UNIVERSITY of PÉCS Characterization of the Vestibular NADPH
Total Page：16
File Type：pdf, Size：1020Kb
UNIVERSITY OF PÉCS Biological Doctoral School Characterization of the Vestibular NADPH Oxidase Enzyme Complex PhD Thesis PÉTER KISS PÉCS, 2009 1 UNIVERSITY OF PÉCS Biological Doctoral School Characterization of the Vestibular NADPH Oxidase Enzyme Complex PhD Thesis PÉTER KISS Supervisors : Dr. Joseph Zabner M.D. Dr. Kerepesi Ildikó, PhD PÉCS, 2009 2 1. Table of Contents 1. Table of Contents.....................................................................................3 2. Glossary and Abbreviations .....................................................................5 3. Introduction ..............................................................................................7 3.1 Reactive oxygen species......................................................................7 3.1.1 Reactive oxygen species in biology ...............................................7 3.1.2 Reactive oxygen species produced by NADPH oxidases..............9 3.1.2.1 Superoxide ..............................................................................9 3.1.2.2 Hydrogen peroxide................................................................10 3.1.2 The source of free radicals ..........................................................12 3.1.2.1 Endogenous free radicals......................................................13 3.1.2.1.1 Mitochondrial sources of free radicals ............................13 3.1.2.2 Extramitochondrial ROS sources ..........................................13 3.1.2.2.1 Cytochrome P-450 enzymes ..........................................13 3.1.2.2.2 Peroxisomes...................................................................14 3.1.2.2.3 NADPH oxidases............................................................14 3.2 The NADPH oxidase enzyme family ................................................15 3.2.1 Historical overview of ROS-generating NADPH oxidases............15 3.2.2 Nox family enzymes in mammals ................................................18 2.2.2.1 NOX2 ....................................................................................21 2.2.2.2 NOX1 ....................................................................................23 2.2.2.3 NOX3 ....................................................................................24 2.2.2.4 NOX4 ....................................................................................25 2.2.2.5 NOX5 ....................................................................................26 2.2.2.6 DUOX....................................................................................27 3.2.3 Subunits and Regulatory Proteins ...............................................29 2.2.3.1 p22phox ................................................................................29 3.2.3.2 Organizer subunits: NOXO1, p47phox ..................................31 3.2.3.3 Activator subunits: NOXA1 and p67phox ..............................32 3.2.3.4 p40phox ................................................................................33 3.2.3.5 Rac GTPases........................................................................34 3.3 Anatomy of the inner ear ....................................................................35 3.3.1 Vestibule......................................................................................36 3.3.1.1 Vestibular Sacs.........................................................................37 4. Aims of the present study ......................................................................39 5. Materials and methods...........................................................................40 5.1 Mouse lines and genotyping ................................................40 5.2 Conventional and real-time RT-PCR....................................41 5.3 Generation of Noxo1 transgenic mice ..................................42 5.4 Functional testing of the auditory system .............................43 5.4 Functional testing of the auditory system .............................43 5.5 Histology, immunohistochemistry, and scanning electron microscopy..........................................................................43 5.6 Transmission electron microscopy.......................................44 5.7 X-ray photoelectron spectroscopy........................................44 5.8 In situ hybridization ..............................................................45 5.9 Cell culture, DNA constructs, transfection, and superoxide detection .............................................................................45 5.10 Generation of NOXA1 knockout mouse model ..................46 3 5.11 p22phox deficient mouse strain .........................................47 5.12 p22phox