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1
Paunescu, Karina. "DNA-Stabilität und Thioredoxin-Thioredoxin-Reduktase im Zellkern". [S.l.] : [s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=969680333.
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Osborne, Leisa Jane. "Characterisation of Thioredoxin Dimers: A Biochemical Study". Thesis, Griffith University, 2011. http://hdl.handle.net/10072/365531.
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In addition to the conserved active site cysteines that are responsible for the classical
redox activity of thioredoxins (Trx’s), vertebrate Trx’s contain an additional three
conserved cysteines at position 62, 69 and 73. These structural cysteines are known to
be subjected to a variety of post translational modifications including dimerisation that
are believed to contribute to the regulation and diversity of function of vertebrate Trx’s.
Reports of the formation of “disulphide linked dimers” have been a long standing
observation since the earliest studies on vertebrate Trx’s, however detailed studies on
dimerisation have been limited in number and the extent of characterisation achieved.
Despite the potential for a diversity of dimeric forms with different structural and
functional properties there has been a common assumption arising from the literature
(despite some evidence to the contrary) that all dimers so far described are much the
same and all are redox inactive.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Science
Science, Environment, Engineering and Technology
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3
Missirlis, Fanis. "Functional characterization of novel thioredoxin reductase and thioredoxin peroxidase in Drosophila". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2002. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp05/NQ65830.pdf.
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4
Shah, Fenil. "Thioredoxin and its Target Proteins: Thioredoxin Expression under Different Oxygen Conditions". Thesis, Griffith University, 2011. http://hdl.handle.net/10072/367670.
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Thioredoxin is an antioxidant protein that performs multiple functions in the
intracellular and extracellular environment of cells. Thioredoxin is highly expressed in
cancer cells, especially more metastatic and aggressive cancers. Previous studies have
demonstrated a functional role for thioredoxin in cancer cell invasion, however little
information is currently available regarding the role of thioredoxin in the invasive
process. In order to perform these and other functions, thioredoxin interacts with several
different protein partners. The primary aim of this project was to identify previously
unknown binding partners for thioredoxin, particularly those involved in the cancer cell
invasion process, on the cell surface of breast cancer cells.
In order to identify the binding partners for thioredoxin, a kinetic trapping protocol was
employed. Constructs that expressed a thioredoxin mutant wherein the second cysteine
of the active site (Cys35) is changed to a serine, were utilized to perform trapping
experiments. The trapping mutants remain covalently linked to the target protein
(because they lack the second cysteine to resolve the mixed disulfide bond formed),
allowing for purification of resulting thioredoxin–substrate complexes. One of the
known protein partners for thioredoxin is Methionine Sulfoxide Reductase A (MsrA).
Kinetic trapping experiments were performed to determine which of the four cysteine
residues within the MsrA protein directly bound to thioredoxin. The experiments were
performed using various MsrA cysteine mutants and determined that thioredoxin can
bind to either the 3rd or 4th cysteine residue within the MsrA sequence.
The kinetic trapping protocol was also used to attempt to identify previously unknown
binding partners for thioredoxin on the surface of MDA-MB-231 breast cancer cells.
Cell-surface trapping resulted in a single band visualized on anti-thioredoxin western
blots, which indicates the recovery of a protein partner. However, the high number of
cells used to get a positive result made it unrealistic to attempt even higher cell numbers
which would be required in order to trap enough protein to identify the unknown protein
partner.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Sciences
Science, Environment, Engineering and Technology
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5
Gregory, Mary Sarah-Jane, i n/a. "Thioredoxin and Oxidative Stress". Griffith University. School of Health Science, 2004. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20040301.082639.
