Verfügbarkeit: Lessons from the interaction of Importin 5 with SUMO2

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Bibliographische Detailangaben
Beteilige Person:	Richter, Sebastian (VerfasserIn)
Format:	Hochschulschrift/Dissertation Buch
Sprache:	Englisch
Veröffentlicht:	Heidelberg [2017]
Schlagwörter:	Hochschulschrift
Links:	http://d-nb.info/1151916870/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030187669&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA
Umfang:	224 Seiten Illustrationen, Diagramme 30 cm

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245	1	0	\|a Lessons from the interaction of Importin 5 with SUMO2 \|b novel insights into isoform-specific SUMO interaction motifs \|c presented by Sebastian Mario Richter, M.Sc.
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502			\|b Dissertation \|c Ruperto-Carola University of Heidelberg \|d 2017
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Datensatz im Suchindex

DE-BY-UBR_call_number	24/W17473
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DE-BY-UBR_location	UB Magazin
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adam_text	TABLE OF CONTENTS ABSTRACT...................................................................................................................... 1 ZUSAMMENFASSUNG................................................................................................. 3 TABLE OF CONTENTS................................................................................................ 5 FIGURES.......................................................................................................................... 9 TABLES.......................................................................................................................... 11 ABBREVIATIONS........................................................................................................ 13 1 INTRODUCTION...................................................................................................... 19 1.1 SUMOYLATION.................................................................................................................................................19 1.1.1 THE SMALL UBIQUITIN-RELATED MODIFIER SUMO....................................................................................... 19 1.1.2 SUMO CONJUGATION AND DE-CONJUGATION MACHINERIES.......................................................................... 21 1.1.3 PATHWAYS AND CONSEQUENCES OF SUMOYLATION .................................................................................... 27 1.1.4 CLASSES OF SUMO BINDING MOTIFS (SBM S)...........................................................................................31 1.1.5 THE SUMO INTERACTION MOTIF.................................................................................................................32 1.2 NUCLEOCYTOPLASMIC TRANSPORT......................................................................................................................37 1.2.1 REGULATION BY THE SMALL GTPASE RAN AND THE RANBP2 COMPLEX........................................................37 1.2.2 IMPORTIN-SS-LIKE NUCLEAR TRANSPORT RECEPTORS..........................................................................................39 1.2.3 THE NUCLEAR IMPORT RECEPTOR IMPORTIN 5 ................................................................................................43 1.2.4 LINKS BETWEEN SUMOYLATION AND NUCLEAR TRANSPORT............................................................................ 47 1.3 AIMS OF THIS THESIS........................................................................................................................................ 48 2 RESULTS................................................................................................................... 49 2.1 SUMOYLATION OF IMPORTIN 5 IN VIVO AND IN VITRO .................................................................................. 49 2.1.1 IMPORTIN SS AND IMPORTIN 5 ARE ENDOGENOUSLY SUMOYLATED ............................................................... 49 2.1.2 OXIDATIVE STRESS REDUCES THE ENDOGENOUS SUMOYLATION OF IMPORTIN 5 ............................................. 51 2.1.3 CHARACTERIZATION OF RECOMBINANT IMPORTIN 5 ........................................................................................ 52 2.1.4 IMPORTIN 5 IS SUMOYLATED BY PIAS1 AND THE RANBP2 COMPLEX IN VITRO..........................................54 2.1.5 IMPORTIN 5 SUMOYLATION BY PIAS1 IS BLOCKED BY RANGTP OR A SPECIFIC CARGO..............................56 2.1.6 IN VITRO SUMOYLATION OF IMPORTIN 5 BY PIAS1 DEPENDS ON THE LYSINE 637........................................58 2.1.7 IN VITRO SUMOYLATION BY THE RANBP2 COMPLEX MAINLY DEPENDS ON K 755 ..........................................63 2.1.8 IN VIVO SUMOYLATION OF IMPORTIN 5 IS BOOSTED BY NEM ..................................................................... 65 2.1.9 NEM BOOSTS THE IN VITRO SUMOYLATION OF IMPORTIN 5 ..........................................................................69 2.2 IMPORTIN 5 IS A SUM02-SPECIFIC BINDING PROTEIN.................................................................................. 73 2.2.1 IMPORTIN 5 BINDS SUMO WITH A HIGH SPECIFICITY FOR SUM 02..............................................................73 2.2.2 SUM02 INTERACTION DEPENDS ON A V 64 OALL MOTIF IN THE HI 5 INSERT .................................................. 74 2.2.3 V 64 O ALL AND ADJACENT NEGATIVE CHARGES FORM A CLASS I SUMO INTERACTION M OTIF ............................. 75 2.2.4 RANGTP BINDING, BUT NOT CARGO BINDING, PREVENTS SUM02-INTERACTION.............................................78 2.2.5 PHOSPHOMIMETIC MUTANTS IN IMPORTIN 5 INCREASE SUM02 BINDING .................................................... 80 2.2.6 BOTH IMPORTIN-5-SPECIFIC INSERTS CONTRIBUTE TO SUM02 BINDING.........................................................81 2.2.7 LYS32 AND THR37 IN SUM02 ARE INVOLVED IN IMPORTIN 5 BINDING.......................................................82 2.2.8 GLU49 IN SUMOL IS A MAIN DETERMINANT FOR ISOFORM-SPECIFIC BINDING .............................................. 