Dissertation zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) im Fach Biologie „Identification and Characterization of Interferon-J Induced Ubiquitinated Newly Synthesized Proteins“ Eingereicht an der Mathematisch-Naturwissenschaftlichen Fakultät I Humboldt-Universität zu Berlin von Frau Diplom-Chemikerin Anne Wiemhoefer Präsident der Humboldt-Universität zu Berlin: Prof. Dr. Jan-Hendrik Olbertz Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I: Prof. Dr. Andreas Herrmann Gutachter: 1. Prof. Dr. Peter-Michael Kloetzel 2. Prof. Dr. Wolfgang Lockau 3. Prof. Dr. Wolfgang Dubiel Tag der mündlichen Prüfung: 29.06.2011 Inhaltsverzeichnis Zusammenfassung ................................................................................................ i Abstract ............................................................................................................... iii 1 Introduction .................................................................................................... 1 1.1 Ubiquitination ............................................................................................ 3 1.1.1 Ubiquitin ........................................................................................ 3 1.1.2 Process of ubiquitination ............................................................... 4 1.1.3 Endoplasmatic reticulum associated degradation system .............. 8 1.1.4 Ubiquitin chains and functions .................................................... 10 1.2 Process of deubiquitination ..................................................................... 11 1.3 The proteasome and degradation of ubiquitinated proteins .................... 13 1.3.1 Recruitment of ubiquitinated proteins to the proteasome............ 13 1.3.2 Constitutive proteasome 20S core complex ................................ 14 1.3.3 Proteasomal activators ................................................................. 15 1.3.4 Proteasome subtypes ................................................................... 19 1.4 Influence of interferon-J on ubiquitination ............................................. 20 1.4.1 Influence of interferon-Jon the cell ............................................ 20 1.4.2 Influence of interferon-J on ubiquitin proteasome system .......... 20 1.5 Aims of this research study ..................................................................... 23 2 Materials and Methods ................................................................................ 25 2.1 Equipments and materials ....................................................................... 25 2.2 Molecular biological methods ................................................................. 30 2.2.1 Polymerase chain reaction (PCR) ................................................ 30 2.2.2 Agarose based gel electrophoresis ............................................... 31 2.2.3 Ligation of the PCR products into a TOPO® vector ................... 32 2.2.4 Competent E.coli cells ................................................................. 32 2.2.5 Transformation ............................................................................ 33 2.2.6 Plasmid DNA isolation and sequencing ...................................... 33 2.2.7 Digestion via restriction enzymes ............................................... 34 2.2.8 Ligation into pEGFP-N3 vector .................................................. 34 2.3 Human cell culture methods .................................................................... 34 2.3.1 Cell lines, buffers and media ....................................................... 34 2.3.2 Subcultivation .............................................................................. 36 2.3.3 Transfection and generation of stable transfectants ..................... 36 2.3.4 “Short” stable isotope labeling with amino acids in cell culture . 37 2.3.5 Interferon-Jinduction of HeLa cells and cell harvesting ............. 39 2.3.6 Determination of protein concentration ....................................... 40 2.4 Isolation of ubiquitin conjugates ............................................................. 40 2.4.1 Preparation of isolation matrices ................................................. 40 2.4.2 Immuno precipitation and affinity purification ............................ 41 2.5 Bioanalytical methods ............................................................................. 42 2.5.1 Preparation of the ubiquitin conjugates for mass spectrometry ... 42 2.5.2 Mass spectrometric analysis ........................................................ 44 2.6 Biochemical methods ............................................................................... 45 2.6.1 Sodiumdodecylsulfoxide-polyacrylamide gel electrophoresis .... 45 2.6.2 Two dimensional gel electrophoresis ........................................... 46 2.6.3 Staining of polyacrylamide gels ................................................... 47 2.6.4 Semidry Western blotting and immuno detection ....................... 48 3 Results ............................................................................................................ 49 3.1 Isolation of ubiquitin conjugates ............................................................. 50 3.1.1 Evaluation of different isolation methods .................................... 50 3.1.2 Decision on the best enrichment method for ubiquitin conjugates51 3.2 Establishment of the cell culture model ................................................... 53 3.2.1 Analysis of transfection efficiency and expression stability ........ 55 3.2.2 Interferon-J influence on the model system ................................. 56 3.3 Analysis of ubiquitin-conjugates via 2D gel electrophoresis .................. 57 3.4 Optimization of mass spectrometric approach ........................................ 59 3.4.1 Digestion of ubiquitin-conjugate with peptidases ........................ 59 3.4.2 Selection of the sample preparation procedure ............................ 62 3.4.3 “Short” stable isotope labeling with amino acids in cell culture . 63 3.4.4 Final optimized experimental approach ....................................... 64 3.5 Results of mass spectrometric ubiquitin conjugate analysis .................... 67 3.5.1 Evaluation of ubiquitination sites ................................................ 70 3.5.2 Candidates for interferon-J influenced ubiquitination ................. 75 3.5.3 Preferences of ubiquitination for newly synthesized proteins ..... 78 3.5.4 Proteasome ubiquitination ........................................................... 79 3.6 Immuno chemical identification of ubiquitin targets .............................. 86 3.6.1 Sequestosome-1 ........................................................................... 87 3.6.2 Cytoplasmatic tryptophanyl t-RNA synthetase ........................... 88 3.6.3 Tripatite motif 21 ......................................................................... 90 3.6.4 Proteasome ATPase regulatory subunit Rpt5 .............................. 91 4 Discussion ...................................................................................................... 95 4.1 Methodic progress in the isolation of the Ub-conjugates ........................ 96 4.1.1 Optimum isolation strategy for the targeted Ub-conjugates ........ 96 4.1.2 Cell culture model for the tagged ubiquitin based isolation ........ 97 4.2 Gel electrophoretic characteristics of ubiquitin conjugates .................... 98 4.3 Innovations in the preparation for mass spectrometry ............................ 98 4.3.1 Ubiquitin-conjugate sample preparation for analysis .................. 98 4.3.2 Optimal quantitative mass spectrometric approach ................... 100 4.4 Evaluation of ubiquitin conjugate analysis ........................................... 102 4.5 Evidence of the interferon-J induced changes in Ub-chain linkages .... 103 4.6 Identified ubiquitin conjugates .............................................................. 107 4.7 Identified interferon-J induced ubiquitination candidates ..................... 107 4.8 Evaluation of the mass spectrometric data ............................................ 110 4.9 Relevance of interferon-J induced identified ubiquitin conjugates ....... 112 4.10Final conclusion and future perspectives .............................................. 117 5 Appendix ..................................................................................................... 123 5.1 Literature ............................................................................................... 123 5.2 Abbreviations ........................................................................................ 133 5.3 Supplementary data ............................................................................... 137 5.3.1 Expression level of tagged ubiquitin and phenotype of cells .... 137 5.3.2 MS2 fragmentations of Ub-linkage indicator peptides .............. 139 5.3.3 Ub-targets and candidates from SILAC experiments ................ 144 Danksagung .................................................................................................. 167 Erklärung ...................................................................................................... 169 i Zusammenfassung Ein Schlüsselprozess in der Immunantwort ist die durch das proinflammatorische Zytokin Interferon-Jinduziertetransiente Akkumulation von neu synthetisierten defekten Proteinen, die durch Anknüpfen