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Masterarbeit / Master's Thesis MASTERARBEIT / MASTER’S THESIS Titel der Masterarbeit / Title of the Master‘s Thesis „The role of TRIM29 in NFκB regulation“ verfasst von / submitted by Richard Wallner BSc angestrebter akademischer Grad / in partial fulfilment of the requirements for the degree of Master of Science (MSc) Wien, 2017 / Vienna 2017 Studienkennzahl lt. Studienblatt / A 066 830 degree programme code as it appears on the student record sheet: Studienrichtung lt. Studienblatt / Masterstudium Molekulare Mikrobiologie, degree programme as it appears on Mikrobielle Ökologie und Immunbiologie the student record sheet: Betreut von / Supervisor: Gijs Versteeg Ph.D. Table of Contents Abstract (deutsch).................................................................................................................................... 4 Abstract ................................................................................................................................................... 6 Introduction ............................................................................................................................................. 8 Structure of the NFκB complex ........................................................................................................... 8 The inhibitor of κB (IκB) .................................................................................................................. 11 Structure and function of the IKK complex ...................................................................................... 11 The role of NEMO ............................................................................................................................ 12 NFκB activation ................................................................................................................................ 12 NFκB in DNA damage response ....................................................................................................... 15 Regulation of NFκB activity ............................................................................................................. 19 Regulation through IκB negative feedback ....................................................................................... 19 Regulation and posttranslational modifications of NFκB ................................................................. 20 Shutting off NFκB activity ................................................................................................................ 21 The family of TRIM proteins ............................................................................................................ 21 TRIM proteins and the immune system ............................................................................................ 24 TRIM29 ............................................................................................................................................. 26 TRIM29 in the DNA damage response ............................................................................................. 27 TRIM29 and p53 ............................................................................................................................... 28 TRIM29 and the Wnt pathway .......................................................................................................... 30 Involvement of TRIM29 in the ERK and JNK pathways ................................................................. 31 TRIM29 and the NFκB pathway ....................................................................................................... 31 Hypothesis ......................................................................................................................................... 33 Results ................................................................................................................................................... 34 TRIM29 is highly expressed in the epidermis of the skin ................................................................. 34 TRIM29 and IKKα/β or NEMO do not interact upon overexpression .............................................. 35 High protein concentrations and low stringency of tested washing buffers led to co-precipitation of NEMO ............................................................................................................................................... 36 NEMO non-specifically interacts with TRIM proteins ..................................................................... 37 TRIM29 and the IKKs do not interact when overexpressed in Cos7 or HeLa cells ......................... 38 Endogenous TRIM29 and NEMO do not interact in HaCaT cells .................................................... 39 TRIM29 and IKKα/β or NEMO do not interact when overexpressed in HaCaTs or keratinocytes .. 40 TRIM29 and NEMO do not interact when stimulated with TNFα or DNA damaging reagents ....... 41 TRIM29 is localized in the cytoplasm of HaCaTs, HeLas, keratinocytes and in overexpressing HEK-293T cells ................................................................................................................................. 42 P a g e | 2 Establishing an assay to investigate NFκB activation through TRIM29 ........................................... 43 TNFα and LPS induce NFκB activation in MEFs and RAW264.7 cells .......................................... 44 None of our tested stimuli did induce NFκB activation in HaCaTs or keratinocytes ....................... 46 Trim29 is efficiently knocked-out in HaCaTs and keratinocytes using CRISPR/Cas9 ..................... 46 TRIM29 ablation did not affect survival of HaCaT cells against UV radiation ................................ 48 Discussion ............................................................................................................................................. 52 Methods ................................................................................................................................................. 60 Cell culture ........................................................................................................................................ 60 Antibodies and reagents .................................................................................................................... 60 Keratinocyte isolation ........................................................................................................................ 60 Cloning and plasmid construction ..................................................................................................... 60 Transformation .................................................................................................................................. 61 Transfection ....................................................................................................................................... 61 Lenti-virus production and viral transduction ................................................................................... 61 Immunoprecipitation ......................................................................................................................... 61 SDS-Page and Western Blot .............................................................................................................. 62 Immunofluorescence ......................................................................................................................... 62 MTT assay ......................................................................................................................................... 62 References ............................................................................................................................................. 63 P a g e | 3 Abstract (deutsch) Das angeborene Immunsystem ist die erste Verteidigungslinie gegen eindringende Pathogene und führt seine Funktion durch eine Reihe sehr vielseitiger Funktionswege aus. Diese werden aktiviert durch das Erkennen bestimmter molekularer Strukturen, genannt „Pathogen-assoziirte molekulare Muster“, welche von vielen Pathogenen geteilt werden. Sie werden erkannt durch verschiedene membrangebundene und zytoplasmische Rezeptoren, die alle spezifisch für eine bestimmte Art von Muster sind und zur Aktivierung des angeborenen Immunsystems zur Bekämpfung der Bedrohung führen. Der NFκB Signalweg ist einer der zentralsten und wichtigsten dieser Signalwege und verbindet immunologische Funktionen, wie die Produktion von entzündlichen Zytokinen, mit anderen Funktionen wie zelluläres Überleben, Wachstum und Apoptose. Er wird ausgelöst durch die Aktivierung einiger immunrelevanter Rezeptoren, aber auch durch andere Einflüsse, wie zum Beispiel DNA-Schäden. Unter Ruhebedingungen ist der NFκB Komplex von seinem Inhibitor, dem Inhibitor von κB (IκB), gebunden, der ihn im Zytoplasma beschlagnahmt und so daran hindert seine Funktionen als Transkriptionsfaktor auszuführen. Nach Aktivierung des Signalweges wird der IκB durch den Inhibitor von κB Kinase (IKK) Komplex phosphoryliert, was zu dessen Ubiquitin abhängigen Abbau führt und dem NFκB Komplex ermöglicht in den Zellkern zu translozieren. Nach dieser Translokation arbeitet NFκB nicht bloß als Transkriptionsfaktor zur Verteidigung gegen angreifende Pathogene, sondern auch für Zell Homeostase, Wachstum und die Reparator von beschädigter DNA. Er ist daher nicht bloß während Infektionen wichtig, sondern auch bei Krebs und zur DNA Reparatur. Da eine Fehlregulation schreckliche Folgen haben kann, welche von Immunschwäche
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