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The experiments described in this thesis involve the expression and characterisation of recombinant truncated thioredoxin (tTrx) and the potential involvement that thioredoxin (Trx) has in the cellular responses to oxidative stress. Truncated Trx (80 amino acids) was expressed from a plasmid containing the ORF for tTrx that had been introduced into E.coli BL-21(DE3) cells. The protein was initially extracted using a combination of high concentrations of urea, high pH levels, and multiple sonification steps to remove the tTrx from inclusion bodies formed during expression. This procedure produced a stable solution of tTrx. Purification of tTrx from this protein solution required anion exchange chromatography followed by gel permeation in a HPLC system to obtain fully purified, recombinant tTrx which allowed further characterisation studies to be undertaken. An initial investigation into tTrx was performed to determine some basic physical, biochemical and functional aspects of this hitherto relatively undefined protein. Analysis by sedimentation equilibrium indicated that freshly prepared tTrx forms a single species with a molecular weight of 18.8kDa. This value indicates that recombinant tTrx naturally forms a dimer in solution that was shown to be non-covalent in nature and stable in solution. The capacity of tTrx to reduce protein disulphide bonds was determined using the insulin reduction assay. Results show that tTrx lacks this particular redox ability. The rate of oxidisation at 4 degrees C was analysed using free thiol determination, sedimentation equilibrium and SDS-PAGE patterning. Results indicated a steady rise in the degree of oxidation of tTrx over an eight day period. After six days the oxidated protein consistently displayed the presence of intramolecular disulphide bonds. Covalently-linked disulphide dimers and higher molecular weight oligomers were detectable after eight days oxidation. An investigation of the reducing capacity of the basic Trx system determined that fully oxidised tTrx was unable to act alone as a substrate for thioredoxin reductase (TR). However, when reduced Trx was added to the system, it appeared capable of acting as an electron donor to the oxidised tTrx in order to reduce disulphide groups. Recombinant tTrx was successfully radiolabelled with Trans 35S-methionine/cysteine for use in cell association studies. No evidence was found to indicate the presence of a receptor for tTrx on either MCF-7 or U-937 cells. Findings suggest that a low level of non-specific binding of tTrx to these cell lines rather than a classical ligand-binding mechanism occurs thus suggesting the absence of a cell surface receptor for tTrx. The role that Trx may play in the cellular responses to oxidative stress was also investigated. The chemical oxidants hydrogen peroxide (H2O2) and diamide were used to establish an in vitro model of oxidative stress for the choriocarcinoma cytotrophoblast cell line JEG-3. Cellular function was assessed in terms of membrane integrity, metabolic activity and the ability to synthesis new DNA following exposure to these oxidants. Results indicated that both agents were capable of causing cells to undergo oxidative stress without inducing immediate apoptosis or necrosis. Initially, JEG-3 cells exposed to 38μM or 75μM H2O2 or 100μM diamide were shown to display altered cell metabolism and DNA synthesis without loss to cell viability or membrane integrity. Cells were also shown to be capable of some short-term recovery but later lapsed into a more stressed state. Expression levels of Trx were studied to determine whether this type of chemical stress caused a change in intercellular protein levels. Both cELISA and western blotting results indicated that only cells exposed to 100μM diamide displayed any significant increase in Trx protein levels after 6 or 8hrs exposure to the oxidant. Further studies over a longer time-frame were also performed. These found that when JEG-3 cells were exposed to 18μM H2O2 or 200μM diamide over 12-48hrs, a positive correlation between increasing endogenous Trx protein levels and a decline in cell proliferation was observed. Cytotrophoblast cells, which are responsible for implantation and placentation, are susceptible to oxidative stress in vivo and their anti-oxidant capacity is fundamental to the establishment of pregnancy. The findings obtained during these studies suggest that Trx plays a role in this process.
6
Gregory, Mary Sarah-Jane. "Thioredoxin and Oxidative Stress". Thesis, Griffith University, 2004. http://hdl.handle.net/10072/367183.
Pełny tekst źródłaStreszczenie:
The experiments described in this thesis involve the expression and characterisation of recombinant truncated thioredoxin (tTrx) and the potential involvement that thioredoxin (Trx) has in the cellular responses to oxidative stress.
Truncated Trx (80 amino acids) was expressed from a plasmid containing the ORF for tTrx that had been introduced into E.coli BL-21(DE3) cells. The protein was initially extracted using a combination of high concentrations of urea, high pH levels, and multiple sonification steps to remove the tTrx from inclusion bodies formed during expression. This procedure produced a stable solution of tTrx. Purification of tTrx from this protein solution required anion exchange chromatography followed by gel permeation in a HPLC system to obtain fully purified, recombinant tTrx which allowed further characterisation studies to be undertaken.
