This student paper was written as an assignment in the graduate course

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1 77:222 Spring 2003 Free Radicals in Biology and Medicine Page 0 This student paper was written as an assignment in the graduate course Free Radicals in Biology and Medicine (77:222, Spring 2003) offered by the Free Radical and Radiation Biology Program B-180 Med Labs The University of Iowa Iowa City, IA Spring 2003 Term Instructors: GARRY R. BUETTNER, Ph.D. LARRY W. OBERLEY, Ph.D. with guest lectures from: Drs. Freya Q. Schafer, Douglas R. Spitz, and Frederick E. Domann The Fine Print: Because this is a paper written by a beginning student as an assignment, there are no guarantees that everything is absolutely correct and accurate. In view of the possibility of human error or changes in our knowledge due to continued research, neither the author nor The University of Iowa nor any other party who has been involved in the preparation or publication of this work warrants that the information contained herein is in every respect accurate or complete, and they are not responsible for any errors or omissions or for the results obtained from the use of such information. Readers are encouraged to confirm the information contained herein with other sources. All material contained in this paper is copyright of the author, or the owner of the source that the material was taken from. This work is not intended as a threat to the ownership of said copyrights.

2 lxiao TrxR 1 Thioredoxin Reductase (TrxR) By Ling Xiao B-180 Medical Laboratories Free Radical and Radiation Biology program The University of Iowa Iowa City, IA Tel: 319/ Fax: 319/ ling-xiao@uiowa.edu For 77:222, Spring 2003 Due Date: March 13, 2003 Paper III Abbreviations FAD: flavin adenine dinucleotide (oxidized form) GR: glutathione disulfide reductase GSH: glutathione (reduced form) GSSG: glutathione (oxidized form) hgr: human glutathione disulfide reductase NADPH: nicotinamide dinucleotide phosphate (reduced form) ROS: reactive oxygen species SeCys: selenocysteine TrxRs: thioredoxin reductases Trxs: thioredoxins

3 lxiao TrxR 2 Table of contents Page number Abstract 2 Introduction 2 Structure of TrxR 3 Catalytic mechanism 4 Physiological functions 6 Summary 9 References 10 Abstract Mammalian thioredoxin reductases (TrxRs) are a class of Se-containing pyridine nucleotide-disulfide oxidoreductases. TrxRs have a conserved -CVNVGC- active site, located on the FAD binding domain, and a C-terminal -Cys-SeCys- active site, which are both essential for TrxR enzymatic activity. Mammalian TrxRs have been found to locate in mitochondria and cytoplasm. For instance, human TrxR1 was showed to localized on cytoplasm and human TrxR2 was localized on mitochondria. Human TrxRs have broad substrates but the main substrate is thioredoxins (Trx). The TrxRs are involved in cell growth, protection against oxidant stress, redox signaling, and various diseases. Introduction The thioredoxin reductases (TrxRs) are members of the flavoprotein family of pyridine nucleotide-disulfide oxidoreductases that include glutatione reductase, mercuric ion reductase,

4 lxiao TrxR 3 and lipoamide dehydrogenase [1]. Members of this family are homodimeric flavoproteins that each monomer contains one redox-active disulfide, FAD prosthetic group and a NADPH binding domain (Figures 1, 2). Sequencing and coloning show mammalian TrxRs are highly similar to GR (glutathione disulfide reductase) [2]. The mammalian TrxRs have broad substrate specificity. It not only can reduce oxidazed thioredoxins (Trxs), a group of small ubiquitous redox-active peptides which are necessary for redox regulation of cell signaling [3], but also selenite, lipid hydroperoxides, vitamin K, and H 2 O 2 [6]. The physiological function of TrxR is very complicated. There are a lot of arguments showing that TrxR is involved in Redox signaling-regulation [3], protection of oxidation stress [11,13], cell growth and death [12] and diseases [6]. This review mainly focuses on the structure of TrxR and the underlying catalytic mechanisms of TrxR as well as the physiological functions of TrxR. Structure of TrxR The mammalian thioredoxin reductase gene has been coloned and sequenced, and it showed highly similar to GR [2,4]. The mass of monomer TrxR from mammalian species is around 55 kda, compared with 35 kda of TrxR from prokaryotes, E. coli, yeast, or plants [10]. The predicted domain structure of TrxR is show in Figure 1, which contains FAD bind domain, a functional disulfide/dithiol, NADPH binding domain, and a penultimate C-terminal selenocysteine residue in each subunit [7]. The catalytic site of human TrxR, -CVNVGC-, is also found in human GR and is located on the FAD binding domain of the enzyme, whereas the catalytic site of E. coli TrxR, -CATC-, is located on the NADPH binding domain. The C- terminal contains a penultimate SeCys residue within the unique sequence, -GC-SeCG-, which is conserved in all mammlian TrxR [2, 4, 7]. The SeCys residue is essential for TrxR catalytic

