Driving Forces Stabilizing Cellular Prion Protein (PrpC) Monomers and Dimers on the Cell Surface

ABSTRACTThe search for antiprion compounds has been encouraged by the fact that transmissible spongiform encephalopathies (TSEs) share molecular mechanisms with more prevalent neurodegenerative pathologies, such as Parkinson's and Alzheimer's diseases. Cellular prion protein (PrPC) conversion into protease-resistant forms (protease-resistant PrP [PrPRes] or the scrapie form of PrP [PrPSc]) is a critical step in the development of TSEs and is thus one of the main targets in the screening for antiprion compounds. In this work, three trimethoxychalcones (compounds J1, J8, and J20) and one oxadiazole (compound Y17), previously identifiedin vitroto be potential antiprion compounds, were evaluated through different approaches in order to gain inferences about their mechanisms of action. None of them changed PrPCmRNA levels in N2a cells, as shown by reverse transcription-quantitative real-time PCR. Among them, J8 and Y17 were effective in real-time quaking-induced conversion reactions using rodent recombinant PrP (rPrP) from residues 23 to 231 (rPrP23–231) as the substrate and PrPScseeds from hamster and human brain. However, when rPrP from residues 90 to 231 (rPrP90–231), which lacks the N-terminal domain, was used as the substrate, only J8 remained effective, indicating that this region is important for Y17 activity, while J8 seems to interact with the PrPCglobular domain. J8 also reduced the fibrillation of mouse rPrP23–231seeded within vitro-produced fibrils. Furthermore, most of the compounds decreased the amount of PrPCon the N2a cell surface by trapping this protein in the endoplasmic reticulum. On the basis of these results, we hypothesize that J8, a nontoxic compound previously shown to be a promising antiprion agent, may act by different mechanisms, since its efficacy is attributable not only to PrP conversion inhibition but also to a reduction of the PrPCcontent on the cell surface.

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Mapping the Prion Protein Using Recombinant Antibodies

Journal of Virology ◽

10.1128/jvi.72.11.9413-9418.1998 ◽

1998 ◽

Vol 72 (11) ◽

pp. 9413-9418 ◽

Cited By ~ 150

Author(s):

R. Anthony Williamson ◽

David Peretz ◽

Clemencia Pinilla ◽

Hadyn Ball ◽

Raiza B. Bastidas ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Cell Surface ◽

Prion Protein ◽

Disease Process ◽

Cellular Prion Protein ◽

Epitope Region ◽

Molecular Events ◽

C Terminus ◽

Phage Display Libraries ◽

Recombinant Molecule

ABSTRACT The fundamental event in prion disease is thought to be the posttranslational conversion of the cellular prion protein (PrPC) into a pathogenic isoform (PrPSc). The occurrence of PrPC on the cell surface and PrPSc in amyloid plaques in situ or in aggregates following purification complicates the study of the molecular events that underlie the disease process. Monoclonal antibodies are highly sensitive probes of protein conformation which can be used under these conditions. Here, we report the rescue of a diverse panel of 19 PrP-specific recombinant monoclonal antibodies from phage display libraries prepared from PrP deficient (Prnp0/0) mice immunized with infectious prions either in the form of rods or PrP 27-30 dispersed into liposomes. The antibodies recognize a number of distinct linear and discontinuous epitopes that are presented to a varying degree on different PrP preparations. The epitope reactivity of the recombinant PrP(90-231) molecule was almost indistinguishable from that of PrPC on the cell surface, validating the importance of detailed structural studies on the recombinant molecule. Only one epitope region at the C terminus of PrP was well presented on both PrPC and PrPSc, while epitopes associated with most of the antibodies in the panel were present on PrPCbut absent from PrPSc.

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The 37/67kDa laminin receptor (LR) inhibitor, NSC47924, affects 37/67kDa LR cell surface localization and interaction with the cellular prion protein

Scientific Reports ◽

10.1038/srep24457 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 10

Author(s):

Daniela Sarnataro ◽

Anna Pepe ◽

Gennaro Altamura ◽

Imma De Simone ◽

Ada Pesapane ◽

...

Keyword(s):

Cell Surface ◽

Prion Protein ◽

Cellular Prion Protein ◽

Laminin Receptor ◽

Surface Localization ◽

Cell Surface Localization

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Cell-surface retention of PrPC by anti-PrP antibody prevents protease-resistant PrP formation

Journal of General Virology ◽

10.1099/vir.0.80113-0 ◽

2004 ◽

Vol 85 (11) ◽

pp. 3473-3482 ◽

Cited By ~ 63

Author(s):

Chan-Lan Kim ◽

Ayako Karino ◽

Naotaka Ishiguro ◽

Morikazu Shinagawa ◽

Motoyoshi Sato ◽

...

