaller vibrational frequency and larger spin-orbit splitting. The {Alpha}{prime}{sup 2}{Delta} states of both ScO and YO show very strong photon energy-dependent detachment cross sections. Four similar photoelectron features were observed for the dioxides with those of YO{sub 2}{sup {minus}} having lower binding energies. A second isomer due to an O{sub 2} complex was also observed for Sc and Y. Broad and featureless spectra were observed for the higher oxides. At least two isomers were present for the higher oxides, one with low and one with high binding energies.« less

Ab initio Study of Transition metal binding to the Prion Protein

NASA Astrophysics Data System (ADS)

Cox, Daniel L.; Singh, Rajiv R. P.; Pan, Jianping

2004-03-01

Fundamental understanding of the prion protein (PrP) is of critical public health importance in view of mad cow and chronic wasting diseases. In recent years, it has been shown that the normal form (PrP^c) binds copper^1), and the structure of the copper binding domain has been elaborated. Hypotheses about toxicity associated with binding of other metals (notably manganese) have been put forward, Accordingly, using the ab initio SIESTA density functional theory code^2), we calculated the binding energy E_B(M) of M-(PrP) complexes relative to initially uncomplexed M ions, with M=Cu,Ni,Zn,Mn and (PrP)^* the minimal binding domain. The binding energy trend is E_B(Ni)>E_B(Cu)>E_B(Zn)>E_B(Mn), consistent with recent experiments apart from the surprising stability of Ni. We will also present preliminary results for binding of initially complexed M ions. *-Supported by U.S. DOE, Office of Basic Energy Sciences, Division of Materials Research 1) G.S. Jackson et al., Proc. Nat. Acad. Sci. (USA) 98, 8531 (2001). 2) P. Ordejón, et al., Phys. Rev. B53, R10441 (1996); J.M. Soler et al., J. Phys. Cond. Matt. 14, 2745 (2002).

Principal network analysis: identification of subnetworks representing major dynamics using gene expression data

PubMed Central

Kim, Yongsoo; Kim, Taek-Kyun; Kim, Yungu; Yoo, Jiho; You, Sungyong; Lee, Inyoul; Carlson, George; Hood, Leroy; Choi, Seungjin; Hwang, Daehee

2011-01-01

Motivation: Systems biology attempts to describe complex systems behaviors in terms of dynamic operations of biological networks. However, there is lack of tools that can effectively decode complex network dynamics over multiple conditions. Results: We present principal network analysis (PNA) that can automatically capture major dynamic activation patterns over multiple conditions and then generate protein and metabolic subnetworks for the captured patterns. We first demonstrated the utility of this method by applying it to a synthetic dataset. The results showed that PNA correctly captured the subnetworks representing dynamics in the data. We further applied PNA to two time-course gene expression profiles collected from (i) MCF7 cells after treatments of HRG at multiple doses and (ii) brain samples of four strains of mice infected with two prion strains. The resulting subnetworks and their interactions revealed network dynamics associated with HRG dose-dependent regulation of cell proliferation and differentiation and early PrPSc accumulation during prion infection. Availability: The web-based software is available at: http://sbm.postech.ac.kr/pna. Contact: dhhwang@postech.ac.kr; seungjin@postech.ac.kr Supplementary information: Supplementary data are available at Bioinformatics online. PMID:21193522

Genetics of Prion Disease in Cattle

PubMed Central

Murdoch, Brenda M.; Murdoch, Gordon K.

2015-01-01

Bovine spongiform encephalopathy (BSE) is a prion disease that is invariably fatal in cattle and has been implicated as a significant human health risk. As a transmissible disease of livestock, it has impacted food safety, production practices, global trade, and profitability. Genetic polymorphisms that alter the prion protein in humans and sheep are associated with transmissible spongiform encephalopathy susceptibility or resistance. In contrast, there is no strong evidence that nonsynonymous mutations in the bovine prion gene (PRNP) are associated with classical BSE (C-BSE) disease susceptibility, though two bovine PRNP insertion/deletion polymorphisms, in the putative region, are associated with susceptibility to C-BSE. However, these associations do not explain the full extent of BSE susceptibility, and loci outside of PRNP appear to be associated with disease incidence in some cattle populations. This article provides a review of the current state of genetic knowledge regarding prion diseases in cattle. PMID:26462233

Frameshifted prion proteins as pathological agents: quantitative considerations.