5 lxiao TrxR 4 activity [5, 7]. The proposed model of two subunits interaction is show in Figure 2. This mode was based on homology to GR. It shows that the Cys-SeCys residues of one subunit are close to the redox-active disulfide/dithiol of the second subunit. Electrons can be transferred from the redox-active disulfide/dithiol to the SeCys residue that is the active site to reduce the substrates [5,7]. Figure 1:Domain structure of human TrxR (htrxr1, htrxr2), E. coli TrxR (etrxr) and human glutathione reductase (hgr). The catalytic-site sequences are indicated by triangles and the position of the C-terminal penultimate SeCys residue is shown for the two human TrxRs. Adapted from [6]. Catalytic mechanism Numerous evidences supported that selenolthiol form (Cys-SeCys) was the actual activity site of the enzyme for reduction of the substates [5, 7, 8]. The enzyme would be inactivated if Cys-SeCys was truncated or SeCys was replaced by Ser residue [5, 7, 9]. The mechanisms for

6 lxiao TrxR 5 mammalian TrxR to reduce Trx were proposed in Figure 2 and Figure 3. During the reaction, the NADPH binds to the NADPH binding domain of TrxR, the electron flow from NADPH through FAD to reduce the conserved catalytic-site, -CVNVGC-, disulfide bond of TrxR. Then the thioldissulfide exchang results in oxidation of the CVNVGC- site and reduction of the C-terminal Cys-SeCys (selenol) site. The reduced Cys-SeCys transfers electrons to a substrate. So the active-site disulfide group maintains the selenol site in the reduced state by getting electrons from NADPH. Figure 2: Proposed model of mammlian TrxR based on homology to GR. ( upper) model of GR with FAD, NADPH, and interface domains. Glutathione disulfide bound is indicated as well as the activiesite (black circle). (lower) Model of mammalian TrxR. The elongation with 16-residues extends from the interface, positioning SeCys 498 and Cys 497 in one subunit adjacent to Cys 59 and Cys 64 of the other subunit. Adapted from [7].

7 lxiao TrxR 6 Figure 3. Postulated mechanism of mammalian TrxR. The catalytic reaction starts by reduction of the selenenylsulfide to the selenolate anion which get electrons from NADPH (A). The selenolate anion (-Se - ) attacks the disulfide of Trx (B). Then the enzyme-trx-mixed selenylsulfide is attacked by Cys 497 to regernerate the selenylsulfide (C). The selenenylsulfide will be redused by the Cys 59 (D). Then the disulfide between Cys 59 and Cys 64 get electrons from NADPH via FAD to recover the enzyme (E). Adapted from [7]. Physiological functions The biological function of TrxRs is very complicated. And some of the major physiological functions of mammalian thioredoxin system are summarized in Figure 4. We will briefly discuss the involvement of TrxR in protection of oxidation stress [11,13], cell growth and death [12], and Redox signaling-regulation [3].

8 lxiao TrxR 7 Figure 4: Graphic summary of major physiological function of the mammalian Thioredoxin system. Reduction of hydrogen peroxide and regeneration of the active site in glutathione peroxidase are reactions that can also be directlu catalyzed by TrxR. Thioredoxin-dependent reduction of hydrogen peroxide is highly efficient in conjunction with the activities of peroxiredoxins and to some extent glutathione peroxidases, although the latter also function without involvement of the thioredoxin system (via GSH and GR). Adapted from [11]. Protection of oxidation stress Reactive oxygen species (ROS) is part of product of normal metabolism [14]. The targets of ROS damage include all major groups of biomolecules, such as DNA, lipids, and proteins. And ROS-mediated tissue damage can result in cell death, mutation and carcinogenesis. Mammalian cells have antioxidant systems to protect them from the lethal damage caused by excessive ROS formation. These cellular antioxidant systems can be divided into enzymatic and non-enzymatic groups. The non-enzymatic antioxidant groups include some small compounds such as vitamin C and E. The enzymatic groups mainly include SOD, catalase, GPx, and TrxR. One of the striking characteristics of TrxR is that it has low substrate specificity. TrxR can reduce not only oxidized Trx, but also dehydroascorbic acid, lipid hydroperoxides (ROOH), and α-lipoic acid (Figure 4). The other characteristic of TrxR is that it can direct reduce different protein disulfides, many low molecular weight disulfide compounds, and nondisulfide compounds. And many antioxidants can be regenerated by these reduction reactions. Among

