scholarly journals Quaternary structural convergence and structural diversification of prion assemblies at the early replication stage

2019 ◽  
Author(s):  
Angélique Igel-Egalon ◽  
Florent Laferrière ◽  
Mohammed Moudjou ◽  
Mathieu Merzach ◽  
Tina Knäpple ◽  
...  
Keyword(s):  
Quaternary Structure ◽  
Prion Diseases ◽  
Mechanistic Explanation ◽  
Structural Rearrangement ◽  
Structural Heterogeneity ◽  
Cellular Prion Protein ◽  
Replication Stage ◽  
Early Replication ◽  
Structural Diversification

AbstractAggregation of misfolded forms from host-encoded proteins is key to the pathogenesis of a number of neurodegenerative disorders, including prion diseases, Alzheimer’s disease and Parkinson’s disease. In prion diseases, the cellular prion protein PrPCcan misfold into PrPScand auto-organize into conformationally distinct assemblies or strains. A plethora of observations reports the existence of PrPScstructural heterogeneity within prion strains, suggesting the emergence and coevolution of structurally distinct PrPScassemblies during prion replication in controlled environment. Such PrPScdiversification processes remain poorly understood. Although central to prion host-adaptation, structural diversification of PrPScassemblies is also a key issue for the formation of PrP conformers involved in neuronal injury. Here, we characterized the evolution of the PrPScquaternary structure during prion replicationin vivoand inbona fidecell-free amplification assays. Regardless of the strain studied, the early replication stage conduced to the preferential formation of small PrPScoligomers, thus highlighting a quaternary structural convergence phenomenon. Their evolutionary kinetics revealed the existence of a PrPC-dependent secondary templating pathway in concert with a structural rearrangement. This secondary templating pathway provides, for the first time, a mechanistic explanation for prion structural diversification during replication, a key determinant for prion adaptation on further transmission, including to other host species. The uncovered processes are also key for a better understanding of the accumulation mechanisms of other misfolded assemblies believed to propagate by a prion-like process.

scholarly journals Early stage prion assembly involves two subpopulations with different quaternary structures and a secondary templating pathway

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Angélique Igel-Egalon ◽  
Florent Laferrière ◽  
Mohammed Moudjou ◽  
Jan Bohl ◽  
Mathieu Mezache ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Parental Strain ◽  
Early Stage ◽  
Prion Diseases ◽  
Structural Heterogeneity ◽  
Self Organized ◽  
Sequential Processes ◽  
Quaternary Structures ◽  
Underlying Processes ◽  
Structural Diversification

Abstract The dynamics of aggregation and structural diversification of misfolded, host-encoded proteins in neurodegenerative diseases are poorly understood. In many of these disorders, including Alzheimer’s, Parkinson’s and prion diseases, the misfolded proteins are self-organized into conformationally distinct assemblies or strains. The existence of intrastrain structural heterogeneity is increasingly recognized. However, the underlying processes of emergence and coevolution of structurally distinct assemblies are not mechanistically understood. Here, we show that early prion replication generates two subsets of structurally different assemblies by two sequential processes of formation, regardless of the strain considered. The first process corresponds to a quaternary structural convergence, by reducing the parental strain polydispersity to generate small oligomers. The second process transforms these oligomers into larger ones, by a secondary autocatalytic templating pathway requiring the prion protein. This pathway provides mechanistic insights into prion structural diversification, a key determinant for prion adaptation and toxicity.

scholarly journals Toxicological Evaluation of Anti-Scrapie Trimethoxychalcones and Oxadiazoles

2015 ◽  
Vol 87 (2 suppl) ◽  
pp. 1421-1434 ◽  
Author(s):  
CLAUDIA P. FIGUEIREDO ◽  
NATALIA C. FERREIRA ◽  
GISELLE F. PASSOS ◽  
ROBSON DA COSTA ◽  
FERNANDA S. NEVES ◽  
...  
Keyword(s):  
Prion Protein ◽  
Aromatic Compounds ◽  
Prion Diseases ◽  
Intraperitoneal Administration ◽  
Neuroblastoma Cells ◽  
Cellular Prion Protein ◽  
Transmissible Spongiform Encephalopathies ◽  
Toxicological Evaluation ◽  
Spongiform Encephalopathies

