Characterisation of atypical Rho GTPases of the RhoBTB family and their binding partners [Elektronische Ressource] / vorgelegt von Kristína Schenková

Jiraskovlastimil

Sujets

• Biologie

Informations

• Publié par: universitat_zu_koln
• Publié le: 01 janvier 2010
• Nombre de lectures: 30
• Langue: English
• Poids de l'ouvrage: 17 Mo

Extrait

Characterisation of atypical
Rho GTPases of the RhoBTB family
and their binding partners

Inaugural-Dissertation
zur
Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultät
der Universität zu Köln


vorgelegt von

Kristína Schenková
aus Nitra, Slowakei


Köln 2010




















Berichterstatter/in: Prof. Dr. Angelika A. Noegel
Prof. Dr. Jürgen Dohmen





Tag der mündlichen Prüfung: 30. Juni 2010




Die vorliegende Arbeit wurde in der Zeit von November 2006 bis Mai 2010 unter
Anleitung von Prof. Dr. Angelika A. Noegel und der Betreuung von PD Dr. Francisco
Rivero am Institut für Biochemie I der Medizinischen Fakultät der Universität zu Köln und
Centre for Biomedical Research, Hull York Medical School, University of Hull,
Großbritannien angefertigt.
2Acknowledgement

I would like to thank all people that that by any means contribute to the successful
completion of this dissertation thesis. Especially I would like to thank:

- PD Dr. Francisco Rivero for the opportunity to work in his group on the interesting
topic, for sharing his knowledge and experience, for never ending helpfulness, for long
discussions and for the opportunity to present my research at various international
meetings.

- Prof. Dr. Angelika A. Noegel for the opportunity to conduct my work in her renowned
institute.

- Prof. Dr. Jürgen Dohmen, Prof. Dr. Matthias Hammerschmidt and Prof. Dr. Ludwig
Eichinger for their willingness and time to read this thesis and participate in the
committee during my exam.

- Prof. Dr. Pontus Aspenström for providing RhoBTB constructs and one cullin
construct, Prof. Dr. Reinhard Fässler for providing kindlin constructs, Dr. Manabu
Furukawa for providing cullin constructs, Prof. Dr. Martin Bähler for myosin construct
and to Dr. Michael Gmachl for providing the ubiquitin construct.

- All my colleagues and co-workers in the Institute for Biochemistry I, Medical Faculty,
University of Cologne and in the Centre for Biomedical Research, Hull York Medical
School, University of Hull for help and advices, especially to Anja and Nils. Thank you
for the great time in Hull ☺!

- My family for support.

- Vlasta for encouragement, emotional support and faith in me.

3Table of contents


1 INTRODUCTION........................................................................................ 8

1.1 The Ras superfamily of small GTPases...................................................... 8
1.2 The Rho-family............................................................................................. 9
1.3 RhoBTB proteins.......................................................................................... 10
1.3.1 Structure of RhoBTB proteins........................................................................ 11
1.3.1.1 The GTPase domain....................................................................................... 11
1.3.1.2 The proline-rich region................................................................................... 12
1.3.1.3 The BTB domain............................................................................................ 13
1.3.1.4 The C-terminal region.................................................................................... 14
1.3.2 Expression of RhoBTB proteins..................................................................... 14
1.3.3 Function of RhoBTB pr15
1.3.3.1 RhoBTB, cell growth, and apoptosis.............................................................. 15
1.3.3.2 RhoBTB and chemokine expression.............................................................. 16
1.3.3.3 RhoBTB and vesicle transport........................................................................ 16
1.3.3.4 RhoBTB and the actin filament system.......................................................... 17
1.3.4 RhoBTB in human diseases............................................................................ 18
1.4 RhoBTB proteins and proteasome-dependent degradation ................... 19
1.4.1 General overview of the proteasome-dependent degradation pathway ........ 19
1.4.2 Cullin dependent E3 ligases .......................................................................... 21
1.4.3 RhoBTB proteins as adaptors of Cul3-dependent ubiquitin ligases ............. 22
1.5 MUF1/LRRC41 ........................................................................................... 23
1.5.1 The SOCS-box .............................................................................................. 24
1.5.2 The LRR ....................................................................................................... 25
1.6 The kindlin protein family ......................................................................... 26
1.6.1 The role of kindlins in integrin activation .................................................... 27
1.6.2 Expression and localisation of kindlins ........................................................ 28
1.6.3 Kindlin in human diseases ............................................................................ 29
1.7 Uev1 .............................................................................................................. 29
1.8 Aims of the study ......................................................................................... 31

2 MATERIAL AND METHODS ................................................................. 32

2.1 Material ....................................................................................................... 32
2.1.1 Cell lines and strains ..................................................................................... 32
2.1.2 Vectors .......................................................................................................... 32
2.1.3 Oligonucleotides for siRNA ......................................................................... 32
2.1.4 Oligonucleotides for RT-PCR ...................................................................... 33
2.1.5 Oligonucleotides for PCR ............................................................................. 33
2.1.6 Constructs ..................................................................................................... 34
2.1.7 Enzymes ........................................................................................................ 35
2.1.8 Antibodies and fluorescent dyes ................................................................... 36
2.1.8.1 Primary antibodies ........................................................................................ 36
2.1.8.2 Secondary antibodies .................................................................................... 36
2.1.8.3 Fluorescent dyes ........................................................................................... 36
2.1.9 Inhibitors ....................................................................................................... 36
2.1.10. Transfection reagents .................................................................................... 37
42.1.11. Antibiotics ..................................................................................................... 37
2.1.12 Molecular weight markers ............................................................................ 37
2.1.13 Chemicals ..................................................................................................... 38
2.1.14 Kits ............................................................................................................... 38
2.1.15 Laboratory material ...................................................................................... 38
2.2 Sterilisation ................................................................................................. 39
2.3 Cell culture methods ................................................................................... 39
2.3.1 Defrosting of mammalian cell stocks ........................................................... 39
2.3.2 Passaging of mammalian cells ...................................................................... 40
2.3.3 Transfection of mammalian cells .................................................................. 40
2.3.4 Drug treatment .............................................................................................. 40
2.3.5 Determination of protein stability ................................................................. 40
2.3.6 Gene silencing .............................................................................................. 41
2.3.7 Cryostocks preparation ................................................................................. 41
2.4 Bacterial culture methods .......................................................................... 42
2.4.1 Media for bacterial cells cultivation ...............................