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 Prion Diseases: A Unique Transmissible Agent or a Model for Neurodegenerative Diseases?

Biomolecules ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 207
Author(s):  
Diane L. Ritchie ◽  
Marcelo A. Barria
Keyword(s):  
Public Health ◽  
Neurodegenerative Diseases ◽  
Prion Protein ◽  
Neurodegenerative Disorders ◽  
Prion Diseases ◽  
Experimental Models ◽  
Misfolded Proteins ◽  
Current Evidence

The accumulation and propagation in the brain of misfolded proteins is a pathological hallmark shared by many neurodegenerative diseases such as Alzheimer’s disease (Aβ and tau), Parkinson’s disease (α-synuclein), and prion disease (prion protein). Currently, there is no epidemiological evidence to suggest that neurodegenerative disorders are infectious, apart from prion diseases. However, there is an increasing body of evidence from experimental models to suggest that other pathogenic proteins such as Aβ and tau can propagate in vivo and in vitro in a prion-like mechanism, inducing the formation of misfolded protein aggregates such as amyloid plaques and neurofibrillary tangles. Such similarities have raised concerns that misfolded proteins, other than the prion protein, could potentially transmit from person-to-person as rare events after lengthy incubation periods. Such concerns have been heightened following a number of recent reports of the possible inadvertent transmission of Aβ pathology via medical and surgical procedures. This review will provide a historical perspective on the unique transmissible nature of prion diseases, examining their impact on public health and the ongoing concerns raised by this rare group of disorders. Additionally, this review will provide an insight into current evidence supporting the potential transmissibility of other pathogenic proteins associated with more common neurodegenerative disorders and the potential implications for public health.

scholarly journals PrP P102L and Nearby Lysine Mutations Promote Spontaneous In Vitro Formation of Transmissible Prions

2017 ◽  
Vol 91 (21) ◽  
Author(s):  
Allison Kraus ◽  
Gregory J. Raymond ◽  
Brent Race ◽  
Katrina J. Campbell ◽  
Andrew G. Hughson ◽  
...  
Keyword(s):  
Prion Protein ◽  
Prion Diseases ◽  
Transmissible Spongiform Encephalopathy ◽  
Clinical Signs ◽  
Protein Aggregates ◽  
Structural Features ◽  
Spongiform Encephalopathy ◽  
Specific Sequence

ABSTRACT Accumulation of fibrillar protein aggregates is a hallmark of many diseases. While numerous proteins form fibrils by prion-like seeded polymerization in vitro, only some are transmissible and pathogenic in vivo. To probe the structural features that confer transmissibility to prion protein (PrP) fibrils, we have analyzed synthetic PrP amyloids with or without the human prion disease-associated P102L mutation. The formation of infectious prions from PrP molecules in vitro has required cofactors and/or unphysiological denaturing conditions. Here, we demonstrate that, under physiologically compatible conditions without cofactors, the P102L mutation in recombinant hamster PrP promoted prion formation when seeded by minute amounts of scrapie prions in vitro. Surprisingly, combination of the P102L mutation with charge-neutralizing substitutions of four nearby lysines promoted spontaneous prion formation. When inoculated into hamsters, both of these types of synthetic prions initiated substantial accumulation of prion seeding activity and protease-resistant PrP without transmissible spongiform encephalopathy (TSE) clinical signs or notable glial activation. Our evidence suggests that PrP's centrally located proline and lysine residues act as conformational switches in the in vitro formation of transmissible PrP amyloids. IMPORTANCE Many diseases involve the damaging accumulation of specific misfolded proteins in thread-like aggregates. These threads (fibrils) are capable of growing on the ends by seeding the refolding and incorporation of the normal form of the given protein. In many cases such aggregates can be infectious and propagate like prions when transmitted from one individual host to another. Some transmitted aggregates can cause fatal disease, as with human iatrogenic prion diseases, while other aggregates appear to be relatively innocuous. The factors that distinguish infectious and pathogenic protein aggregates from more innocuous ones are poorly understood. Here we have compared the combined effects of prion seeding and mutations of prion protein (PrP) on the structure and transmission properties of synthetic PrP aggregates. Our results highlight the influence of specific sequence features in the normally unstructured region of PrP that influence the infectious and neuropathogenic properties of PrP-derived aggregates.

scholarly journals Signal sequence insufficiency contributes to neurodegeneration caused by transmembrane prion protein

2010 ◽  
Vol 188 (4) ◽  
pp. 515-526 ◽  
Author(s):  
Neena S. Rane ◽  
Oishee Chakrabarti ◽  
Lionel Feigenbaum ◽  
Ramanujan S. Hegde
Keyword(s):  
Prion Protein ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Signal Sequences ◽  
Hydrophobic Domain ◽  
Sequence Variations ◽  
Sufficient Magnitude ◽  
The Impact

Protein translocation into the endoplasmic reticulum is mediated by signal sequences that vary widely in primary structure. In vitro studies suggest that such signal sequence variations may correspond to subtly different functional properties. Whether comparable functional differences exist in vivo and are of sufficient magnitude to impact organism physiology is unknown. Here, we investigate this issue by analyzing in transgenic mice the impact of signal sequence efficiency for mammalian prion protein (PrP). We find that replacement of the average efficiency signal sequence of PrP with more efficient signals rescues mice from neurodegeneration caused by otherwise pathogenic PrP mutants in a downstream hydrophobic domain (HD). This effect is explained by the demonstration that efficient signal sequence function precludes generation of a cytosolically exposed, disease-causing transmembrane form of PrP mediated by the HD mutants. Thus, signal sequences are functionally nonequivalent in vivo, with intrinsic inefficiency of the native PrP signal being required for pathogenesis of a subset of disease-causing PrP mutations.

scholarly journals Neuroprotective role of PrPC against kainate-induced epileptic seizures and cell death depends on the modulation of JNK3 activation by GluR6/7–PSD-95 binding

2011 ◽  
Vol 22 (17) ◽  
pp. 3041-3054 ◽  
Author(s):  
Patricia Carulla ◽  
Ana Bribián ◽  
Alejandra Rangel ◽  
Rosalina Gavín ◽  
Isidro Ferrer ◽  
...  
Keyword(s):  
Prion Protein ◽  
Knockout Mice ◽  
Neuronal Excitability ◽  
Epileptic Seizures ◽  
Cellular Prion Protein ◽  
Transmissible Spongiform Encephalopathies ◽  
Spongiform Encephalopathies ◽  
Glycosyl Phosphatidylinositol

Cellular prion protein (PrPC) is a glycosyl-phosphatidylinositol–anchored glycoprotein. When mutated or misfolded, the pathogenic form (PrPSC) induces transmissible spongiform encephalopathies. In contrast, PrPC has a number of physiological functions in several neural processes. Several lines of evidence implicate PrPC in synaptic transmission and neuroprotection since its absence results in an increase in neuronal excitability and enhanced excitotoxicity in vitro and in vivo. Furthermore, PrPC has been implicated in the inhibition of N-methyl-d-aspartic acid (NMDA)–mediated neurotransmission, and prion protein gene (Prnp) knockout mice show enhanced neuronal death in response to NMDA and kainate (KA). In this study, we demonstrate that neurotoxicity induced by KA in Prnp knockout mice depends on the c-Jun N-terminal kinase 3 (JNK3) pathway since Prnpo/oJnk3o/o mice were not affected by KA. Pharmacological blockage of JNK3 activity impaired PrPC-dependent neurotoxicity. Furthermore, our results indicate that JNK3 activation depends on the interaction of PrPC with postsynaptic density 95 protein (PSD-95) and glutamate receptor 6/7 (GluR6/7). Indeed, GluR6–PSD-95 interaction after KA injections was favored by the absence of PrPC. Finally, neurotoxicity in Prnp knockout mice was reversed by an AMPA/KA inhibitor (6,7-dinitroquinoxaline-2,3-dione) and the GluR6 antagonist NS-102. We conclude that the protection afforded by PrPC against KA is due to its ability to modulate GluR6/7-mediated neurotransmission and hence JNK3 activation.

