Mechanistic analysis of the Hsp90 machinery: Effects of cochaperones, posttranslational modifications and clients:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
München
Verl. Dr. Hut
2014
|
| Ausgabe: | 1. Aufl. |
| Schriftenreihe: | Biochemie
|
| Schlagworte: | |
| Online-Zugang: | Inhaltstext Inhaltsverzeichnis |
| Beschreibung: | X, 153 S. Ill., graph. Darst. 210 mm x 148 mm, 258 g |
| ISBN: | 9783843917841 3843917841 |
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| 100 | 1 | |a Rehn, Alexandra Beatrice |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Mechanistic analysis of the Hsp90 machinery: Effects of cochaperones, posttranslational modifications and clients |c Alexandra Beatrice Rehn |
| 250 | |a 1. Aufl. | ||
| 263 | |a 201506 | ||
| 264 | 1 | |a München |b Verl. Dr. Hut |c 2014 | |
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Datensatz im Suchindex
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CONTENTS
1 SNMMARY 1
2 INTRODUCTION 3
2.1 THE MOLECULAR CHAPERONE HSP90 3
2.1.1 MEMBERS AND STRUCTURE OF THE HSP90 FAMILY 3
2.1.2 ATPASE CYCLE OF HSP90 6
2.2 HSP90 COCHAPERONES 8
2.2.1 TPR-COCHAPERONES 8
2.2.2 THE NON-TPR-COCHAPERONE P23 9
2.2.2.1 STRUCTURE OF P23 9
2.2.2.2 INTERACTION WITH HSP90 11
2.2.2.3 HSP90 INDEPENDENT FUNCTIONS 12
2.2.2.3.1 P23 AS AN INDEPENDENT CHAPERONE 13
2.2.2.3.2 NUCLEAR FUNCTIONS OF P23 13
2.2.3 THE INTERPLAY OF HSP90 COCHAPERONES 14
2.3 POSTTRANSLATIONAL MODIFICATIONS OF HSP90 16
2.3.1 PHOSPHORYLATION, ACETYLATION AND FURTHER PTMS 16
2.3.2 METHYLATION - A NEWLY DISCOVERED PTM OF HSP90 20
2.4 THE POLIOVIRUS PRECURSOR PI- A NOVEL HSP90 CLIENT 22
3 OBJECTIVES 25
3.1 ANALYSIS OF THE CHARGED LINKER OF HSP90 25
3.2 ANALYSIS OF THE C-TERMINAL DOMAIN OF P23 26
3.3 ANALYSIS OF A METHYLATION POSITION IN HSP90 28
3.4 ANALYSIS OF THE HSP90 CLIENT PI 29
V
HTTP://D-NB.INFO/106041323X
CONTENTS
4 RESULTS 31
4.1 ANALYSIS OF THE CHARGED LINKER 31
4.1.1 SUPPLEMENTATION OF THE LINKER AFFECTS THE INTRINSIC ATPASE
ACTIVITY 31
4.1.2 CHARGED LINKER SUPPLEMENTATIONS MODULATE THE ACTIONS OF THE
COCHAPERONES AHAL, SBAL AND STIL 31
4.1.3 SUPPLEMENTATION OF THE CHARGED LINKER INFLUENCES CELL VIABILITY
AND MORPHOLOGY 33
4.1.4 CL MUTANTE ARE SENSITIVE TOWARDS HEAT STRESS 35
4.1.5 CL SUPPLEMENTATIONS SHOW CHANGED SENSITIVITY TOWARDS THE
HSP90 INHIBITOR RADICICOL 36
4.1.6 CHARGED LINKER IS INVOLVED IN CLIENT ACTIVATION IN VIVO 37
4.2 ANALYSIS OF THE C-TERMINAL DOMAIN OF THE HSP90 COCHAPERONE P23 .
40
4.2.1 THE CHAPERONE ACTIVITY OF P23 IS ENGRAINED IN THE CHARGED C-
TERMINAL SEGMENT OF THE TAIL 40