An altered form of the cellular prion protein, the PrPScor PrPRes, is implicated in the occurrence of the still untreatable transmissible spongiform encephalopathies. We have previously synthesized and characterized aromatic compounds that inhibit protease-resistant prion protein (PrPRes) accumulation in scrapie-infected cells. These compounds belong to different chemical classes, including acylhydrazones, chalcones and oxadiazoles. Some of the active compounds were non-toxic to neuroblastoma cells in culture and seem to possess drugable properties, since they are in agreement with the Lipinski´s rule of 5 and present desirable pharmacokinetic profiles as predicted in silico. Before the evaluation of the in vivo efficacy of the aromatic compounds in scrapie-infected mice, safety assessment in healthy mice is needed. Here we used Swiss mice to evaluate the acute toxicity profile of the six most promising anti-prionic compounds, the 2,4,5-trimethoxychalcones (J1, J8, J20 and J35) and the 1,3,4-oxadiazoles (Y13 and Y17). One single oral administration (300 mg/kg) of J1, J8, J20, J35, Y13 and Y17 or repeated intraperitoneal administration (10 mg/kg, 3 times a week, for 4 weeks) of J1, J8 and J35, did not elicit toxicity in mice. We strongly believe that the investigated trimethoxychalcones and oxadiazoles are interesting compounds to be further analyzed in vivo against prion diseases.

scholarly journals Expression and characterisation of fully posttranslationally modified cellular prion protein in Pichia pastoris

2013 ◽  
Vol 394 (11) ◽  
pp. 1475-1483
Author(s):  
Jendrik Marbach ◽  
Peter Zentis ◽  
Philipp Ellinger ◽  
Henrik Müller ◽  
Eva Birkmann
Keyword(s):  
Plasma Membrane ◽  
Pichia Pastoris ◽  
Prion Protein ◽  
Prion Diseases ◽  
Phosphatidyl Inositol ◽  
Cellular Prion Protein ◽  
Glycosylation Pattern ◽  
Signal Sequences

Abstract Prion diseases are fatal neurodegenerative diseases which occur as sporadic, genetic, and transmissible disorders. A molecular hallmark of prion diseases is the conformational conversion of the host-encoded cellular form of the prion protein (PrPC) into its misfolded pathogenic isoform (PrPSc). PrPSc is the main component of the pathological and infectious prion agent. The study of the conversion mechanism from PrPC to PrPSc is a major field in prion research. PrPC is glycosylated and attached to the plasma membrane via its glycosyl phosphatidyl inositol (GPI)-anchor. In this study we established and characterised the expression of fully posttranslationally modified mammalian Syrian golden hamster PrPC in the yeast Pichia pastoris using native PrPC-specific N- and C-terminal signal sequences. In vivo as well as in vitro-studies demonstrated that the signal sequences controlled posttranslational processing and trafficking of native PrPC, resulting in PrPC localised in the plasma membrane of P. pastoris. In addition, the glycosylation pattern of native PrPC could be confirmed.

scholarly journals Structural Basis of Prion Protein Conformation Conversion Inhibition

2014 ◽  
Vol 70 (a1) ◽  
pp. C812-C812
Author(s):  
Pravas Baral ◽  
Mridula Swayampakula ◽  
Manoj Rout ◽  
Leo Spyracopoulos ◽  
Adriano Aguzzi ◽  
...  
Keyword(s):  
Prion Protein ◽  
Conformational Transition ◽  
Prion Diseases ◽  
Mammalian Species ◽  
Binding Pocket ◽  
Structural Rearrangement ◽  
Cellular Prion Protein ◽  
Molecular Structures ◽  
Structural Basis ◽  
Scrapie Strains