Keyword(s):

Cell Surface ◽

Prion Protein ◽

Flow Cytometric Analysis ◽

Degradation Pathway ◽

Surface Flow ◽

Cellular Prion Protein ◽

Terminal Portion ◽

Persistently Infected ◽

Prion Propagation ◽

In Cells

The C-terminal portion of the prion protein (PrP), corresponding to a protease-resistant core fragment of the abnormal isoform of the prion protein (PrPSc), is essential for prion propagation. Antibodies to the C-terminal portion of PrP are known to inhibit PrPSc accumulation in cells persistently infected with prions. Here it was shown that, in addition to monoclonal antibodies (mAbs) to the C-terminal portion of PrP, a mAb recognizing the octapeptide repeat region in the N-terminal part of PrP that is dispensable for PrPSc formation reduced PrPSc accumulation in cells persistently infected with prions. The 50 % effective dose was as low as ∼1 nM, and, regardless of their epitope specificity, the inhibitory mAbs shared the ability to bind cellular prion protein (PrPC) expressed on the cell surface. Flow cytometric analysis revealed that mAbs that bound to the cell surface during cell culture were not internalized even after their withdrawal from the growth medium. Retention of the mAb–PrPC complex on the cell surface was also confirmed by the fact that internalization was enhanced by treatment of cells with dextran sulfate. These results suggested that anti-PrP mAb antagonizes PrPSc formation by interfering with the regular PrPC degradation pathway.

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Cell-surface prion protein interacts with glycosaminoglycans

Biochemical Journal ◽

10.1042/bj20020773 ◽

2002 ◽

Vol 368 (1) ◽

pp. 81-90 ◽

Cited By ~ 102

Author(s):

Tao PAN ◽

Boon-Seng WONG ◽

Tong LIU ◽

Ruliang LI ◽

Robert B. PETERSEN ◽

...

Keyword(s):

Cell Surface ◽

Prion Protein ◽

Chondroitin Sulphate ◽

Divalent Cations ◽

Cellular Prion Protein ◽

Amino Acid Residues ◽

Heparin Binding ◽

Wild Type ◽

N Terminus ◽

Surface Receptor

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.

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A soluble derivative of PrPC activates cell-signaling and regulates cell physiology through LRP1 and the NMDA receptor

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013779 ◽

2020 ◽

Vol 295 (41) ◽

pp. 14178-14188

Author(s):

Elisabetta Mantuano ◽

Pardis Azmoon ◽

Michael A. Banki ◽

Michael S. Lam ◽

Christina J. Sigurdson ◽

...

Keyword(s):

Nervous System ◽

Cell Migration ◽

Cell Surface ◽

Cell Signaling ◽

Prion Protein ◽

Src Family Kinases ◽

Cellular Prion Protein ◽

Trk Receptors ◽

Extracellular Signal Regulated Kinase ◽

Extracellular Signal

Cellular prion protein (PrPC) is a widely expressed glycosylphosphatidylinositol-anchored membrane protein. Scrapie prion protein is a misfolded and aggregated form of PrPC responsible for prion-induced neurodegenerative diseases. Understanding the function of the nonpathogenic PrPC monomer is an important objective. PrPC may be shed from the cell surface to generate soluble derivatives. Herein, we studied a recombinant derivative of PrPC (soluble cellular prion protein, S-PrP) that corresponds closely in sequence to a soluble form of PrPC shed from the cell surface by proteases in the A Disintegrin And Metalloprotease (ADAM) family. S-PrP activated cell-signaling in PC12 and N2a cells. TrkA was transactivated by Src family kinases and extracellular signal–regulated kinase 1/2 was activated downstream of Trk receptors. These cell-signaling events were dependent on the N-methyl-d-aspartate receptor (NMDA-R) and low-density lipoprotein receptor-related protein-1 (LRP1), which functioned as a cell-signaling receptor system in lipid rafts. Membrane-anchored PrPC and neural cell adhesion molecule were not required for S-PrP–initiated cell-signaling. S-PrP promoted PC12 cell neurite outgrowth. This response required the NMDA-R, LRP1, Src family kinases, and Trk receptors. In Schwann cells, S-PrP interacted with the LRP1/NMDA-R system to activate extracellular signal–regulated kinase 1/2 and promote cell migration. The effects of S-PrP on PC12 cell neurite outgrowth and Schwann cell migration were similar to those caused by other proteins that engage the LRP1/NMDA-R system, including activated α2-macroglobulin and tissue-type plasminogen activator. Collectively, these results demonstrate that shed forms of PrPC may exhibit important biological activities in the central nervous system and the peripheral nervous system by serving as ligands for the LRP1/NMDA-R system.