PubMed

Wills, Peter R

2013-05-21

A quantitatively consistent explanation for the titres of infectivity found in a variety of prion-containing preparations is provided on the basis that the ætiological agents of transmissible spongiform encephalopathy comprise a very small population fraction of prion protein (PrP) variants, which contain frameshifted elements in their N-terminal octapeptide-repeat regions. A mechanism for the replication of frameshifted prions is described and calculations are performed to obtain estimates of the concentration of these PrP variants in normal and infected brain, as well as their enrichment in products of protein misfolding cyclic amplification. These calculations resolve the lack of proper quantitative correlation between measures of infectivity and the presence of conformationally-altered, protease-resistant variants of PrP. Experiments, which could confirm or eventually exclude the role of frameshifted variants in the ætiology of prion disease, are suggested. Copyright © 2013 Elsevier Ltd. All rights reserved.

Spontaneous Generation of Infectious Prion Disease in Transgenic Mice

PubMed Central

Castilla, Joaquín; Pintado, Belén; Gutiérrez-Adan, Alfonso; Andréoletti, Olivier; Aguilar-Calvo, Patricia; Arroba, Ana-Isabel; Parra-Arrondo, Beatriz; Ferrer, Isidro; Manzanares, Jorge; Espinosa, Juan-Carlos

2013-01-01

We generated transgenic mice expressing bovine cellular prion protein (PrPC) with a leucine substitution at codon 113 (113L). This protein is homologous to human protein with mutation 102L, and its genetic link with Gerstmann–Sträussler–Scheinker syndrome has been established. This mutation in bovine PrPC causes a fully penetrant, lethal, spongiform encephalopathy. This genetic disease was transmitted by intracerebral inoculation of brain homogenate from ill mice expressing mutant bovine PrP to mice expressing wild-type bovine PrP, which indicated de novo generation of infectious prions. Our findings demonstrate that a single amino acid change in the PrPC sequence can induce spontaneous generation of an infectious prion disease that differs from all others identified in hosts expressing the same PrPC sequence. These observations support the view that a variety of infectious prion strains might spontaneously emerge in hosts displaying random genetic PrPC mutations. PMID:24274622

Transmission of scrapie prions to primate after an extended silent incubation period.

PubMed

Comoy, Emmanuel E; Mikol, Jacqueline; Luccantoni-Freire, Sophie; Correia, Evelyne; Lescoutra-Etchegaray, Nathalie; Durand, Valérie; Dehen, Capucine; Andreoletti, Olivier; Casalone, Cristina; Richt, Juergen A; Greenlee, Justin J; Baron, Thierry; Benestad, Sylvie L; Brown, Paul; Deslys, Jean-Philippe

2015-06-30

Classical bovine spongiform encephalopathy (c-BSE) is the only animal prion disease reputed to be zoonotic, causing variant Creutzfeldt-Jakob disease (vCJD) in humans and having guided protective measures for animal and human health against animal prion diseases. Recently, partial transmissions to humanized mice showed that the zoonotic potential of scrapie might be similar to c-BSE. We here report the direct transmission of a natural classical scrapie isolate to cynomolgus macaque, a highly relevant model for human prion diseases, after a 10-year silent incubation period, with features similar to those reported for human cases of sporadic CJD. Scrapie is thus actually transmissible to primates with incubation periods compatible with their life expectancy, although fourfold longer than BSE. Long-term experimental transmission studies are necessary to better assess the zoonotic potential of other prion diseases with high prevalence, notably Chronic Wasting Disease of deer and elk and atypical/Nor98 scrapie.

Transmission of scrapie prions to primate after an extended silent incubation period

PubMed Central

Comoy, Emmanuel E.; Mikol, Jacqueline; Luccantoni-Freire, Sophie; Correia, Evelyne; Lescoutra-Etchegaray, Nathalie; Durand, Valérie; Dehen, Capucine; Andreoletti, Olivier; Casalone, Cristina; Richt, Juergen A.; Greenlee, Justin J.; Baron, Thierry; Benestad, Sylvie L.; Brown, Paul; Deslys, Jean-Philippe