transgenic mouse strain ......................................48 5.13 Nitroblue Tetrazolium test (NBT)........................................48 6. Results...................................................................................................49 6.1 I. NOXO1 is the physiological organizer subunit of the vestibular NADPH oxidase complex...............................................................49 6.1.1 Identification of a Noxo1 mutant mouse strain .........................49 6.1.2 NOXO1-deficient mice unable to balance ................................52 6.1.3 Absence of otoconia mineralization and accumulation of OC- 90/95 containing conglomerates in the inner ear of ‘head slant’ mice..........................................................................................54 6.1.4 Noxo1 mRNA is widely expressed in the embryonic inner ear.58 6.1.5 The hslt mutation of Noxo1 prevents its biochemical function..60 6.1.6 Analysis of the Ca2+, Na+, K+, and Cl- content of the endo- and perilymph by X-Ray Photoelectron Spectrometer.....................62 6.1.7 Lactoperoxidase expression in the utricle ................................64 6.1.8 Secretion of recombinant mouse OC-90 by transfected HEK293T cells..........................................................................66 6.1.9 Noxo1 transgene rescues ‘head slant’ phenotype....................67 6.2 II. Functional deletion of NOXA1 gene in mice results in non- functional NOX3 complex ..............................................................70 6.3 III. Identification of a p22phox deficient mouse strain .....................73 7. Discussion..............................................................................................76 8. Summary................................................................................................79 9. References.............................................................................................82 10. Publications............................................................................................96 11. Acknowledgements................................................................................97 4 2. Glossary and Abbreviations AD Activation domain ABR Auditory-evoked brainstem responses AIR Autoinhibitory region BAC Bacterial artificial clone CBA Chicken β-actin promoter cDNA Complementary deoxyribonucleic acid CSF Cerebrospinal fluid CGD Chronic granulomatous disease cM Centi Morgan CMV Cytomegalovirus COOH Carboxyl group CYBA Cytochrome b558 subunit alpha CYBB Cytochrome b558 subunit beta CYPs Cytochrome P-450 enzymes DUOXA1 and DUOXA2 DOUX maturation factors 1 and 2 DUOX Dual Oxidase EBV Epstein-Barr virus ED Embryonic day EDS Energy Dispersive Spectroscopy EPO Eosinophil peroxidase EST Expressed Sequence Tag FAD/FMN Flavin adenine dinucleotide/ Flavin mononucleotide GFP Green fluorescence protein Gfer Growth factor erv1-like hslt Head slant HPLC High performance liquid chromatography IgG Immunoglobulin G kDa Kilo daltons LPO Lactoperoxidase Mox-1 Mitogenic oxidase 1 mRNA Messenger ribonucleic acid MPO Myeloperoxidase NOS Nitric oxide synthase NOX NADPH oxidase NOH-1 NADPH oxidase homologue 1 NOXA1 NOX Activator 1 5 NOXO1 NOX Organizer 1 PAC P1 artificial clone PHOX Phagocyte NADPH oxidase PMA Phorbol 12-myristate 13-acetate PX Phox domains PB1 Phox and Bem1 domain PCR Polymerase chain reaction P Postnatal day PRR Proline-rich region PKC Protein Kinase C RNS Reactive nitrogen species ROS Reactive oxygen species RSS Reactive sulfur species SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis sPLA2 Secretory phospholipase A2 SH3 Src homology 3 domain SOD Superoxide dismutase TPO Thyroid peroxidase TPR Tetratricopeptide repeat Tbl3 Transducin (beta)-like 3 XPS X-ray photoelectron spectroscopy 6 3. Introduction 3.1 Reactive oxygen species 3.1.1 Reactive oxygen species in biology The presence of reactive oxygen species (ROS) in biological materials was discovered more than 50 years ago [1]. Soon thereafter, Denham Harman hypothesized that oxygen radicals can be formed as by-products of enzymatic reactions in vivo . In 1956, he described free radicals as a Pandora’s box of evils that may account for gross cellular damage, mutagenesis, cancer, and, last but not least, the degenerative process of biological aging [2, 3]. ROS have historically been viewed as a harmful but unavoidable consequence of an aerobic lifestyle. Considerable interest in ROS comes from related pathologies including atherosclerosis, hypertension, ischemia-reperfusion injury, inflammation, cystic fibrosis, cancer, type-2 diabetes, Parkinson’s disease, Alzheimer’s disease, and other neurodegenerative diseases. Defining the aspect of aging lies in the progressive vicious cycle in which oxidative stress plays a major role. Oxidative stress arises from a significant