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Mechanistische Aspekte Der Evolution Von Lipoxygenasen

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Mechanistische Aspekte Der Evolution Von Lipoxygenasen Aus dem Institut für Biochemie der Medizinischen Fakultät Charité – Universitätsmedizin Berlin DISSERTATION Mechanistische Aspekte der Evolution von Lipoxygenasen zur Erlangung des akademischen Grades Doctor medicinae (Dr. med.) vorgelegt der Medizinischen Fakultät Charité – Universitätsmedizin Berlin von Susan Adel aus Köln Datum der Promotion: 09.12.2016 Meinen Eltern. 2 Inhaltsverzeichnis Inhaltsverzeichnis .................................................................................................... 2 Abstrakt ..................................................................................................................... 7 Abstract ..................................................................................................................... 9 Abkürzungsverzeichnis ......................................................................................... 11 1 Einleitung ............................................................................................................. 16 1.1 Definition von Lipoxygenasen und biologische Wirkprinzipien ............................. 16 1.2 Lipoxygenase-Reaktion ........................................................................................ 17 1.3 Eigenschaften von Lipoxygenasen ...................................................................... 18 1.3.1 Struktur von Lipoxygenasen .............................................................................. 18 1.3.2 Kinetische Besonderheiten von Lipoxygenasen ................................................ 19 1.3.3 Regulation von Lipoxygenasen ......................................................................... 20 1.3.4 Reaktionsspezifität von Lipoxygenasen ............................................................ 22 1.4 Klassifizierung von Lipoxygenasen und Enzymnomenklatur ................................ 25 1.4.1 Spezifitätsbasiertes Klassifizierungskonzept ..................................................... 25 1.4.2 Sequenzbasiertes Klassifizierungskonzept ....................................................... 26 1.5 Evolution von Lipoxygenasen .............................................................................. 27 1.5.1 Einteilung irdischer Lebewesen ........................................................................ 27 1.5.2 Lipoxygenasen in Bakterien .............................................................................. 27 1.5.3 Mögliches Vorkommen von Lipoxygenasen in Archaeen .................................. 28 1.5.4 Lipoxygenasen in Eukaryonten ......................................................................... 29 1.5.4.1 Vorkommen von Lipoxygenasen in pflanzlichen Organismen ........................ 29 1.5.4.2 Vorkommen von Lipoxygenasen in Protozoen und niederen Metazoen ........ 30 1.5.4.3 Vorkommen von Lipoxygenasen in Chordaten .............................................. 31 1.5.4.3.1 Entwicklung der ALOX15 während der Säugetierevolution ......................... 34 1.5.4.3.2 Vorkommen von Lipoxygenasen in Hominiden und Menschen ................... 35 1.6 Biologische Rolle von Lipoxygenasen .................................................................. 36 1.6.1 Physiologische Funktionen von Lipoxygenasen ................................................ 37 1.6.1.1 Zelldifferenzierung und Organogenese .......................................................... 37 1.6.1.2 Zellproliferation und Apoptose ....................................................................... 39 1.6.1.3 Hirnentwicklung und interneuronale Kommunikation ..................................... 40 1.6.1.4 Hämostase ..................................................................................................... 40 1.6.2 Pathophysiologische Funktionen von Lipoxygenasen ....................................... 41 3 1.6.2.1 Entzündungsreaktion ..................................................................................... 41 1.6.2.2 Lungenerkrankungen (Asthma bronchiale, ARDS) ........................................ 43 1.6.2.3 Nierenerkrankungen ....................................................................................... 44 1.6.2.4 Herz-Kreislauf-Erkrankungen ......................................................................... 45 1.6.2.5 Karzinogenese ............................................................................................... 46 1.6.2.6 Neurologische Erkrankungen ......................................................................... 49 1.6.2.7 Metabolische Erkrankungen ........................................................................... 51 1.7 Fragestellung ....................................................................................................... 52 2 Materialien und Methoden .................................................................................. 54 2.1 Materialien ........................................................................................................... 54 2.1.1 Chemikalien ...................................................................................................... 54 2.1.2 Nährmedien, Puffer und Lösungen ................................................................... 55 2.1.3 Enzyme und Kits ............................................................................................... 57 2.1.4 Plasmide und Oligonukleotide ........................................................................... 57 2.1.5 Nukleotid- und Proteinsequenzen ..................................................................... 60 2.1.6 Bakterienstämme .............................................................................................. 61 2.1.7 Eukaryontische Zellstämme .............................................................................. 61 2.1.8 Geräte ............................................................................................................... 61 2.2 Molekularbiologische Methoden ........................................................................... 62 2.2.1 Klonierungen und Ligationen ............................................................................ 62 2.2.2 Ortsgerichtete Mutagenese ............................................................................... 64 2.2.3 Transformation von E. coli ................................................................................ 65 2.2.4 Prokaryontische Expressionen mit E. coli ......................................................... 65 2.2.5 Eukaryontische Expressionen der zbfALOX5 in HEK293-Zellen ...................... 67 2.2.6 Enzymaufreinigung ........................................................................................... 67 2.2.7 Western Blot ..................................................................................................... 68 2.2.8 Membranbindungsassay ................................................................................... 69 2.2.9 Aktivitätsassay .................................................................................................. 70 2.3 Analytische Methoden .......................................................................................... 71 2.3.1 Analyse des Produktspektrums mittels HPLC ................................................... 71 2.3.2 Statistische Datenauswertung ........................................................................... 72 2.4 Recherchestrategien ............................................................................................ 72 3 Ergebnisse ........................................................................................................... 75 3.1 Bakterielle Lipoxygenasen ................................................................................... 75 4 3.2 Lipoxygenasen in Archaeen ................................................................................. 77 3.3 Charakterisierung der zbfALOX5 ......................................................................... 77 3.3.1 Expression der zbfALOX5 in pro- und eukaryontischen Expressionssystemen 82 3.3.1.1 Reaktionsspezifität der bakteriell exprimierten zbfALOX5 .............................. 83 3.3.1.2 Reaktionsspezifität der eukaryontisch exprimierten zbfALOX5 ...................... 84 3.3.2 Versuche zur Anwendbarkeit Volumenhypothese auf die zbfALOX5 ................ 85 3.3.2.1 Sloane-Mutanten ............................................................................................ 86 3.3.2.1.1 Einfachmutante A424F ................................................................................ 87 3.3.2.1.2 Einfachmutante N425M ............................................................................... 88 3.3.2.1.3 Einfachmutante N425F ............................................................................... 89 3.3.2.1.4 Zweifachmutante A424I+N425M ................................................................. 91 3.3.2.1.5 Normalphasen-HPLC interessanter Sloane-Mutanten ................................ 92 3.3.2.1.6 Enantioselektivität dominant 8-lipoxygenierender zbfALOX5-Mutanten ...... 93 3.3.2.2 Kombinierte Mutationen der Sloane- und Borngräber-Determinanten ........... 94 3.3.2.2.1 Zweifachmutante F359W+N425M ............................................................... 95 3.3.2.2.2 Dreifachmutante F359W+A424I+N425M .................................................... 95 3.3.2.2.3 Vierfachmutante F359W+A424I+N425M+A603I ......................................... 97 3.3.2.3 Enantioselektivität der 15-lipoxygenierenden zbfALOX5-Mutanten ............... 98 3.3.2.4
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