Prion diseases are fatal neurodegenerative diseases that affect humans and other animals. A conformational transition of the cellular prion protein, PrPC, into an infectious isoform, PrPSc, is the central event leading to aggregation and the fatal progression of these diseases. One of the therapeutic approaches for the prion diseases is the use of pharmacological chaperones. These molecules can stabilize the prion protein in its native conformation and can arrest the disease progression. Tricyclic phenothiazine compounds exhibit anti-prion activity; however, the underlying molecular mechanism of PrPSc inhibition remains elusive. We have determined the molecular structures of promazine and chlorpromazine bound to the mouse prion protein (moPrP) by forming crystals of the ternary complexes of the POM1 Fab:moPrP:promazine and the POM1 Fab:moPrP:chlorpromazine. The structures were solved by X-ray crystallography to resolutions of 1.9 Å and 2.2 Å, respectively. The small molecules are bound in a novel binding pocket formed at the intersection of the structured and the unstructured domains of the moPrP. Promazine binding induces a structural rearrangement of a portion of the unstructured region proximal to the first β-strand, β1, through the formation of a "hydrophobic anchor". We demonstrate that these molecules, promazine in particular, allosterically stabilize the misfolding initiator-motifs such as C-terminus of helix, α2, the α2-α3 loop as well as the polymorphic β2-α2 loop. Hence, the stabilization effects of the phenothiazine derivatives on initiator-motifs, induce a PrPC isoform that potentially resists oligomerization. Subtle structural differences are observed in the so-called initiator-motifs of the prion proteins that belong to many different mammalian species, and these diversities may possibly explain the generation of wide variety of scrapie strains in prion diseases.

scholarly journals Quaternary Structure Changes for PrPSc Predate PrPC Downregulation and Neuronal Death During Progression of Experimental Scrapie Disease

2020 ◽  
Vol 58 (1) ◽  
pp. 375-390
Author(s):  
Ghazaleh Eskandari-Sedighi ◽  
Leonardo M. Cortez ◽  
Jing Yang ◽  
Nathalie Daude ◽  
Klinton Shmeit ◽  
...  
Keyword(s):  
Quaternary Structure ◽  
Prion Diseases ◽  
Disease Process ◽  
Neuronal Loss ◽  
Cross Sectional Study ◽  
Cellular Prion Protein ◽  
Proteinase K ◽  
Disease Pathogenesis ◽  
Cross Sectional ◽  
Cellular Analysis

AbstractPrion diseases are fatal neurodegenerative diseases in mammals with the unique characteristics of misfolding and aggregation of the cellular prion protein (PrPC) to the scrapie prion (PrPSc). Although neuroinflammation and neuronal loss feature within the disease process, the details of PrPC/PrPSc molecular transition to generate different aggregated species, and the correlation between each species and sequence of cellular events in disease pathogenesis are not fully understood. In this study, using mice inoculated with the RML isolate of mouse-adapted scrapie as a model, we applied asymmetric flow field-flow fractionation to monitor PrPC and PrPSc particle sizes and we also measured seeding activity and resistance to proteases. For cellular analysis in brain tissue, we measured inflammatory markers and synaptic damage, and used the isotropic fractionator to measure neuronal loss; these techniques were applied at different timepoints in a cross-sectional study of disease progression. Our analyses align with previous reports defining significant decreases in PrPC levels at pre-clinical stages of the disease and demonstrate that these decreases become significant before neuronal loss. We also identified the earliest PrPSc assemblies at a timepoint equivalent to 40% elapsed time for the disease incubation period; we propose that these assemblies, mostly composed of proteinase K (PK)–sensitive species, play an important role in triggering disease pathogenesis. Lastly, we show that the PK-resistant assemblies of PrPSc that appear at timepoints close to the terminal stage have similar biophysical characteristics, and hence that preparative use of PK-digestion selects for this specific subpopulation. In sum, our data argue that qualitative, as well as quantitative, changes in PrP conformers occur at the midpoint of subclinical phase; these changes affect quaternary structure and may occur at the threshold where adaptive responses become inadequate to deal with pathogenic processes.

scholarly journals Heterogeneity and Architecture of Pathological Prion Protein Assemblies: Time to Revisit the Molecular Basis of the Prion Replication Process?

Viruses ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 429 ◽  
Author(s):  
Angélique Igel-Egalon ◽  
Jan Bohl ◽  
Mohammed Moudjou ◽  
Laetitia Herzog ◽  
Fabienne Reine ◽  
...  
Keyword(s):  
Prion Protein ◽  
Current Knowledge ◽  
Prion Diseases ◽  
Structural Heterogeneity ◽  
Cellular Prion Protein ◽  
Protein Assemblies ◽  
Replication Process ◽  
Data Points ◽  
Autocatalytic Process ◽  
Β Sheet