9 lxiao TrxR 8 them are selenium-containing enzymes and compounds, ascorbic acid (vitamin C) and α- tocopherol (vitamin E), lipoic acid, and ubiquinone (coenzyme Q/Q10) [11]. Many electrophilic compounds act as inhibitors of TrxR. These include arsenicals, 4- vinyl pyridine, iodo-acetic acid, and gold compounds used in treatment of rheumatoid arthritis. These inhibitors decrease TrxR activity, and hence decrease the regeneration of those antioxidants by TrxR. And the direct consequence of this inhibition is that cell has less total antioxidant capacity and leads to significant oxidative stress (Figure 5). Figure 5: Scheme of the effect of TrxR inhibition. Inhibition of TrxR will increase product of ROS, GSSG level, secretion of Trx, expression of NF-kB dependent protein, expression of ASK-1 and so on. The net result is significant intracellular oxidative stress. Increased oxidation of thioredoxin may lead to apoptosis via increased ASK-1 activity. Adapted from [11] Cell growth and death The redox state and activity of Trx are controlled by TrxR. And TrxR control cell growth and death mainly through Trx. Trx has been shown to exert redox control over some transcription factors to modulate their binding ability to DNA and hence regulate gene transcription. Transcription factors regulated by thioredoxin include NF- К B, the glucocorticoid

10 lxiao TrxR 9 receptor, and AP-1 (Fos/Jun heterodimer) through redox factor 1 (Ref-1). These transcriptional factors will induce cell proliferation. Evidences showed that Trx also acted as an apoptosis inhibitor [12]. Transfection of WEHI7.2 mouse thymoma-derive cells with human trx cdna is able to prevent apoptosis induced by dexamethasone [12]. Redox signaling-regulation ROS, together with Trx, is thought to participate in redox signaling. A model has been proposed to describe the role of TrxR (TR) in oxidative stress signaling (Figure 6). ROS leads to direct oxidation of TrxR and thus decrease the TrxR activity. The oxidation of Trx affects the Trx target proteins. In addition, ROS direct other proteins such as Trx. The whole system can be recycled as a result of subsequent increased expression of TrxR. Figure 6. Model for the role of TrxR in redox regulation of cell signaling. Adapted from [3]. Summary TrxR is an important antioxidant to prevent cells from ROS-induced damage, which is primarily through reduction of Trx, although Trx is not the only substrates of TrxR. The central

11 lxiao TrxR 10 role is played by thioredoxin system for redox control cell function and protection against oxidative damage is evident and more results will be unraveled by future studies. References 1. Williams, Jr., C. H. (1995) In chemistry and Biochemistry of Flavoenzymes, ed Muller, F. pp Zhong, L., Arner, E. S. J., Ljung, J., Aslund, F., Holmgren, A. (1998) Rat and calf thioredoxin reductase are homologous to Glutathione reductase with a carboxyl-terminal elongation containing a conserved catalytically active penultimate selenocysteinr residue. J Biol Chem. 273: Sun, Q., Wu, Y., Zappacosta, F., Jeang, K., Lee, B. J., Hatfield, D.L., Gladyshev, V. N. (1999) Redox regulation of cell signaling by selenocysteine in mammalian thioredixin reductases. J Biol Chem. 274: Gasdaska, P.Y., Gasdaska, J. R., Cochran, S., Powis, G. (1995) Cloning and sequencing of a human thioredoxin reductase. FEBS Letters 373: Zhong, L., Holmgren, A. (2000) Essential role of selenium in the catalytic activity of mammalian thioredox reductase revealed by characterization of recombinant enzyme with selenocysteine mutations. J Biol Chem.275: Mustacich, D., Powis, G. (2000) Thioredoxin Reductase. Biochem J. 346: Zhong, L., Arner, E. S. J., Homgren, A. (2000) Structure and mechanism of mammalian thioredoxin reductase: The activity site is a redox-active selenolthiol/selenylsulfide formed from the conserved cysteine- selenocysteine sequence. PNAS. 97: Berggren, M., Gallegos, A., Gasdaska, J., Powis, G. (1997) Cellular thioredoxin reductase activity is regulated by selenium. Anticancer Res. 17: Lee, S., Bar-Noy, S., Kwon, J., Levine, R. L., Stadtman, T. C., Rhee, S. G. (2000) Mammalian thioredoxin reductase: Oxidation of the C-terminal cysteine/selenocysteine active site forms a thioselenide, and replascement of selenium with sulfur markedly reduces catalytic activity. PNAS. 97: Holmgren, A.(1989) Thioredoxin and glutaredoxin systems. J Biol Chem. 264: Nordberg, J., Arner, E. S. J. (2001) Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radical Biol Med. 31: Powis, G., Gasdaska, J. R., Gasdaska, P. Y., Berggern, M. (1997) Selenium and the thioredoxin redox system: Effects on cell growth and Death. Oncology Res. 9: Ueno, M., Masutani, H., Arai, R. J., Yamauchi, A., Hirota, K., Sakai, T. Inamoto, T., Yamaoka, Y., Yodoi, J., Nikaido, T. (1990) Thioredoxin-dependent Redox Regulation of p53-mediated p21 Activition. J Biol Chem. 274: Cadenas, E. (1989) Biochemistry of oxygen toxicity. Annu Rev Biochem. 58: