The cellular prion protein interacts with and promotes the activity of Na,K-ATPases

The prion protein (PrP) is best known for its ability to cause fatal neurodegenerative diseases in humans and animals. Here, we revisited its molecular environment in the brain using a well-developed affinity-capture mass spectrometry workflow that offers robust relative quantitation. The analysis confirmed many previously reported interactions. It also pointed toward a profound enrichment of Na,K-ATPases (NKAs) in proximity to cellular PrP (PrPC). Follow-on work validated the interaction, demonstrated partial co-localization of the ATP1A1 and PrPC, and revealed that cells exposed to cardiac glycoside (CG) inhibitors of NKAs exhibit correlated changes to the steady-state levels of both proteins. Moreover, the presence of PrPC was observed to promote the ion uptake activity of NKAs in a human co-culture paradigm of differentiated neurons and glia cells, and in mouse neuroblastoma cells. Consistent with this finding, changes in the expression of 5’-nucleotidase that manifest in wild-type cells in response to CG exposure can also be observed in untreated PrPC-deficient cells. Finally, the endoproteolytic cleavage of the glial fibrillary acidic protein, a hallmark of late-stage prion disease, can also be induced by CGs, raising the prospect that a loss of NKA activity may contribute to the pathobiology of prion diseases.

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Dimerization of the cellular prion protein inhibits propagation of scrapie prions

Journal of Biological Chemistry ◽

10.1074/jbc.ra117.000990 ◽

2018 ◽

Vol 293 (21) ◽

pp. 8020-8031 ◽

Cited By ~ 2

Author(s):

Anna D. Engelke ◽

Anika Gonsberg ◽

Simrika Thapa ◽

Sebastian Jung ◽

Sarah Ulbrich ◽

...

Keyword(s):

Transgenic Mice ◽

Prion Protein ◽

Conformational Transition ◽

Prion Diseases ◽

Neuroblastoma Cells ◽

Direct Interaction ◽

Cellular Prion Protein ◽

Dominant Negative ◽

Dimerization Domain ◽

Hydrophobic Domain

A central step in the pathogenesis of prion diseases is the conformational transition of the cellular prion protein (PrPC) into the scrapie isoform, denoted PrPSc. Studies in transgenic mice have indicated that this conversion requires a direct interaction between PrPC and PrPSc; however, insights into the underlying mechanisms are still missing. Interestingly, only a subfraction of PrPC is converted in scrapie-infected cells, suggesting that not all PrPC species are suitable substrates for the conversion. On the basis of the observation that PrPC can form homodimers under physiological conditions with the internal hydrophobic domain (HD) serving as a putative dimerization domain, we wondered whether PrP dimerization is involved in the formation of neurotoxic and/or infectious PrP conformers. Here, we analyzed the possible impact on dimerization of pathogenic mutations in the HD that induce a spontaneous neurodegenerative disease in transgenic mice. Similarly to wildtype (WT) PrPC, the neurotoxic variant PrP(AV3) formed homodimers as well as heterodimers with WTPrPC. Notably, forced PrP dimerization via an intermolecular disulfide bond did not interfere with its maturation and intracellular trafficking. Covalently linked PrP dimers were complex glycosylated, GPI-anchored, and sorted to the outer leaflet of the plasma membrane. However, forced PrPC dimerization completely blocked its conversion into PrPSc in chronically scrapie-infected mouse neuroblastoma cells. Moreover, PrPC dimers had a dominant-negative inhibition effect on the conversion of monomeric PrPC. Our findings suggest that PrPC monomers are the major substrates for PrPSc propagation and that it may be possible to halt prion formation by stabilizing PrPC dimers.

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Toxicological Evaluation of Anti-Scrapie Trimethoxychalcones and Oxadiazoles

Anais da Academia Brasileira de Ciências ◽

10.1590/0001-3765201520140712 ◽

2015 ◽

Vol 87 (2 suppl) ◽

pp. 1421-1434 ◽

Cited By ~ 1

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.

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Neurotropic influenza A virus infection causes prion protein misfolding into infectious prions in neuroblastoma cells

Scientific Reports ◽

10.1038/s41598-021-89586-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Hideyuki Hara ◽

Junji Chida ◽

Keiji Uchiyama ◽

Agriani Dini Pasiana ◽

Etsuhisa Takahashi ◽

...

