Understanding the functional role of the cyclophilin domain of RanBP2/Nup358
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
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[2017]
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| 100 | 1 | |a Iachia y Baca, Blanca Ana |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Understanding the functional role of the cyclophilin domain of RanBP2/Nup358 |c presented by M.Sc. Blanca Ana Iachia y Baca |
| 264 | 1 | |a Heidelberg |c [2017] | |
| 300 | |a xiv, 210 Seiten |b Illustrationen, Diagramme |c 30 cm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Ruperto-Carola Univerity of Heidelberg |d 2017 | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 856 | 4 | 2 | |m B:DE-101 |q application/pdf |u http://d-nb.info/1144992087/04 |3 Inhaltsverzeichnis |
| 856 | 4 | 2 | |m DNB Datenaustausch |q application/pdf |u http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030485957&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |3 Inhaltsverzeichnis |
| 943 | 1 | |a oai:aleph.bib-bvb.de:BVB01-030485957 | |
Datensatz im Suchindex
| _version_ | 1819639047096631296 |
|---|---|
| adam_text | CONTENTS
A
BSTRACT........................................................................................................................
XI
ZUSAMMENFASSUNG........................................................................................................XIII
1 INTRODUCTION 1
1.1 NUCLEAR PORE
COMPLEXES......................................................................................
1
1.1.1 NUCLEOCYTOPLASMIC TRANSPORT
................................................................. 2
1.2 THE NUCLEOPORIN
RANBP2...................................................................................
5
1.2.1 THE RANBP2/RANGAPL COMPLEX IS A DISASSEMBLY MACHINERY .... 8
1.2.2 THE RANBP2 COMPLEX IS A SUMO E3 LIGASE
........................................
9
1.2.3 RANBP2 IS A PROLYL CIS/TRANS ISOMERASE
.................................................
12
1.3 AIM OF THIS W
ORK..................................................................................................
20
2 RESULTS 21
2.1 INVESTIGATION OF RANBP2CYP BINDING PARTNERS
.................................................
21
2.1.1 PULL-DOWN/MASS SPECTROMETRY ANALYSIS ALLOWS IDENTIFICATION OF
POTEN
TIAL BINDING
PARTNERS................................................................................
21
2.1.2 GOTERM ANALYSIS OF THE RANBP2CYP INTERACTOME
............................... 28
2.1.3 VALIDATION OF PUTATIVE BINDING PARTNERS
.................................................
30
2.2 FILAMINS AS BINDING PARTNERS AND AS POTENTIAL SUBSTRATES OF
RANBP2CYP .
.
. 33
2.2.1 FILAMIN A INTERACTS WITH RANBP2CYP
....................................................
35
2.2.2 FLNA BINDING TO RANBP2CYP DOES NOT DEPEND ON RANBP2CYP S AC
TIVE SITE OR ON FLNA S CRSPRO
................................................................
46
2.2.3 CHEMICAL CROSS-LINKING ANALYSIS SUGGESTS DIFFERENT INTERACTING
SURFACES
ON
RANBP2CYP.........................................................................................
50
2.2.4 ATTEMPTS TO MEASURE THE ACTIVITY OF RANBP2CYP
............................... 56
2.3 THP1 ARANBP2CYP AS A MODEL TO STUDY PHYSIOLOGY OF CELLS LACKING
RANBP2CYP 58
2.3.1 THPLARANBP2CYP GENERAL FEATURES
....................................................
58
2.3.2 FILAMIN A CANNOT BE DETECTED BY IMMUNOBLOTTING AND IMMUNOFLUO
RESCENCE IN
THP1......................................................................................
64
2.3.3 THPLARANBP2CYP AND WILD TYPE SHOW SOME DIFFERENCES IN NUCLEO
CYTOPLASMIC
TRANSPORT.............................................................................
66
2.3.4 COMPARISON OF THE PROTEOME OF THPLARANBP2CYP AND WILD TYPE . 70
2.3.5 SUMOYLATION IN THPLARANBP2CYP AND WILD TYPE
CELLS................... 74
2.4 EDC3 AND UPF1 AS POTENTIAL SUMO SUBSTRATES AND RANBP2CYP BINDING
PARTNERS..................................................................................................................
