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Peroxiredoxins are a family of multifunctional enzymes that are able to protect the cell against oxidative stress. Peroxiredoxins form part of a recently discovered peroxide scavenging system along with thioredoxin, thioredoxin reductase and sulfiredoxin. This study describes the purification of a recombinant human peroxiredoxin II from human erythrocytes. The original recombinant clone contained a point mutation at the fourth residue from glycine to valine and a number of problems were encountered with aggregation during purification. Reverting back to the original amino acid sequence allowed the protein to be purified and concentrated without aggregation, as well as leading to over-expression in the same oligomeric state as the native sample from blood. This study also describes the over-expression and purification of the human peroxiredoxin II protein in the intermolecular disulfide form as well as the subsequent crystallisation and X-ray diffraction studies. The crystal structure for this form of the protein was obtained to 3.3 Å resolution revealing the peroxiredoxin to be in the decameric form. In addition conformational changes in the protein that are necessary for formation of the intermolecular disulfide between the peroxidatic (Cys52) and resolving cysteine (Cys172) have been observed. The structure also revealed that these movements did not interfere with the dimer:dimer interface as had been previously suggested. This then allows the disulfide to be seen within the decameric form of peroxiredoxin. The production of covalent complexes formed between peroxiredoxin and sulfiredoxin, and peroxiredoxin and thioredoxin was also investigated. Complexes were stabilised by using DTNB to form a covalent bond between specific cysteine residues. The complex binding results from size exclusion chromatography showed that decameric peroxiredoxin bound to sulfiredoxin in a 1:5 ratio and decameric peroxiredoxin bound to thioredoxin in a 1:10 ratio. Cloning, over-expression and purification of the selenocysteine containing enzyme thioredoxin reductase was achieved. A minimal selenocysteine insertion sequence was added to the 3’ end of the DNA sequence to drive selenocysteine insertion in place of the typical stop UGA codon. The activity of this protein was found to be low but was greatly increased when co-expressed with a plasmid containing the selA, selB and selC genes. Although the activity of this co-expressed thioredoxin reductase was ~20% of the native enzyme activity, it was comparable to the activity of other recombinant forms of the enzyme. These studies report the purification of all of the proteins necessary to reform the peroxiredoxin system and allow the production of a working assay for peroxiredoxin activity. Together with the first report for a structure of a decameric disulfide form of human peroxiredoxin II a greater insight into the peroxiredoxin system has been obtained.

Trichomonas vaginalis is an anaerobic, parasitic protozoan, responsible for trichomoniasis, the world’s most common, non-viral sexually transmitted infection. Lacking many of the defenses present in other organisms to combat oxidative stress, Trichomonas vaginalis relies extensively on the thioredoxin system—NADPH, thioredoxin reductase, and thioredoxin—as a means to protect against exposure to excess oxygen. Current trichomoniasis treatment relies exclusively on the 5-nitroimidazole drugs, but fear of drug-resistant strains and allergic reactions to 5-nitroimidazole treatment necessitate the discovery of a new treatment method for trichomoniasis. Previous research has shown that auranofin, an FDA-approved drug, was effective at inhibiting activity of one of Trichomonas vaginalis’ isoforms of thioredoxin reductase (of which the organism has five total). Our research showed that only two of the isoforms were transcribed and expressed at high levels, and that both of these isoforms were susceptible to auranofin treatment. Not only that, these two isoforms were also shown to be susceptible to various auranofin analogs, having comparable or lower IC50 values. Further tests on these analogs might show that they are actually better treatment candidates if they exhibit less symptoms than auranofin. Experiments examining how mRNA and protein levels were modulated in response to two different concentrations of auranofin treatment showed that while some isoforms show increased levels, no one isoform experienced any drastic changes. Together, this data suggests that further studies should focus on these two most highly expressed isoforms of thioredoxin reductase.

