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Methionine Sulfoxide Reductases: Studies on the Reducing METHIONINE SULFOXIDE REDUCTASES: STUDIES ON THE REDUCING REQUIREMENTS AND ROLE IN THE METABOLISM OF SULINDAC by David J. Brunell A Dissertation Submitted to the Faculty of The Charles E. Schmidt College of Science in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Florida Atlantic University Boca Raton, FL August 2009 ACKNOWLEDGEMENTS I would like to express my deepest appreciation to my committee chair and mentor, Dr. Herbert Weissbach, for his faith in me as a scientist and his patience, guidance and unselfish dedication through all the long hours it took to complete my studies. I would also like to thank my other committee members, Drs. Nathan Brot, David Binninger, Howard Prentice and Keith Brew for their thoughtful criticism and time away from busy schedules and Dr. Daphna Sagher for passing along to me her considerable expertise gained through many years of experience in the lab. My students Chan Le, Anna ToiGB, Diana Navarro and Shari Selesky not only made contributions to the research but also extended their sincere friendship at a time when it was needed and appreciated. Special thanks also go to the other lab members who contributed to the research including Dr. Ian Moench and Alex Kreymerman. Most especially, I would like to thank my wife Kateri for her patience and support and my mother Ruth Brunell for instilling in me from a young age a deep curiosity about how the world works. iii ABSTRACT Author: David J. Brunell Title: Methionine Sulfoxide Reductases: Studies on the Reducing Requirements and Role in the Metabolism of Sulindac Institution: Florida Atlantic University Dissertation Advisor: Dr. Herbert Weissbach Degree: Doctor of Philosophy Year: 2009 The methionine sulfoxide reductase (Msr) enzymes catalyze the reduction of methionine sulfoxide (Met(O)) to methionine. The Msr enzymes protect cells against oxidative stress and may have a role in aging. The MsrA family of enzymes reduces stereospecifically the S epimer of free and protein-bound Met(O) while the MsrB family reduces the R epimer of Met(O) in proteins. It has been generally accepted, primarily from studies on MsrA, that the biological reductant for the Msr enzymes is thioredoxin (Trx), although high levels of dithiothreitol (DTT) can be used as the reductant in vitro. In contrast, certain MsrB enzymes show less than 10% of the activity with Trx as compared to DTT. This raises the possibility that in animal cells Trx may not be the direct hydrogen donor for the MsrB enzymes. Studies with bovine liver extracts have shown that thionein, the apoprotein of metallothionein, can function as a reductant for iv the Msr proteins. Certain selenium compounds such as selenocystamine and selenocystine can also serve as potent reducing agents for the Msr enzymes. Since an increased activity of Msr enzymes can reduce the level of oxidative damage in tissues, compounds that could activate Msr may have therapeutic potential. A high-throughput screening assay has been developed to screen large chemical libraries to find activators of MsrA, as well as specific inhibitors that could be useful research tools. This study will be done in collaboration with The Scripps Florida Research Institute. Sulindac was originally developed as a non-steroidal anti-inflammatory drug but has also shown efficacy in the treatment of certain cancers. The S epimer of sulindac is known to be reduced by MsrA, but the enzymes responsible for reduction of the R epimer are not known. An activity has been purified from rat liver which is capable of reducing the R epimers of sulindac, free Met(O) and a dabsylated Met(O) substrate, the latter suggesting that this enzyme may have properties similar to the MsrB enzymes. The oxidation of the epimers of sulindac to sulindac sulfone has also been characterized, and the members of the cytochrome P450 family involved in the oxidation have been identified. v METHIONINE SULFOXIDE REDUCTASES: STUDIES ON THE REDUCING REQUIREMENTS AND ROLE IN THE METABOLISM OF SULINDAC List of Tables......................................................................................................................x List of Figures...................................................................................................................xii 1 INTRODUCTION..........................................................................................................1 1.1 Reactive oxygen species, oxidative damage and aging...........................................1 1.2 Role of the methionine sulfoxide reductases...........................................................3 1.3 Genetic studies on the role of the Msr enzymes......................................................6 1.4 Mechanism of action of the Msr enzymes...............................................................9 1.5 Reducing system for the MsrB enzymes...............................................................12 1.6 Substrate specificity of the Msr Enzymes.............................................................13 1.7 Msr as a catalytic antioxidant................................................................................13 1.8 Assays for Msr activity..........................................................................................15 1.9 Msr and disease......................................................................................................17 1.10 Sulindac and its relationship to the Msr system..................................................19 1.11 Objectives of the current study............................................................................22 2 MATERIALS AND METHODS..................................................................................23 2.1 Materials................................................................................................................23 2.2 Expression and purification of enzymes................................................................23 2.3 Colorimetric DABS assay for Msr activity...........................................................24 vi 2.4 Purification of metallothionein (MT) from bovine liver.......................................25 2.5 Preparation of thionein (T) and T(O) and assay of T(O) reduction by Trx...........26 2.6 Analysis of zinc content.........................................................................................27 2.7 Separation of the sulindac epimers.......................................................................27 2.8 Preparation of plasma samples from sulindac-treated animals..............................27 2.9 Assay of sulindac reduction ..................................................................................28 2.10 Purification of sulindac-R reductase from rat liver.............................................28 2.11 Assay of sulindac oxidation via cytochrome P450 enzymes...............................29 2.12 Induction of sulindac oxidation in human hepatocytes .....................................30 3 RESULTS.....................................................................................................................31 3.1 Studies on the reducing requirements for the Msr enzymes..................................31 3.1.1 Reduced Trx is not an efficient reducing agent for hMsrB2 and hMsrB3....31 3.1.2 Zn-MT in the presence of EDTA can serve as a reducing agent for Msr......32 3.1.3 T can function in the Msr system in the absence of EDTA...........................38 3.1.4 Trx can reduce T(O)......................................................................................39 3.1.5 Selenocystamine (SeCm) enhances activity with mammalian Msr enzymes........................................................................................................42 3.1.6 SeCm is reduced by Trx reductase and can directly reduce the Msr enzymes.........................................................................................................44 3.1.7 Selenocystine enhances MsrB activity but is not directly reduced by Trx reductase.................................................................................................45 3.1.8 Thionein can reduce SeCm............................................................................46 vii 3.2 A high-throughput screening assay for MsrA........................................................50 3.2.1 Development of the assay.............................................................................50 3.2.2 Miniaturization of the assay..........................................................................55 3.2.3 Follow up of active compounds....................................................................59 3.2.4 Optimization of any lead compounds............................................................61 3.2.5 Problems using a coupled system to measure NADPH oxidation................61 3.2.6 Absorbance at 340 nm of chemicals in the library........................................62 3.2.7 Elimination of non-specific activators or inhibitors......................................62 3.3 The metabolism of sulindac...................................................................................64 3.3.1 Sulindac metabolites detected in rat tissues ................................................64 3.3.2 MsrA knockout mice can reduce both sulindac epimers in vivo...................65 3.3.3 Liver homogenates from MsrA knockout mice do not reduce sulindac-S....66 3.3.4 Partial purification of sulindac-R reductase from rat liver............................68 3.3.5 Reducing requirements
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