Activation mechanism of the AAA+ chaperone ClpB/Hsp104:
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2012
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | III, 105 S. Ill., graph. Darst. |
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| 100 | 1 | |a Seyffer, Fabian |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Activation mechanism of the AAA+ chaperone ClpB/Hsp104 |c [presented by Fabian Seyffer] |
| 264 | 1 | |c 2012 | |
| 300 | |a III, 105 S. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
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| 502 | |a Heidelberg, Univ., Diss., 2012 | ||
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| adam_text | IMAGE 1
TABLE O F CONTENTS
TABLE OF CONTENTS
1. SUMMARY 1
1.1. SUMMARY (ENGLISH VERSION) 1
1.2. ZUSAMMENFASSUNG (DEUTSCHE VERSION) 3
2. INTRODUCTION 6
2.1. MOLECULAR CHAPERONES - THE CELLULAR PROTEIN QUALITY CONTROL 6
2.1.1. PROTEIN FOLDING IN THE CELL 6
2.1.2. CLASSIFICATION AND CONCEPT O F MOLECULAR CHAPERONES 7
2.1.3. CHAPERONES PROTECT PROTEINS FROM MISFOLDING AND AGGREGATION 8
2.1.4. CHAPERONES ORGANIZE AND SOLUBILISE PROTEIN AGGREGATES 9
2.1.5. CHAPERONES ASSIST COMPLEX ASSEMBLY AND DISASSEMBLY 11
2.1.6. AAA+ PROTEINS ARE INVOLVED IN PROTEOLYSIS 12
2.2. THE HSP70-SYSTEM 13
2.2.1. HSP70S AND THEIR HSP40 AND NEF CO-CHAPERONES 13
2.2.2. FUNCTIONS O F THE FLSP70-SYSTEM IN THE CELLULAR CHAPERONE NETWORK
15
2.3. CLPB/HSPL04 - A HIGHLY SPECIALIZED HSPLOO/CLP PROTEIN 16
2.3.1. P-LOOP NTPASES, THE AAA + SUPERFAMILY AND HSP100/CLP PROTEINS 16
2.3.2. STRUCTURE AND FUNCTION O F CLPB/HSP104 19
2.3.3. REGULATION O F THE ATPASE ACTIVITIES O F CIPB AND HSP104 23
2.3.4. COOPERATION BETWEEN CLPB/HSP104 AND THE HSP70-SYSTEM 26
2.4. MACROMOLECULAR SUBSTRATES O F HSPLOO/CLP PROTEINS 28
2.4.1. AMORPHOUS AGGREGATES AS A RESULT O F CELLULAR STRESS 28
2.4.2. AMYLOID FIBRILS AND THE MODEL SUBSTRATE HYPF-N 29
2.4.3. VIPA/VIPB TUBULES AND THE TYPE VI SECRETION SYSTEM 30
3. AIMS O F THIS WORK 32
4. MATERIALS 33
4.1. EQUIPMENT 33
4.2. SOFTWARE 33
4.3. EXPENDABLE ITEMS 34
4.4. PRIMER, PLASMIDS AND STRAINS 34
4.4.1. PRIMER 34
4.4.2. PLASMIDS 35
4.4.3. STRAINS 36
4.5. CHEMICALS 36
4.5.1. GENERAL CHEMICALS AND MEDIA 36
I
HTTP://D-NB.INFO/1025876083
IMAGE 2
TABLE O F CONTENTS
4.5.2. COLUMN MATERIALS 36
4.5.3. SPECIAL CHEMICALS FOR PROTEIN ASSAYS AND ELECTRON MICROSCOPY 36
4.6. KITS 37
4.7. ANTIBODIES, ENZYMES AND OTHER PROTEINS 37
4.8. D N A AND PROTEIN SIZE STANDARDS 37
4.9. GROWTH MEDIA 37
4.10. ANTIBIOTICS 38
5. METHODS 39
5.1. MOLECULAR BIOLOGY METHODS 39
5.1.1. MOLECULAR CLONING 39
5.1.2. AGAROSE GEL ELECTROPHORESIS 39
5.1.3 PREPARATION O F CHEMICALLY COMPETENT CELLS AND TRANSFORMATION 39
5.2. PROTEIN PURIFICATION AND GENERATION O F HYPF-N AGGREGATES 39
5.2.1. CIPVB/CLPVH 39
5.2.2. VIPA/VIPB AND VIPAA/IPB-YFP 41
5.2.3. CLPB/HSP104 41
5.2.4. DNAK 42
5.2.5. HYPF-N PURIFICATION AND AGGREGATE PREPARATION 43
5.3. PROTEIN ANALYSIS 44
5.3.1. BRADFORD COLORIMETRIC PROTEIN QUANTIFICATION 44
5.3.2. SDS-PAGE 44
5.3.3. COOMASSIE-STAINING O F SDS GELS 44
5.3.4. SILVER-STAINING O F SDS GELS 45
5.3.5. WESTERNBLOT 45
5.3.6. MASS-SPECTROMETRY 46
5.3.7. NEGATIVE STAINING TRANSMISSION ELECTRON MICROSCOPY 46
5.4. BIOCHEMICAL ASSAYS 46
5.4.1. DISASSEMBLY O F VIPAA/IPB TUBULES 46
5.4.2. BINDING O F CIPVB/CLPVH TO VIPAA/IPB-TUBULES 47
5.4.3. MDH DISAGGREGATION 47
5.4.4. FLUORESCENCE O F FREE AND VIPB-COUPLED YFP 47
5.4.5. VBAP DEGRADATION ASSAY 48
5.4.6. REACTIVATION O F AGGREGATED LUCIFERASE 48
5.4.7. ATPASE ACTIVITY ASSAY 49
5.4.8. RESOLUBILIZATION O F HYPF-N AGGREGATES AND OTHER AMYLOIDS 49
5.4.9. HSP40 BINDING TO HYPF-N AGGREGATES 50
6. RESULTS AND DISCUSSION 51
6.1. CLPB HAS L O W BASAL SUBSTRATE DISASSEMBLY ACTIVITY 51
II
IMAGE 3
TABLE O F CONTENTS
6.1.1. MOTIVATION 51
6.1.2. CIPVB RECOGNIZES AND DISASSEMBLES VIPAA/IPB TUBULES 51
