Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München How the mammalian endoplasmic reticulum handles aggregation-prone β-sheet proteins Lisa Vincenz-Donnelly geb. Vincenz aus Velbert, Deutschland 2016 Contents Contents Erklärung.................................................................................................................................... 2 Contents...................................................................................................................................... 3 Summary .................................................................................................................................... 6 Introduction ................................................................................................................................ 8 Protein folding and molecular chaperones ............................................................................. 8 Proteostasis ........................................................................................................................... 12 The endoplasmic reticulum .................................................................................................. 13 Protein translocation into the ER.......................................................................................... 14 The special folding environment of the ER.......................................................................... 15 ER protein folding factors .................................................................................................... 17 The calnexin/calreticulin system....................................................................................... 18 The BiP chaperone system................................................................................................ 19 The GRP94 chaperone ...................................................................................................... 22 Degradation of ER proteins .................................................................................................. 23 Cellular stress responses....................................................................................................... 27 The cytosolic stress response ............................................................................................ 27 The unfolded protein response.......................................................................................... 27 Protein aggregation toxicity.................................................................................................. 33 Conformational diseases caused by mutant ER proteins .................................................. 34 Mutations in ERQC factors can cause diseases ................................................................ 38 ER stress responses in disease .......................................................................................... 38 ER quality control in aging ............................................................................................... 39 Aims of the study ..................................................................................................................... 41 Aim of part 1......................................................................................................................... 41 Aim of part 2......................................................................................................................... 44 Materials and Methods............................................................................................................. 46 Materials ............................................................................................................................... 46 Chemicals.......................................................................................................................... 46 Antibodies ......................................................................................................................... 50 Enzymes............................................................................................................................ 51 Bacterial strains................................................................................................................. 52 3 Contents Mammalian cell lines........................................................................................................ 52 Buffers............................................................................................................................... 52 Polyacrylamide Bis-Tris gels............................................................................................ 53 Media ................................................................................................................................ 53 Kits.................................................................................................................................... 54 Other materials and Instruments ....................................................................................... 54 Plasmids ............................................................................................................................ 57 siRNAs.............................................................................................................................. 58 Softwares........................................................................................................................... 58 Methods ................................................................................................................................ 59 Production of chemically competent E. coli ..................................................................... 59 Plasmid preparation .......................................................................................................... 59 PCR amplification and purification of PCR products....................................................... 60 DNA Restriction Digestion and Ligation.......................................................................... 60 Cloning of expression plasmids ........................................................................................ 60 Cell culture and transfections............................................................................................ 61 Immunofluorescence imaging........................................................................................... 61 Immunoblotting................................................................................................................. 62 Solubility analysis............................................................................................................. 63 Cell viability assay............................................................................................................ 64 Analysis of secreted proteins ............................................................................................ 64 Cycloheximide chase ........................................................................................................ 65 SILAC labelling ................................................................................................................ 65 Sample preparation for SILAC-MS analysis .................................................................... 65 Sample preparation for label-free MS analysis................................................................. 66 LC-MS/MS ....................................................................................................................... 67 Analysis of MS data.......................................................................................................... 67 Fluorescence-activated cell sorting (FACS) ..................................................................... 68 siRNA knockdowns .......................................................................................................... 68 Luciferase assays............................................................................................................... 68 Deglycosylation ................................................................................................................ 69 Analysis of Q97 inclusions by fluorescence microscopy ................................................. 69 Results ...................................................................................................................................... 70 Part 1 - How the ER handles aggregation-prone β-sheet proteins........................................ 70 4 Contents Targeting an aggregation-prone β-protein to the ER ........................................................ 70 ER-β proteins are retained in the ER ................................................................................ 73 Targeting β-protein to the ER reduces toxicity and aggregation propensity .................... 76 ER-β17 is also retained in a detergent-soluble state in stably expressing cell lines......... 82 Analysis of the ER-β23 interactome ................................................................................. 84 ER-β23 interacts with a distinct set of ER chaperones and ERAD factors ...................... 86 ER-β23 accumulates at levels exceeding those of interacting chaperones ....................... 90 ER-β23 inhibits UPR induction ........................................................................................ 91 ER-β23 is not glycosylated ..............................................................................................