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Und Meprin く Unter Besonderer Berücksichtigung Pathologischer Umstände in Der Menschlichen Dermis

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Und Meprin く Unter Besonderer Berücksichtigung Pathologischer Umstände in Der Menschlichen Dermis Funktionelle Charakterisierung der Metalloendopeptidasen Meprin g und Meprin く unter besonderer Berücksichtigung pathologischer Umstände in der menschlichen Dermis DISSERTATION ZUR ERLANGUNG DES GRADES DOKTOR DER NATURWISSENSCHAFTEN AM FACHBEREICH BIOLOGIE DER JOHANNES GUTENBERG-UNIVERSITÄT MAINZ BERND BRUNS GEB. AM 01.04.1975 IN MÜNSTER MAINZ, 2011 Die im Rahmen dieser Arbeit erzielten Ergebnisse wurden in zwei Veröffentlichungen detailliert beschrieben. Im Folgenden werden diese und weiterführende Arbeiten erläutert und zusammengefasst. Mainz, März 2011 I Verzeichnisse Inhalt 1. Zusammenfassung ........................................................................................................ 1 2. Einleitung ....................................................................................................................... 2 2.1 Metzinkine ............................................................................................................... 2 2.1.1 Astacine ........................................................................................................... 3 2.1.2 Adamalysine ...................................................................................................14 2.1.3 Matrix-Metalloproteasen ..................................................................................15 2.2 Kollagene und Kollagenassemblierung ...................................................................16 2.2.1 Biosynthese und posttranslationale Modifikation fibrillärer Kollagene ..............16 2.3 Stratifin ...................................................................................................................17 2.4 IGFBP3 ..................................................................................................................18 2.5 FGF19 ....................................................................................................................19 3. Material und Methoden .................................................................................................20 3.1 RNA Isolation und RT-PCR ....................................................................................20 3.2 Agarose Gelelektrophorese ....................................................................................21 3.3 Proteinexpression und Insektenzellkultur ...............................................................22 3.3.1 Baculovirus-Expressionssystem ......................................................................22 3.3.2 Hitzeschocktransformation und Transposition .................................................23 3.3.3 Bakterienkultivierung .......................................................................................24 3.3.4 Plasmidisolierung ............................................................................................25 3.3.5 Bacmidisolierung und –überprüfung ................................................................25 3.3.6 Insektenzellkultur ............................................................................................26 3.3.7 Transfektion mit rekombinanten Bacmiden und Virenamplifikation ..................26 3.3.8 Proteinreinigung und Aktivierung von Meprin und .....................................27 3.4 Zellkultur humaner Zelllinien ...................................................................................30 3.4.1 Aufzucht der verwendeten Zelllinien ................................................................30 3.4.2 Passage der HDFa-und HaCats ......................................................................31 3.4.3 HaCat/HDFa Kokultur ......................................................................................31 3.5 MMP1-Aktivitätsassays ..........................................................................................32 3.6 Substratassays .......................................................................................................33 3.7 Zymographie ..........................................................................................................33 4. Ergebnisse ...................................................................................................................34 4.1 Spaltung von MMP1 und Stratifin durch Meprine ....................................................34 4.2 Vergleich der Spaltung von pro-MMP1 und aktiviertem MMP1 durch Meprine .......35 4.3 Einfluss der Substrate auf die MMP1-Expression in dermalen Fibroblasten ...........36 4.4 Detektion MMP1- und MMP9-Aktivität im Zellkulturüberstand ................................39 II Verzeichnisse 4.4.1 Einfluss der Substrate. ....................................................................................39 4.4.2 Einfluss der Meprine........................................................................................41 5. Diskussion ....................................................................................................................43 5.1 MMP1-Spaltung durch Meprin führt zu dessen Funktionsverlust: ...........................43 5.2 Stratifin ...................................................................................................................44 5.3 IGFBP3 ..................................................................................................................45 5.4 FGF19 ....................................................................................................................46 5.5 Meprin ....................................................................................................................46 6. Literaturliste ..................................................................................................................50 7. Anhang .........................................................................................................................59 7.1 Veröffentlichung 1 ..................................................................................................... i 7.2 Veröffentlichung 2 .................................................................................................... ii 8. Eidesstattliche Erklärung ............................................................................................... iii III Verzeichnisse Abbildungsverzeichnis Abbildung 1: Einteilung der Metalloproteasen ( nach GOMIS-RÜTH, 2003) .......................... 2 Abbildung 2: Katalysemechanismus von Astacin (nach Grams et al.,1996): .......................... 3 Abbildung 3: Domänenstruktur der Meprine: ......................................................................... 4 Abbildung 4: Spaltspezifitäten für Meprin und Meprin . ..................................................... 7 Abbildung 5: Detektion von Meprin und Meprin in der menschlichen Haut....................... 9 Abbildung 6: Dorsale und ventrale Sicht eines Limulus polyphemus (www.infovisual.info). 10 Abbildung 7: Domänenstruktur der humanen Tolloide ......................................................... 12 Abbildung 8: BMP1 Spaltspezifität. ...................................................................................... 13 Abbildung 9: Domänenstruktur der Adamalysine ................................................................. 15 Abbildung 10: Überblick Baculovirus-Expressionssystem .................................................... 23 Abbildung 11: Prinzip der Nickel-NTA-Affinitätschromatographie ........................................ 29 Abbildung 12: Schematische Darstellung des HaCat/HDFa Kokultur-Systems .................... 32 Abbildung 13: In vitro Spaltung von Stratifin und pro-MMP1 durch Meprine. ....................... 34 Abbildung 14: MMP1-Aktivität in Abhängigkeit von de Abfolge der Aktivierung und Spaltung durch Meprin und .......................................................................................................... 36 Abbildung 15: RT-PCR mit MMP1,- Meprin und GAPDH-Primern .................................... 37 Abbildung 16: Western Blot: MMP1-Expession im Zellkulturüberstand- und Zelllysat von HDFa-Zellen ........................................................................................................................ 38 Abbildung 17: Relative Kollagenase-Aktivität und MMP9-Aktivität im Zellkulturüberstand von Fibroblasten. ....................................................................................................................... 40 Abbildung 18: Relative Kollagenase-Aktivität im Zellkulturüberstand von Fibroblasten nach Inkubation mit Meprin und Meprin ................................................................................. 41 Abbildung 19: Übersicht über den Einfluss von Stratifin und epidermalen Faktoren auf die Aktivität von Plasmin, MMP1, MMP3 und MMP9. Abbildung 20: Überblick zur hypohetischen Meprin-vermittelten MMP1-und MMP9- Expression durch Interleukin 1 und Interleukin 18 induzierte MAPK- Signaltransduktionskaskaden:. ............................................................................................ 48 Verzeichnisse IV Tabellenverzeichnis Tabelle 1: PCR-Protokoll ......................................................................................................20 Tabelle 2: Reagenzien für die Agarose-Gelelektrophorese ...................................................21 Tabelle 3: Materialien für die Bakterienkultivierung ...............................................................24 Tabelle 4: Bacmid-PCR-Ansatz (25 µl) .................................................................................25 Tabelle 5: PCR-Protokoll zur Überprüfung der Bacmide
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