85 2.2.9 SUMOS FLEXIBLE N-TERMINUS AS AN INHIBITOR OF CLASS I SUMO INTERACTIONS......................................89 2.2.10 MUTATIONS IN THE N-TERMINI OF SUMO CAN AUGMENT SIM INTERACTIONS..............................................93 2.2.11 THE YEAST ORTHOLOG OF IMPORTIN 5 - KAPL21P - IS A SUMO-BINDING PROTEIN .................................... 97 3 DISCUSSION...........................................................................................................101 3.1 SUM02-SPECIFIC SIMS IN IMPORTIN 5 AND BEYOND..................................................................................102 3.1.1 SUMO ISOFORM DISCRIMINATION BY GLU49 IN SUM OL........................................................................ 103 3.1.2 REGULATION OF ISOFORM-SPECIFIC BINDING OF IMPORTIN 5 AND KAPL21P................................................107 3.2 SUMOS N-TERMINUS - A LID FOR THE SIM BINDING GROOVE?....................................................................109 3.2.1 POSSIBLE REGULATORY ROLES FOR SUMO*S N-TERMINUS............................................................................ 111 3.3 SYNERGY BETWEEN H15 AND H18 - SUMO-SANDWICH OR -CHAINS? ......................................................... 112 3.4 IMPORTIN 5 - AN IMPORT RECEPTOR FOR SUMOYLATED PROTEINS?...............................................................115 3.4.1 SUMO BINDING IN SUB-NUCLEAR TARGETING OF CARGO PROTEINS ............................................................... 117 3.5 SUMOYLATION OF IMPORTIN 5 - LOCATIONS AND CONSEQUENCES...............................................................118 3.5.1 SUMOYLATION OF IMPORTIN 5 - NUCLEUS VS. CYTOPLASM......................................................................119 3.5.2 FUNCTIONAL CONSEQUENCES OF IMPORTIN 5 SUMOYLATION......................................................................121 3.6 THE NEM EFFECT - NATURAL ELECTROPHILES AND IMMUNE RESPONSE .......................................................... 122 3.6.1 REGULATION OF IMPORTIN 5 SUMOYLATION BY ELECTROPHILES............................................................... 124 3.7 FUTURE PERSPECTIVES..................................................................................................................................... 125 4 APPENDIX - SUMOYLATION OF THE SMALL GTPASE RAN 127 4.1 INTRODUCTION.................................................................................................................................................. 127 4.1.1 NUCLEAR IMPORT OF RAN IS MEDIATED BY NTF2 ..................................................................................... 127 4.1.2 SUMOYLATION OF THE SMALL GTPASE RAN BY THE RANBP2 COMPLEX...................................................128 4.1.3 AIMS........................................................................................................................................................128 4.2 RESULTS............................................................................................................................................................131 4.2.1 RAN SUMOYLATION BY RANBP2 DEPENDS ON RAN-BINDING DOMAINS...................................................131 4.2.2 COMPLEX FORMATION WITH NTF2 PREVENTS RAN SUMOYLATION............................................................ 132 4.2.3 LYSL30 AND LYSL52 ARE THE MAIN SUMO ACCEPTOR SITES IN R AN........................................................135 4.2.4 NTF2 BLOCKS RAN SUMOYLATION BY PREVENTING INTERACTION WITH RANBDS ....................................... 137 4.3 DISCUSSION..................................................................................................................................................... 141 4.3.1 RANBD-DEPENDENT SUMOYLATION - RANGTP VS. RANGDP..................................................................141 4.3.2 CHAPERONING RANGDP - A NEW ROLE FOR NTF2.....................................................................................142 5 MATERIAL & METHODS....................................................................................145 5.1 MATERIALS....................................................................................................................................................... 145 5.1.1 TECHNICAL EQUIPMENT.............................................................................................................................145 5.1.2 CONSUMABLES......................................................................................................................................... 148 5.1.3 SOFTWARE.................................................................................................................................................149 5.1.4 CHEMICALS, REAGENTS AND KITS................................................................................................................ 150 5.1.5 BUFFERS AND STOCK SOLUTIONS.................................................................................................................. 153 5.1.6 ANTIBODIES..............................................................................................................................................157 5.1.7 PEPTIDES..................................................................................................................................................159 5.1.8 PLASMIDS.................................................................................................................................................160 5.1.9 RECOMBINANT PROTEINS...........................................................................................................................165 5.1.10 CELL LINES..............................................................................................................................................169 5.1.11 DNA OLIGONUCLEOTIDES........................................................................................................................170 5.2 MOLECULAR BIOLOGICAL METHODS................................................................................................................... 