Molecular and phenotypic analysis of Opitz syndrome patients and characterization of the relationship between the RBCC protein MID1 and the tumor suppressor CYLD:
Gespeichert in:
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2008
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| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | 159 S. Ill., graph. Darst. |
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| 245 | 1 | 0 | |a Molecular and phenotypic analysis of Opitz syndrome patients and characterization of the relationship between the RBCC protein MID1 and the tumor suppressor CYLD |c von Joyce So |
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Datensatz im Suchindex
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| adam_text | Titel: Molecular and phenotypic analysis of Opitz syndrome patients and characterization of the relationshi
Autor: So, Joyce
Jahr: 2008
Table of Contents —.,
1 Introduction 5
1.1 Opitz syndrome 5
1.1.1 Opitz syndrome phenotype 5
1.1.2 Phenotypic variability in Opitz syndrome 5
1.2 MIDI 6
1.2.1 MIDI gene and mutations 6
1.2.2 MIDI protein and domains 8
1.2.3 MIDI function 11
1.2.3.1 MIDI and microtubules 12
1.2.3.2 MIDI and ubiquitinatlon 14
1.2.3.3 MIDI and PP2A regulation 16
1.2.3.3.1 PP2A targets 4EBP1 17
1.2.3.3.2 PP2A targets S6 kinase ...18
1.2.3.3.3 PP2A targets GLI3 19
1.3 Phenotypic overlap of Opitz syndrome with other genetic
syndromes 20
1.3.1 Common syndromic features 20
1.3.2 Basal cell nevus syndrome 21
1.3.2.1 Basal cell nevus syndrome phenotype and overlap with Opitz
syndrome 21
1.3.2.2 PTCH1 gene and mutations 22
1.3.3 Cylindromatosis 23
1.3.3.1 Cylindromatosis phenotype and overlap with basal cell nevus
syndrome -. 24
1.3.3.2 GUI is upregulated in cylindromas 24
1.4 CYLD 25
1.4.1 CYLD gene and mutations 25
1.4.2 CYLD protein and domains 25
1.4.2.1 Identified binding domains in CYLD 29
1.4.3 CYLD function 30
1.4.3.1 CYLD in the NF-kB pathway..... 30
1.4.3.2 CYLD in apoptosis 33
1.4.3.3 CYLD at the microtubules 34
1.5 Microtubules 34
1.5.1 Microtubuie structure and function 34
1.5.2 Post-translational modifications of tubulin 36
1.5.3 Microtubule-associated proteins... 37
1.5.4 Agents affecting microtubuie stability and dynamics 38
1.6 Aim of the study 38
1
2 Materials and Methods • 40
40
2.1 Materials *
2.1.1 Reagents and buffers * ,
2.1.2 Cell lines and transfection reagents 43
2.1.3 Antibodies AA
2.1.4 Knockdown constructs ¦•• 46
2.1.5 Expression constructs 46
2.2 Methods 48
2.2.1 Patients 48
2.2.2 MIDI mutation analysis 48
2.2.3 DNA transformation 49
2.2.4 Small-scale DNA preparation 50
2.2.5 Large-scale DNA preparation 50
2.2.6 Protein quantitation 50
2.2.7 Expression constructs 51
2.2.8 Cell culture and transfections 51
2.2.9 SDS-polyacrylamide gel electrophoresis 52
2.2.10 Western blot 52
2.2.11 Immunofluorescence microscopy 53
2.2.12 Microtubule assembly assay 53
2.2.13 Semi-quantitative RT-PCR 54
2.2.14 Nocodazole withdrawal 54
2.2.15 No codazole challenge 55
2.2.16 Detergent extraction assay 55
2.2.17 Protein extraction for evaluation of S6 kinase and 4EBP1
phosphorylation 56
2.2.18 Apoptosis assay 56
2.2.19 Trypan blue exclusion 57
2.2.20 Immunoprecipitation to evaluate TRAF2 ubiquitination 57
2.2.21 Statistical analysis 58
3 Results 59
3.1 Opitz syndrome is phenotypically variable 59
3.1.1 Novel MIDI mutations identified in X-linked Opitz syndrome
patients 59
3.1.2 X-linked Opitz syndrome patients show inter- and infra-familial
phenotyplc variability 59
3.1.3 Phenotypic spectrum of patients with MIDI mutations differs from that
of patients without MIDI mutations 65
3.1.4 Phenotypic spectrum of patient series mild compared to
literature ., 68
3.2 Opitz syndrome shows phenotypic overlap with other syndromes... 70
3.2.1 Patient with phenotypic features of Opitz syndrome that overlap with
Wolf-Hirschhorn syndrome..... 70
2
3.2.2 Terminal 4p deletion with Xp22.31 duplication identified by array
CGH 72
3.3 The tumour suppressor CYLD may act as a modifying factor to account
for phenotypic variability by opposing the function of MIDI 75
3.3.1 CYLD associates with microtubules 76
3.3.1.1 CYLD is found at the microtubules 76
3.3.1.2 C-terminally truncated CYLD mutant associates with
microtubules 79
3.3.2 CYLD affects microtubule stability 79
3.3.2.1 CYLD knockdown stabilizes microtubules against
nocodazole 79
3.3.2.1.1 CYLD knockdown is achieved with siRNA and shRNA....... 80
3.3.2.1.2 CYLD knockdown increases microtubule recovery rate after
nocodazole treatment 81
3.3.2.2 CYLD knockdown increases microtubule resistance to
nocodazole 84
3.3.2.3 CYLD knockdown increases microtubule stability in the
presence of nocodazole 84
3.3.2.4 CYLD over-expression decreases microtubule resistance to
nocodazole 86
3.3.2.5 Stabilization of microtubules by CYLD knockdown is rescued by
expression of siRNA target site-mutated CYLD 87
3.3.3 CYLD interacts with MIDI at the microtubules 88
3.3.3.1 CYLD interacts with MIDI 89
3.3.3.2 CYLD and MIDI co-localize at the microtubules 90
3.3.4 CYLD and MIDI have opposing effects at the microfubules 91
3.3.4.1 MIDI knockdown is achieved by siRNA 91
3.3.4.2 MIDI knockdown rescues CYLD knockdown effects on
microtubule resistance to nocodazole 92
3.3.5 CYLD regulates PP2A function in an opposing manner to MIDI... 94
3.3.5.1 CYLD knockdown decreases dephosphorylation of the PP2A
target S6 kinase, with rescue by simultaneous MIDI
knockdown 94
3.3.5.2 CYLD knockdown increases nuclear localization of GLI3, with
rescue by simultaneous MIDI knockdown 95
3.3.5.3 CYLD over-expression decreases nuclear localization of GLI3,
while C-terminally truncated CYLD mutant does not..... 97
3.3.6 MIDI affects apoptosis activation in an opposing manner to
CYLD 98
3.3.6.1 MIDI knockdown decreases cell viability with NF-kB activation
and TNFa-induced apopfosis, with rescue by simultaneous CYLD
knockdown • 98
3.3.7 Alpha4 affects apoptosis activation in an opposing manner to
CYLD 100
3.3.7.1 Alpha4 knockdown is achieved by siRNA 100
3
1
3.3.7.2 Alpha4 knockdown decreases cell viability with NF-kB
activation and TNFa-induced apoptosis, with rescue by
simultaneous CYLD knockdown 101
3.3.8 MIDI regulates the NF-kB pathway through ubiquitination of TRAF2, a
known CYLD ubiquitin hydrolase target 102
3.3.8.1 MIDI knockdown decreases ubiquitination of TRAF2 102
3.3.8.2 CYLD knockdown rescues MIDI knockdown effect on
ubiquitination of TRAF2 103
4 Discussion 105
4.1 Opitz syndrome is phenotypically variable 105
4.1.1 Opitz syndrome patients can be less severely affected than
previously reported 105
4.1.2 The Opitz syndrome phenotype shows inter- and intra-familial
variability 106 |
4.2 Opitz syndrome phenotypically overlaps with other syndromes 107 j
4.2.1 Opitz syndrome and Wolf-Hirschhorn syndrome have phenotypic j
overlap 107 |
4.2.2 Opitz syndrome phenotypically overlaps with tumour predisposition ,
syndromes 110
4.3 CYLD is a MIDI antagonist 111
4.3.1 CYLD associates with and destabilizes microtubules 111
4.3.2 CYLD interacts with MIDI at the microtubules 115
4.3.3 CYLD regulates PP2A activity 117
4.3.4 CYLD regulates localization of the Sonic hedgehog member
GU3 119
4.3.5 MIDI promotes apoptotic resistance through the NF-kB
pathway 120
4.4 CYLD and MIDI as modifying factors 121
4.4.1 CYLD may be a modifying factor in Opitz syndrome 121
4.4.2 MIDI may be a modifying factor in tumour predisposition 122
4.5 A model for the CYLD-MID1 relationship 123
4.6 Outlook..... 125
5 Summary 127
6 Zusammenfassung 129
7 Abbreviations.. 132
8 References... !35
9 Acknowledgements/Danksagung 154
4
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| spelling | So, Joyce Verfasser aut Molecular and phenotypic analysis of Opitz syndrome patients and characterization of the relationship between the RBCC protein MID1 and the tumor suppressor CYLD von Joyce So 2008 159 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Berlin, Freie Univ., Diss., 2008 Ubiquitin-Protein-Ligase (DE-588)4398417-4 gnd rswk-swf Signaltransduktion (DE-588)4318717-1 gnd rswk-swf Erbkrankheit (DE-588)4015106-2 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Erbkrankheit (DE-588)4015106-2 s Signaltransduktion (DE-588)4318717-1 s Ubiquitin-Protein-Ligase (DE-588)4398417-4 s DE-604 HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=017020797&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | So, Joyce Molecular and phenotypic analysis of Opitz syndrome patients and characterization of the relationship between the RBCC protein MID1 and the tumor suppressor CYLD Ubiquitin-Protein-Ligase (DE-588)4398417-4 gnd Signaltransduktion (DE-588)4318717-1 gnd Erbkrankheit (DE-588)4015106-2 gnd |
| subject_GND | (DE-588)4398417-4 (DE-588)4318717-1 (DE-588)4015106-2 (DE-588)4113937-9 |
| title | Molecular and phenotypic analysis of Opitz syndrome patients and characterization of the relationship between the RBCC protein MID1 and the tumor suppressor CYLD |
| title_auth | Molecular and phenotypic analysis of Opitz syndrome patients and characterization of the relationship between the RBCC protein MID1 and the tumor suppressor CYLD |
| title_exact_search | Molecular and phenotypic analysis of Opitz syndrome patients and characterization of the relationship between the RBCC protein MID1 and the tumor suppressor CYLD |
| title_full | Molecular and phenotypic analysis of Opitz syndrome patients and characterization of the relationship between the RBCC protein MID1 and the tumor suppressor CYLD von Joyce So |
| title_fullStr | Molecular and phenotypic analysis of Opitz syndrome patients and characterization of the relationship between the RBCC protein MID1 and the tumor suppressor CYLD von Joyce So |
| title_full_unstemmed | Molecular and phenotypic analysis of Opitz syndrome patients and characterization of the relationship between the RBCC protein MID1 and the tumor suppressor CYLD von Joyce So |
| title_short | Molecular and phenotypic analysis of Opitz syndrome patients and characterization of the relationship between the RBCC protein MID1 and the tumor suppressor CYLD |
| title_sort | molecular and phenotypic analysis of opitz syndrome patients and characterization of the relationship between the rbcc protein mid1 and the tumor suppressor cyld |
| topic | Ubiquitin-Protein-Ligase (DE-588)4398417-4 gnd Signaltransduktion (DE-588)4318717-1 gnd Erbkrankheit (DE-588)4015106-2 gnd |
| topic_facet | Ubiquitin-Protein-Ligase Signaltransduktion Erbkrankheit Hochschulschrift |
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