4.2.2 THE CHAPERONE-ACTIVE MOTIF MUST BE SEPARATED FROM THE CORE
DOMAIN BY A SPACER 40
4.2.3 THE UNSTRUCTURED P23 TM DISPLAYS CHAPERONE ACTIVITY ON ITS
OWN 42
4.2.4 ADDITION OF THE TAIL TVANSFORMS AN INACTIVE DOMAIN INTO A CHAP
ERONE 44
4.2.5 THE GGA-, QL- AND CHAPERONE-ACTIVE-REGIONS ARE DISPENSABLE
FOR THE INHIBITION OF THE HSP90 ATPASE 44
4.2.6 THE KWVDED MOTIF IS RESPONSIBLE FOR THE INHIBITION OF THE
HSP90 ATPASE 45
4.2.7 THE KWVDED MOTIF IS CRUCIAL FOR HSP90 BINDING 47
4.2.8 THE KWVDED MOTIF IS IMPORTANT FOR PROGRESSION THROUGH THE
HSP90 CYCLE 48
4.2.9 THE ISOLATED TAIL IS ABLE TO PARTIALLY RELEASE AHAL FROM HSP90 50
4.3 ANALYSIS OF A METHYLATION SITE IN HSP90 51
4.3.1 THE POSITIVE CHARGE OF THE METHYLATION POSITION REGULATES THE
ATPASE ACTIVITY OF HSP90 51
4.3.2 CO CHAPERONE-INDUCED REARRANGEMENTS OF HSP90 ARE AFFECTED BY
THE REPLACEMENT OF THE LYSINE RESIDUE 52
4.3.3 REPLACEMENT OF THE LYSINE RESIDUE BY A NEGATIVELY CHARGED
AMINO ACID CAUSES STRUCTURAL RESTRAINTS IN HSP90 54
VI
CONTENTS
4.3.4 ANALYSIS OF THE METHYLATION POSITION IN VIVO 55
4.3.5 STRESS RESPONSES ARE AFFECTED BY THE SUBSTITUTION OF THE METHY
LATION POSITION 60
4.3.6 THE METHYLATION POSITION PLAYS A CRUCIAL ROLE IN CLIENT PROTEIN
ACTIVATION 61
4.4 ANALYSIS OF THE HSP90 CLIENT PI 64
4.4.1 EXPRESSION OF PI IN S. CEREVISIAE 64
4.4.2 BBCPRESSION OF THE VIRAL SUBUNITS IN S. CEREVISIAE 65
4.4.3 EXPRESSION OF VPO CAUSES A TAXIC PHENOTYPE IN S. CEREVISIAE . . 66
4.4.4 HIGHER PLOIDY LEADS TO RESISTANCE OF VPO TAXICITY 67
4.4.5 A GENETIC SCREEN REVEALED ENHANCERS OF VPO TAXICITY 68
4.4.6 EXPRESSION OF PI AND ITS SUBUNITS IN E. COLI 70
5 DISCUSSION 73
5.1 FUNCTIONAL ANALYSIS OF THE CHARGED LINKER OF HSP90 73
5.1.1 SUPPLEMENTATION OF THE CL HACILITATES N-TERMINAL DIMERIZATION 73
5.1.2 SUPPLEMENTATION OF AA 264-272 CAUSES CONFORMATIONAL RESTRAINTS 74
5.1.3 IN VIVO FUNCTIONS OF DOCKED/UNDOCKED STATES OF THE CL . 75
5.1.4 MODEL OF CL STATES DUERING THE HSP90 CYCLE 75
5.2 ANALYSIS OF THE HSP90 COCHAPERONE P23 77
5.2.1 THE FUNCTION OF P23 AS AN INDEPENDENT CHAPERONE 77
5.2.2 THE ROLE OF THE C-TERMINAL TAIL IN ATPASE INHIBITION 80
5.2.3 THE INTERPLAY BETWEEN THE P23 MUTANTS AND AHAL IN THE HSP90
CYCLE 82
5.3 ANALYSIS OF THE METHYLATION SITE IN HSP90 83
5.3.1 THE METHYLATION POSITION IS SENSITIVE TO CHANGES CONCEMING THE
CHARGE 83
5.3.2 HUMAN SYSTEM IS MORE SUSCEPTIBLE FOR CHANGES IN THE
METHYTAR
TION POSITION 85
5.3.3 THE METHYLATION SITE IS A CONSERVED C-TERMINAL SWITCH POINT IN
HSP90 85
5.3.4 FUTUIE INVESTIGATIONS 86
5.4 ANALYSIS OF THE HSP90 CLIENT PI 88
5.4.1 PI IS INSOLUBLE IN S. CEREVISIAE AND E. COLI 88