An initial investigation into tTrx was performed to determine some basic physical, biochemical and functional aspects of this hitherto relatively undefined protein. Analysis by sedimentation equilibrium indicated that freshly prepared tTrx forms a single species with a molecular weight of 18.8kDa. This value indicates that recombinant tTrx naturally forms a dimer in solution that was shown to be non-covalent in nature and stable in solution. The capacity of tTrx to reduce protein disulphide bonds was determined using the insulin reduction assay. Results show that tTrx lacks this particular redox ability.
The rate of oxidisation at 4 degrees C was analysed using free thiol determination, sedimentation equilibrium and SDS-PAGE patterning. Results indicated a steady rise in the degree of oxidation of tTrx over an eight day period. After six days the oxidated protein consistently displayed the presence of intramolecular disulphide bonds. Covalently-linked disulphide dimers and higher molecular weight oligomers were detectable after eight days oxidation.
An investigation of the reducing capacity of the basic Trx system determined that fully oxidised tTrx was unable to act alone as a substrate for thioredoxin reductase (TR). However, when reduced Trx was added to the system, it appeared capable of acting as an electron donor to the oxidised tTrx in order to reduce disulphide groups.
Recombinant tTrx was successfully radiolabelled with Trans 35S-methionine/cysteine for use in cell association studies. No evidence was found to indicate the presence of a receptor for tTrx on either MCF-7 or U-937 cells. Findings suggest that a low level of non-specific binding of tTrx to these cell lines rather than a classical ligand-binding mechanism occurs thus suggesting the absence of a cell surface receptor for tTrx.
The role that Trx may play in the cellular responses to oxidative stress was also investigated. The chemical oxidants hydrogen peroxide (H2O2) and diamide were used to establish an in vitro model of oxidative stress for the choriocarcinoma cytotrophoblast cell line JEG-3. Cellular function was assessed in terms of membrane integrity, metabolic activity and the ability to synthesis new DNA following exposure to these oxidants. Results indicated that both agents were capable of causing cells to undergo oxidative stress without inducing immediate apoptosis or necrosis. Initially, JEG-3 cells exposed to 38μM or 75μM H2O2 or 100μM diamide were shown to display altered cell metabolism and DNA synthesis without loss to cell viability or membrane integrity. Cells were also shown to be capable of some short-term recovery but later lapsed into a more stressed state.
Expression levels of Trx were studied to determine whether this type of chemical stress caused a change in intercellular protein levels. Both cELISA and western blotting results indicated that only cells exposed to 100μM diamide displayed any significant increase in Trx protein levels after 6 or 8hrs exposure to the oxidant. Further studies over a longer time-frame were also performed. These found that when JEG-3 cells were exposed to 18μM H2O2 or 200μM diamide over 12-48hrs, a positive correlation between increasing endogenous Trx protein levels and a decline in cell proliferation was observed. Cytotrophoblast cells, which are responsible for implantation and placentation, are susceptible to oxidative stress in vivo and their anti-oxidant capacity is fundamental to the establishment of pregnancy. The findings obtained during these studies suggest that Trx plays a role in this process.Thesis (Masters)
Master of Philosophy (MPhil)
School of Health Sciences
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7
Björkhem, Bergman Linda. "Thioredoxin reductase and selenium in carcinogenesis and multidrug resistance /". Stockholm, 2004. http://diss.kib.ki.se/2004/91-7349-954-4/.
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8
Zhong, Liangwei. "Selenium in mammalian thioredoxin reductase /". Stockholm, 2000. http://diss.kib.ki.se/2000/91-628-4243-9/.
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9
Callister, Matthew Eric James. "Thioredoxin and the inflammatory response". Thesis, Imperial College London, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.414905.
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10
Rozell, Björn. "Immunohistochemical studies of the thioredoxin system". Göteborg : Dept. of Histology, University of Göteborg, 1987. http://catalog.hathitrust.org/api/volumes/oclc/17242526.html.
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11
Sze, Jun Hui. "Targeting Thioredoxin Reductase in Multiple Myeloma". Thesis, Griffith University, 2020. http://hdl.handle.net/10072/392857.
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Multiple myeloma (MM) is a clonal plasma B-cell neoplasm characterised by the presence of uncontrolled proliferation of antibody-secreting plasma cells in the bone marrow. Despite therapeutic advancements, MM remains incurable due to its low median survival rate and nearly all patients will eventually relapse regardless of the frontline regimen they receive. Thus, alternative therapeutic strategies that target the pathogenic and resistance mechanisms of MM need to be developed to improve overall survival in MM patients.
