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Die Funktion Von Plakophilin 1 Und Plakophilin 3 in Der Regulation Der Translation

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Die Funktion Von Plakophilin 1 Und Plakophilin 3 in Der Regulation Der Translation Die Funktion von Plakophilin 1 und Plakophilin 3 in der Regulation der Translation Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Naturwissenschaftlichen Fakultät I – Biowissenschaften – der Martin-Luther-Universität Halle-Wittenberg, vorgelegt von Frau Christina Ann-Susann Kießling geb. am 22.02.1985 in Plauen Gutachter: Prof. Dr. Mechthild Hatzfeld Prof. Dr. Elmar Wahle Prof. Dr. Rudolf Leube Tag der öffentlichen Verteidigung: 26.02.2014 So eine Arbeit wird eigentlich nie fertig, man muss sie für fertig erklären, wenn man nach Zeit und Umständen das mögliche getan hat. (Johann Wolfgang von Goethe, 1787) für Stephan Inhaltsverzeichnis 1. Einleitung ................................................................................................................ 1 1.1. Plakophiline – Mitglieder der Armadillo-Proteinfamilie .............................................. 1 1.2. Die Funktion der Plakophiline ................................................................................... 3 1.2.1. Die Funktion der Plakophiline in der Zelladhäsion .................................................... 3 1.2.2. Die Funktion der Plakophiline in der Signaltransduktion ........................................... 6 1.2.3. Die duale Funktion der Plakophiline in der Kanzerogenese ...................................... 7 1.3. Die Translation ......................................................................................................... 9 1.3.1. Die cap-abhängige Initiation der Translation ............................................................ 9 1.3.2. Die IRES-vermittelte Initiation der Translation ........................................................ 13 1.3.3. Translation und Kanzerogenese ............................................................................. 15 1.4. Zielstellung der Arbeit ............................................................................................ 16 2. Material und Methoden ........................................................................................ 18 2.1. Materialien ............................................................................................................. 18 2.1.1. Chemikalien und Enzyme ....................................................................................... 18 2.1.2. Bakterienstämme ................................................................................................... 18 2.1.3. Hefestämme ........................................................................................................... 19 2.1.4. Zelllinien ................................................................................................................. 20 2.1.5. Chromatographie-Säulen ....................................................................................... 20 2.1.6. Radiochemikalien ................................................................................................... 20 2.2. Molekularbiologische Methoden ............................................................................. 20 2.2.1. Isolierung von RNA ................................................................................................ 20 2.2.2. Reverse Transkription ............................................................................................ 21 2.2.3. Polymerase-Kettenreaktion (PCR) ......................................................................... 22 2.2.4. Quantitative RT-PCR (qRT-PCR) ........................................................................... 22 2.2.5. Agarose-Gelelektrophorese von Nukleinsäuren ..................................................... 24 2.2.6. Reinigung von Nukleinsäuren ................................................................................. 24 2.2.7. Klonierung von Expressionsplasmiden ................................................................... 24 2.2.8. Restriktionsverdau ................................................................................................. 24 2.2.9. Ligation .................................................................................................................. 25 2.2.10. Herstellung von chemokompetenten Bakterien ...................................................... 25 2.2.11. Herstellung von elektrokompetenten Bakterien ...................................................... 26 2.2.12. Transformation von chemokompetenten Bakterien mit Plasmid-DNA ..................... 26 2.2.13. Transformation von elektrokompetenten Bakterien mit Plasmid-DNA ..................... 26 2.2.14. Präparation von Plasmid-DNA aus Bakterien ......................................................... 27 2.2.15. Präparation von Plasmid-DNA aus Hefen............................................................... 27 2.2.16. Sequenzierung von Plasmid-DNA .......................................................................... 28 2.3. Zellbiologische Methoden ....................................................................................... 28 2.3.1. Kultivierung humaner und muriner Zellen ............................................................... 28 2.3.2. Einfrieren und Auftauen von Zellen ........................................................................ 29 2.3.3. Transfektion humaner und muriner Zellen .............................................................. 29 2.3.3.1. Calcium-Phosphat-Präzipitation ............................................................................. 29 2.3.3.2. Lipofektion .............................................................................................................. 30 2.3.3.3. Nukleofektion ......................................................................................................... 30 2.3.4. Immunfluoreszenz .................................................................................................. 31 2.3.5. Fluoreszenz-in-situ-Hybridisierung (FISH) .............................................................. 31 2.3.6. Bimolekulare Fluoreszenzkomplementation (BiFC) ................................................ 32 2.3.7. Proximity Ligation Assay (PLA) .............................................................................. 33 2.3.8. Messung der Zellproliferation mittels BrdU-Einbau ................................................. 33 2.3.9. Bestimmung der Zellzahl und Vitalität .................................................................... 33 2.4. Biochemische Methoden ........................................................................................ 34 2.4.1. Isolierung von Proteinen ......................................................................................... 34 2.4.2. Bestimmung der Proteinkonzentration .................................................................... 34 2.4.3. SDS-PAGE ............................................................................................................ 34 2.4.4. Coomassie-Färbung von Polyacrylamid-Gelen ...................................................... 35 2.4.5. Western Blot .......................................................................................................... 35 35 2.4.6. Metabolische Markierung von Zellen mit L-[ S]-Methionin ..................................... 36 2.4.7. Expression von GST- bzw. His6-Fusionsproteinen ................................................. 37 2.4.8. Reinigung von Plakophilin 3 ................................................................................... 38 2.4.9. Präzipitation von transient exprimierten FLAG- bzw. - GFP-Fusionsproteinen ....... 39 2.4.10. GST-pulldown ........................................................................................................ 39 2.4.11. Präzipitation mit 7-Methyl-GTP-Sepharose ............................................................ 40 2.4.12. Hefe-Dihybrid-System ............................................................................................ 40 2.4.13. Dichtegradientenzentrifugation ............................................................................... 41 2.4.14. Microarray-Analyse ................................................................................................ 42 2.4.15. Analyse der Translation in vitro .............................................................................. 43 2.4.16. Statistische Auswertung ......................................................................................... 43 3. Ergebnisse ............................................................................................................ 44 3.1. Die Funktion von Plakophilin 1 und Plakophilin 3 in der Regulation der Translation ............................................................................................................. 44 3.1.1. Plakophilin 3 stimuliert die globale Proteinsynthese ............................................... 44 3.1.2. Der Einfluss von Plakophilin 3 auf die Translation in vitro ....................................... 45 3.1.3. Plakophilin 3 beeinflusst die Proliferation und die Zellgröße ................................... 47 3.2. Identifizierung neuer Plakophilin-3-Interaktionspartner ........................................... 50 3.3. Plakophilin 1 und Plakophilin 3 sind Bestandteil des Translationsinitiationskomplexes ............................................................................ 51 3.3.1. Plakophilin 3 kann mit 7-Methyl-GTP-Sepharose präzipitiert werden ..................... 51 3.3.2. Plakophilin 1 und Plakophilin 3 präzipitieren mit Komponenten des
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