Keyword(s):

Prion Protein ◽

Influenza A Virus ◽

Protein Misfolding ◽

Influenza A ◽

Prion Diseases ◽

Neuroblastoma Cells ◽

Cellular Prion Protein ◽

Infected Cells ◽

Hit And Run

AbstractMisfolding of the cellular prion protein, PrPC, into the amyloidogenic isoform, PrPSc, which forms infectious protein aggregates, the so-called prions, is a key pathogenic event in prion diseases. No pathogens other than prions have been identified to induce misfolding of PrPC into PrPSc and propagate infectious prions in infected cells. Here, we found that infection with a neurotropic influenza A virus strain (IAV/WSN) caused misfolding of PrPC into PrPSc and generated infectious prions in mouse neuroblastoma cells through a hit-and-run mechanism. The structural and biochemical characteristics of IAV/WSN-induced PrPSc were different from those of RML and 22L laboratory prions-evoked PrPSc, and the pathogenicity of IAV/WSN-induced prions were also different from that of RML and 22L prions, suggesting IAV/WSN-specific formation of PrPSc and infectious prions. Our current results may open a new avenue for the role of viral infection in misfolding of PrPC into PrPSc and formation of infectious prions.

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Pharmacological inactivation of the prion protein by targeting a folding intermediate

Communications Biology ◽

10.1038/s42003-020-01585-x ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Giovanni Spagnolli ◽

Tania Massignan ◽

Andrea Astolfi ◽

Silvia Biggi ◽

Marta Rigoli ◽

...

Keyword(s):

Prion Protein ◽

Prion Diseases ◽

Cellular Prion Protein ◽

Surface Glycoprotein ◽

Folding Intermediate ◽

Biological Regulation ◽

Cell Surface Glycoprotein ◽

Folding Intermediates ◽

Protein Levels ◽

Small Molecule Ligands

AbstractRecent computational advancements in the simulation of biochemical processes allow investigating the mechanisms involved in protein regulation with realistic physics-based models, at an atomistic level of resolution. These techniques allowed us to design a drug discovery approach, named Pharmacological Protein Inactivation by Folding Intermediate Targeting (PPI-FIT), based on the rationale of negatively regulating protein levels by targeting folding intermediates. Here, PPI-FIT was tested for the first time on the cellular prion protein (PrP), a cell surface glycoprotein playing a key role in fatal and transmissible neurodegenerative pathologies known as prion diseases. We predicted the all-atom structure of an intermediate appearing along the folding pathway of PrP and identified four different small molecule ligands for this conformer, all capable of selectively lowering the load of the protein by promoting its degradation. Our data support the notion that the level of target proteins could be modulated by acting on their folding pathways, implying a previously unappreciated role for folding intermediates in the biological regulation of protein expression.

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Scrapie prion proteins accumulate in the cytoplasm of persistently infected cultured cells.

The Journal of Cell Biology ◽

10.1083/jcb.110.6.2117 ◽

1990 ◽

Vol 110 (6) ◽

pp. 2117-2132 ◽

Cited By ~ 192

Author(s):

A Taraboulos ◽

D Serban ◽

S B Prusiner

Keyword(s):

Cultured Cells ◽

Prion Diseases ◽

Neuroblastoma Cells ◽

Brain Cell ◽

Cellular Prion Protein ◽

Intracellular Accumulation ◽

Golgi Stack ◽

Glycosyl Phosphatidylinositol ◽

The Central Nervous System

The cellular prion protein (PrPC) is a sialoglycoprotein anchored to the external surface of cells by a glycosyl phosphatidylinositol moiety. During scrapie, an abnormal PrP isoform designated PrPSc accumulates, and much evidence argues that it is a major and necessary component of the infectious prion. Based on the resistance of native PrPSc to proteolysis and to digestion with phosphatidylinositol-specific phospholipase C as well as the enhancement of PrPSc immunoreactivity after denaturation, we devised in situ immunoassays for the detection of PrPSc in cultured cells. Using these immunoassays, we identified the sites of PrPSc accumulation in scrapie-infected cultured cells. We also used these immunoassays to isolate PrPSc-producing clones from a new hamster brain cell line (HaB) and found an excellent correlation between their PrPSc content and prion infectivity titers. In scrapie-infected HaB cells as well as in scrapie-infected mouse neuroblastoma cells, most PrPSc was found to be intracellular and most localized with ligands of the Golgi marker wheat germ agglutinin. In one scrapie-infected HaB clone, PrPSc also localized extensively with MG-160, a protein resident of the medial-Golgi stack whereas this colocalization was not observed in another subclone of these cells. Whether the sites of intracellular accumulation of PrPSc are limited to a few subcellular organelles or they are highly variable remains to be determined. If the intracellular accumulation of PrPSc is found in the cells of the central nervous system, then it might be responsible for the neuronal dysfunction and degeneration which are cardinal features of prion diseases.

