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Interactions of Prion Proteins and PrP-derived Peptides in Scrapie infection
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Prion diseases are fatal and incurable spongiform encephalopathies that occur amongst mammals. The central pathological event is the misfolding of the cellular prion protein (PrPC) into an amyloid, neurotoxic isoform called scrapie (PrPSc). PrPSc is the main, or sole, constituent of infectious prions. PrPSc resists cellular degradation and also induces misfolding of PrPC via a process called conversion. Conversion seems to be an endocytotic event implicating auxiliary cellular cofactors interacting with PrPC and/or PrPSc. The aim of this thesis is to decipher and modulate key events involved in prion conversion and cytopathology, by studying persistently scrapie infected murine neuronal cell cultures. This work shows that cell penetrating peptides derived from the prion protein (PrP-CPPs) can suppress cellular PrPSc levels. The PrP-CPPs assert these actions on two prion strains regardless of peptide configuration and do not inhibit any PrP-interaction with heparan sulfate (HS) proteoglycans (PG). A polybasic motif in the PrP-CPPs may interact with PrPSc, but the anti-prion effect is controlled by a signal peptide sequence. The PrP-CPPs represents a novel form of prion antagonizing compound. Prion-induced alterations in protein expression, cellular localization, activity and metabolism, designate putative mediators of disease or neuroprotective defence mechanisms. We report on interplay between the HSPG glypican-1 (Gpc-1) and scrapie-infection. Gpc-1 is aberrantly distributed in scrapie-infected cells and HS degradation by autocatalytic deaminative cleavage is elevated, suggestively in order to restrain PrPSc levels. Additionally, we demonstrate that scrapie-infection elevates the activity of Src family kinase members Src and Fyn, in part by affecting Src expression and Fyn membrane distribution. This causes an uncontrolled tyrosine phosphorylation which could contribute to neuronal loss in vivo.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2010. , 132 p.
Keyword [en]
Spongiform encephalopathy, Creutzfeldt-Jakob disease, Amyloidosis, Neurodegeneration, Cell penetrating peptide, Protein Transduction Domain, Heparan sulfate, Proteoglycan, Glypican, Src family kinase, Fyn
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-45736ISBN: 978-91-7447-179-3 (print)OAI: oai:DiVA.org:su-45736DiVA: diva2:369696
Public defence
2010-12-21, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defence the following paper was unpublished and had a status as follows: Paper nr. 5: ManuscriptAvailable from: 2010-11-29 Created: 2010-11-10 Last updated: 2010-12-03Bibliographically approved
List of papers
1. Antiprion properties of prion protein-derived cell-penetrating peptides
Open this publication in new window or tab >>Antiprion properties of prion protein-derived cell-penetrating peptides
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2008 (English)In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 22, no 7, 2177-2184 p.Article in journal (Refereed) Published
Abstract [en]

In prion diseases, the cellular prion protein (PrPC) becomes misfolded into the pathogenic scrapie isoform (PrPSc) responsible for prion infectivity. We show here that peptides derived from the prion protein N terminus have potent antiprion effects. These peptides are composed of a hydrophobic sequence followed by a basic segment. They are known to have cell-penetrating ability like regular cell-penetrating peptides (CPPs), short peptides that can penetrate cellular membranes. Healthy (GT1–1) and scrapie-infected (ScGT1–1) mouse neuronal hypothalamic cells were treated with various CPPs, including the prion protein-derived CPPs. Lysates were analyzed for altered protein levels of PrPC or PrPSc. Treatment with the prion protein-derived CPPs mouse mPrP1–28 or bovine bPrP1–30 significantly reduced PrPSc levels in prion-infected cells but had no effect on PrPC levels in noninfected cells. Further, presence of prion protein-derived CPPs significantly prolonged the time before infection was manifested when infecting GT1–1 cells with scrapie. Treatment with other CPPs (penetratin, transportan-10, or poly-L-arginine) or prion protein-derived peptides lacking CPP function (mPrP23–28, mPrP19–30, or mPrP23–50) had no effect on PrPSc levels. The results suggest a mechanism by which the signal sequence guides the prion protein-derived CPP into a cellular compartment, where the basic segment binds specifically to PrPSc and disables formation of prions.—Löfgren, K., Wahlström, A., Lundberg, P., Langel, U., Gräslund, A., and Bedecs, K. Antiprion properties of prion protein-derived cell-penetrating peptides.