183 5.2.1 CULTURING, STORAGE AND TRANSFORMATION OF BACTERIAL CELLS .................................................................... 183 5.2.2 POLYMERASE CHAIN REACTION TECHNIQUES................................................................................................ 183 5.2.3 RESTRICTION DIGESTION OF DN A .............................................................................................................. 184 5.2.4 AGAROSE GEL ELECTROPHORESIS AND DNA EXTRACTION...............................................................................184 5.2.5 DE-PHOSPHORYLATION OF LINEARIZED PLASMIDS........................................................................................ 185 5.2.6 LIGATION OF DNA FRAGMENTS................................................................................................................. 185 5.2.7 PLASMID DNA PREPARATION.................................................................................................................... 185 5.2.8 DNA SEQUENCING.............. ...................................................................................................................186 5.3 BIOCHEMICAL METHODS................................................................................................................................... 187 5.3.1 DETERMINATION OF PROTEIN CONCENTRATION............................................................................................... 187 5.3.2 SDS-PAGE..............................................................................................................................................188 5.3.3 COOMASSIE STAINING................................................................................................................................ 188 5.3.4 IMMUNOBLOT.............................................................................................................................................189 5.3.5 IN VITRO SUMOYLATION ASSAYS WITH RECOMBINANT PROTEINS...................................................................189 5.3.6 MASS SPECTROMETRIC ANALYSIS OF SUMOYLATED IMPORTIN 5 ...................................................................190 5.3.7 NEM-TREATMENT OF NUCLEAR TRANSPORT RECEPTORS ................................................................................... 190 5.3.8 PULL-DOWN ASSAYS.................................................................................................................................... 190 5.3.9 SUMO-IMMUNOPRECIPITATION................................................................................................................ 191 5.3.10 ISOTHERMAL TITRATION CALORIMETRY..........................................................................................................192 5.3.11 GEL FILTRATION BINDING ASSAYS............................................................................................................... 193 5.3.12 EXPRESSION AND PURIFICATION OF RECOMBINANT PROTEINS.......................................................................193 5.4 CELL BIOLOGICAL METHODS................................................................................................................................200 5.4.1 CULTIVATION AND STORAGE OF HUMAN CELL LINES........................................................................................ 200 5.4.2 SEMI-PERMEABILIZATION OF HELA CELLS...................................................................................................201 5.4.3 IN VITRO SUMOYLATION WITH SEMI-PERMEABILIZED CELLS ....................................................................... 201 5.4.4 IN VITRO IMPORT ASSAYS............................................................................................................................ 202 5.4.5 TRANSFECTION........................................................................................................................................... 202 5.4.6 IMMUNOFLUORESCENCE............................................................................................................................. 203 REFERENCES.............................................................................................................. 205 SCIENTIFIC CONTRIBUTIONS.............................................................................. 221 ACKNOWLEDGMENTS............................................................................................ 223
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spellingShingle	Richter, Sebastian Lessons from the interaction of Importin 5 with SUMO2 novel insights into isoform-specific SUMO interaction motifs
subject_GND	(DE-588)4113937-9
title	Lessons from the interaction of Importin 5 with SUMO2 novel insights into isoform-specific SUMO interaction motifs
title_auth	Lessons from the interaction of Importin 5 with SUMO2 novel insights into isoform-specific SUMO interaction motifs
title_exact_search	Lessons from the interaction of Importin 5 with SUMO2 novel insights into isoform-specific SUMO interaction motifs
title_full	Lessons from the interaction of Importin 5 with SUMO2 novel insights into isoform-specific SUMO interaction motifs presented by Sebastian Mario Richter, M.Sc.
title_fullStr	Lessons from the interaction of Importin 5 with SUMO2 novel insights into isoform-specific SUMO interaction motifs presented by Sebastian Mario Richter, M.Sc.
title_full_unstemmed	Lessons from the interaction of Importin 5 with SUMO2 novel insights into isoform-specific SUMO interaction motifs presented by Sebastian Mario Richter, M.Sc.
title_short	Lessons from the interaction of Importin 5 with SUMO2
title_sort	lessons from the interaction of importin 5 with sumo2 novel insights into isoform specific sumo interaction motifs
title_sub	novel insights into isoform-specific SUMO interaction motifs
topic_facet	Hochschulschrift
url	http://d-nb.info/1151916870/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030187669&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA
work_keys_str_mv	AT richtersebastian lessonsfromtheinteractionofimportin5withsumo2novelinsightsintoisoformspecificsumointeractionmotifs

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