Cancer cells generally have higher metabolic demands due to their high proliferative nature and as such, production of intracellular by-products including free reactive oxygen species (ROS) are also increased, which can cause oxidative cell death. Hence, cancer cells exploit antioxidant molecules to maintain intracellular redox homeostasis and to prevent further cell damage from the anticancer drugs that induce oxidative stress. One of the major antioxidant systems is the thioredoxin (Trx) system, which is comprised of thioredoxin (Trx), thioredoxin reductase (TrxR), and nicotinamide adenine dinucleotide phosphate (NADPH), all of which as one can scavenge ROS, reduce oxidised proteins and redox-sensitive molecules, and regulate transcription factors. Myeloma cells have been shown to have high Trx and TrxR expression, which correlates with increased cell proliferation, evasion of programmed cell death and chemoresistance.
Gold compounds have a high affinity for thiol and selenol groups, which makes the Trx system vulnerable to these compounds. In this study, TrxR was targeted in myeloma cells with gold(I) compounds, including auranofin and [Au(d2pype)2]Cl. [Au(d2pype)2]Cl exhibited significant inhibition on TrxR activity in both in vitro and in vivo models, which led to induction of ROSmediated cell death in myeloma cells. [Au(d2pype)2]Cl also completely abrogated the tumourigenic capacity of myeloma cells in clonogenic assays, whereas auranofin was less effective. The expression of the MYC oncogene, which drives myeloma progression, was also downregulated in both in vitro and in vivo models when treated with [Au(d2pype)2]Cl.
Auranofin and [Au(d2pype)2]Cl were further evaluated in 2D co-culture assays to determine their efficacy in overcoming environment mediated drug resistance (EMDR) in myeloma cells that are co-cultured with bone marrow stromal cells (BMSCs) in normoxic (20% O2) and hypoxic (1% O2) conditions. [Au(d2pype)2]Cl generally displayed much better efficacy by exhibiting lower IC50 values as compared to auranofin when used on either the attached or non-attached myeloma cells in both normoxia and hypoxia. Based on the potency that [Au(d2pype)2]Cl displayed over auranofin in these co-culture assays, [Au(d2pype)2]Cl was selected as a tool for subsequent studies on the role of the Trx system in the molecular mechanisms that contribute to drug resistance in MM.
Cell adhesion mediated drug resistance (CAM-DR) occurs from the intercellular adhesion between myeloma cells and the bone marrow microenvironment (BMME). Several studies have indicated CAM-DR is associated with the activation of NF-ĸB and causes a subset of myeloma cells attached to BMSCs and/or extracellular matrix (ECM) to be chemo-resistant prior to MM therapy. In this study, inhibition of TrxR with [Au(d2pype)2]Cl decreased the adhesive capabilities of myeloma cells to BMSCs and was accompanied by downregulation of integrin Very Late Activation Antigen-4 (VLA-4), which is an important cell adhesion molecule responsible for CAM-DR in MM. [Au(d2pype)2]Cl treatment also significantly downregulated protein levels of the p65 subunit of NF-ĸB as well as expression of NF-ĸB-regulated genes. Moreover, the expression of VLA-4 was markedly decreased when NF-ĸB was inhibited by BAY 11-7082, inhibitor of IĸB kinase (IKK), similar to results obtained when [Au(d2pype)2]Cl was used. These findings suggest that the Trx system may affect the expression of VLA-4 in myeloma cells by regulating the NF-ĸB signalling pathway.
Based on the results obtained with VLA-4, the role of another cell adhesion molecule, mucin 1 (MUC1) was evaluated in this study. MUC1 is aberrantly expressed in MM and is involved in oncogenesis as well as regulation of ROS. Since there is a possible link between the redox cellular balance and MUC1, this study investigated if inhibition of TrxR could affect MUC1 expression in myeloma cells. Results showed that inhibition of TrxR decreased the expression of MUC1 in myeloma cells that were co-cultured with BMSCs. Moreover, the adhesion of myeloma cells to BMSCs was significantly decreased following the inhibition of MUC1 with either siRNA or MUC1 peptide inhibitor, GO201. Treatment of myeloma cells with [Au(d2pype)2]Cl resulted in decreased expression of NF-ĸB and Sp1, which coincided with a decreased expression of MUC1.
