Oxidative thiol modifications in pro- and eukaryotic organisms [Elektronische Ressource] / vorgelegt von Nicolas Brandes

Oxidative Thiol Modifications
in Pro- and Eukaryotic Organisms






Dissertation zur Erlangung des
naturwissenschaftlichen Doktorgrades
der Julius-Maximilians-Universität Würzburg



vorgelegt von
Nicolas Brandes
aus Hildesheim




Ann Arbor, 2010
I

Oxidative Thiol Modifications
in Pro- and Eukaryotic Organisms






Dissertation zur Erlangung des
naturwissenschaftlichen Doktorgrades
der Julius-Maximilians-Universität Würzburg



vorgelegt von
Nicolas Brandes
aus Hildesheim




Ann Arbor, 2010

II




Eingereicht am: 21.01.2010

Mitglieder der Prüfungskommission
Vorsitzender: Prof. Dr. Th. Dandekar
Gutachter: Prof. Dr. R. Benz
Gutachter: Prof. Dr. U. Jakob


Tag des Promotionskolloquiums: 10.03.2010


Doktorurkunde ausgehändigt am: ………………………..










III




PER ASPERA AD ASTRA (SENECA)

IV




Erklärung


Hiermit erkläre ich, dass diese Arbeit bisher von mir weder an der Julius-Maximilians-
Universität Würzburg noch einer anderen wissenschaftlichen Einrichtung zum
ZZwweecckkee ddeerr PPrroommoottiioonn eeiinnggeerreeiicchhtt wwuurrddee..

Weiter erkläre ich, dass ich diese Arbeit selbständig verfasst und keine anderen als
die darin angegebenen QQuueelllleenn uunndd Hilfsmittel benutzt habe.

Ferner erkläre ich, dass ich früher weder akademische Grade erworben habe oder zu
erwerben versucht habe.



17. Januar 2010
(ORT, DATUM) (NICOLAS BRANDES)



V




List of Publications


Brandes, N., S. Schmitt and U. Jakob (2009)
“Thiol-Based Redox Switches in Eukaryotic Proteins”
Antioxid Redox Signal. May, 11(5): 997-1014.


Brandes, N., A. Rinck, L.I. Leichert and U. Jakob (2007)
“Nitrosative stress treatment of E. coli targets distinct set of thiol-containing proteins”
Mol Microbiol. November, 66(4): 901-14


Stegmeier, J. F., G. Polleichtner, N. Brandes, C. Hotz, and C. Andersen (2006)
“Importance of the Adaptor (Membrane Fusion) Protein Hairpin Domain for the
Functionality of Multidrug Efflux Pumps“
Biochemistry, 45 (34), pp 10303–10312

VI
Co-auutthhoorr ssttaatteemmeennttss
Brandes, N., S. Schmitt and U. Jakob (2009)
“Thiol-Based Redox Switches in Eukaryotic Proteins”
Antioxid Redox Signal. May, 11(5): 997-1014.

The majority of the manuscript was written by NB with only minor contributions from SS. U.J.
supervised and commented on the work.



Nicolas Brandes Sebastian Schmitt



Ursula Jakob





Brandes, N., A. Rinck, L.I. Leichert and U. Jakob (2007)
“Nitrosative stress treatment of E. coli targets distinct set of thiol-containing proteins”
Mol Microbiol. November, 66(4): 901-14

The majority of experiments were performed by NB, including two-dimensional gel analysis, enzyme
assays, UV absorbance and mass spectrometry. AR performed cloning and protein purification. NB,
LIL and UJ analyzed data. NB wrote the manuscript, supervised by LIL and UJ. Research was
designed by UJ.



Nicolas Brandes Andrea Rinck



Lars I. Leichert Ursula Jakob
VII
Table of Contents
Erklärung ................................................................................................................... IV
List of Publications ...................................................................................................... V
Co-author statements ................................................................................................ VI
Table of Contents ..................................................................................................... VII
Abbreviations ........................................................................................................... XIII
Summary .......................................................................................................... 1
I. Summary .......................................................................................................... 1
II. Zusammenfassung ........................................................................................... 4
1 Introduction ............................................................................................... 7
1.1 Reactive oxygen species and oxidative stress .......................................... 8
1.2 Cysteine thiols — Central components of redox-sensitive
nanoswitches ................................................................................................ 9
1.3 Changing gene transcription to protect cells against oxidative
stress ........................................................................................................... 12
1.4 Yap1 — Changing the redox state means switching homes................... 12
1.4.1 Yap1 — Member of a two-component redox relay ................................. 13
1.4.2 Redox-mediated fine-tuning of Yap1’s functional activity ....................... 15
1.4.3 Activation of Yap1 — More than one way to get the response ............... 16
1.4.4 Yap1 — A prototype for emerging concepts in redox regulation? .......... 17
1.4.5 Redox theme with variations — The Nrf2-Keap1 connection ................. 18
Table of Contents VIII
1.5 GapDH — Putting cellular metabolism under redox-control .................. 19
1.5.1 Re-Routing the metabolic flux to protect cells against oxidative
damage .................................................................................................. 20
1.5.2 ROS-mediated GapDH aggregation and its role in apoptosis ................ 22
1.5.3 Role of GapDH’s redox-regulation in signaling ....................................... 23
1.5.4 GapDH — One of many redox-regulated metabolic enzymes................ 25
1.6 PTP1B — Redox regulation of eukaryotic signal transduction
cascades ...................................................................................................... 26
1.6.1 PTP1B — Using cyclic sulfenamide formation as redox switch .............. 27
1.6.2 Overoxidation of PTP1B — More than a dead-end product? ................. 29
1.6.3 Other redox-regulated signal transduction cascades in
eukaryotes .............................................................................................. 30
1.7 Concluding remarks ................................................................................... 31
2 Nitrosative Stress Treatment of E. coli Targets Distinct Set of
Thiol-containing Proteins ........................................................................34
2.1 Introduction ................................................................................................. 35
2.2 Material and Methods ................................................................................. 37
2.2.1 Bacterial strains and culture conditions .................................................. 37
2.2.2 Plasmid construction .............................................................................. 37
2.2.3 Differential thiol trapping ........................................................................ 38
2.2.3.1 Sample preparation and differential thiol trapping .......................... 38
2.2.3.2 Staining of the gels, storage phosphor autoradiography,
and image analysis ........................................................................ 39
2.2.3.3 Data analysis and identification of proteins from 2D gels ............... 39
2.2.4 Purification of Glutamate Synthase (GOGAT) ........................................ 40
2.2.5 Enzyme assays ...................................................................................... 40
2.2.6 UV absorbance and analysis of GOGAT’s thiol status using
mass spectrometry ................................................................................. 41
Table of Contents IX
2.3 Results and Discussion ............................................................................. 42
2.3.1 Global analysis of the thiol-disulfide state of E. coli proteins upon
DEA/NO treatment ................................................................................. 42
2.3.2 NO-treatment causes oxidative thiol modification in a distinct set
of E. coli proteins .................................................................................... 46
2.3.3 Identification of essential RNS-sensitive proteins involved in
DEA/NO-induced growth inhibition ......................................................... 48
2.3.4 Activity of IlvC is affected by DEA/NO treatment in vivo ......................... 50
2.3.5 Glutamate Synthase — A protein specifically sensitive to
nitrosative stress .............................................................