Thioredoxin (Trx) and thioredoxin reductase (TrxR) along with an electron donor form a thioredoxin system. Such systems are widely distributed among the organisms belonging to the three domains of life. It is one of the major disulfide reducing systems, which provides electrons to several enzymes, such as ribonucleotide reductase, methionine sulfoxide reductase and glutathione peroxidase to name a few. It also plays an important role in combating oxidative stress and redox regulation of metabolism. Trx is a small redox protein, about 12 kDa in size, with an active site motif of Cys-X-X-Cys. The reduction of the disulfide in Trx is catalyzed by TrxR. Two types of thioredoxin reductases are known, namely NADPH thioredoxin reductase (NTR) with NADPH as the electron donor and ferredoxin thioredxoin reductase (FTR) which depends on reduced ferredoxin as electron donor. Although NTR is widely distributed in the three domains of life, it is absent in some archaea, whereas FTRs are mostly found in plants, photosynthetic eukaryotes, cyanobacteria, and some archaea. The thioredoxin system has been well studied in plants, mammals, and a few bacteria, but not much is known about the archaeal thioredoxin system. Our laboratory has been studying the thioredoxin systems of methanogenic archaea, and a major focus has been on Methanocaldococcus jannaschii, a deeply rooted archaeon that has two Trxs and one TrxR. My thesis research concerns the thioredoxin system of the late evolving members of the group which are exposed to oxygen more frequently than the deeply rooted members of the group, and have several Trxs and TrxRs. Methanosarcina mazei is one such organism, whose thioredoxin system is composed of one NTR, two FTRs, and five Trx homologs. Characterization of the components of a thioredoxin system sets the basis to further explore its function. I have expressed in Escherichia coli and purified the five Trxs and three TrxRs of M. mazei. I have shown the disulfide reductase activities in MM_Trx1 and MM_Trx5 by their ability to reduce insulin with DTT as the electron donor, and that in MM_Trx3 through the reduction of DTNB by this protein with NADPH as the electron donor, and in the presence of NTR as the enzyme. MM_Trx3 was found to be the only M. mazei thioredoxin to accept electrons through the NTR, and to form a complete Trx - NTR system. The Trx - FTR systems are well studied in plants, and such a system is yet to be defined in archaea. I have proposed a mechanism of action for one of the FTRs. FTR2 harbors a rubredoxin domain, and this unit is the only rubredoxin in this organism. Superoxide reductase, an enzyme that reduces superoxide radical to hydrogen peroxide without forming oxygen, utilizes rubredoxin as the direct electron source and this enzyme is found in certain anaerobes, including Methanosarcina species. Thus, it is possible that FTR2 provides electrons via a Trx to the superoxide reductase of M. mazei. This activity will define FTR2 as a tool in combating oxidative stress in M. mazei. In my thesis research I have laid a foundation to understand a complex thioredoxin system of M. mazei, to find the role of each Trx and TrxR, and to explore their involvement in oxidative stress and redox regulation.

Abstract: Aluminum (Al) and cadmium (Cd) are non-redox active metal ions of agricultural importance. Both are able to induce oxidative damage as a mechanism of toxicity. To increase our understanding of the mechanisms of Al and Cd toxicity and tolerance in plants, the potential role of antioxidant enzymes of the thioredoxin system, peroxiredoxins and glutaredoxins in Al and Cd tolerance was investigated in yeast (Saccharomyces cerevisiae) and Arabidopsis thaliana. Single and multiple mutants defective in genes of the thioredoxin system, peroxiredoxins and glutaredoxins in yeast were used to identify relevant genes in Al and Cd tolerance. A mutant defective in cytoplasmic thioredoxin reductase (TRR1) showed the most hypersensitive phenotype and increased levels of lipid peroxidation upon Al and Cd exposure. Transcript levels of TRR1 increased with increasing concentrations of Al and Cd. Complementation of the trr1Δ mutant with the wild-type TRR1 gene was able to restore growth to WT levels in the presence of Al and Cd. These results suggest that thioredoxin reductases are relevant genes in Al and Cd tolerance in yeast and that these genes might be also relevant in Al and Cd tolerance in plants. The role of NADPH-thioredoxin reductases (NTRs) in Al and Cd tolerance was further investigated in Arabidopsis thaliana. Among the mutants studied in Arabidopsis, only lines defective in NTRs (NTRA and NTRB) showed reduced growth compared to WT upon Al and Cd exposure. NTRA and NTRB mutants also showed increased levels of Al- and Cd-induced lipid peroxidation compared to WT. However, NTRA and NTRB transcript levels did not show a clear induction with increasing concentrations of Al and Cd. NTRA and NTRB were overexpressed in Arabidopsis to test whether increased expression of NTRs confers increased tolerance to Al and Cd. Only lines overexpressing NTRA showed increased transcript, protein and enzyme activity levels. However, these lines did not show increased tolerance to Al and Cd. My results suggest that NTRs might not be important ROS scavengers upon Al and Cd exposure, but they may play a role as a signal transducer and modulate other antioxidant enzymes.