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A glycolipid-anchored prion protein is endocytosed via clathrin-coated pits.

The Journal of Cell Biology ◽

10.1083/jcb.125.6.1239 ◽

1994 ◽

Vol 125 (6) ◽

pp. 1239-1250 ◽

Cited By ~ 169

Author(s):

S L Shyng ◽

J E Heuser ◽

D A Harris

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Prion Protein ◽

Transmembrane Protein ◽

Primary Cultures ◽

Neuroblastoma Cells ◽

Cellular Prion Protein ◽

Coated Vesicles ◽

Coated Pits ◽

Hypertonic Medium

The cellular prion protein (PrPc) is a glycolipid-anchored, cell surface protein of unknown function, a posttranslationally modified isoform of which PrPSc is involved in the pathogenesis of Creutzfeldt-Jakob disease, scrapie, and other spongiform encephalopathies. We have shown previously that chPrP, a chicken homologue of mammalian PrPC, constitutively cycles between the cell surface and an endocytic compartment, with a transit time of approximately 60 min in cultured neuroblastoma cells. We now report that endocytosis of chPrP is mediated by clathrin-coated pits. Immunogold labeling of neuroblastoma cells demonstrates that the concentration of chPrP within 0.05 microns of coated pits is 3-5 times higher than over other areas of the plasma membrane. Moreover, gold particles can be seen within coated vesicles and deeply invaginated coated pits that are in the process of pinching off from the plasma membrane. ChPrP is also localized to coated pits in primary cultures of neurons and glia, and is found in coated vesicles purified from chicken brain. Finally, internalization of chPrP is reduced by 70% after neuroblastoma cells are incubated in hypertonic medium, a treatment that inhibits endocytosis by disrupting clathrin lattices. Caveolae, plasmalemmal invaginations in which several other glycolipid-anchored proteins are concentrated, are not seen in neuroblastoma cells analyzed by thin-section or deep-etch electron microscopy. Moreover, these cells do not express detectable levels of caveolin, a caveolar coat protein. Since chPrP lacks a cytoplasmic domain that could interact directly with the intracellular components of clathrin-coated pits, we propose that the polypeptide chain of chPrP associates with the extracellular domain of a transmembrane protein that contains a coated pit internalization signal.

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Induction of cellular prion protein gene expression by copper in neurons

AJP Cell Physiology ◽

10.1152/ajpcell.00160.2005 ◽

2006 ◽

Vol 290 (1) ◽

pp. C271-C281 ◽

Cited By ~ 44

Author(s):

Lorena Varela-Nallar ◽

Enrique M. Toledo ◽

Luis F. Larrondo ◽

Ana L. B. Cabral ◽

Vilma R. Martins ◽

...

Keyword(s):

Cell Surface ◽

Prion Protein ◽

Transcriptional Activation ◽

Cortical Neurons ◽

Conformational Transition ◽

Prion Diseases ◽

Cellular Prion Protein ◽

Proteinase K ◽

Glycosylation Pattern ◽

Mobility Shift

Prion diseases are caused by the conformational transition of the native α-helical cellular prion protein (PrPC) into a β-sheet pathogenic isoform. However, the normal physiological function of PrPC remains elusive. We report herein that copper induces PrPC expression in primary hippocampal and cortical neurons. PrPC induced by copper has a normal glycosylation pattern, is proteinase K-sensitive and reaches the cell surface attached by a glycosyl phosphatidylinositol anchor. Immunofluorescence analysis revealed that copper induces PrPC levels in the cell surface and in an intracellular compartment that we identified as the Golgi complex. In addition, copper induced the activity of a reporter vector driven by the rat PrPC gene ( Prnp) promoter stably transfected into PC12 cells, whereas no effect was observed in glial C6 clones. Also cadmium, but not zinc or manganese, upregulated Prnp promoter activity in PC12 clones. Progressive deletions of the promoter revealed that the region essential for copper modulation contains a putative metal responsive element. Although electrophoretic mobility shift assay demonstrated nuclear protein binding to this element, supershift analysis showed that this is not a binding site for the metal responsive transcription factor-1 (MTF-1). The MTF-1-independent transcriptional activation of Prnp is supported by the lack of Prnp promoter activation by zinc. These findings demonstrate that Prnp expression is upregulated by copper in neuronal cells by an MTF-1-independent mechanism, and suggest a metal-specific modulation of Prnp in neurons.

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