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 In vitro conversion and seeded fibrillization of posttranslationally modified prion protein

2011 ◽  
Vol 392 (5) ◽  
Author(s):  
Jan Stöhr ◽  
Kerstin Elfrink ◽  
Nicole Weinmann ◽  
Holger Wille ◽  
Dieter Willbold ◽  
...  
Keyword(s):  
Prion Protein ◽  
Posttranslational Modifications ◽  
Prion Diseases ◽  
Chinese Hamster Ovary Cells ◽  
Sodium Dodecyl ◽  
Chinese Hamster ◽  
Full Length ◽  
Proteinase K

AbstractThe conversion of the cellular isoform of the prion protein (PrPC) into the pathologic isoform (PrPSc) is the key event in prion diseases. To study the conversion process, anin vitrosystem based on varying the concentration of low amounts of sodium dodecyl sulfate (SDS) has been employed. In the present study, the conversion of full-length PrPCisolated from Chinese hamster ovary cells (CHO-PrPC) was examined. CHO-PrPCharbors native, posttranslational modifications, including the GPI anchor and two N-linked glyco-sylation sites. The properties of CHO-PrPCwere compared with those of full-length and N-terminally truncated recombinant PrP. As shown earlier with recombinant PrP (recPrP90-231), transition from a soluble α-helical state as known for native PrPCinto an aggregated, β-sheet-rich PrPSc-like state could be induced by dilution of SDS. The aggregated state is partially proteinase K (PK)-resistant, exhibiting a cleavage site similar to that found with PrPSc. Compared to recPrP (90-231), fibril formation with CHO-PrPCrequires lower SDS concentrations (0.0075%), and can be drastically accelerated by seeding with PrPScpurified from brain homogenates of terminally sick hamsters. Our results show that recPrP 90-231 and CHO-PrPC behave qualitatively similar but quantitatively different. Thein vivosituation can be simulated closer with CHO-PrPCbecause the specific PK cleave site could be shown and the seed-assisted fibrillization was much more efficient.

scholarly journals Prion protein attenuates excitotoxicity by inhibiting NMDA receptors

2008 ◽  
Vol 181 (3) ◽  
pp. 551-565 ◽  
Author(s):  
Houman Khosravani ◽  
Yunfeng Zhang ◽  
Shigeki Tsutsui ◽  
Shahid Hameed ◽  
Christophe Altier ◽  
...  
Keyword(s):  
Nmda Receptors ◽  
Prion Protein ◽  
Hippocampal Neurons ◽  
Neuronal Excitability ◽  
Physiological Role ◽  
Cellular Prion Protein ◽  
Glutamate Excitotoxicity ◽  
Null Mouse

It is well established that misfolded forms of cellular prion protein (PrP [PrPC]) are crucial in the genesis and progression of transmissible spongiform encephalitis, whereas the function of native PrPC remains incompletely understood. To determine the physiological role of PrPC, we examine the neurophysiological properties of hippocampal neurons isolated from PrP-null mice. We show that PrP-null mouse neurons exhibit enhanced and drastically prolonged N-methyl-d-aspartate (NMDA)–evoked currents as a result of a functional upregulation of NMDA receptors (NMDARs) containing NR2D subunits. These effects are phenocopied by RNA interference and are rescued upon the overexpression of exogenous PrPC. The enhanced NMDAR activity results in an increase in neuronal excitability as well as enhanced glutamate excitotoxicity both in vitro and in vivo. Thus, native PrPC mediates an important neuroprotective role by virtue of its ability to inhibit NR2D subunits.