Prions are proteinaceous infectious agents responsible for a range of neurodegenerative diseases in animals and humans. Prion particles are assemblies formed from a misfolded, β-sheet rich, aggregation-prone isoform (PrPSc) of the host-encoded cellular prion protein (PrPC). Prions replicate by recruiting and converting PrPC into PrPSc, by an autocatalytic process. PrPSc is a pleiomorphic protein as different conformations can dictate different disease phenotypes in the same host species. This is the basis of the strain phenomenon in prion diseases. Recent experimental evidence suggests further structural heterogeneity in PrPSc assemblies within specific prion populations and strains. Still, this diversity is rather seen as a size continuum of assemblies with the same core structure, while analysis of the available experimental data points to the existence of structurally distinct arrangements. The atomic structure of PrPSc has not been elucidated so far, making the prion replication process difficult to understand. All currently available models suggest that PrPSc assemblies exhibit a PrPSc subunit as core constituent, which was recently identified. This review summarizes our current knowledge on prion assembly heterogeneity down to the subunit level and will discuss its importance with regard to the current molecular principles of the prion replication process.

scholarly journals OPA1 overexpression ameliorates mitochondrial cristae remodeling, mitochondrial dysfunction, and neuronal apoptosis in prion diseases

2019 ◽  
Vol 10 (10) ◽  
Author(s):  
Wei Wu ◽  
Deming Zhao ◽  
Syed Zahid Ali Shah ◽  
Xixi Zhang ◽  
Mengyu Lai ◽  
...  
Keyword(s):  
Mitochondrial Dysfunction ◽  
Neuronal Apoptosis ◽  
Prion Diseases ◽  
Cellular Prion Protein ◽  
Primary Neurons ◽  
Mitochondrial Network ◽  
Mitochondrial Dynamic ◽  
Structure Damage

Abstract Prion diseases caused by the cellular prion protein (PrPC) conversion into a misfolded isoform (PrPSc) are associated with multiple mitochondrial damages. We previously reported mitochondrial dynamic abnormalities and cell death in prion diseases via modulation of a variety of factors. Optic atrophy 1 (OPA1) is one of the factors that control mitochondrial fusion, mitochondrial DNA (mtDNA) maintenance, bioenergetics, and cristae integrity. In this study, we observed downregulation of OPA1 in prion disease models in vitro and in vivo, mitochondria structure damage and dysfunction, loss of mtDNA, and neuronal apoptosis. Similar mitochondria findings were seen in OPA1-silenced un-infected primary neurons. Overexpression of OPA1 not only alleviated prion-induced mitochondrial network fragmentation and mtDNA loss, decrease in intracellular ATP, increase in ADP/ATP ratio, and decrease in mitochondrial membrane potential but also protected neurons from apoptosis by suppressing the release of cytochrome c from mitochondria to cytosol and activation of the apoptotic factor, caspase 3. Our results demonstrated that overexpression of OPA1 alleviates prion-associated mitochondrial network fragmentation and cristae remodeling, mitochondrial dysfunction, mtDNA depletion, and neuronal apoptosis, suggesting that OPA1 may be a novel and effective therapeutic target for prion diseases.

scholarly journals Characterization of mesenchymal stem cells in sheep naturally infected with scrapie

2015 ◽  
Vol 96 (12) ◽  
pp. 3715-3726 ◽  
Author(s):  
Diego R. Mediano ◽  
David Sanz-Rubio ◽  
Rosa Bolea ◽  
Belén Marín ◽  
Francisco J. Vázquez ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Protein Misfolding ◽  
Prion Diseases ◽  
Transcript Level ◽  
Cellular Prion Protein ◽  
Jakob Disease

