Studies on the interaction of the cellular and the pathogenic isoforms of the prion protein in the presence of the lipid membrane [Elektronische Ressource] / vorgelegt von Agnieszka Salwierz

Agi Lindegren

Sujets

• Biologie
• Gesundheit

Informations

• Publié par: heinrich-heine-universitat_dusseldorf
• Publié le: 01 janvier 2009
• Nombre de lectures: 44
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

Studies on the interaction of the cellular and the
pathogenic isoforms of the prion protein in the
presence of the lipid membrane


Inaugural-Dissertation



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


vorgelegt von

Agnieszka Salwierz
aus Kościerzyna, Polen






Düsseldorf, 2009




























Angefertigt im Institut für Physikalische Biologie
Heinrich-Heine-Universität
Düsseldorf
Abgabedatum 02. Juni 2009
Tag der mündlichen Prüfung 02. Juli 2009
Betreuer Prof. Dr. D. Riesner
2. Gutachter Prof. Dr. D. Willbold









Eidesstattliche Erklärung
Hiermit erkläre ich an Eides statt, dass ich diese Arbeit selbständig verfasst und keine anderen
als die angegebenen Quellen und Hilfsmittel verwendet, sowie Zitate kenntlich gemacht habe.

Düsseldorf, 02.06.2009

Agnieszka Salwierz


I

Table of contents

Table of contents ............................................................................................................................ I
Abbreviations .............................. IV
Table of figures ............................................................................................................................. V

Table of contents ............................................................................................................................ I
1 Introduction ............................................................................................................. 1
1.1 Transmissible spongiform encephalopathies ........................ 1
1.1.1 Scrapie ....................................................................................................................... 2
1.1.2 Bovine Spongiform Encephalopathy (BSE) ............................. 2
1.1.3 Creutzfeldt-Jakob disease (CJD) ............................................................................... 3
1.2 Molecular basics and characteristics of prion protein ......... 4
1.2.1 Identification of the infectious agent ......................................................................... 4
1.2.2 Structural characteristics of prion protein . 5
1.2.3 Biosynthesis and cellular trafficking of prion protein ............................................... 8
1.2.4 Cellular localization of prion protein ........................................ 9
Sc1.2.5 Subcellular sites of PrP formation ........................................ 11
C1.3 Normal cellular function of PrP ......................................... 12
1.4 in vitro conversion systems ................................................... 14
1.5 Prion replication hypotheses ................................................ 15
1.6 Aims of the thesis ................................................................... 19
2 Materials and Methods ......................................................... 21
2.1 Chemicals ............................................................................... 21
II

2.2 Solutions and buffers ............................................................................................ 21
2.3 Lipids ...................................................... 22
2.3.1 Raft-like lipid composition ................................................................ 22
2.4 Chinese hamster ovary (CHO) - cell culture ...................... 23
2.4.1 Selection and amplification systems ....................................................................... 23
2.4.2 CHO-cells culture medium ...................................................................................... 24
C2.5 Purification of CHO-PrP .................... 25
C2.5.1 Solubilization of CHO-PrP ................................................................ 25
2+2.5.2 Copper immobilized metal-chelate affinity chromatography (Cu -IMAC) .......... 26
2.5.3 Immunopurification ................................................................................................. 26
2.5.4 Concentrating step ... 27
C2.6 Preparation of CHO-PrP aggregates ................................................................. 27
Sc2.7 Selective precipitation of PrP ............ 27
2.8 Preparation of prion rods ..................................................................................... 28
2.8.1 Lipid extraction ....................................................................................................... 28
2.9 Preparation of insulin fibrils ................ 29
2.10 Preparation of small unilamellar vesicles (SUVs) .............................................. 29
2.10.1 Vesicles manufacture .............................................................................................. 30
2.11 Differential ultracentrifugation ........................................... 30
2.12 Protein gel electrophoresis (SDS-PAGE) ............................................................ 31
2.13 Silver staining of protein gels ............................................... 32
2.14 Dot blot .................................................................................. 32
2.15 Semi-dry blot ......................................... 33
2.16 Immunologic protein detection ............................................ 33
2.17 Kinetic analysis of molecular interactions .......................................................... 34
2.17.1 Total internal reflection and evanescent field ......................... 34
2.17.2 Surface plasmon ...................................................................................................... 36 III

2.17.3 Biacore set-up .......................................................................................................... 36
2.17.4 Sensor chip L1 ......... 37
3 Results .................................................................................................................... 39
C3.1 Purification of CHO-PrP .................... 39
3.1.1 Optimization steps ................................................................................................... 40
3.1.2 Immobilized metal chelate affinity chromatography (IMAC) 41
3.1.3 Immunopurification ................................................................................................. 42
3.2 Formation of a lipid bilayer.................................................................................. 44
C3.3 Saturation of the lipid bilayer with CHO-PrP 46
C3.4 Interaction of aggregated proteins with membrane-anchored PrP ................ 48
3.4.1 Interaction studies with non-infectious aggregates ................................................. 49
3.4.2 Interaction studies with infectious protein aggregates ............ 52
3.4.3 Interaction studies with prion rods .......................................................................... 56
3.5 Control studies for the specificity ........ 61
3.6 Influence of the prion rods preparations ............................................................ 63
4 Discussion ............................................................................................................... 68
4.1 Complexity of the multi-component in vitro system ........... 69
C Sc4.2 PrP as a potential receptor for PrP ................................................................. 77
4.3 Outlook ................................................................................................................... 80
5 Summary ................ 83
6 Zusammenfassung ................................................................................................. 85
7 References ............................................................................................................ VII

IV

Abbreviations
BSE bovine spongiform encephalopathy
CBS citrate-buffered saline
CHO-cells Chinese hamster ovary cells
C CCHO-PrP Chinese hamster ovary PrP
CJD Creutzfeldt-Jakob-disease
DMPC di-myristol-phosphatidylcholine
EDTA ethylene diamine tetra acetic acid
GPI-anchor glycosyl-phospatidyl-inositol-anchor
IMAC immobilized metal-chelate affinity chromatography
kDa kilodalton
MLV multilamellar vesicle
NaPTA sodium phosphotungstate
PK proteinase K
PrP prion protein
CPrP cellular prion protein
ScPrP disease-associated form of PrP
ScPrP 27-30 PK-resistant fragment of PrP
recPrP (90-231) recombinant PrP with the amino acid sequence of PrP 27-30
SDS sodium-dodecyl-sulfate
SDS-PAGE sodium-dodecyl-sulfate polyacrylamide gel electrophoresis
SHa Syrian Gold hamster
SPR surface plasmon resonance
SUV small unilamellar vesicle
TIR total internal reflection
TSE transmissible spongiform encephalopathy