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 Mutated but Not Deleted Ovine PrPCN-Terminal Polybasic Region Strongly Interferes with Prion Propagation in Transgenic Mice

2015 ◽  
Vol 90 (3) ◽  
pp. 1638-1646 ◽  
Author(s):  
Manal Khalifé ◽  
Fabienne Reine ◽  
Sophie Paquet-Fifield ◽  
Johan Castille ◽  
Laetitia Herzog ◽  
...  
Keyword(s):  
Amino Acid ◽  
Prion Protein ◽  
Major Effect ◽  
Cellular Prion Protein ◽  
Spongiform Encephalopathy ◽  
Structural Variations ◽  
Prion Propagation ◽  
Polybasic Region

ABSTRACTMammalian prions are proteinaceous infectious agents composed of misfolded assemblies of the host-encoded, cellular prion protein (PrP). Physiologically, the N-terminal polybasic region of residues 23 to 31 of PrP has been shown to be involved in its endocytic trafficking and interactions with glycosaminoglycans or putative ectodomains of membrane-associated proteins. Several recent reports also describe this PrP region as important for the toxicity of mutant prion proteins and the efficiency of prion propagation, bothin vitroandin vivo. The question remains as to whether the latter observations made with mouse PrP and mouse prions would be relevant to other PrP species/prion strain combinations given the dramatic impact on prion susceptibility of minimal amino acid substitutions and structural variations in PrP. Here, we report that transgenic mouse lines expressing ovine PrP with a deletion of residues 23 to 26 (KKRP) or mutated in this N-terminal region (KQHPH instead of KKRPK) exhibited a variable, strain-dependent susceptibility to prion infection with regard to the proportion of affected mice and disease tempo relative to findings in their wild-type counterparts. Deletion has no major effect on 127S scrapie prion pathogenesis, whereas mutation increased by almost 3-fold the survival time of the mice. Deletion marginally affected the incubation time of scrapie LA19K and ovine bovine spongiform encephalopathy (BSE) prions, whereas mutation caused apparent resistance to disease.IMPORTANCERecent reports suggested that the N-terminal polybasic region of the prion protein could be a therapeutic target to prevent prion propagation or toxic signaling associated with more common neurodegenerative diseases such as Alzheimer's disease. Mutating or deleting this region in ovine PrP completes the data previously obtained with the mouse protein by identifying the key amino acid residues involved.

scholarly journals Decrease in Skin Prion-Seeding Activity of Prion-Infected Mice Treated with a Compound Against Human and Animal Prions: a First Possible Biomarker for Prion Therapeutics

2021 ◽  
Author(s):  
Mingxuan Ding ◽  
Kenta Teruya ◽  
Weiguanliu Zhang ◽  
Hae Weon Lee ◽  
Jue Yuan ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Prion Protein ◽  
Therapeutic Efficacy ◽  
Protein Misfolding ◽  
Prion Diseases ◽  
Inhibitory Effect ◽  
Cellular Prion Protein ◽  
Cellulose Ethers ◽  
Wasting Disease

AbstractPrevious studies have revealed that the infectious scrapie isoform of prion protein (PrPSc) harbored in the skin tissue of patients or animals with prion diseases can be amplified and detected through the serial protein misfolding cyclic amplification (sPMCA) or real-time quaking-induced conversion (RT-QuIC) assays. These findings suggest that skin PrPSc-seeding activity may serve as a biomarker for the diagnosis of prion diseases; however, its utility as a biomarker for prion therapeutics remains largely unknown. Cellulose ethers (CEs, such as TC-5RW), widely used as food and pharmaceutical additives, have recently been shown to prolong the lifespan of prion-infected mice and hamsters. Here we report that in transgenic (Tg) mice expressing hamster cellular prion protein (PrPC) infected with the 263K prion, the prion-seeding activity becomes undetectable in the skin tissues of TC-5RW-treated Tg mice by both sPMCA and RT-QuIC assays, whereas such prion-seeding activity is readily detectable in the skin of untreated mice. Notably, TC-5RW exhibits an inhibitory effect on the in vitro amplification of PrPSc in both skin and brain tissues by sPMCA and RT-QuIC. Moreover, we reveal that TC-5RW is able to directly decrease protease-resistant PrPSc and inhibit the seeding activity of PrPSc from chronic wasting disease and various human prion diseases. Our results suggest that the level of prion-seeding activity in the skin may serve as a useful biomarker for assessing the therapeutic efficacy of compounds in a clinical trial of prion diseases and that TC-5RW may have the potential for the prevention/treatment of human prion diseases.

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