Rho GTPases comprise a subgroup of the Ras superfamily of GTPases that controls a wide range of cellular processes by participating in processes such as actin cytoskeleton organization, phagocytosis, cell adhesion, cell-cycle progression and cell survival (Reviewed by Ridley, 2001). In budding yeast, Rho5 is a less characterized member of the Rho family of GTPase, and it shares considerable homology (45%) with Rac GTPase (another member of the Rho family of GTPases). One of the better-established functions of Rac is activation of NADPH oxidase in neutrophils that is required for killing microbes (Abo et al., 1991). The Rho family of proteins have a unique “Rho insert region” and this domain has been implicated in Rac mediated NADPH oxidase activation (Freeman et al., 1996). Historically, these oxidase were thought to function specifically in immune cells. However, in recent years, studies have shown that superoxides and ROS (reactive oxygen species) have important roles as signaling molecules, and they regulate a number of biological processes (reviewed by Werner, 2004, Veal et al., 2007). Although there are a considerable number of reports on the events associated with the oxidative stress response, and the role of Rho GTPases in the production of ROS, the mechanism of sensing and oxidant-mediated signaling remains to be fully established.In this study, the role of the Rho5 GTPase in hydrogen peroxide (H2O2)-induced cell death was explored. The presence of functional Rho5 in cells was found to confer sensitivity to various oxidants, including H2O2. Cells lacking Rho5 (i.e. Rho5-GTP bound form) exhibited much less cell death when exposed to H2O2, suggesting the presence of a Rho5-mediated cell death program. This Rho5-mediated cell death process was triggered by H2O2 and exhibited some hallmarks of mammalian apoptosis such as ROS accumulation and DNA fragmentation. Additionally, Rho5 was also found to interact with Trr1 (Thioredoxin reductase), an important component of the thioredoxin antioxidant system. Interestingly, Rho1 GTPase another member of the Rho family of GTPases was found to facilitate cell survival upon exposure to H2O2. These observations suggest that Rho GTPases control different cell fates depending on the oxidative stress level in promoting cell survival or cell death. This study uncovers a new role of a Rho GTPase in oxidant-induced cell death in yeast. This work also provides evidence for Rho5 interaction with Trr1, which is a novel link between a Rho GTPase and an antioxidant component.This study raises important questions about the extent to which the apoptotic cell death machinery is conserved from yeast through higher multicellular systems, and also about the benefits that a unicellular organism like yeast gains by retaining this function. This study has opened avenues to explore the above possibilities and hopefully identify more intracellular players functioning in the oxidative stress response and cell death processes. Identification of players that have homologous partners in higher systems will help determine the extent to which these cellular processes are conserved from yeast to humans.

