Lcms-Based Analysis Explains the Basis of Oxidative Resistance in Selenium-Containing Thioredoxin Reductase
Total Page：16
File Type：pdf, Size：1020Kb
University of Vermont UVM ScholarWorks Graduate College Dissertations and Theses Dissertations and Theses 2021 Lcms-Based Analysis Explains The Basis Of Oxidative Resistance In Selenium-Containing Thioredoxin Reductase Daniel Haupt University of Vermont Follow this and additional works at: https://scholarworks.uvm.edu/graddis Part of the Analytical Chemistry Commons, and the Biochemistry Commons Recommended Citation Haupt, Daniel, "Lcms-Based Analysis Explains The Basis Of Oxidative Resistance In Selenium-Containing Thioredoxin Reductase" (2021). Graduate College Dissertations and Theses. 1422. https://scholarworks.uvm.edu/graddis/1422 This Thesis is brought to you for free and open access by the Dissertations and Theses at UVM ScholarWorks. It has been accepted for inclusion in Graduate College Dissertations and Theses by an authorized administrator of UVM ScholarWorks. For more information, please contact [email protected]. LCMS-BASED ANALYSIS EXPLAINS THE BASIS OF OXIDATIVE RESISTANCE IN SELENIUM-CONTAINING THIOREDOXIN REDUCTASE A Thesis Presented by Daniel J. Haupt to The Faculty of the Graduate College of The University of Vermont In Partial Fulfillment of the Requirements For the Degree of Master of Science Specializing in Chemistry August, 2021 Defense Date: April 21, 2021 Thesis Examination Committee: Robert J. Hondal, Ph.D., Advisor Michael J. Previs, Ph.D., Chairperson Giuseppe A. Petrucci, Ph.D. Severin T. Schneebeli, Ph.D. Cynthia J. Forehand, Ph.D., Dean of the Graduate College ABSTRACT Selenocysteine (Sec) is referred to as the 21st proteogenic amino acid and is found in place of the redox-sensitive amino acid cysteine (Cys) in a small number of proteins. Sec and Cys carry out similar chemistry and are structural isomers save for a single atom difference; the former contains selenium (Se), while the latter contains sulfur (S) in the identical position. Sec poses a high bioenergetic cost for its synthesis and subsequent incorporation into protein not shared by Cys. Since Sec’s discovery in 1976, scientists have debated why certain proteins express Sec while others express Cys. In recent years, it has been shown that redox-active enzymes expressing Sec exhibit a distinct biological advantage over their Cys-containing counterparts. Sec-containing enzymes retain their catalytic activities when exposed to highly oxidizing conditions, while analogous Cys- containing enzymes become significantly inactivated. This thesis examines the enzyme thioredoxin reductase (TrxR), an essential regulator of cellular redox homeostasis. TrxR is expressed in higher-order organisms such as humans and other mammals with a penultimate Sec residue in its C-terminal redox center, while lower-order organisms such as Drosophila melanogaster (D. melanogaster) express TrxR with Cys. Using spectrophotometric activity assays, we show that the presence of Sec preserves thioredoxin reductase’s ability to reduce its canonical substrate, thioredoxin (Trx), in the presence of hydrogen peroxide. Herein, we use mass spectrometry analysis to provide a biophysical basis for this phenomenon through the characterization and quantitation of the TrxR’s two redox centers that drive its mechanism of action. Our findings show that Sec confers superior resistance to oxidation-induced inactivation not because Sec better resists irreversible hyperoxidation but instead TrxR’s redox centers to better retain their reductive abilities. In D. melanogaster’s Cys-containing TrxR, we show that a significant loss of its redox center’s reductive ability coincides with the loss of its enzymatic activity. DEDICATION To my parents, Robert and Sandra; my first teachers. ii ACKNOWLEDGEMENTS The work of this thesis would not be possible without the effort and guidance of others. I am extremely grateful to my advisor, Rob Hondal, for his mentorship and unwavering encouragement. I want to thank my former advisor, Dwight Matthews, who trained me as a mass spectrometrist and opened my eyes to its possibilities. I want to thank Emma Ste. Marie, Karat Sidhu, Bruce O’Rourke, Michael Previs, and Neil Wood for their contributions to my research efforts and always going above and beyond to help me. Special thanks to Scott Tighe and Wolfgang Dostmann for being the firsts to believe in me and helping me to become a better scientist. iii TABLE OF CONTENTS DEDICATION................................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................ iii LIST OF FIGURES ......................................................................................................... vi LIST OF TABLES .......................................................................................................... vii Chapter 1: SELENOCYSTEINE: THE 21st AMINO ACID ........................................ 