78
2.4.1 EDC3 CAN BE IN VITRO
SUMOYLATED........................................................... 78
2.5 DEFINITION OF A RANBP2CYP S SUBSTRATE BINDING M
OTIF
.....................................
81
2.6 NUCLEOPORINS ARE POTENTIAL SUBSTRATES OF RANBP2CYP
.....................................
86
2.6.1 MAPPING OF THE INTERACTION BETWEEN RANBP2CYP AND NUCLEOPORINS . .
86
3 DISCUSSION 91
3.1 POTENTIAL PHYSIOLOGICAL ROLES OF RANBP2CYP - INSIGHTS FROM THE
INTERACTOME . 91
3.2 RANBP2CYP AS DOCKING SITE OR ISOMERASE?
.......................................................
92
3.3 FILAMIN A IS AN IDEAL SUBSTRATE-BINDING PARTNER FOR THE RANBP2CYP
............
93
3.4 EDC3 AND UPF1 AND THE MRNA MACHINERY AT THE NUCLEAR PORE COMPLEX . .
95
3.5 ATTEMPTS TO DEFINE A SUBSTRATE MOTIF: IN SILICO ANALYSIS OF HIV-1
CAPSID
PROTEIN AND FILAMIN A
.........................................................................................
97
3.5.1 NUCLEOPORINS ENRICHMENT IN THE MASS SPEC LIST AND MOTIF ANALYSIS .
. 99
3.6 NUCLEOCYTOPLASMIC TRANSPORT IN THP1 CELLS LACKING THE CYCLOPHILIN
DOMAIN
OF RANBP2
..............................................................................................................100
3.7 FUTURE
PERSPECTIVES..................................................................................................104
4 MATERIALS AND METHODS 107
4.1 MATERIALS
.................................................................................................................107
4.1.1 CHEMICALS, REAGENTS AND
KITS......................................................................107
4.1.2 M
EDIA.........................................................................................................109
4.1.3 BUFFERS, STOCK SOLUTIONS AND STANDARDS
.................................................
109
4.1.4
CONSUMABLES............................................................................................
112
4.1.5 OLIGONUCLEOTIDES
......................................................................................112
4.1.6
PLASMIDS......................................................................................................119
4.1.7 ANTIBODIES
...............................................................................................125
4.1.8 RECOMBINANT
PROTEINS.............................................................................126
4.1.9
PEPTIDES.....................................................................................................128
4.1.10 CELL LINES
..................................................................................................128
4.1.11 TECHNICAL
EQUIPMENT................................................................................129
4.1.12
SOFTWARE.....................................................................................................129
4.2 MOLECULAR BIOLOGY
METHODS...................................................................................130
4.2.1 TRANSFORMATION OF COMPETENT BACTERIA
.................................................
130
4.2.2 POLYMERASE CHAIN REACTION (PCR)
..........................................................
130
4.2.3 RESTRICTION OF DNA BY ENDONUCLEASES
....................................................
130
4.2.4 LIGATION OF DNA
FRAGMENTS....................................................................131
4.2.5 SITE-DIRECTED
MUTAGENESIS.......................................................................131
4.2.6 GIBSON ASSEMBLY CLONING
.......................................................................132
4.2.7 AGAROSE GEL ELECTROPHORESIS AND DNA FRAGMENTS ISOLATION
..................
132
4.2.8 PLASMID ISOLATION FROM BACTERIA
.............................................................
132
4.2.9 DNA
SEQUENCING......................................................................................133
4.3 BIOCHEMICAL
METHODS............................................................................................133
4.3.1 SDS-PAGE AND PROTEIN ANALYSIS
..........................................................
133
4.3.2 EXPRESSION OF RECOMBINANT PROTEINS
.......................................................
134
4.3.3 PURIFICATION OF RECOMBINANT PROTEINS
....................................................
135
4.3.4 PULL DOWN
ASSAY.........................................................................................137
4.3.5
CROSS-LINKING............................................................................................141
4.3.6 ISOTHERMAL TITRATION CALORIMETRY (IT
C
)
.................................................