Mesenchymal stem cells (MSCs) can be infected with prions and have been proposed as in vitro cell-based models for prion replication. In addition, autologous MSCs are of interest for cell therapy in neurodegenerative diseases. To the best of our knowledge, the effect of prion diseases on the characteristics of these cells has never been investigated. Here, we analysed the properties of MSCs obtained from bone marrow (BM-MSCs) and peripheral blood (PB-MSCs) of sheep naturally infected with scrapie — a large mammal model for the study of prion diseases. After three passages of expansion, MSCs derived from scrapie animals displayed similar adipogenic, chondrogenic and osteogenic differentiation ability as cells from healthy controls, although a subtle decrease in the proliferation potential was observed. Exceptionally, mesenchymal markers such as CD29 were significantly upregulated at the transcript level compared with controls. Scrapie MSCs were able to transdifferentiate into neuron-like cells, but displayed lower levels of neurogenic markers at basal conditions, which could limit this potential. The expression levels of cellular prion protein (PrPC) were highly variable between cultures, and no significant differences were observed between control and scrapie-derived MSCs. However, during neurogenic differentiation the expression of PrPC was upregulated in MSCs. This characteristic could be useful for developing in vitro models for prion replication. Despite the infectivity reported for MSCs obtained from scrapie-infected mice and Creutzfeldt–Jakob disease patients, protein misfolding cyclic amplification did not detect PrPSc in BM- or PB-MSCs from scrapie-infected sheep, which limits their use for in vivo diagnosis for scrapie.

scholarly journals Structural studies on the folded domain of the human prion protein bound to the Fab fragment of the antibody POM1

2012 ◽  
Vol 68 (11) ◽  
pp. 1501-1512 ◽  
Author(s):  
Pravas Kumar Baral ◽  
Barbara Wieland ◽  
Mridula Swayampakula ◽  
Magdalini Polymenidou ◽  
Muhammad Hafiz Rahman ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Prion Protein ◽  
Prion Diseases ◽  
Passive Immunization ◽  
Antibody Fragment ◽  
Binding Mode ◽  
Cellular Prion Protein ◽  
Fab Fragment ◽  
Human Prion Protein

Prion diseases are neurodegenerative diseases characterized by the conversion of the cellular prion protein PrPcinto a pathogenic isoform PrPsc. Passive immunization with antiprion monoclonal antibodies can arrest the progression of prion diseases. Here, the crystal structure of the Fab fragment of an antiprion monoclonal antibody, POM1, in complex with human prion protein (huPrPc) has been determined to 2.4 Å resolution. The prion epitope of POM1 is in close proximity to the epitope recognized by the purportedly therapeutic antibody fragment ICSM18 Fab in complex with huPrPc. POM1 Fab forms a 1:1 complex with huPrPcand the measuredKdof 4.5 × 10−7 Mreveals moderately strong binding between them. Structural comparisons have been made among three prion–antibody complexes: POM1 Fab–huPrPc, ICSM18 Fab–huPrPcand VRQ14 Fab–ovPrPc. The prion epitopes recognized by ICSM18 Fab and VRQ14 Fab are adjacent to a prion glycosylation site, indicating possible steric hindrance and/or an altered binding mode to the glycosylated prion proteinin vivo. However, both of the glycosylation sites on huPrPcare positioned away from the POM1 Fab binding epitope; thus, the binding mode observed in this crystal structure and the binding affinity measured for this antibody are most likely to be the same as those for the native prion proteinin vivo.

scholarly journals A novel, resistance-linked ovine PrP variant and its equivalent mouse variant modulate the in vitro cell-free conversion of rPrP to PrPres

2006 ◽  
Vol 87 (12) ◽  
pp. 3747-3751 ◽  
Author(s):  
Louise Kirby ◽  
Wilfred Goldmann ◽  
Fiona Houston ◽  
Andrew C. Gill ◽  
Jean C. Manson
Keyword(s):  
Amino Acid ◽  
Prion Diseases ◽  
Cellular Prion Protein ◽  
Transmission Characteristics ◽  
Different Types ◽  
A Cell ◽  
Conversion Efficiencies ◽  
Equivalent Variant

Prion diseases are associated with the conversion of the normal cellular prion protein, PrPc, to the abnormal, disease-associated form, PrPSc. This conversion can be mimicked in vitro by using a cell-free conversion assay. It has recently been shown that this assay can be modified to use bacterial recombinant PrP as substrate and mimic the in vivo transmission characteristics of rodent scrapie. Here, it is demonstrated that the assay replicates the ovine polymorphism barriers of scrapie transmission. In addition, the recently identified ovine PrP variant ARL168Q, which is associated with resistance of sheep to experimental BSE, modulates the cell-free conversion of ovine recombinant PrP to PrPres by three different types of PrPSc, reducing conversion efficiencies to levels similar to those of the ovine resistance-associated ARR variant. Also, the equivalent variant in mice (L164) is resistant to conversion by 87V scrapie. Together, these results suggest a significant role for this position and/or amino acid in conversion.

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