Institut fur¨ Experimentelle Genetik GSF-Forschungszentrum fur¨ Umwelt und Gesundheit, Neuherberg Search for New Steroid Hormone Metabolizing Enzymes: Functional Genomics of the Short-Chain Dehydrogenase/Reductase Superfamily Brigitte D. Keller Vollst¨andiger Abdruck der von der Fakult¨at Wissenschaftszentrum Weihenstephan fur¨ Ern¨ahrung, Landnutzung und Umwelt der Technischen Universit¨at Munc¨ hen zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr. Martin Hrab´e de Angelis Pruf¨ er der Dissertation: 1. Univ.-Prof. Dr. Johannes Buchner 2. Univ.-Prof. Dr. Dr. Adelbert Bacher 3. apl. Prof. Dr. Jerzy Adamski Die Dissertation wurde am 02.06.2006 bei der Technischen Universit¨at Mun¨ chen einge- reicht und durch die Fakult¨at Wissenschaftszentrum Weihenstephan fu¨r Ern¨ahrung, Land- nutzung und Umwelt am 18.10.2006 angenommen. Meinen Eltern Contents Abbreviations . xi Zusammenfassung . 1 Abstract . 3 1. Introduction 5 1.1. The Short-Chain Dehydrogenase/Reductase superfamily . 5 1.1.1. Structural characteristics of SDRs . 6 1.1.2. Enzymatic characteristics and substrates of SDRs . 7 1.1.3. Medical impact of SDR superfamily . 8 1.2. Hormones . 8 1.2.1. Steroid hormones . 10 1.2.2. Distribution and action of steroid hormones . 10 1.2.3. Classes of steroid hormones . 11 1.2.4. Biosynthesis of steroid hormones . 12 1.3. Steroid metabolizing enzymes: Biosynthesis and pre-receptor control of steroid action . 15 1.3.1. 20α-Hydroxysteroid dehydrogenases . 15 1.3.2. 3α-Hydroxysteroid dehydrogenases . 15 1.3.3. 3β-Hydroxysteroid dehydrogenases/isomerases . 15 1.3.4. 11β-Hydroxysteroid dehydrogenases 1 and 2 . 16 1.3.5. 17β-Hydroxysteroid dehydrogenases . 16 1.4. The aim of the study . 17 2. Results 19 2.1. Introducing remarks . 19 2.2. Identification of SDR candidate enzymes and classification as SDRs . 19 2.2.1. Identification of putative SDRs using SDR Finder . 19 2.2.2. Verification of the SDR Finder identified enzymes and classification as SDR enzymes . 23 2.2.3. Identification and verification of MGC4172 and MGC18716 . 24 2.2.4. Annotation of the identified SDR type enzymes to the genome . 24 2.3. Selection of identified SDRs for in-depth characterization . 28 v Contents 2.4. Expression patterns of candidate enzymes . 33 2.4.1. Development of an automated In Silico Northern Blot (ISNB) . 33 2.4.2. Expression analysis of the candidate genes in silico and wet lab . 35 2.4.3. Expression pattern of murine orphan Sdr (mSdr-o) . 37 2.4.4. Expression pattern of human retSDR3 . 38 2.4.5. Expression pattern of rat dhrs7b . 38 2.4.6. Expression pattern of rat dhrs8 . 39 2.4.7. Expression pattern of murine Rdh12 . 40 2.4.8. Expression pattern of human RDH12 . 41 2.4.9. Expression pattern of human RDH13 . 42 2.4.10. Expression pattern of murine Dhrs4 . 43 2.4.11. Expression pattern of human DHRSX . 43 2.4.12. Expression pattern of human WWOX . 44 2.4.13. Expression pattern of murine Wwox . 45 2.4.14. Expression pattern of human MGC4172 . 46 2.4.15. Expression pattern of murine MGC18716 . 47 2.5. First hints to substrate specificity: phylogenetic analyses . 48 2.6. Investigation of substrate specificity: test of steroidogenic substrates . 51 2.6.1. Selection of substrates and establishment of measurement . 51 2.6.2. Establishment of substrate conversion assays . 52 2.6.3. Conduction of conversion assays for enzymes under investigation . 58 2.6.4. Expression plasmids . 60 2.6.5. Test of overexpression . 60 2.6.6. Enzymes not converting the tested substrates . 61 2.6.7. Human retSDR3 oxidizes 17β-estradiol to estrone . 62 2.6.8. Human RDH12, human DHRSX, and murine Dhrs4 reduce dihy- drotestosterone to androstanediol . 63 2.6.9. Murine Dhrs4 has reductive enzymatic activity not only versus di- hydrotestosterone but also towards androstenedione and estrone . 65 2.6.10. Murine Wwox reduces androstenedione to testosterone and andros- terone to androstanediol . 68 2.7. Investigation of subcellular localization . 71 2.7.1. Bioinformatic means to predict subcellular localization . 72 2.7.2. Vectors and system of analysis . 72 2.7.3. Murine Sdr-o is a mitochondrial protein . 