Keyword
scrapie, prion conversion, N terminus, therapy, signal peptide
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-14492 (URN)10.1096/fj.07-099549 (DOI)000257292500010 ()
Available from: 2009-01-12 Created: 2009-01-12 Last updated: 2017-12-13Bibliographically approved
2. Mechanisms of prion antagonization by PrP-derived cell-penetrating peptides
Open this publication in new window or tab >>Mechanisms of prion antagonization by PrP-derived cell-penetrating peptides
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Cell penetrating peptides derived from the prion protein N-terminus (PrP-CPPs) reduce PrPSc levels in prion-infected neuronal cell cultures (1). The PrP-CPPs consist of the hydrophobic PrP signal sequence followed by a basic segment (KKRPKP) and enter cells through raft-dependent macropinocytosis. To decipher the PrP-CPP anti-prion mechanism, different peptide constructs were analyzed for effects on PrPSc levels in GT1-1 neuronal cell cultures infected with either prion strain RML or 22L. For both strains, the PrP-CPPs antagonized the infection, but RML and 22L-infections differed in sensitivity to the PrP-CPP anti-prion effect. We also show that the effect on PrPSc levels does not depend on peptide interaction with any chiral receptor. The signal sequence segment of the PrP-CPPs promotes a specific positioning within the cell where conversion may occur, as signal sequence segment shortening or targeting of the KKRPKP-motif into alternative sub-cellular compartments disrupts the peptide anti-prion effect. Defining the anti-prion mechanism of PrP-CPPs is a matter of establishing how the peptides connect to the prion replicative interface. As the conversion process is poorly understood, the PrP-CPPs represent useful tools to outline the sub-cellular context of prion propagation.

Keyword
Creutzfeldt–Jakob disease, nuclear localization signal, lipid rafts, protein transduction domain
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-44379 (URN)
Available from: 2010-11-11 Created: 2010-11-08 Last updated: 2016-01-29Bibliographically approved
3. Involvement of glypican-1 autoprocessing in scrapie infection.
Open this publication in new window or tab >>Involvement of glypican-1 autoprocessing in scrapie infection.
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2008 (English)In: Europena Journal of Neuroscience, ISSN 1460-9568, Vol. 28, no 5, 964-72 p.Article in journal (Refereed) Published
Abstract [en]

The copper-binding cellular prion protein (PrP(C)) and the heparan sulphate (HS)-containing proteoglycan glypican-1 (Gpc-1) can both be attached to lipid rafts via their glycosylphosphatidylinositol anchors, and copper ions stimulate their cointernalization from the cell surface to endosomes. The prion protein controls cointernalization and delivers copper necessary for S-nitrosylation of conserved cysteines in the Gpc-1 core protein. Later, during recycling through endosomal compartments, nitric oxide can be released from the S-nitroso groups and catalyses deaminative degradation and release of the HS substituents. Here, by using confocal immunofluorescence microscopy, we show that normal PrP(C) and Gpc-1 colocalize inside GT1-1 cells. However, in scrapie-infected cells (ScGT1-1), Gpc-1 protein remained at the cell surface separate from the cellular prion protein. Scrapie infection stimulated Gpc-1 autoprocessing and the generated HS degradation products colocalized with intracellular aggregates of the disease-related scrapie prion protein isoform (PrP(Sc)). Coimmunoprecipitation experiments demonstrated an association between Gpc-1 and PrP(C) in uninfected cells, and between HS degradation products and PrP(Sc) in infected cells. Silencing of Gpc-1 expression or prevention of Gpc-1 autoprocessing elevated the levels of intracellular PrP(Sc) aggregates in infected cells. These results suggest a role for Gpc-1 autoprocessing in the clearance of PrP(Sc) from infected cells.

