Function and clearance of conformers of the prion protein [Elektronische Ressource] / vorgelegt von Janine Monique Muyrers

Nini

Sujets

• Biologie
• Gesundheit

Informations

• Publié par: heinrich-heine-universitat_dusseldorf
• Publié le: 01 janvier 2008
• Nombre de lectures: 17
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Function and Clearance of Conformers of
the Prion Protein


Inaugural-Dissertation

zur
Erlangung des Doktorgrades der
Mathematisch-Naturwissenschaftlichen Fakultät
der Heinrich-Heine-Universität Düsseldorf

vorgelegt von
Janine Monique Muyrers
aus Aachen

Mai 2008


Aus dem Institut für Neuropathologie
der Heinrich-Heine-Universität Düsseldorf









Gedruckt mit der Genehmigung der
Mathematisch-Naturwissenschaftlichen Fakultät der
Heinrich-Heine-Universität Düsseldorf


Referent: PD. Dr. C. Korth

Korreferent: Prof. Dr. D. Willbold

Tag der mündlichen Prüfung: 16.06.2008
Table of contents

Table of contents
1 Introduction ...............................................................................................3
1.1 Prion Protein................................................................................................3
C1.2 Bioconformatics - topological heterogeneity of PrP ...................................6
C1.3 Physiological function of PrP .....................................................................9
1.4 PrP assays ................................................................................................10
Sc 1.4.1 PrP assay10
1.4.2 Topological- and conformational PrP assay ..............................................10
1.4.3 Conformation specific antibodies...............................................................11
1.5 Protein degradation...................................................................................12
1.5.1 The ubiquitin proteasome system (UPS)...................................................12
1.5.2 Lysosomal degradation .............................................................................12
Sc1.5.3 Clearance of PrP ....................................................................................16
1.6 Objectives .................................................................................................17
2 Material and Methods..............................................................................18
2.1 Reagents...................................................................................................18
2.2 DNA methods............................................................................................25
2.2.1 General procedures (DNA)........................................................................25
2.2.2 DNA Constructs.........................................................................................25
2.2.3 Primer........................................................................................................26
2.3 Microbiological culture...............................................................................27
2.3.1 Bacteria .....................................................................................................27
2.3.2 Media27
2.3.3 Preparation of chemical competent E. coli ................................................28
2.3.4 Transformation of chemical competent E.coli (Hanahan, 1991) ...............28
2.4 Protein methods ........................................................................................28
2.4.1 General procedures (protein) ....................................................................28
2.4.2 Preparation of brain homogenate..............................................................28
2.4.3 Immunoprecipitation (IP) ...........................................................................29
2.5 Antibodies .................................................................................................33
2.5.1 33
2.5.2 Production of polyclonal antibodies...........................................................34
2.5.3 Expression of recombinant scFvAb in E. coli.............................................34
2.5.4 Antibody purification..................................................................................35
2.6 Cell culture ................................................................................................37
2.6.1 Cell lines....................................................................................................37
2.6.2 Media and reagents ..................................................................................38
2.6.3 Culture conditions......................................................................................39
2.6.4 Transfection...............................................................................................39
2.6.5 Immunofluorescence of transient transfected N2a ....................................40
2.6.6 Immunohistochemistry ..............................................................................42
2.6.7 TUNEL staining .........................................................................................43
2.6.8 Histoblots ..................................................................................................44
2.7 Animal experiments...................................................................................45
1 Table of contents
2.8 PrP assays................................................................................................ 45
Sc Sc2.8.1 Biochemical detection of PrP (PrP assay) Cell lysates were
proteolyzed at 37°C for 30 min with 20 µg/mL proteinase K. .................... 45
C2.8.2 Conformational PrP assay ...................................................................... 45
Sc2.8.3 PrP inhibition assay in ScN2a cells (compounds).................................. 46
Sc2.8.4 PrP inhibition assay in ScN2a cells (mAb) ............................................. 46
Sc2.8.5 Sizing of PrP aggregates via sucrose gradient centrifugation................ 47
3 Results..................................................................................................... 48
3.1 Topological isoforms of the Prion Protein ................................................. 48
Ntm3.1.1 Characterization of the PrP specific murine mAb19B10....................... 48
Ntm3.2 Expression pattern of PrP..................................................................... 51
3.2.1 Experiments with single chain fragment of mAb 19B10 (scFv19B10)....... 55
Ntm3.2.2 PrP ligands........................................................................................... 60
Ctm3.3 Functional characterization of the PrP specific mAb 19C3................... 63
3.4 Clearance of PrPSc .................................................................................. 72
3.4.1 Rapamycin antagonizes the antiprion effect of tocopherol succinate ....... 72
C Sc3.4.2 The influence of rheb to the conversion of PrP to PrP ......................... 76
3.4.3 The influence of rheb and the dominate negative mutant of rheb on
CPrP expression........................................................................................ 78
Sc3.4.4 The influence of autophagy on the clearance of PrP ............................. 80
4 Discussion............................................................................................... 83
C4.1 Topological isoforms of PrP .................................................................... 84
Sc4.2 Clearance of PrP .................................................................................... 92
5 Abstract ................................................................................................... 98
6 Zusammenfassung ................................................................................. 99
7 References ............................................................................................ 101
8 Abbreviations 110
9 Acknowledgement ................................................................................ 114
10 Erklärung............................................................................................... 115
2 Introduction
1 Introduction
1.1 Prion Protein
Prions (proteinaceous, infectious particles) are unprecedented infectious pathogens
that cause prion diseases. Prion diseases are a group of neurodegenerative
diseases that include Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-
Scheinker syndrome (GSS), fatal familial insomnia and Kuru in humans, bovine
spongiform encephalopathy in cattle and scrapie in sheep. These diseases are
characterized by the triad of spongiform change, neuronal loss and reactive gliosis
(Kretzschmar, et al., 1996). They are caused by the conversion of the normal cellular
C Scisoforms of the prion protein (PrP ) into the scrapie isoform (PrP ) through a
Scposttranslational process Prusiner, 1998, which is stimulated by PrP itself (Fig. 1)
(McKinley, et al., 1983). The conversion is thought to take place either directly at the
plasma membrane or in the early compartments of the endocytic pathway, e.g.
caveolae or rafts, specialized regions in sphingolipids, cholesterol, and glycosyl
phosphaticyl-inositol-anchored (Vey et al., 1996; Naslavsky et al., 1997, Taraboulos
et al., 1995; Nunziante et al., 2003). The exact mechanism of the conversion is still
enigmatic, although two models are currently in consideration. The first model favors
Sca crystallization reactio