The high Mr (~55 kDa) thioredoxin reductases (TR) characteristic of higher eukaryotes are members of the glutathione reductase (GR) family of pyridine nucleotide disulfide oxidoreductases. These homodimeric enzymes catalyze the reduction of a cognate disulfide substrate. During the enzymatic cycle, reducing equivalents pass from NADPH to the conserved active site disulfide via an enzyme-bound FAD and then to the cognate substrate. TRs are unique in the family as electrons are then transferred to the C-terminal active site of the adjacent molecule as part of a 16 amino acid extension (in place of the cognate GR substrate GSSG), prior to transfer to the substrate thioredoxin. Each electron transfer step occurs via thiol-disulfide exchange in a multi-step process mediated by a conserved catalytic acid/base. Mammalian TRs require selenocysteine (Sec) incorporated into the Gly-Cys-Sec-Gly-OH (GCUG) C-terminal tetrapeptide motif, while the TR from Drosophila melanogaster (DmTR) does not, and instead contains a Ser-Cys-Cys-Ser-OH (SCCS) tetrapeptide motif indicating that Sec is not universally necessary to catalyze the reduction of thioredoxin. This project has achieved three major objectives; 1) development of a semisynthetic method for production of mouse mitochondrial TR (mTR3) for structure-function studies, 2) establishment of a new method to study the mechanism of TR by using tetrapeptides in the oxidized form equivalent to the C-terminal active sites as substrates for the truncated forms of both enzymes, 3) determination of the crystal structure of DmTR. The results show that the structure of DmTR explains the biochemical data and has developed a new testable hypothesis in the field for the requirement of Sec in mammalian TR. We demonstrate that the tetrapeptides tested in Aim 2 were all better substrates for DmTR. The data also shows a far greater dependence on Sec for mTR3 than DmTR, which is in agreement with that observed for the collection full-length mutants produced for each enzyme in Aim 1. As this method of investigation is more analogous to the other enzymes of the GR family, the structures of the tetrapeptides determined by NMR spectroscopy were oriented in the active site of the both enzymes using the diglutathione bound in the structure of GR as template. DmTR appears to have a more open active site than observed in the known structure of mTR3. Residues from the helical face of the FAD-domain proximal to the FAD-associated active site are less bulky in DmTR to accommodate the hydroxyls of the serines. This is likely to make the enzyme more amenable for the conformational switching of the SCCS peptide necessary to protonate the leaving group cysteine by the proposed catalytic acid/base. In contrast, mTR3 shows a more restricted interface by incorporating bulkier residues at the interface in conjunction with the smaller Gly residues of the C-terminal sequence GCUG. The tetrapeptides display a conformational preference not suitable for protonation of the first leaving group in mTR3.

Mammalian thioredoxin reductase (TR) contains the rare amino acid selenocysteine (Sec), which is essential for the enzyme’s catalytic activity. Substitution of the catalytic Sec residue for a cysteine (Cys) residue, results in a drop in kcat of 100- fold. Homologous high molecular weight TRs from other eukaryotes such as D. melanogaster and C. elegans, have naturally evolved a Sec to Cys substitution in their active sites and these enzymes function with high catalytic activity without the need for a Sec residue. Thus, various TRs can catalyze an identical reaction with either a Cys or Sec residue. A natural assumption in the field has always been that the lower nucleophilicity of a Cys thiol, relative to the selenol of Sec, is the reason for the much lower activity of the mammalian Cys-containing mutant. However, here I provide an alternative explanation. High Mr TRs contain either a Cys-Cys or Cys-Sec dyad that forms an eight-membered ring in the oxidized state during the redox cycle of the enzyme. These eight-membered ring structures are rare in protein structures, presumably due to the strain induced in the intervening peptide bond between the Cys residues. Here I take a “chemical approach” to studying the enzyme mechanism of TR by breaking it into two pieces. This approach is possible because of TR’s structural and mechanistic similarity to glutathione reductase (GR). In comparison to GR, TR contains an additional thiol-disulfide exchange step resulting from the presence of a sixteen amino acid C-terminal extension containing either a vicinal disulfide bond or vicinal selenylsulfide bond. This additional thiol-disulfide exchange step is in the form of the reduction and opening of the eight-membered ring motif. I have constructed a truncated version of the enzyme lacking the amino acid sequence possessing the ring motif so that I could isolate this ring-opening step from the rest of the catalytic cycle by using peptide disulfides/selenylsulfides as substrates. The results of this study using peptide substrates show that the ring opening step is the step of the catalytic cycle that is most effected by Sec to Cys substitution because the higher pKa of the Cys thiolate in comparison to the Sec selenolate means that the Cys residue must be protonated in this step.

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.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska instututet, 2006. / Härtill 4 uppsatser.

No description available.

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.

No description available.

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.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.