6.1.3. CIPVB TRANSLOCATES VIPB WITH LIMITED UNFOLDING POWER 54
6.2. THE M-DOMAIN REPRESSES SUBSTRATE DISASSEMBLY ACTIVITY O F CLPB 57
6.2.1. MOTIVATION 57
6.2.2. DELETION O F THE M-DOMAIN O F CIPVB UNLEASHES HIGH UNFOLDING
ACTIVITY 57
6.2.3. MUTATIONS IN THE M-DOMAIN EITHER REPRESS O R DEREPRESS THE
ACTIVITY O F CIPVB 58
6.3. CLPB/HSPL04 IS ACTIVATED B Y THE INTERACTION O F THE M-DOMAIN WITH
THE HSP70-
SYSTEM 62
6.3.1. MOTIVATION 62
6.3.2. COMPLETE TUBULE DISINTEGRATION IS ACHIEVED B Y A SYNERGISTIC
COOPERATION BETWEEN
CIPVB/CLPVH AND THE HSP70-SYSTEM 62
6.3.3. EFFECT O F M-DOMAIN DELETIONS AND ALTERATIONS ON CIPVB ACTIVATION
B Y KJE 68
6.4. ACTIVATION O F CLPB RESULTS IN A COORDINATED ATPASE CYCLE 72
6.4.1. MOTIVATION 72
6.4.2. STIMULATION O F THE ATPASE RATE O F CIPVB B Y SUBSTRATE AND KJE
72
6.4.3. THE ACTIVATED STATE O F CIPVB IS POISONED B Y THE INCORPORATION O
F ATPASE-DEFICIENT
SUBUNITS 74
6.5. A A A - 1 AND A A A - 2 O F CLPB HAVE DIFFERENT FUNCTIONS IN THE
UNFOLDING PROCESS 76
6.5.1. MOTIVATION 76
6.5.2. A T P HYDROLYSIS IN AAA-2, BUT NOT IN AAA-1, IS ESSENTIAL FOR
HIGH DISASSEMBLY ACTIVITY. 77
6.5.3. GUANIDINE HYDROCHLORIDE PRIMARILY AFFECTS A T P HYDROLYSIS AT
AAA-1 79
6.6. RECOGNITION AND RESOLUBILIZATION O F AMYLOID FIBRILS BY THE
CLPB/HSPL04/HSP70-
SYSTEM 81
6.6.1. MOTIVATION 81
6.6.2. CLPB HAS LIMITED ACTIVITY TOWARDS HYPF-N AMYLOID FIBRILS AND
HEAT-AGGREGATES 82
6.6.3. DNAJ RECOGNIZES AMYLOID FIBRILS WITH HIGH AFFINITY 84
7. CONCLUSIONS AND OUTLOOK 87
8. LITERATURE 91
8.1. REFERENCES 91
8.2. PUBLICATIONS FROM THIS PHD THESIS 100
9. TABLE O F FIGURES 101
10. ABBREVIATIONS 102
10.1. A M I N O ACIDS, ONE-LETTER CODE 102
10.2. GENERAL ABBREVIATIONS 102
11. ACKNOWLEDGEMENTS (DANKSAGUNG) 104
III
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| any_adam_object | 1 |
| author | Seyffer, Fabian |
| author_facet | Seyffer, Fabian |
| author_role | aut |
| author_sort | Seyffer, Fabian |
| author_variant | f s fs |
| building | Verbundindex |
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| dewey-full | 572.633 |
| dewey-hundreds | 500 - Natural sciences and mathematics |
| dewey-ones | 572 - Biochemistry |
| dewey-raw | 572.633 |
| dewey-search | 572.633 |
| dewey-sort | 3572.633 |
| dewey-tens | 570 - Biology |
| discipline | Biologie |
| format | Thesis Book |
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| language | English |
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| physical | III, 105 S. Ill., graph. Darst. |
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| spelling | Seyffer, Fabian Verfasser aut Activation mechanism of the AAA+ chaperone ClpB/Hsp104 [presented by Fabian Seyffer] 2012 III, 105 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Heidelberg, Univ., Diss., 2012 (DE-588)4113937-9 Hochschulschrift gnd-content DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025235454&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Seyffer, Fabian Activation mechanism of the AAA+ chaperone ClpB/Hsp104 |
| subject_GND | (DE-588)4113937-9 |
| title | Activation mechanism of the AAA+ chaperone ClpB/Hsp104 |
| title_auth | Activation mechanism of the AAA+ chaperone ClpB/Hsp104 |
| title_exact_search | Activation mechanism of the AAA+ chaperone ClpB/Hsp104 |
| title_full | Activation mechanism of the AAA+ chaperone ClpB/Hsp104 [presented by Fabian Seyffer] |
| title_fullStr | Activation mechanism of the AAA+ chaperone ClpB/Hsp104 [presented by Fabian Seyffer] |
| title_full_unstemmed | Activation mechanism of the AAA+ chaperone ClpB/Hsp104 [presented by Fabian Seyffer] |
| title_short | Activation mechanism of the AAA+ chaperone ClpB/Hsp104 |
| title_sort | activation mechanism of the aaa chaperone clpb hsp104 |
| topic_facet | Hochschulschrift |
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