75 2.7.4. Human retSDR3 localizes to the cytoplasm . 76 2.7.5. dhrs7b from rat is localized in the endoplasmic reticulum . 77 2.7.6. Rat dhrs8 is an ER-localized SDR enzyme . 78 vi Contents 2.7.7. Murine Rdh12 is endoplasmic reticulum-localized . 79 2.7.8. Human RDH12 localizes to the ER . 80 2.7.9. Human type 13 retinol dehydrogenase localizes to the mitochondria 81 2.7.10. Murine Dhrs4 is neither a peroxisomal SDR nor mitochondrial but cytoplasmically distributed . 82 2.7.11. Human DHRSX co-localizes with ER-staining . 83 2.7.12. MGC4172 and MGC18716 may both be ER-localized . 84 3. Discussion 87 3.1. General remarks . 87 3.2. The set-up - is it functional genomics? . 87 3.3. Identification of candidate proteins and classification as SDRs . 88 3.3.1. The SDR Finder - friend or foe? . 88 3.3.2. On NCBI NonRedundant Database . 89 3.3.3. Annotation of identified SDRs to chromosomal location . 90 3.4. Analysis of the expression patterns . 92 3.4.1. Need of expression analysis . 92 3.4.2. Experimental expression analysis by Northern Blot . 93 3.4.3. In silico expression analysis . 93 3.5. Use of phylogenetics in predicting substrate specificities . 96 3.6. The substrate conversion assay . 98 3.6.1. Prokaryotic or eukaryotic overexpression? . 98 3.6.2. A steroid hormone conversion assay in living cells - what are the advantages? . 100 3.6.3. Considerations on the cell line used . 100 3.6.4. The conversion assay in living cells - is it in vivo? . 103 3.6.5. Michaelis-Menten kinetics in living cells . 103 3.6.6. Measurement of progestin conversion . 105 3.7. Studies on subcellular localization . 105 3.7.1. Prediction of subcellular localization . 105 3.7.2. Considerations on the experimental design . 106 3.8. Conclusions about the enzymes under investigation . 111 3.8.1. Murine orphan Sdr (Sdr-o) . 111 3.8.2. Human retSDR3 . 113 3.8.3. Rat SDRs under investigation: dhrs7b and dhrs8 . 115 3.8.4. Retinol dehydrogenases under investigation: murine type 12, human types 12 and 13 . 116 3.8.5. Murine Dhrs4 . 118 3.8.6. Human DHRSX . 119 vii Contents 3.8.7. Human and murine WW-box containing oxidoreductases . 120 3.8.8. MGC4172 and MGC18716 . 122 3.9. What else could be done? . 124 3.10. Outlook . 125 4. Methods 127 4.1. Work with E. coli . 127 4.1.1. Culture media . 127 4.1.2. Inhibitory and selective media supplements . 127 4.1.3. Growing of bacteria . 127 4.1.4. Short- and long-term-storage of bacterial culture . 128 4.1.5. Production of competent E. coli and transformation of plasmid DNA 128 4.2. Methods with eukaryotic cell lines . 129 4.2.1. Cultivation of cells . 129 4.2.2. Maintenance of cell culture: Splitting, thawing, freezing, and long- term storage of eukaryotic cells . 130 4.2.3. Transfection of eukaryotic cells . 131 4.2.4. Seeding of cells and transfection in different formats . 131 4.2.5. Immuncytochemical methods for subcellular localization studies . 132 4.3. DNA-based molecular biological methods . 134 4.3.1. Isolation and purification procedures . 134 4.3.2. Measurement and quality assessment of DNA solutions . 135 4.3.3. Cloning strategies . 137 4.3.4. PCR-based methods . 138 4.4. RNA-based methods . 139 4.4.1. Synthesis of RNA by in vitro transcription and digoxigenin labelling 139 4.4.2. Isolation and purification methods . 139 4.4.3. Handling and measurement . 140 4.5. Analysis of Gene Expression . 141 4.5.1. Reverse transcription of mRNA into cDNA . 141 4.5.2. Hybridization of cDNA probes to membrane bound RNA: Northern Blot . 141 4.5.3. Analysis of gene expression in mouse embryos . 142 4.6. Measurement of substrate specificity . 144 4.6.1. Measurement of substrate conversion in living cells . 144 4.6.2. Determination of Michaelis-Menten kinetics . 144 4.6.3. Purification of HPLC samples by solid phase extraction . 146 4.6.4. HPLC measurement . 147 4.7. Bioinformatic methods . 148 viii Contents 4.7.1. Identification of SDR candidate genes . 148 4.7.2. In silico Northern Blot . 148 4.7.3. Alignments and phylogeny . 149 4.7.4. Bioinformatic assessment of subcellular localization . 149 5. Programs, organisms, material 151 5.1. Programs . ..