Table Of ContentDeveloping Tools to Investigate Protein Sulfenylation in Living Cells
by
Stephen E. Leonard
A dissertation submitted in partial fulfillment
Of the requirements for the degree of
Doctor of Philosophy
(Chemical Biology)
In The University of Michigan
2011
Doctoral Committee:
Professor Kate S. Carroll, co-Chair
Professor Anna K. Mapp, co-Chair
Professor Mark A. Saper
Professor Raymond C. Trievel
Stephen E. Leonard
2011
To my wife Jolie, my support and happiness.
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Acknowledgements
First, I would like to thank my mentor, Dr. Kate Carroll, for the opportunity to work in her lab
and for her knowledge, guidance, and support. I would like to acknowledge and thank all of the
Carroll lab members. I have enjoyed working with everyone in the lab. They have always
provided great feedback in experimental design, preparing for talks, and editing my writing.
Specifically I would like to thank my fellow graduate students Candie Paulsen, Devayani Bhave,
Jiyoung Hong, Thu Ha Truong, and Francisco Garcia for the good times and the science. I would
like to thank Dr. Young Ho Seo and Dr. Khalilah Reddie for always being willing to discuss our
work and helping me troubleshoot problem experiments. Also, I would like to thank Jesse Song
for being the hardest working undergraduate scientist I have ever come across.
Outside of the lab I would like to thank Dr. Anna Mapp, Dr. Mark Saper, and Dr. Ray Trievel for
meeting with me as my thesis committee over the past three years. They have all given me
valuable input on my research and been good sounding boards when determining my future
directions. Matthieu Depuydt and Dr. Jean-Francois Collet collaborated with me on a very
successful project and showed me that other people could use the tools that I developed. Dr.
Jason Gestwicki and all of the Gestwicki lab members deserve special thanks for providing me
with space to work and camaraderie when I was separated from my lab.
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Finally, I would like to thank my family for their support and encouragement over the last five
years. Jolie, thank you for making it through graduate school with me and keeping me
motivated and always encouraging me every day. Also, thanks for getting us cats.
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Preface
This thesis is the compilation of published and unpublished work on the development of sulfenic
acid-specific probes to monitor protein cysteine oxidation in living cells. Sulfenic acid is the
oxidation product that forms when a cysteine thiolate reacts with hydrogen peroxide. Protein
sulfenic acid formation is a reversible process that has emerged as a central mechanism for
dynamic post-translational modification in all major protein classes and correlates with disease
states.
In Chapter 1, we discuss chemical and biological features of sulfenic acids while placing them in
the larger context of protein cysteine modifications and review current methods for their study.
This work has been published as a review for which the citation is Leonard S.E. and Carroll, K.S.,
“Chemical 'omics' approaches for understanding protein cysteine oxidation in biology,” (2011)
Curr Opin Chem Biol (2011) Feb;15(1):88-102.
Chapter 2 focuses on the synthesis and evaluation of DAz-1, the first cell-permeable probe
capable of trapping and tagging sulfenic acid modifications directly in cells. The citation for this
article is Reddie, K.G., Seo, Y.H., Muse, W.B. Leonard, S.E. and Carroll, K.S., “A chemical approach
for detecting sulfenic acid-modified proteins in living cells.,” Mol Biosyst. (2008) Jun;4(6):521-31.
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In Chapter 3, we present the development and application of a second generation probe for
sulfenic acids, DAz-2. This analogue exhibits significantly improved potency for detecting
sulfenic acids both in vitro and in cells. DAz-2 was used to conduct a proteomic investigation of
the sulfenome in the HeLa human tumor cell line. This study identified approximately two
hundred proteins that undergo sulfenic acid formation, which are distributed throughout the
cell and function in a diverse array of biological processes. These data are published as Leonard,
S.E., Reddie, K.G., and Carroll, K.S., “Mining the thiol proteome for sulfenic acid modifications
reveals new targets for oxidation in cells,” ACS Chem Biol. (2009) Sep 18;4(9):783-99.
In Chapter 4, DAz-2 was used to examine oxidation of single cysteine-containing proteins in the
Escherichia coli periplasm, an oxidizing environment in which most thiols are involved in
disulfide bonds. Experiments with the DAz-2 probe led to the discovery of two thioredoxin-
related proteins that protect single cysteines from irreversible oxidation to sulfinic and sulfonic
acid, with functional homologues in eukaryotic cells. This work was published as Depuydt, M.,
Leonard, S.E., Vertommen D., Denoncin K., Morsomme P., Wahni K., Messens J., Carroll K.S., and
Collet J.F. “A periplasmic reducing system protects single cysteine residues from oxidation,”
Science (2009) Nov 20;326(5956):1109-11.
Chapter 5 details how chemical probes for detecting sulfenic acids were further refined through
the addition of a binding module to target the protein tyrosine phosphatase (PTP) family of
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enzymes. These probes exhibit significantly improved sensitivity for detecting cysteine oxidation
in this important class of signaling enzymes. This work has been published as Leonard S.E.,
Garcia F.J., Goodsell D.S., Carroll K.S. “Redox-Based Probes for Protein Tyrosine Phosphatases.”
Angew Chem Int Ed Engl. (2011) May 2;50(19):4423-7.
Finally, Chapter 6 is a discussion of future directions for developing and applying cell-permeable
small molecule probes for detecting protein sulfenic acid formation in cells.
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Table of Contents
Dedication ii
Acknowledgements iii
Preface v
List of Figures xvi
List of Schemes xix
List of Tables xx
List of Appendices xxi
List of Abbreviations xxii
Abstract xxvii
Chapter
1. Omics approaches for understanding protein cysteine oxidation in biology
1.1. Abstract 1
1.2. Introduction 2
1.3. Indirect versus direct detection of oxidative cysteine modifications 5
1.4. Lysate versus Cellular analysis of oxidative cysteine modifications 7
1.5. Chemical approaches to detect reactive cysteines 7
1.6. General chemical approaches to detect cysteine oxidation 9
1.7. Direct detection of protein disulfide formation 11
1.8. Chemical approaches to detect protein glutathionylation 12
1.9. Chemical approaches to detect protein nitrosylation 14
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1.10. Chemical and immunochemical approaches to detect protein sulfenylation 18
1.11. Conclusions and future directions 24
1.12. References and recommended reading 26
2. A chemical approach for detecting sulfenic acid-modified proteins in living cells 33
2.1. Abstract 33
2.2. Introduction 34
2.3. Results 39
2.3.1. DAz-1 irreversibly labels sulfenic acid-containing proteins 39
2.3.2. DAz-1 detects sulfenic acid-modified proteins in cell lysate 47
2.3.3. Incorporation of DAz-1 into proteins in cultured human cells 50
2.4. Discussion 56
2.5. Experimental Procedures 57
2.5.1. Preparation, enzymatic assay and chemical modifications of papain 57
2.5.2. NBD-Cl assay and chemical modification of HAS 58
2.5.3. P-biotin labeling of papain and HSA 59
2.5.4. Immunoblotting 59
2.5.5. Cell culture 60
2.5.6. DAz-1 labeling of Jurkat lysate 60
2.5.7. DAz-1 labeling of Jurkat cells 61
2.5.8. DAz-1 labeling of Jurkat cells post-oxidant challenge 61
2.5.9. Chemical Methods 62
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