In conclusion, targeting TrxR with improved gold(I)-based compounds may provide an effective anti-myeloma approach for the future development of MM therapies. Moreover, [Au(d2pype)2]Cl has been shown to be useful for future functional studies that will focus on investigating the mechanisms that are involved in the cellular changes related to the effects from TrxR inhibition.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environment and Sc
Science, Environment, Engineering and Technology
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12
James, Paul Brian Charles. "Investigation into peroxiredoxin and interactions in the peroxiredoxin peroxide scavenging system". Thesis, University of Exeter, 2010. http://hdl.handle.net/10036/3162.
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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.
13
Nalvarte, Ivan. "Functional characterization of cytosolic and mitochondrial thioredoxin reductases /". Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-919-X/.
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Jauregui, Jose. "Auranofin Targets Thioredoxin Reductases in Trichomonas vaginalis". Scholarly Commons, 2017. https://scholarlycommons.pacific.edu/uop_etds/2976.
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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.
15
Damdimopoulos, Anastasios E. "Identification and functional characterization of novel thioredoxin systems /". Stockholm,, 2003. http://diss.kib.ki.se/2003/91-7349-661-8/.
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蕭嘉慧 i Ka-wai Siu. "Identification of biological inhibitors of the mammalian thioredoxin system". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1999. http://hub.hku.hk/bib/B31221634.
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Eckenroth, Brian E. "The Mechanism of High MR Thioredoxin Reductase Investigated by Semisynthesis and Crystallography". ScholarWorks @ UVM, 2007. http://scholarworks.uvm.edu/graddis/73.
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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.
18
Loganathan, Usha R. "Characterization of the thioredoxin system in Methanosarcina mazei". Thesis, Virginia Tech, 2014. http://hdl.handle.net/10919/71334.
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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.Master of Science
19
Matsuo, Yoshiyuki. "Identification of a Novel Thioredoxin-related Transmembrane Protein". Kyoto University, 2001. http://hdl.handle.net/2433/150552.
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Ueno, Masaya. "Thioredoxin-dependent redox regulation of p53 mediated-p21activation". Kyoto University, 2000. http://hdl.handle.net/2433/180842.
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Nishiyama, Akira. "Identification of thioredoxin-binding protein-2/vitamin D_3 up-regulated protein 1 as a nagative regulator of thioredoxin function and expression". Kyoto University, 1999. http://hdl.handle.net/2433/181259.
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Clapper, Erin M. "Investigating Intrinsic and Extrinsic Mechanisms of Tyrosine Kinase Inhibitor Resistance in Chronic Myeloid Leukaemia". Thesis, Griffith University, 2021. http://hdl.handle.net/10072/409643.
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myeloid leukaemia (CML) is a myeloproliferative disorder that is responsible for 15% of all adult leukaemia cases. While the initial stages of CML are relatively mild, the terminal stage of disease, known as blast crisis, has an average survival time of approximately 12 months. CML is caused by a reciprocal chromosomal translocation that results in the production of a constitutively active non-receptor tyrosine kinase, known as bcr-abl. Bcr-abl activates a wide variety of cell proliferation and survival pathways, and this leads to abnormal cell growth and therefore cancer. Due to the involvement of bcr-abl in the progression of CML, most of the treatments for this cancer are bcr-abl specific tyrosine kinase inhibitors (TKIs). Whilst initially effective, various studies have found that resistance to TKIs occurs in 20-50% of CML cases. This is primarily linked to the highly mutagenic nature of bcr-abl. The current strategy to overcome acquired TKI resistance is to prescribe a newer generation of TKI, which is often either partly or completely ineffective. Therefore, to more effectively overcome acquired drug resistance in CML, bcr-abl independent targets need to be identified and investigated. This thesis outlines three distinct cellular systems and assesses their effect on drug resistance in CML, and their potential as targets for future CML treatments.