5.4.2 VPO INDUCES A TAXIC PHENOTYPE IN YEAST 88
VII
CONTENTS
6 MATERIAL AND METHODS 91
6.1 MATERIAL 91
6.1.1 CHEMICALS 91
6.1.2 ENZYMS 94
6.1.3 ANTIBODIES 94
6.1.4 SIZE MARKERS AND KITS 95
6.1.5 INSTRUMENTS AND ADDITIONAL MATERIALS 96
6.1.6 COMPUTER SOFTWARE 98
6.2 ORGANISMS AND CULTIVATION 99
6.2.1 STRAINS 99
6.2.2 MEDIA 100
6.2.3 CULTIVATION TECHNIQUES 101
6.2.3.1 GROWTH AND STORAGE OF E. COLI 101
6.2.3.2 GROWTH AND STORAGE OF S. CEREVISIAE 101
6.3 METHODS IN MOLECULAR BIOLOGY 101
6.3.1 PLASMIDS 101
6.3.1.1 EXPRESSION IN E. COLI 101
6.3.1.2 EXPRESSION IN S. CEREVISIAE 103
6.3.1.3 ADDITIONAL PLASMIDS 106
6.3.2 OLIGONUCLEOTIDES 107
6.3.3 BUFFERS IN MOLECULAR BIOLOGY 109
6.3.4 POLYMERASE-CHAIN-REACTION (PCR) 110
6.3.4.1 GENE AMPLIFICATION 110
6.3.4.2 BIUNT-END-MUT&GENESIS 111
6.3.4.3 LINKER PCR 112
6.3.4.4 COLONY PCR 112
6.3.5 SEPARATION OF DNA BY AGAROSE GEL ELECTROPHORESIS 112
6.3.6 PURIFICATION AND STORAGE OF DNA 113
6.3.7 RESTRICTION, DIGESTION AND LIGATION 113
6.3.8 TRANSFORMATION 114
6.3.8.1 PREPARATION OF COMPETENT E. COLI CELLS 114
6.3.8.2 TRANSFORMATION OF E. COLI 114
6.3.8.3 TRANSFORMATION OF S. CEREVISIAE 114
6.4 PREPARATIVE METHODS 115
6.4.1 BUFFERS 115
VIII
CONTENTS
6.4.2 EXPRESSION KINETICS IN E. COLI 115
6.4.3 GROWTH, INDUCTION AND HARVEST OF E. COLI 116
6.4.4 CELL DISRUPTION OF E. COLI 116
6.4.5 EXPRESSION KINETICS IN S. CEREVISIAE 116
6.4.6 PREPARATION OF YEAST EXTRACTS 117
6.5 METHODS IN PROTEIN PURIFICATION 117
6.5.1 BUFFERS 117
6.5.2 STANDARD PURIFICATION OF PROTEINS WITH TAGS 119
6.6 PROTEIN CHEMICAL METHODS 120
6.6.1 BUFFERS 120
6.6.2 SDS POLYACRYLAMID GEL ELECTROPHORESIS 122
6.6.2.1 COOMASSIE-STAINING OF SDS-GELS 122
6.6.2.2 SILVER-STAINING OF SDS-GELS 122
6.6.3 DETERMINATION OF PROTEIN CONCENTRATION IN LYSATES 122
6.6.3.1 BRADFORD-ASSAY 122
6.6.3.2 BCA-ASSAY 123
6.6.4 IMMUNOBLOTTING 123
6.7 SPECTROSCOPIC METHODS 124
6.7.1 BUFFERS 124
6.7.2 ABSORPTION SPECTROSCOPY (UV-VIS) 124
6.7.3 CIRCULAR DICHROISM SPECTROSCOPY 124
6.7.4 FLUORESCENCE SPECTROSCOPY 125
6.7.4.1 FLUORESCENCE ANISOTROPY 125
6.7.4.2 FOERSTER RESONANCE ENERGY TRANSFER (FRET) 126
6.8 ACTIVITY ASSAYS FOR PROTEINS IN VITRO 127
6.8.1 BUFFERS 127
6.8.2 ATPASE ASSAY WITH AN ATP-REGENERATION SYSTEM 128
6.8.3 THERMAL AGGREGATION ASSAY 128
6.9 ACTIVITY ASSAYS FOR PROTEINS IN VIVO 129
6.9.1 PLASMID SHUFFLING 129
6.9.2 DROP ASSAYS 130
6.9.3 V-SRC ACTIVITY ASSAY 130
6.9.4 GR ACTIVITY ASSAY 130
6.10 FUITHER METHODS 131
6.10.1 SCANNING ELECTRON MICROSCOPY 131
IX
CONTENTS
6.10.2 ANALYTICAL ULTRACENTRIFUGATION 131
BIBLIOGRAPHY 133
X |
