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The Fate of Endoplasmic Reticulum-Targeted Proteins in the Face of Proteasome Failure

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The Fate of Endoplasmic Reticulum-Targeted Proteins in the Face of Proteasome Failure The fate of endoplasmic reticulum-targeted proteins in the face of proteasome failure Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) vorgelegt von: Franziska Höfer an der Mathematisch-Naturwissenschaftliche Sektion Fachbereich Biologie Tag der mündlichen Prüfung: 18. Mai 2015 1. Referent: Prof. Dr. Martin Scheffner 2. Referent: Prof. Dr. Marcus Groettrup 3. Referent: Prof. Dr. Jörg Tatzelt Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-291257 “Imagination is more important than knowledge. For knowledge is limited, whereas imagination embraces the entire world, and all there ever will be to know and understand, stimulating progress, giving birth to evolution. It is, strictly speaking, a real factor in scientific research.” Albert Einstein, 1931 Table of Content 1. SUMMARY .................................................................................................................. 1 2. ZUSAMMENFASSUNG .............................................................................................. 2 3. INTRODUCTION ......................................................................................................... 3 3.1 The endoplasmic reticulum (ER) ......................................................................................................... 3 3.1.1 Composition and function of the endoplasmic reticulum .................................................................. 3 3.1.2 Protein transport across the mammalian ER membrane ................................................................. 3 3.1.3 ER signal peptides and the ER signal peptidase complex ............................................................... 9 3.1.4 ER-associated degradation (ERAD) ............................................................................................... 11 3.1.5 ER under stress: the unfolded protein response, preemptive quality control in the ER and mislocalized proteins. .............................................................................................................................. 16 3.1.6 Antigen processing and the endoplasmic reticulum ....................................................................... 21 3.2 The (immuno-) proteasome ................................................................................................................ 23 3.2.1 Composition and assembly of the mammalian (immuno-) proteasome ......................................... 23 3.2.2 Ubiquitin and the proteasomal protein degradation process .......................................................... 26 3.2.3 Modification and inhibition of the proteasome ................................................................................ 28 3.2.4 Oxidative stress and proteasomes ................................................................................................. 31 3.3 The brain .............................................................................................................................................. 33 3.3.1 Anatomy of the brain ...................................................................................................................... 33 3.3.2 The (immuno-) biology of astrocytes .............................................................................................. 34 3.3.3 The role of the (immuno-) proteasome in the central nervous system (CNS) ................................ 34 4. OBJECTIVES ............................................................................................................ 36 5. MATERIAL & METHODS .......................................................................................... 38 5.1 Prokaryotic cells .................................................................................................................................. 38 5.1.1 Bacterial strain and culture conditions ............................................................................................ 38 5.1.2 Preparation and transformation of competent bacteria .................................................................. 38 5.2 Eukaryotic Cells ................................................................................................................................... 38 5.2.1 Cell lines ......................................................................................................................................... 38 5.2.2 Heat shock of eukaryotic cells ........................................................................................................ 39 5.2.3 Cytokine stimulation of cell lines .................................................................................................... 39 5.2.4 Lymphocytic choriomeningitis virus (LCMV) infection of cells ........................................................ 39 5.2.5 Chemical inhibitors and inducers ................................................................................................... 39 5.2.6 RNA silencing ................................................................................................................................. 40 5.2.7 Transient transfection of eukaryotic cells with DNA plasmids ........................................................ 41 5.2.8 Generation of stable transfected cell lines ..................................................................................... 44 5.2.9 Lysis of cells ................................................................................................................................... 44 5.2.10 Fluorescence-activated cell sorting .............................................................................................. 44 5.2.11 Confocal fluorescence microscopy of cells .................................................................................. 44 5.2.12 Magnetic cell separation ............................................................................................................... 45 5.2.13. Measurement of cell death .......................................................................................................... 45 5.3 Proteins ................................................................................................................................................ 46 5.3.1 Immunoprecipitation (IP) ................................................................................................................ 46 5.3.2 Deglycosylation of proteins ............................................................................................................ 46 5.3.3 Radioactive labeling of proteins with [35S]-methionine/cysteine (pulse-chase) .............................. 47 5.3.4 Separation of proteins with SDS-PAGE ......................................................................................... 47 5.3.6 Analysis of radioactive proteins on SDS-gels................................................................................. 49 5.3.7 Non-equilibrium pH gel electrophoresis (NEPHGE) ....................................................................... 50 5.3.8 Fractionating cellular proteins using osmotic pressure .................................................................. 50 5.3.9 Proteasome activity assay .............................................................................................................. 51 5.3.10 Sucrose gradient density centrifugation ....................................................................................... 51 5.3.11 Purification of antibodies from rabbit sera .................................................................................... 51 5.4. DNA ...................................................................................................................................................... 51 5.4.1 Preparation of DNA ........................................................................................................................ 51 5.4.2 Agarose gel electrophoresis ........................................................................................................... 52 5.4.4 Polymerase Chain Reaction – PCR ............................................................................................... 52 5.4.5 Restriction enzyme digestion ......................................................................................................... 53 5.4.6 Ligation of DNA fragments ............................................................................................................. 53 5.4.7 Site directed mutagenesis of plasmids ........................................................................................... 53 5.4.8 Quantitative real-time polymerase chain reaction (qRT-PCR) ....................................................... 54 5.5 RNA ....................................................................................................................................................... 54 5.5.1 RNA extraction from eukaryotic cells ............................................................................................. 54 5.5.2 Synthesis of cDNA from RNA samples – Reverse Transcriptase Reaction .................................. 54 5.6 Animals ................................................................................................................................................. 54 5.6.1 Mice ................................................................................................................................................ 54
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