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Influence of Ubiquinol/Ubiquinone on Energy Metabolism and DNA Repair Enzyme Hogg1 in Mitochondria of Human Skin Fibroblasts

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Influence of Ubiquinol/Ubiquinone on Energy Metabolism and DNA Repair Enzyme Hogg1 in Mitochondria of Human Skin Fibroblasts Influence of ubiquinol/ubiquinone on energy metabolism and DNA repair enzyme hOGG1 in mitochondria of human skin fibroblasts Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr.rer.nat.) vorgelegt von Schniertshauer, Daniel an der Mathematisch-Naturwissenschaftliche Sektion Fachbereich Biologie Konstanz, 2020 Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-2-1e3p13av7exa93 Tag der mündlichen Prüfung: 23.09.2020 1. Referent: Prof. Dr. Alexander Bürkle 2. Referent: Prof. Dr. Jörg Bergemann II "All our science, measured against reality, is primitive and childlike - and yet it´s the most precious thing we have." Albert Einstein for my parents III Unless otherwise indicated, parts of the present work are from articles published in the following journals: Schniertshauer D, Gebhard D, van Beek H, Nöth Vivien, Schon J, Bergemann J. The activity of the DNA repair enzyme hOGG1 can be directly modulated by ubiquinol. DNA Repair (Amst). 2020 Jan 3;87:102784. DOI: 10.1016/j.dnarep.2019.102784. Schniertshauer D, Gebhard D, Bergemann J. A new Efficient Method for Measuring Oxygen Consumption Rate Directly ex vivo in Human Epidermal Biopsies. Bio Protoc. 2019; DOI:10.21769/BioProtoc.3185. Schniertshauer D, Gebhard D, Bergemann J. Age-Dependent Loss of Mitochondrial Function in Epithelial Tissue Can Be Reversed by Coenzyme Q10. J Aging Res. 2018; 2018:6354680. Schniertshauer D, Müller S, Mayr T, Sonntag T, Gebhard D, Bergemann J. Accelerated Regeneration of ATP Level after Irradiation in Human Skin Fibroblasts by Coenzyme Q10. Photochem Photobiol. 2016; 92(3):488-94. Gebhard D, Kirschbaum K, Matt K, Müller S, Schniertshauer D, Bergemann J. Biologie von Alterungsvorgängen - Rolle der mitochondrialen Funktion am Modellsystem Haut. FHprofUnt2012 "MitoFunk". Abschlussbericht 2015; DOI:10.2314/GBV:867965967. IV Table of Contents List of abbreviations................................................................................................ IX Summary ................................................................................................................ XIII Zusammenfassung ................................................................................................. XV 1. Introduction ........................................................................................................... 1 1.1 Anatomy and function of the human skin .......................................................... 1 1.1.1 Influence and protection of the skin against ultraviolet radiation ................ 3 1.2 Mitochondrial function and genetics .................................................................. 4 1.2.1 Respiratory chain (oxidative phosphorylation) ............................................ 6 1.2.2 Mitochondria in the process of ageing ........................................................ 8 1.3 Reactive oxygen species ................................................................................... 9 1.3.1 Formation of ROS and oxidative stress ...................................................... 9 1.4 DNA damage ................................................................................................... 11 1.4.1 Oxidative DNA damage ............................................................................ 12 1.4.2 7,8-dihydro-8-oxoguanine ......................................................................... 13 1.5 DNA repair mechanisms.................................................................................. 14 1.5.1 Direct DNA repair ..................................................................................... 15 1.5.2 Postreplicative repair (mismatch repair) ................................................... 16 1.5.3 Nucleotide excision repair ........................................................................ 16 1.5.4 Base excision repair ................................................................................. 18 1.5.5 Mitochondrial DNA Repair ........................................................................ 22 1.6 The human 8-oxoguanine DNA glycosylase 1 ................................................. 23 1.6.1 Gene localization and structure ................................................................ 24 1.6.2 Isoforms of hOGG1 .................................................................................. 25 1.6.3 DNA repair mechanism by hOGG1 .......................................................... 26 1.7. Coenzyme Q10 ................................................................................................ 29 1.7.1 Chemical composition and properties ....................................................... 29 1.7.2 Biosynthesis and intracellular transport .................................................... 30 1.7.3 The role of coenzyme Q10 in the energy metabolism of eukaryotic cells .. 32 1.7.4 The role of coenzyme Q10 in the prevention of oxidative DNA damage .... 33 1.8 Clinical relevance ............................................................................................ 34 1.9 Aims of this thesis ........................................................................................... 36 2. Materials .............................................................................................................. 38 2.1 Antibiotics ........................................................................................................ 38 2.2 Cell Culture ...................................................................................................... 38 V 2.3 Cell Culture Medium ........................................................................................ 38 2.4 Chemicals and Reagents ................................................................................ 39 2.5 Buffers and Solutions ...................................................................................... 42 2.6 Enzymes.......................................................................................................... 45 2.7. Coenzyme Q10 formulations ........................................................................... 45 2.8 Reporter oligonucleotides ................................................................................ 45 2.9 Primer .............................................................................................................. 46 2.10 Molecular Biological Kits................................................................................ 46 2.11 Laboratory Equipment ................................................................................... 47 2.12 Consumables ................................................................................................ 49 2.13 Software ........................................................................................................ 51 3. Methods ............................................................................................................... 53 3.1 Cell culture ...................................................................................................... 53 3.1.1 Sample collection / Ethical aspects .......................................................... 53 3.1.2 Isolation of skin fibroblasts ....................................................................... 55 3.1.3 Culturing conditions .................................................................................. 56 3.1.4 Subculturing.............................................................................................. 56 3.1.5 Cell number determination ....................................................................... 57 3.1.6 Cryopreservation of Cells ......................................................................... 57 3.1.7 Thawing of cryopreserved cells ................................................................ 58 3.2 Test compound supplementation .................................................................... 58 3.3 Irradiation of cells ............................................................................................ 59 3.4 Isolation and lysis of mitochondria ................................................................... 60 3.5 Cell viability assay ........................................................................................... 60 3.6 ATP bioluminescence assay ........................................................................... 61 3.7 ROS- / mtROS-Assay ...................................................................................... 63 3.8 Mitochondrial membrane potential (∆ψm) measurements ............................... 64 3.9 Gene expression analysis ............................................................................... 65 3.10 Mitochondrial respiration measurements - Sample preparation .................... 66 3.10.1 Monolayer cell culture ............................................................................. 66 3.10.2 Epidermis................................................................................................ 66 3.11 Mitochondrial respiration measurements ................................................... 68 3.12 Base excision repair assay ............................................................................ 72 3.12.1 Reporter oligonucleotides ....................................................................... 72 3.12.2 Measurement of base excision repair ..................................................... 72 VI 3.13 Co-immunoprecipitation ...............................................................................
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