2015-01-01

Classical bovine spongiform encephalopathy (c-BSE) is the only animal prion disease reputed to be zoonotic, causing variant Creutzfeldt-Jakob disease (vCJD) in humans and having guided protective measures for animal and human health against animal prion diseases. Recently, partial transmissions to humanized mice showed that the zoonotic potential of scrapie might be similar to c-BSE. We here report the direct transmission of a natural classical scrapie isolate to cynomolgus macaque, a highly relevant model for human prion diseases, after a 10-year silent incubation period, with features similar to those reported for human cases of sporadic CJD. Scrapie is thus actually transmissible to primates with incubation periods compatible with their life expectancy, although fourfold longer than BSE. Long-term experimental transmission studies are necessary to better assess the zoonotic potential of other prion diseases with high prevalence, notably Chronic Wasting Disease of deer and elk and atypical/Nor98 scrapie. PMID:26123044

Measurements of energetic electron distributions in uv and ir laser plasmas. [0. 35 AND 1. 05. mu. M; 5 X 10/sup 13/ to 2 x 10/sup 15/ W/cm/sup 2/

DOE Office of Scientific and Technical Information (OSTI.GOV)

Keck, R.L.

1985-06-01

Measurements have been made of the x-ray continuum produced by plasmas irradiated with 0.35 and 1.05 ..mu..m laser light over an intensity range of 5 x 10/sup 13/ to 2 x 10/sup 15/ W/cm/sup 2/. From the x-ray continuum, which was measured over a range of 1.5 to 300 keV, both the temperature of and fractional energy in any supra-thermal electron distributions can be obtained. The measurements show the presence of a very high temperature (20 to 60 keV) electron distribution with either 0.35 or 1.05 ..mu..m irradiation. This component, which is attributed to the presence of the two-plasmon decaymore » instability, is observed above an intensity of approximately 10/sup 14/ W/cm/sup 2/ at 1.05 ..mu..m and contains less than 0.1% of the incident laser energy. With 0.35 ..mu..m irradiation, the intensity at which this component is observed is approximately a factor of 3 higher. At 1.05 ..mu..m, this very high temperature component appears in addition to a third, 2 to 7 keV, component attributed to resonance absorption. 38 refs., 37 figs., 6 tabs.« less

Catalytic ionic hydrogenation of ketones using tungsten or molybdenum catalysts with increased lifetimes

DOEpatents

Bullock, R. Morris; Kimmich, Barbara F. M.; Fagan, Paul J.; Hauptman, Elisabeth

2003-09-02

The present invention is a process for the catalytic hydrogenation of ketones and aldehydes to alcohols at low temperatures and pressures using organometallic molybdenum and tungsten complexes and the catalyst used in the process. The reactants include a functional group which is selected from groups represented by the formulas R*(C.dbd.O)R' and R*(C.dbd.O)H, wherein R* and R' are selected from hydrogen or any alkyl or aryl group. The process includes reacting the organic compound in the presence of hydrogen and a catalyst to form a reaction mixture. The catalyst is prepared by reacting Ph.sub.3 C.sup.+ A.sup.- with a metal hydride. A.sup.- represents an anion and can be BF.sub.4.sup.-, PF.sub.6.sup.-, CF.sub.3 SO.sub.3.sup.- or Bar'.sub.4.sup.-, wherein Ar'=3,5-bis(trifluoromethyl)phenyl. The metal hydride is represented by the formula: HM(CO).sub.2 [.eta..sup.5 :.eta..sup.1 --C.sub.5 H.sub.4 (XH.sub.2).sub.n PR.sub.2 ] wherein M represents a molybdenum (Mo) atom or a tungsten (W) atom; X is a carbon atom, a silicon atom or a combination of carbon (C) and silicon (Si) atoms; n is any positive integer; R represents two hydrocarbon groups selected from H, an aryl group and an alkyl group, wherein both R groups can be the same or different. The metal hydride is reacted with Ph.sub.3 C.sup.+ A.sup.- either before reacting with the organic compound or in the reaction mixture.

Novel epitopes identified by Anti-PrP monoclonal antibodies produced following immunization of Prnp0/0 Balb/cJ mice with purified scrapie prions

USDA-ARS?s Scientific Manuscript database

Prions, or infectious proteins, cause a class of uniformly fatal neurodegenerative diseases. Prions are composed solely of an aberrantly folded isoform(PrPSc)of a normal cellular protein (PrPC). Shared sequence identity of PrPSc with PrPC has limited the detection sensitivity of immunochemical assay...