The first system that was investigated in this thesis was the thioredoxin (Trx) system, which is involved in maintaining redox homeostasis within the cell. Upregulation of the Trx system has been associated with increased progression and poor prognosis in other cancers. In this thesis it was observed that Trx1 expression was increased in both CML cell lines and CML patient samples, compared to non-cancerous controls. Furthermore, it was found that Trx1 expression was increased in CML cells that were resistant to imatinib treatment, compared to imatinib sensitive cells. Additionally, an enzymatic inhibitor of Trx1, known as thioredoxin interacting protein (TXNIP) was downregulated in CML cells compared to non-cancerous cells, and was also downregulated in imatinib resistant CML cells.
It was found that inhibiting a key element of the Trx system, thioredoxin reductase (TrxR), using chemical and specific siRNA inhibitors resulted in a decrease in the activity and expression of bcr-abl. TrxR inhibition also resulted in decreased protein expression of MYC, which is reported to regulate the transcription of bcr-abl, suggesting that downregulation of MYC could be the mechanism by which TrxR inhibitors decreased bcr-abl expression. Moreover, this thesis found that TrxR inhibition by chemical inhibitors effectively overcame imatinib induced drug resistance, further demonstrating the promising anti-cancer ability of these compounds. Inversely, the inhibition of bcr-abl by four distinct TKIs as well as bcr-abl specific siRNA resulted in decreased expression of both Trx1 and TrxR1, and decreased TrxR activity in CML cells. The inhibition of bcr-abl by TKIs is not the direct cause of apoptosis in CML, it is instead the downregulation of the downstream targets of bcr-abl; this led to the hypothesis that the inhibition of the Trx system is partly responsible for TKI induced apoptosis.
Another mechanism of TKI resistance in CML that was investigated in this thesis was hypoxia, which is defined physiologically to be oxygen levels below 3%. An example of a hypoxic environment within the body is the bone marrow, where CML cells spend a large portion of their lifespan. When cells enter hypoxia, their biology changes in a multitude of ways and this has been linked to drug resistance in many cancers, including CML. The hypoxia inducible factor 1 (HIF-1) pathway is almost always linked to hypoxia-induced drug resistance. The HIF-1 pathway is only active in hypoxia and upregulates the expression of many downstream targets. In this study it was observed that CML cell growth in hypoxia was significantly decreased compared to normoxia, while cell viability and apoptosis levels remained unchanged. Furthermore, the expression of several cyclins was decreased in hypoxia, and this is likely why cell proliferation was slowed. MTT proliferation assays used to assess cell proliferation demonstrated that TKIs were far less effective in hypoxia compared to normoxia.
The Trx system was also observed to be downregulated in hypoxia. However, when cells underwent reoxygenation (incubated in hypoxia and then returned to normoxia for a short period), it was found that expression of the Trx system was significantly increased, which was due to the increase in reactive oxygen species (ROS) levels. Finally, TrxR inhibitors were shown to retain most of their efficacy under low oxygen conditions, in contrast to TKIs which were ineffective in hypoxia. The final aspect of hypoxia investigated was the decreased bcr-abl protein expression observed in hypoxia, which was shown to occur via the activity of the HIF-1 pathway. Using RNA immunoprecipitation, it was found this was specifically through the downregulation of the ribosomal protein RPS6 in hypoxia, as RPS6 mediates the translation of bcr-abl.
The impact of ATP binding cassette (ABC) transporters on TKI resistance in CML was also assessed. ABC transporters induce drug resistance by exporting compounds out of the cell before they can have their full effect. The upregulation of ABC transporters has been associated with increased drug resistance in various cancers. An aim of this thesis was to identify an ABC transporter that had not previously been associated with drug resistance in CML. Using publicly available RNAseq data, it was found that multidrug resistance protein 4 (MRP4) was upregulated in CML patients that were unresponsive to imatinib, compared to imatinib responsive patients. Further studies showed that MRP4 was upregulated in CML cell lines compared to non-cancerous controls, as well as in CML patients in the blast crisis phase of the disease compared to healthy donors.
