Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences presented by Master of Science – Chemical Engineering Michael Lang born in Mulhouse (France) Oral-examination: ……………………………… Identification and characterization of interaction partners of Heat Shock Transcription Factor 1 during the cellular heat shock response Referees: Prof. Dr. Matthias Mayer Prof. Dr. Georg Stoecklin Table of Contents Table of Contents .................................................................................................................................... 5 Abstract ................................................................................................................................................... 8 Zusammenfassung ................................................................................................................................. 10 Chapter 1. Introduction ......................................................................................................................... 12 1.1 Chaperone proteins ................................................................................................................... 12 1.2 The Heat Shock response .......................................................................................................... 13 1.3 Heat Shock transcription Factor 1 ............................................................................................. 14 1.3.1 The different domains of HSF1 .......................................................................................... 14 1.3.2 Localization of HSF1........................................................................................................... 16 1.3.3 HSF1-mediated transactivation of heat shock genes ........................................................ 16 1.3.4 Regulation of the HSF1 activation-attenuation cycle ........................................................ 17 1.3.3.1 Activation of HSF1 ............................................................................................................. 17 1.3.3.2 Attenuation of the heat shock response ........................................................................... 19 1.3.5 Other roles of HSF1 ........................................................................................................... 20 1.4 Overview of Hsp70’s role and mechanism of action ................................................................. 21 1.4.1 Hsp70s’ functions in the cell .............................................................................................. 21 1.4.2 Mechanism of action of Hsp70 .......................................................................................... 23 1.4.3 J-domains proteins and nucleotide exchange factors ....................................................... 24 Chapter 2. Aim of the thesis .................................................................................................................. 26 Chapter 3. Results.................................................................................................................................. 27 3.1 Establishment of a HSF1 overexpressing cell line ........................................................................... 27 3.2 Identification of HSF1-binding proteins .......................................................................................... 31 3.3 Bag2 and Bag4 interact directly with HSF1 ..................................................................................... 33 3.4 In vitro characterization of Bag2 and Bag4 ..................................................................................... 38 3.4.1 Purification of Bag2, Bag4 and Hsc70. ...................................................................................... 38 3.4.2 Secondary structure analysis of Bag2 and Bag4 ....................................................................... 40 3.4.3 Thermal stability of Bag2 and Bag4 .......................................................................................... 41 3.4.4 Bag2 and Bag4 act as nucleotide exchange factors of Hsp70 .................................................. 43 3.4.5 Bag2 and Bag4 increase luciferase refolding rates................................................................... 45 3.5 Bag2 and Bag4 are positive regulators of the heat shock response ............................................... 47 Chapter 4. Discussion ............................................................................................................................ 52 4.1 Implications of the eventual interaction partners identified .......................................................... 52 4.2 Bag2 and Bag4 interact with HSF1 and Hsp70 ................................................................................ 54 5 4.3 Bag2 and Bag4 as in vitro nucleotide exchange factor ................................................................... 56 4.4 Bag2 and Bag4 tune up the heat shock response ........................................................................... 57 Chapter 5. Material and Method .......................................................................................................... 61 5.1 Materials .......................................................................................................................................... 61 5.1.1 Bacterial strains and plasmids .................................................................................................. 61 5.1.1.1 E.coli K12 strains ................................................................................................................ 61 5.1.1.2 Plasmids ............................................................................................................................. 61 5.1.2 Mammalian cell lines ................................................................................................................ 62 5.1.3 Oligodesoxynucleotides ........................................................................................................... 62 5.1.4 Chemicals, enzymes and kits .................................................................................................... 63 5.2 Common solutions and buffers ....................................................................................................... 66 5.3 Methods .......................................................................................................................................... 67 5.3.1 Cell culture and transfection .................................................................................................... 67 5.1.1.1 Cultivation of cells ............................................................................................................. 67 5.1.1.2 Transfection ....................................................................................................................... 67 5.1.1.3 Cryo-conservation of cultivated cells ................................................................................ 68 5.3.2 Molecular biology techniques .................................................................................................. 68 5.3.2.1 Cloning techniques ............................................................................................................ 68 5.3.2.2 Quantitative real-time polymerase chain reaction ........................................................... 70 5.3.3 Immunoprecipitation and Western Blot .................................................................................. 71 5.3.3.1 Immunoprecipitation ........................................................................................................ 72 5.3.3.2 SDS-PAGE and immunoblotting ......................................................................................... 73 5.3.4 Analysis by Shotgun Proteomics .............................................................................................. 73 5.3.5 Protein expression and purification ......................................................................................... 76 5.3.5.1 Purification of Bag4 Bag Domain ....................................................................................... 77 5.3.5.2 Purification of Bag2 full length .......................................................................................... 77 5.3.5.3 Purification of Bag2_BD ..................................................................................................... 78 5.3.5.4 Purification of Hsc70 ......................................................................................................... 78 5.3.5.5 Determination of the protein concentration .................................................................... 78 5.3.6 Biochemical assays ................................................................................................................... 78 5.3.6.1 Luciferase refolding assay ................................................................................................. 78 5.3.6.2 Luciferase assay on cell lysates ......................................................................................... 79 5.3.6.3 Kinetics of ADP-release ..................................................................................................... 80 5.3.7 Circular Dichroism ...................................................................................................................