Place, publisher, year, edition, pages
Wiley InterScience (Blackwell), 2008
Keyword
heparan sulphate, nitric oxide, prion, proteoglycan, recycling
Identifiers
urn:nbn:se:su:diva-14843 (URN)10.1111/j.1460-9568.2008.06386.x (DOI)000258729200013 ()18717736 (PubMedID)
Available from: 2009-01-12 Created: 2009-01-12 Last updated: 2010-11-12Bibliographically approved
4. Increased Src kinase level results in increased protein tyrosine phosphorylation in scrapie-infected neuronal cell lines.
Open this publication in new window or tab >>Increased Src kinase level results in increased protein tyrosine phosphorylation in scrapie-infected neuronal cell lines.
2006 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 580, no 11, 2603-8 p.Article in journal (Refereed) Published
Abstract [en]

We have studied how prion infection may affect the Src kinase activity in three different neuronal cell lines, ScGT1 and ScN2a, where ScGT1 were generated in our laboratory. By immunoblotting, using clone 28 - a monoclonal antibody recognizing active Src, we have found a 32+/-6.3% and 75+/-7.7% elevation in Src activity in ScGT1 and ScN2a cells, respectively, compared to uninfected cells. Immunocomplex in vitro kinase assay confirmed the increased Src activity. The increased Src kinase activity in scrapie-infected cells was further shown to correlate to an increased level of Src protein. In addition, an important increase in the protein tyrosine phosphorylation signal was observed in ScGT1 and ScN2a cells, which was further shown to be Src-dependent, as treatment with PP2 - a Src family kinase specific inhibitor, reversed the protein tyrosine phosphorylation profile. Abnormal Src-kinase activation and subsequent protein tyrosine phosphorylation may be key elements in the neuropathology of the prion diseases.

Place, publisher, year, edition, pages
Elsevier, 2006
Keyword
Animals, Cell Line, Mice, Neurons/drug effects/*metabolism, Phosphorylation, Phosphotyrosine/*metabolism, Prions/*metabolism, Protein Binding, Protein Kinase Inhibitors/pharmacology, Pyrimidines/pharmacology, Scrapie/enzymology/*metabolism/pathology, Substrate Specificity, src-Family Kinases/antagonists & inhibitors/*metabolism
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-14845 (URN)10.1016/j.febslet.2006.03.092 (DOI)16647068 (PubMedID)
Available from: 2008-11-06 Created: 2008-11-06 Last updated: 2017-12-13Bibliographically approved
5. Altered membrane distribution and increased Fyn activity in scrapie-infected neuronal cells
Open this publication in new window or tab >>Altered membrane distribution and increased Fyn activity in scrapie-infected neuronal cells
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The suggested cause of Prion diseases is conversion of the cellular prion protein (PrPC) into aberrant scrapie prion protein (PrPSc) isoform triggered by the latter. PrPC is localized to membrane rafts, subdomains which are implicated in conversion. In this report we have analyzed the expression, plasma membrane distribution and activity of the raft associative Src family kinase member Fyn, in scrapie-infected neuronal cell lines. As a result of prion infection, the specific tyrosine kinase activity of Fyn is increased, although the protein level of Fyn kinase is reduced. This results in an increased overall tyrosine phosphorylation in the infected cells. Interestingly, prion infection also induced a membrane redistribution of Fyn from non-raft to raft-membrane subdomains, following the distribution of PrPSc, as shown by immunoblotting of flotation-fractions after density gradient centrifugation of Triton X-100 extracted cell extracts. This indicates a persistent Fyn activation, possibly due to clustering of intracellular Fyn kinases as a result of PrPSc association to membrane rafts. An increased Fyn kinase activity followed by a dramatic increase in tyrosine phosphorylation may cause and/or contribute to synaptic disorganization and loss which are fundamental features of prion disease.

Keyword
Creutzfelt-Jacobs disease, detergent resistant membranes, lipid rafts, GT1, RML
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Neurochemistry and Neurotoxicology
Identifiers
urn:nbn:se:su:diva-44380 (URN)
Available from: 2010-11-11 Created: 2010-11-08 Last updated: 2010-11-12Bibliographically approved

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