The effect of MRP4 activity on TKI efficacy was then assessed by specifically inhibiting MRP4 and examining differences in cell growth induced by TKIs. Using MTT proliferation and apoptosis assays, it was found that MRP4 inhibition increased the efficacy of both ponatinib and dasatinib, suggesting that MRP4 may be involved in the export of these TKIs out of the cell. Since MRP4 is reportedly under transcriptional control of Nrf2 (which also regulates expression of the Trx system), this thesis also aimed to examine any link between the Nrf2/Trx system and MRP4. Activation of Nrf2 resulted in increased MRP4 expression and activity, however, TrxR inhibitors also induced a similar response. This is because inhibiting the Trx system results in an increase in ROS, which therefore induces the activation of Nrf2. Inhibition of MRP4 also increased the efficacy of TrxR inhibitors. Inhibition of MRP4 may therefore be a promising co-treatment for current or future CML chemotherapeutics.
It was shown in this thesis that MRP4 expression was upregulated in hypoxia in a HIF-1 dependant manner. Upon further investigation, it was found that this upregulation was potentially mediated by the increased expression of adenylyl cyclase 6 in hypoxia, as cAMP accumulation is a major regulator of MRP4 expression. Reoxygenation of CML cells resulted in an increase of MRP4 expression, and this was thought to be due to the increase in Nrf2 expression observed in these conditions. These two results show that changes in the oxygenation state of the cell influence the expression of MRP4 and could potentially lead to increased drug resistance. This was further investigated by inhibiting MRP4 in hypoxia and examining the effect of this on TKI efficacy. MRP4 inhibition sensitised CML cells in hypoxia to both ponatinib and dasatinib. This result reiterates the potential of using MRP4 inhibitors as a co-treatment with other CML chemotherapeutics.
Overall, this thesis outlined the effect of the Trx system, hypoxia and MRP4 on TKI resistance in CML, as well as the interactions between these systems and with bcr-abl. Due to the prevalence of bcr-abl dependant forms of drug resistance in CML, alternative treatments need to be investigated and utilised. It was found that the inhibition of the Trx system using TrxR specific chemical inhibitors was able to overcome both acquired TKI resistance and hypoxia induced drug resistance. Furthermore, specifically inhibiting MRP4 activity increased the efficacy of clinically used TKIs, as well as TrxR inhibitors. MRP4 inhibition was also able to sensitise CML cells to TKIs in hypoxia. These results demonstrate that MRP4 inhibitors may also make promising co-treatments for the management of CML.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environment and Sc
Science, Environment, Engineering and Technology
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McKown, Richard Dwayne. "Localization and partial immunological characterization of Fasciola hepatica Thioredoxin". Texas A&M University, 2004. http://hdl.handle.net/1969.1/1401.
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This study reports the localization and partial characterization of thioredoxin from the parasitic trematode Fasciola hepatica. Snails (Pseudosuccinia columella) were raised in culture and infected with F. hepatica so that Western blotting and immunohistochemical techniques could be utilized to determine the presence of thioredoxin in different stages of the parasites development. The results of these experiments showed that thioredoxin was present in the tegument, gut epithelium, excretory canal epithelium and sperm, of the adult parasite as well as in the tegument and gut of the redia and cercaria intermediate stages. In situ hybridization was used to determine the localization and possible differential mRNA expression of two different F. hepatica thioredoxin isotypes (Fh2020.A and Fh2020.SL) in the adult parasite. The in situ hybridization results showed that both isotypes are expressed in the tegument and gut epithelium. Fh2020.A stains with a greater intensity possibly demonstrating a difference in the amount of expression between the two isotypes.
Recombinant F. hepatica thioredoxin expressed in bacteria using the pMAL Protein Fusion and Expression System was used to test its affects on the production of super oxide anion by murine peritoneal macrophages, bovine monocyte-derived macrophages and bovine whole blood neutrophils, and nitric oxide production by mouse peritoneal macrophages and bovine monocyte-derived macrophages. The results of the cellular assays were not definitive due to the fact that the maltose binding protein (MBP) moiety of the recombinant thioredoxin, when tested alone, increased production of nitric oxide by bovine monocyte-derived macrophages. Consequently, since the MBP could not be effectively separated from the thioredoxin portion of the recombinant, allowing the thioredoxin affects to be tested independently, no true conclusions regarding its affects on the host immune cells tested could be drawn.
This is the first report of the localization of thioredoxin in both the adult F. hepatica as well as in specific intermediate stages of the parasite. These studies demonstrate the possible affects that a protein tag can have on experimental results and demonstrate how such data may be interpreted when a non-cleaved recombinant protein is used in cellular or other assays when compared to native or cleaved recombinant proteins.
