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Untersuchungen Zum Funktionellen Einfluss Von Einzelnukleotidvariationen in Humanen Lipoxygenasen Und Anderen Genen Des LOX-Stoffwechsels

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Untersuchungen Zum Funktionellen Einfluss Von Einzelnukleotidvariationen in Humanen Lipoxygenasen Und Anderen Genen Des LOX-Stoffwechsels Untersuchungen zum funktionellen Einfluss von Einzelnukleotidvariationen in humanen Lipoxygenasen und anderen Genen des LOX-Stoffwechsels DISSERTATION zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.) eingereicht im Fachbereich Biologie, Chemie, Pharmazie der Freien Universität Berlin vorgelegt von Diplom-Biochemiker Thomas Horn aus Berlin 2014 Diese Arbeit wurde zwischen dem 01.01.2011 und 31.12.2013 am Institut für Biochemie der Medizinischen Fakultät der Charité – Universitätsmedizin Berlin unter der Leitung von Prof. Dr. sc. med. Hartmut Kühn angefertigt. 1. Gutachter: Prof. Dr. Rupert Mutzel 2. Gutachter: Prof. Dr. Hartmut Kühn Disputation am 18.03.2014 Danksagung Mein besonderer Dank gilt Herrn Prof. Dr. Hartmut Kühn für die freundliche Überlassung des Arbeitsthemas. Außerdem möchte ich mich bei ihm für die gute wissenschaftliche Betreuung und die erfolgreiche Zusammenarbeit bedanken. Er war stets offen für Diskussionen, Fragen und Probleme, was für das erfolgreiche Gelingen dieser Arbeit entscheidend war. Ich danke Herrn Dr. Igor Ivanov und Frau Dr. Almerinda Di Venere für die Durchführung der CD- und Fluoreszenzmessungen. Insbesondere möchte ich mich bei Igor für seine stetige Unterstützung bedanken. Er hat mir alle wichtigen Dinge über die experimentelle Herstellung, Reinigung und Charakterisierung von Lipoxygenasen beigebracht. Danke auch für das Lesen der Manuskripte sowie der Arbeit. Daneben bedanke ich mich recht herzlich bei Frau Dr. Astrid Borchert für die stets interessanten wissenschaftlichen Gespräche, die schönen Mittagspausen und ihre immer fortwährende moralische Unterstützung. Ich bedanke mich auch bei Frau Dr. Dagmar Heydeck und Herrn Dr. Christoph Ufer für die stets gute wissenschaftliche Beratung und ihre Hilfe bei den wissenschaftlichen Arbeiten. Ich danke Frau Sabine Stehling und Frau Monika Anton für die hervorragende technische Unterstützung und ihre viele Geduld mit mir. Ansonsten möchte ich mich noch bei Frau Katharina Hofheinz, Frau Susan Adel und Patrick Scheerer für die gute wissenschaftliche Zusammenarbeit bedanken. Ich danke zudem bei Herrn Kumar Reddy Kakularam, Ass. Prof. Dr. Aparoy Polamarasetty und Prof. Dr. Pallu Reddanna aus dem Institut für Lebenswissenschaften der Universität von Hyderabad (Indien) für ihre Unterstützung bei den bioinformatischen Arbeiten. Sie haben mir die wichtigsten Dinge in Sachen Strukturmodellierung und Docking beigebracht. Außerdem möchte ich bei Kumar und Aparoy sowie allen Mitglieder der AG Reddanna für die tolle Zeit in Indien bedanken. Zudem danke ich Frau Dr. Sophia Wuest und Prof. Dr. Hersberger von Universität Zürich (Schweiz) für die Durchführung der klinischen Studie. Zu guter Letzt möchte ich mich natürlich auch bei meiner Familie, vor allem bei meinen lieben Eltern, für die während der Studien- und Promotionszeit immer fortwährende finanzielle und moralische Unterstützung bedanken. Ohne euch hätte ich das nicht geschafft. Auch meiner Freundin Christiane danke ich für ihre liebevolle Art und ihre Unterstützung. Inhaltsverzeichnis Abkürzungsverzeichnis 1. EINLEITUNG .................................................................................................... 1 1.1. Vorkommen und Klassifizierung von Lipoxygenasen ................................ 1 1.1.1. Reaktionsmechanismus von Lipoxygenasen ............................................. 2 1.1.2. Vorkommen von LOXn und evolutive Aspekte ........................................... 4 1.1.3. Nomenklatur von Lipoxygenasen ............................................................... 7 1.1.4. Biologische Bedeutung von Lipoxygenasen ............................................... 7 1.1.4.1. Entzündung und Asthma bronchiale .......................................... 7 1.1.4.2. Kanzerogenese ......................................................................... 9 1.1.4.3. Atherosklerose ........................................................................ 12 1.1.4.4. Epidermale Differenzierung ..................................................... 14 1.2. Strukturelle Grundlagen der Lipoxygenasereaktion ................................. 15 1.2.1. Kristallstruktur der Lipoxygenasen ........................................................... 15 1.2.2. N-terminale Domäne ............................................................................... 16 1.2.3. C-terminale Domäne ............................................................................... 17 1.2.4. Grundlagen der Stereo- und Positionsspezifität von Lipoxygenasen........ 20 1.2.4.1. Positionsspezifität von Lipoxygenasen ................................... 