Some ABCA3 mutations elevate ER stress and initiate apoptosis of lung epithelial cells Nina Weichert Aus der Kinderklinik und Kinderpoliklinik im Dr. von Haunerschen Kinderspital der Ludwig-Maximilians-Universität München Direktor: Prof. Dr. med. Dr. sci. nat. Christoph Klein Some ABCA3 mutations elevate ER stress and initiate apoptosis of lung epithelial cells Dissertation zum Erwerb des Doktorgrades der Humanmedizin an der Medizinischen Fakultät der Ludwig-Maximilians-Universität zu München Vorgelegt von Nina Weichert aus Heidelberg 2011 Mit Genehmigung der Medizinischen Fakultät der Universität München 1. Berichterstatter: Prof. Dr. Matthias Griese 2. Berichterstatter: Prof. Dr. Dennis Nowak Mitberichterstatter: Priv. Doz. Dr. Angela Abicht Prof. Dr. Michael Schleicher Mitbetreuung durch den promovierten Mitarbeiter: Dr. Suncana Kern Dekan: Herr Prof. Dr. med. Dr. h. c. Maximilian Reiser, FACR, FRCR Tag der mündlichen Prüfung: 24.11.2011 Table of Contents 1.Abstract ................................................................................................................... 1 2.Zusammenfassung................................................................................................. 2 3.Intoduction .............................................................................................................. 3 3.1 Pediatric interstitial lung disease ............................................................................... 3 3.1.1 Epidemiology of pILD............................................................................................... 3 3.1.2 Classification of pILD ............................................................................................... 4 3.1.3 Genetic surfactant dysfunction disorders................................................................. 5 3.1.4 Clinical diagnostics and therapy of pILD.................................................................. 6 3.1.5 Prognosis of pILD .................................................................................................... 6 3.2 ATP- binding cassette protein A3 (ABCA3) ............................................................... 7 3.2.1 ABC transporters ..................................................................................................... 7 3.2.2 General on the ABCA3 protein ................................................................................ 8 3.2.3 Function of ABCA3 .................................................................................................. 8 3.2.3.1 Localization and lamellar body biogenesis ........................................... 8 3.2.3.2 ABCA3 is a lipid transporter.................................................................. 9 3.2.3.3 Categorization of ABCA3 mutations ..................................................... 9 3.2.4 ABCA3 in lung disease .......................................................................................... 10 3.2.4.1 Early and late onset of lung disease ................................................... 10 3.2.4.2 Genotype-phenotype interplay............................................................ 11 3.2.4.3 Outer stressor ..................................................................................... 11 3.2.4.4 Histopathological pattern .................................................................... 11 3.2.4.5 Therapy............................................................................................... 12 3.2.5 ABCA3 mutations in this study............................................................................... 12 3.3 How misfolded proteins disturb cell homeostasis.................................................. 14 3.3.1 Function of the endoplasmic reticulum .................................................................. 14 3.3.2 Induction of the quality control system by unfolded proteins ................................. 15 3.3.3 Induction of apoptosis ............................................................................................ 17 3.4 ER stress and apoptosis contribute to disease pathogenesis .............................. 18 3.5. An objective ...............................................................................................................20 4. Materials ............................................................................................................... 21 4.1 Chemicals.................................................................................................................... 21 4.2 Equipment ...................................................................................................................21 4.3 Enzymes and kits ....................................................................................................... 22 4.4 Primers ........................................................................................................................ 23 I 4.5 Vectors ........................................................................................................................ 24 4.6 Antibodies ................................................................................................................... 25 4.7 Bacterial strains and cell lines .................................................................................. 27 5. Methods................................................................................................................ 27 5.1 Molecular biological methods ................................................................................... 27 5.1.1 Cloning strategy: Generation of pUB6-ABCA3-HA vector ..................................... 27 5.1.2 Site-directed point mutagenesis............................................................................. 28 5.1.3 DNA- sequencing................................................................................................... 29 5.1.4 E.coli DH5 culture .................................................................................................. 29 5.1.5 Generation of competent E.coli DH5 .................................................................... 29 5.1.6 Transformation of E.coli DH5 ................................................................................ 30 5.1.7 Plasmid-DNA isolation ........................................................................................... 30 5.1.8 Restriction .............................................................................................................. 30 5.2 Mamalian cell culture ................................................................................................. 31 5.2.1 Media and growth conditions ................................................................................. 31 5.2.2 Mycoplasma testing ............................................................................................... 31 5.2.3 Transfection of A549 cells...................................................................................... 31 5.3 Biochemical methods ................................................................................................ 31 5.3.1 Whole cell lysates preparation ............................................................................... 31 5.3.2 Crude membrane preparation................................................................................ 32 5.3.3 Determination of protein concentration .................................................................. 32 5.3.4 Deglycosylation-assay ........................................................................................... 32 5.3.5 SDS-Polyacrylamide Gel Electrophoreses (SDS-PAGE) ...................................... 33 5.3.6 Western Blotting..................................................................................................... 33 5.3.7 Liposome preparation and NBD-lipid uptake ......................................................... 34 5.3.8 RNA isolation from A549 cells ............................................................................... 34 5.3.9 RT-PCR ................................................................................................................. 34 5.3.10 Densitrometric analysis of the intensity of protein and DNA bands ..................... 35 5.3.11 Immunofluorescence............................................................................................ 35 5.3.12 FACS analyses .................................................................................................... 36 5.3.13 Statistical analyses .............................................................................................. 36 6. Results.................................................................................................................. 37 6.1 Creating a model system to analyse the effect of ABCA3 mutations on alveolar type II cells´homeostasis ................................................................................................. 37 6.1.1 Generation of pUB6-hABCA3 vector and site-directed point mutagenesis............ 37 6.1.2 Optimization of transfection procedure in A549 cell line ........................................ 39 6.2 General characterization of R43L, R280C and L101P ABCA3 mutations.............. 40 II 6.2.1 Localization and trafficking..................................................................................... 41 6.2.2 ABCA3 WT and mutant protein processing and maturation in A549 cells............. 44 6.2.3 Glycosylation of ABCA3 protein............................................................................