1 1.1 SELENIUM IN BIOLOGY ...................................................................................... 1 1.2 PROPERTIES OF CYSTEINE AND SELENOCYSTEINE ................................... 5 1.2.1 Similarities Between Sulfur and Selenium ......................................................... 5 1.2.2 Differences Between Sulfur and Selenium ......................................................... 5 1.2.3 Mechanism for Selenocysteine Synthesis and Incorporation ............................. 7 1.3 WHY NATURE CHOSES SELENOCYSTEINE .................................................... 9 1.3.1 The electrophilic character of selenocysteine helps selenoproteins resist oxidative inaction ...................................................................................................... 10 1.4 THIOREDOXIN-THIOREDOXIN REDUCTASE SYSTEM ............................... 12 1.4.1 General Function .............................................................................................. 15 1.4.2 Two forms of TrxRs ......................................................................................... 17 1.4.3 High-Mr Thioredoxin Reductase Mechanism of Action ................................... 18 1.4.4 Oxidation of Thioredoxin Reductase ................................................................ 19 Chapter 2: ASSESSING THE ABILITY OF SEC-TRXR TO RESIST OXIDATION-INDUCED INACTIVATION ............................................................... 23 2.1 INTRODUCTION ................................................................................................... 23 2.2 METHODS.............................................................................................................. 25 2.3 RESULTS................................................................................................................ 30 2.3.1 Spectrophotometric thioredoxin reductase activity assays ............................... 33 2.3.2 Detection of DmTrxR redox centers in the absence of E. coli Trx .................. 34 2.3.3 Detection of mTrxR redox centers in the absence of E. coli Trx ..................... 39 2.3.4 Detection of DmTrxR redox centers in the presence of E. coli Trx ................. 43 2.3.5 Detection of mTrxR redox centers in the presence of E. coli Trx .................... 46 2.4 CONCLUSIONS ..................................................................................................... 50 iv REFERENCES ................................................................................................................ 59 APPENDICES ................................................................................................................. 65 A. ABBREVIATIONS ............................................................................................ 65 B. MASS SPECTRA AND CHEMICAL STRUCTURES OF PEPTIDE SPECIES DETECTED BY MS ..................................................................................................... 67 v LIST OF FIGURES Figure Page Figure 1. Structures of selenocysteine and cysteine .......................................................... 2 Figure 2. Quaternary complex involved in selenocysteine incorporation ......................... 8 Figure 3. Cycling of oxidized and reduced selenium-containing and sulfur- containing species ........................................................................................... 11 Figure 4. Mammalian thioredoxin reductase (mTrxR) structure ..................................... 14 Figure 5. Drosophila melanogaster thioredoxin reductase (DmTrxR) structure. ........... 15 Figure 6. Thioredoxin (Trx)/thioredoxin reductase (TrxR) antioxidant system .............. 17 Figure 7. Mechanistic overview of mTrxR and DmTrxR ................................................ 19 Figure 8. Oxidative modification and repair of cysteine and selenocysteine .................. 22 Figure 9. H2O2 knockdown of TrxR E. coli thioredoxin reductase activities .................. 34 Figure 10. DmTrxR N-terminal redox center response to H2O2 in the absence of E. coli Trx ................................................................................................... 36 Figure 11. DmTrxR C-terminal redox center response to H2O2 in the absence of E. coli Trx ....................................................................................................... 38 Figure 12. mTrxR N-terminal redox center response to H2O2 in the absence of E. coli Trx ....................................................................................................... 40 Figure 13. mTrxR C-terminal redox center response to H2O2