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| spelling | Rehn, Alexandra Beatrice Verfasser aut Mechanistic analysis of the Hsp90 machinery: Effects of cochaperones, posttranslational modifications and clients Alexandra Beatrice Rehn 1. Aufl. 201506 München Verl. Dr. Hut 2014 X, 153 S. Ill., graph. Darst. 210 mm x 148 mm, 258 g txt rdacontent n rdamedia nc rdacarrier Biochemie Zugl.: München, Technische Universität, Diss., 2014 Posttranslationale Änderung (DE-588)4204069-3 gnd rswk-swf Hitzeschock-Proteine (DE-588)4255487-1 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Hitzeschock-Proteine (DE-588)4255487-1 s Posttranslationale Änderung (DE-588)4204069-3 s DE-604 X:MVB text/html http://deposit.dnb.de/cgi-bin/dokserv?id=4815608&prov=M&dok_var=1&dok_ext=htm Inhaltstext DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=028151495&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Rehn, Alexandra Beatrice Mechanistic analysis of the Hsp90 machinery: Effects of cochaperones, posttranslational modifications and clients Posttranslationale Änderung (DE-588)4204069-3 gnd Hitzeschock-Proteine (DE-588)4255487-1 gnd |
| subject_GND | (DE-588)4204069-3 (DE-588)4255487-1 (DE-588)4113937-9 |
| title | Mechanistic analysis of the Hsp90 machinery: Effects of cochaperones, posttranslational modifications and clients |
| title_auth | Mechanistic analysis of the Hsp90 machinery: Effects of cochaperones, posttranslational modifications and clients |
| title_exact_search | Mechanistic analysis of the Hsp90 machinery: Effects of cochaperones, posttranslational modifications and clients |
| title_full | Mechanistic analysis of the Hsp90 machinery: Effects of cochaperones, posttranslational modifications and clients Alexandra Beatrice Rehn |
| title_fullStr | Mechanistic analysis of the Hsp90 machinery: Effects of cochaperones, posttranslational modifications and clients Alexandra Beatrice Rehn |
| title_full_unstemmed | Mechanistic analysis of the Hsp90 machinery: Effects of cochaperones, posttranslational modifications and clients Alexandra Beatrice Rehn |
| title_short | Mechanistic analysis of the Hsp90 machinery: Effects of cochaperones, posttranslational modifications and clients |
| title_sort | mechanistic analysis of the hsp90 machinery effects of cochaperones posttranslational modifications and clients |
| topic | Posttranslationale Änderung (DE-588)4204069-3 gnd Hitzeschock-Proteine (DE-588)4255487-1 gnd |
| topic_facet | Posttranslationale Änderung Hitzeschock-Proteine Hochschulschrift |
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