141
4.3.7 IN VITRO
SUMOYLATION................................................................................142
4.3.8
HA-IMMUNOPRECIPITATION..........................................................................142
IX
4.3.9 SUMO-IMMUNOPRECIPITATION
..................
4.3.10 CRYSTALISATION TRIAL - SAMPLE
PREPARATION.................................................143
4.3.11 TRANSPORT
ASSAY............................................................................................144
4.3.12 MASS SPECTROMETRY -
APPENDIX................................................................145
4.4 CELL BIOLOGY METHODS FOR MAMMALIAN
CELLS..........................................................146
4.4.1 CULTURING OF
CELLS.........................................................................................146
4.4.2 CELL CYCLE SYNCHRONIZATION
.........................................................................
147
4.4.3 TRANSIENT TRANSFECTION OF MAMMALIAN
CELLS..............................................147
4.4.4
IMMUNO-FLUORESCENCE...................................................................................147
4.4.5 PROXIMITY LIGATION ASSAY (PLA
)................................................................148
4.4.6 SILAC CULTIVATION OF THP1
CELLS.............................................................149
4.4.7 GROWTH
CURVE...............................................................................................149
4.4.8 BRDU AND PROPIDIUM IODIDE LABELLING
.................................
149
5 APPENDIX 151
5.1 SUPPLEMENTARY D
A
TA
...............................................................................................151
5.1.1 CROSS-LINKING OPTIMISATION
.........................................................................
151
5.1.2 CROSS-LINKING PEPTIDE LIST
.........................................................................
153
5.1.3 CRYSTALLISATION
............................................................................................154
5.1.4 THP1 CLONES
...............................................................................................154
5.1.5 PROTEOME ANALYSIS
......................................................................................155
6 BIBLIOGRAPHY 187
LIST OF FIGURES 203
LIST OF TABLES 207
|
| any_adam_object | 1 |
| author | Iachia y Baca, Blanca Ana |
| author_facet | Iachia y Baca, Blanca Ana |
| author_role | aut |
| author_sort | Iachia y Baca, Blanca Ana |
| author_variant | y b b a i ybba ybbai |
| building | Verbundindex |
| bvnumber | BV045095278 |
| ctrlnum | (OCoLC)1012432362 (DE-599)DNB1144992087 |
| discipline | Biologie |
| format | Thesis Book |
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| genre | (DE-588)4113937-9 Hochschulschrift gnd-content |
| genre_facet | Hochschulschrift |
| id | DE-604.BV045095278 |
| illustrated | Illustrated |
| indexdate | 2024-12-24T06:49:08Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-030485957 |
| oclc_num | 1012432362 |
| open_access_boolean | |
| owner | DE-355 DE-BY-UBR |
| owner_facet | DE-355 DE-BY-UBR |
| physical | xiv, 210 Seiten Illustrationen, Diagramme 30 cm |
| publishDate | 2017 |
| publishDateSearch | 2017 |
| publishDateSort | 2017 |
| record_format | marc |
| spellingShingle | Iachia y Baca, Blanca Ana Understanding the functional role of the cyclophilin domain of RanBP2/Nup358 |
| subject_GND | (DE-588)4113937-9 |
| title | Understanding the functional role of the cyclophilin domain of RanBP2/Nup358 |
| title_auth | Understanding the functional role of the cyclophilin domain of RanBP2/Nup358 |
| title_exact_search | Understanding the functional role of the cyclophilin domain of RanBP2/Nup358 |
| title_full | Understanding the functional role of the cyclophilin domain of RanBP2/Nup358 presented by M.Sc. Blanca Ana Iachia y Baca |
| title_fullStr | Understanding the functional role of the cyclophilin domain of RanBP2/Nup358 presented by M.Sc. Blanca Ana Iachia y Baca |
| title_full_unstemmed | Understanding the functional role of the cyclophilin domain of RanBP2/Nup358 presented by M.Sc. Blanca Ana Iachia y Baca |
| title_short | Understanding the functional role of the cyclophilin domain of RanBP2/Nup358 |
| title_sort | understanding the functional role of the cyclophilin domain of ranbp2 nup358 |
| topic_facet | Hochschulschrift |
| url | http://d-nb.info/1144992087/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030485957&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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