20 1.2.4.2. Stereospezifität von Lipoxygenasen ....................................... 22 1.3. Genetische Variationen (SNPs, Mutationen) in LOXn und ihre Funktion in der Pathogenese von verschiedenen Erkrankungen ..................................... 24 1.4. Ziele der Arbeit ........................................................................................... 28 2. MATERIAL UND METHODEN ....................................................................... 30 2.1. Material ......................................................................................................... 30 2.1.1. Chemikalien ............................................................................................. 30 2.1.2. Nährmedien und Pufferlösungen ............................................................. 31 2.1.3. Kits und Enzyme...................................................................................... 33 2.1.4. Plasmide und Oligonukleotide ................................................................. 33 2.1.5. Bakterienstämme ..................................................................................... 34 2.2. Molekularbiologische und proteinchemische Methoden .......................... 34 2.2.1. Klonierung und Ligation ........................................................................... 35 2.2.2. Plasmid-DNA Transformation in Bakterien ............................................... 35 2.2.3. Ortsgerichtete Mutagenese ..................................................................... 35 2.2.4. Rekombinante Proteinherstellung und Reinigung von verschiedenen LOX- Isoformen ................................................................................................ 36 2.2.5. Eisenbestimmung der über FPLC gereinigten Enzympräparationen mit Atomabsorptionsspektroskopie ................................................................ 37 2.3. Analytische Methoden ................................................................................. 37 2.3.1. HPLC basierter LOX-Aktivitätsassay ....................................................... 37 2.3.2. HPLC-Analytik ......................................................................................... 38 2.3.2.1. Reverse Phase HPLC (RP-HPLC) .......................................... 38 2.3.2.2. Straight Phase HPLC (SP-HPLC) ........................................... 38 2.3.2.3. Chiral Phase HPLC (SP-HPLC) .............................................. 39 2.3.2.4. Methylierung mit Diazomethan ................................................ 39 2.3.3. Photometrischer LOX-Aktivitätsassay ...................................................... 39 2.3.4. Proteinbestimmung .................................................................................. 40 2.3.5. Denaturierende Polyacrylamid-Gelelektrophorese ................................... 40 2.3.6. Western Blot ............................................................................................ 40 2.3.7. Quantifizierung der Proteinexpression ..................................................... 41 2.4. Biophysikalische Methoden ........................................................................ 41 2.4.1. Fluoreszenzspektroskopie ....................................................................... 41 2.4.2. Circulardichroismus (CD) ......................................................................... 41 2.4.3. Thermal shift Assay ................................................................................. 41 2.5. Bioinformatische Methoden ........................................................................ 42 2.5.1. In silico Mutagenese ................................................................................ 42 2.5.2. Homologiemodellierung ........................................................................... 43 2.5.3. Visualisierung und Strukturanalyse .......................................................... 43 2.5.4. Analyse von LOX-Sequenzen und genetischen Variationen .................... 44 3. ERGEBNISSE ................................................................................................ 44 3.1. Funktionelle Untersuchungen zum Einfluss von genetischen Variationen (SNPs, Mutationen) in verschiedenen humanen Lipoxygenasen .................. 45 3.1.1. Humane Arachidonat 15-Lipoxgenase (hALOX15) .................................. 46 3.1.2. Humane Arachidonat 5-Lipoxygenase (hALOX5) .................................... 51 3.1.3. Humane Arachidonat 12-Lipoxgenase (hALOX12) .................................. 54 3.1.4. Humane Arachidonat 15-Lipoxgenase Typ II (hALOX15B) ...................... 57 3.2. Molekulare Ursachen für die Inaktivität der natürlich vorkommenden hALOX15 Gly422Glu Variation ......................................................................... 61 3.2.1. Die Gly422Glu Mutation ist eine fehlgefaltete Enzymvariante. ................
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