Thioredoxin reductase (TrxR) catalyses the reduction of thioredoxin (Trx) by NADPH. Like other members of the pyridine nucleotide-disulfide family, TrxR is a homodimer. The catalytically active unit in the enzyme from Drosophila melanogaster (DmTrxR) consists of three redox centers: FAD and an N-terminal Cys-57/Cys-62 redox-active disulfide from one monomer, and a Cys-489’/Cys-490’ C-terminal redox-active disulfide from the second monomer. Because dipteran insects such as D. melanogaster lack glutathione reductase, glutathione disulfide must be reduced by Trx, making DmTrxR particularly important in this organism. DmTrxR is used as a model for the enzyme from a malaria vector, Anopheles gambiae. Based on the structures and mechanisms of other family members, a dyad of His-464’ and Glu-469’ acts as the acid-base catalyst of the dithiol-disulfide interchange reactions required in the catalysis of DmTrxR. The functions of His-464’ and Glu-469’ in the catalytic mechanism of DmTrxR were investigated. His-464’ was shown to be critical to catalysis by DmTrxR; thus, H464’Q retains only 2% of the wild-type activity. The pH dependence of Vmax for wild-type DmTrxR has apparent pKa values of 6.4 and 9.3, whereas H464’Q DmTrxR has an observable pKa only at 6.4, indicating that the pKa at pH 9.3 is contributed by His-464’. The macroscopic pKa at pH 6.4 has been assigned to Cys-57 and Cys-490’; the thiolate of Cys-57 is the nucleophile in the internal dithiol-disulfide interchange reaction and the thiolate of Cys-490’ is the nucleophile in the reduction of Trx. The rates of both the reductive and oxidative half reactions are markedly smaller in H464’Q DmTrxR than those of wild-type enzyme, indicating that His-464’ is involved in both half reactions. The pH dependence of the steady-state kinetics shows that the basicity of His-464’ decreases in the glutamate variants, as predicted. The reductive half-reactions of two glutamate variants are slower than those of wild-type enzyme. Malaria causes serious public health problems in the world. It is hoped that differences among TrxRs from human, Plasmodium falciparum (the causative agent) and Diptera (the vector) will be useful for developing differential inhibitors, useful as prophylactics.

Arsenic exposure, through various routes, is associated with the development of cancer of the skin, lung, liver, kidney, and bladder. Treatment of cells in culture with trivalent arsenicals has been shown to increase reactive oxygen species (ROS). In particular, monomethylarsonous acid (MMAIII), a trivalent metabolite of arsenite, is highly cytotoxic and possibly carcinogenic. Three trivalent arsenicals; arsenite, arsenic trioxide (ATO), and MMAIII, are also known inhibitors of the selenoprotein thioredoxin reductase (TrxR). Selenium, an essential micronutrient in mammals, is needed in the form of selenocysteine for activity of this enzyme and other selenoproteins. TrxR is part of a key component of the cell's ability to defend against ROS. It has been speculated that TrxR is also involved directly in selenium metabolism, but this has yet to be demonstrated in vivo. The promoter region of the gene encoding the cytosolic TrxR (TrxR1) also contains an antioxidant responsive element (ARE). The ARE is activated by the transcription factor, Nrf2, which is governed by the Nrf2/Keap1 response, and can be triggered by certain oxidants. ATO and arsenite both inhibited incorporation of selenium into selenoproteins. Auranofin, a gold chemotherapeutic inhibitor of TrxR1, also inhibited selenoprotein synthesis. These results seem to support the hypothesis that TrxR1 is needed for selenoprotein synthesis. However, siRNA mediated reduction of TrxR1 did not block incorporation of selenium into selenoproteins. It is likely that ATO and auranofin are forming As-Se and Au-Se complexes, respectively. We also found that exposure of primary lung fibroblasts (WI-38) to MMAIII led to increased synthesis of TrxR1. This increase was dependent on the activation of transcription of the TrxR1 gene, specifically mediated through the ARE element. These results indicate exposure to MMAIII induces the Nrf2 response. The results obtained in these studies aid in both our understanding of the carcinogenic potential of arsenic as well as give new insight into the mechanism of action of emerging cancer drugs.