Identification of novel cytosolic thioredoxin-1 target proteins in mammalian cells by mechanism-based kinetic trapping [Elektronische Ressource] / presented by Lars Weingarten

Identification of novel cytosolic thioredoxin-1 target
proteins in mammalian cells by mechanism-based
kinetic trapping


Dissertation

submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruprecht-Karls University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences



presented by



Dipl.-Biol. Lars Weingarten

born in Hamburg, Germany


Oral examination: December 11, 2008























First Referee: Prof. Dr. Thomas Rausch
Heidelberg Institute of Plant Sciences

Second Referee: PD Dr. Ursula Klingmüller
German Cancer Research Center, Heidelberg
Acknowledgements

First of all, I would like to thank my supervisor Tobias Dick for fruitful discussions, the
considerable leeway he gave me and the opportunity to work in his lab. I highly
appreciate his honest way of doing science.

I thank Thomas Rausch and Ursula Klingmüller for being the referees of this thesis.

I am grateful to all current and former lab members for help, advice and the nice
atmosphere. Especially, I would like to thank Ulla Schwertassek for her expertise in
kinetic trapping and Yves Balmer for helpful discussions on redox biochemistry. I
would like to acknowledge Monique Winkler and Gabriele Kuntz for excellent
technical assistance and Thomas Ruppert for performing mass spectrometry
measurements.

I thank Johanna Engelhard and Martina Kegel for critical proof-reading of this thesis.

Many thanks to all the people who provided antibodies and other reagents for the
analysis of various proteins identified in the proteomic screen. I would like to thank
Ingrid Hoffmann and Onur Cizmecioglu for their great support with cell cycle
reagents.

Moreover, I would like to thank Herman Bujard and Hans Clevers for providing
plasmids for doxycycline-dependent gene expression.

Special thanks to Peter Krammer and his lab not only for their help in apoptosis-
related questions but also for the great time in the beginning of my thesis. I thank
Dirk Brenner for helpful advice on protein biochemistry and Inna Lavrik for the
collaboration on caspases.

Thanks to Mareike for things that cannot be expressed in words.

Finally, I would like to specially thank my parents, my brother and my grandmother to
whom I dedicate this work.
Table of contents
Table of contents
Summary ................................................................................................................... 1
Zusammenfassung................................................................................................... 2
1 Introduction ....................................................................................................... 3
1.1 Oxygen and its reactive species............................................................................................. 3
1.2 Redox regulation: Control of protein activity by thiol modifications ................................. 5
1.2.1 Redox regulation of protein tyrosine phosphatases .......................................................... 6
1.2.2 ation of protein kinases .................................................................................. 8
1.3 Thiol-disulfide oxidoreductases ........................................................................................... 10
1.3.1 The glutathione/glutaredoxin system............................................................................... 10
1.3.2 The thioredoxin system.................................................................................................... 11
1.3.3 Forms and localization of thioredoxins ............................................................................ 12
1.3.4 Structure of Trx1 .............................................................................................................. 14
1.3.5 Catalytic mechanism of Trx1 15
1.3.6 Regulation of Trx1 expression and activity...................................................................... 15
1.3.7 Biological functions of Trx1.............................................................................................. 18
1.3.8 Trx1 in human diseases................................................................................................... 21
1.4 Objective of this work ............................................................................................................ 24
2 Materials........................................................................................................... 26
2.1 Chemicals................................................................................................................................ 26
2.2 Laboratory equipment............................................................................................................26
2.3 Buffers ..................................................................................................................................... 27
2.4 Antibodies ............................................................................................................................... 30
2.5 Media for eukaryotic cells and bacteria ............................................................................... 31
2.6 Biological material.................................................................................................................. 32
2.7 Expression vectors ................................................................................................................ 33
2.8 Enzymes and kits ................................................................................................................... 34
2.9 Software .................................................................................................................................. 34
3 Methods 35
3.1 Methods in molecular biology............................................................................................... 35
3.1.1 Small scale isolation of plasmid DNA from E.coli............................................................ 35

x Table of contents
3.1.2 Large scale isolation of plasmid DNA from E.coli............................................................ 35
3.1.3 Restriction digest of DNA................................................................................................. 35
3.1.4 Polymerase chain reaction (PCR) ................................................................................... 35
3.1.5 Site-directed mutagenesis ............................................................................................... 36
3.1.6 Agarose gel electrophoresis ............................................................................................ 37
3.1.7 DNA extraction from agarose gels 37
3.1.8 Ligation of DNA fragments 37
3.1.9 Cloning of expression vectors.......................................................................................... 37
3.1.10 Transformation of E.coli................................................................................................... 38
3.2 Methods in cell biology.......................................................................................................... 38
3.2.1 Cultivation of cell lines ..................................................................................................... 38
3.2.2 Cryoconservation of cell lines 39
3.2.3 Transfection of eukaryotic cell lines................................................................................. 39
3.2.4 Retroviral transduction of eukaryotic cells ....................................................................... 39
3.2.5 Inducible short-hairpin RNA (shRNA)-mediated knockdown of Trx1 .............................. 40
3.3 Methods in protein biochemistry.......................................................................................... 41
3.3.1 Purification of recombinant proteins from E.coli .............................................................. 41
3.3.2 Immunoprecipitation of protein complexes...................................................................... 42
3.3.3 Mechanism-based kinetic trapping .................................................................................. 42
3.3.4 SDS polyacrylamide gel electrophoresis (SDS-PAGE)................................................... 43
3.3.5 2D diagonal SDS-PAGE (non-reducing/reducing)........................................................... 43
3.3.6 Colloidal Coomassie staining........................................................................................... 44
3.3.7 Silver staining................................................................................................................... 44
3.3.8 Immunoblotting ................................................................................................................ 44
3.3.9 Stripping of immunoblots ................................................................................................. 45
3.3.10 Mass spectrometry .......................................................................................................... 45
3.3.11 Caspase-