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Prion protein and prion disease at a glance

Journal of Cell Science ◽

10.1242/jcs.245605 ◽

2021 ◽

Vol 134 (17) ◽

Author(s):

Caihong Zhu ◽

Adriano Aguzzi

Keyword(s):

Prion Protein ◽

Prion Disease ◽

Neurodegenerative Disorders ◽

Prion Diseases ◽

Amyloid Β ◽

Cellular Prion Protein ◽

Future Studies ◽

Associated Proteins ◽

Main Component ◽

Conformational Conversion

ABSTRACT Prion diseases are neurodegenerative disorders caused by conformational conversion of the cellular prion protein (PrPC) into scrapie prion protein (PrPSc). As the main component of prion, PrPSc acts as an infectious template that recruits and converts normal cellular PrPC into its pathogenic, misfolded isoform. Intriguingly, the phenomenon of prionoid, or prion-like, spread has also been observed in many other disease-associated proteins, such as amyloid β (Aβ), tau and α-synuclein. This Cell Science at a Glance and the accompanying poster highlight recently described physiological roles of prion protein and the advanced understanding of pathogenesis of prion disease they have afforded. Importantly, prion protein may also be involved in the pathogenesis of other neurodegenerative disorders such as Alzheimer's and Parkinson's disease. Therapeutic studies of prion disease have also exploited novel strategies to combat these devastating diseases. Future studies on prion protein and prion disease will deepen our understanding of the pathogenesis of a broad spectrum of neurodegenerative conditions.

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Overexpression of quality control proteins reduces prion conversion in prion-infected cells

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.002754 ◽

2018 ◽

Vol 293 (41) ◽

pp. 16069-16082 ◽

Cited By ~ 4

Author(s):

Simrika Thapa ◽

Basant Abdulrahman ◽

Dalia H. Abdelaziz ◽

Li Lu ◽

Manel Ben Aissa ◽

...

Keyword(s):

Quality Control ◽

De Novo ◽

Prion Diseases ◽

Neuroblastoma Cells ◽

Cellular Prion Protein ◽

Control Mechanisms ◽

Cellular Mechanisms ◽

Prion Propagation ◽

Infected Cells

Prion diseases are fatal infectious neurodegenerative disorders in humans and other animals and are caused by misfolding of the cellular prion protein (PrPC) into the pathological isoform PrPSc. These diseases have the potential to transmit within or between species, including zoonotic transmission to humans. Elucidating the molecular and cellular mechanisms underlying prion propagation and transmission is therefore critical for developing molecular strategies for disease intervention. We have shown previously that impaired quality control mechanisms directly influence prion propagation. In this study, we manipulated cellular quality control pathways in vitro by stably and transiently overexpressing selected quality control folding (ERp57) and cargo (VIP36) proteins and investigated the effects of this overexpression on prion propagation. We found that ERp57 or VIP36 overexpression in persistently prion-infected neuroblastoma cells significantly reduces the amount of PrPSc in immunoblots and prion-seeding activity in the real-time quaking-induced conversion (RT-QuIC) assay. Using different cell lines infected with various prion strains confirmed that this effect is not cell type– or prion strain–specific. Moreover, de novo prion infection revealed that the overexpression significantly reduced newly formed PrPSc in acutely infected cells. ERp57-overexpressing cells significantly overcame endoplasmic reticulum stress, as revealed by expression of lower levels of the stress markers BiP and CHOP, accompanied by a decrease in PrP aggregates. Furthermore, application of ERp57-expressing lentiviruses prolonged the survival of prion-infected mice. Taken together, improved cellular quality control via ERp57 or VIP36 overexpression impairs prion propagation and could be utilized as a potential therapeutic strategy.

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