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Chiu, Joyce Biotechnology & Biomolecular Sciences Faculty of Science UNSW. "Protein engineering of DNA polymerase I: thioredoxin dependent processivity". Awarded by:University of New South Wales. School of Biotechnology and Biomolecular Sciences, 2005. http://handle.unsw.edu.au/1959.4/23077.
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DNA polymerases are found in a diverse range of organisms, prokaryotes, eukaryotes, viruses and bacteriophage. T7 DNA polymerase is a replicative enzyme from E. coli bacteriophage T7. It relies on the thioredoxin binding domain (TBD) of phage gene 5 protein (gp5) and E. coli thioredoxin (Trx) for processive replication of phage DNA. Although T7 DNA polymerase is processive, it is also thermolabile. In order to design a thermostable and processive DNA polymerase, the structural stabilities of the TBD and Trx were studied in respect to their binding affinity and affect on enzyme processivity. An artificial operon was designed for coexpression of subunits of T7 DNA polymerase. By means of a 9??His-tag at the amino terminus of gp5, T7 DNA polymerase complex was purified by one-step nickel-agarose chromatography, with subunits gp5 and Trx co-eluting in a one to one molar ratio. Purified T7 DNA polymerase was assayed for polymerase activity, processivity and residual activity and compared to the commercial T7 DNA polymerase. The two enzymes were not identical with commercial T7 DNA polymerase being less processive at 37??C. Mass spectrometry of the two enzymes identified a mutation of Phe102 to Ser in the Trx subunit (TrxS102) of commercial T7 DNA polymerase. The Ser102 mutation, was found near the carboxyl terminal helix of Trx. TrxS102 was less stable than wild type Trx. In the study of the TBD structural stability, a hybrid polymerase was constructed by inserting the TBD motif into the homologous position in the Stoffel fragment of Taq DNA polymerase. The hybrid enzyme was coexpressed with Trx from an artificial operon; however, the TBD inserted retained a mesophilic binding affinity to Trx. The chimeric polymerase required 100 molar excess of Trx for processive polymerase activity at 60??C. TBD structural deformation at elevated temperatures was hypothesized to be the cause of the change in the subunit stoichiometry. Mutagenesis of TBD would be required to increase its thermostability. An efficient, rapid high throughput mutagenesis method (SLIM) was invented and would be appropriate for further studies.
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Ren, Bin. "Crystallographic studies on redox enzymes containing the thioredoxin fold /". Stockholm, 1999. http://diss.kib.ki.se/1999/91-628-3302-2/.
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Lee, Chi-wai. "Impact of gestational diabetes mellitus on placental thioredoxin system". Click to view the E-thesis via HKUTO, 2007. http://sunzi.lib.hku.hk/HKUTO/record/B39558897.
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Leaver, Susannah. "Intracellular and extracellular thioredoxin implications for the inflammatory response". Thesis, Imperial College London, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.537565.
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Lee, Chi-wai, i 李志慧. "Impact of gestational diabetes mellitus on placental thioredoxin system". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2007. http://hub.hku.hk/bib/B39558897.
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Takashima, Yuichiro. "Differential expression of glutaredoxin and thioredoxin during monocytic differentiation". Kyoto University, 2000. http://hdl.handle.net/2433/151447.
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Eftekharpour, Eftekhar. "Glutathione dependent and thioredoxin dependent peroxidase systems in neural cells". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp05/NQ63863.pdf.
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Ho, Ian-ian. "Does Ras/MEK signaling stimulate the expression of thioredoxin reductase? /". View the Table of Contents & Abstract, 2007. http://sunzi.lib.hku.hk/hkuto/record/B38348123.
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Hall, Gareth A. F. "Structural and functional analysis of thioredoxin and associated inhibitor complexes". Thesis, University of Nottingham, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.495589.
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The thioredoxin redox system, consisting of the thioredoxin protein, thioredoxin reductase and NADPH, is found in both prokaryotic and eukaryotic cells. This essential redox system is known to be important in a multitude of biological functions, including cell cycle regulation and maintainuig an intracellular reduced state. The crystallisation of thioredoxin and thioredoxin-complex structures, covered in this study, has allowed for a detailed analysis of the active site region for the application of drug targeting. understanding of the specific requirements that thioredoxin has for its target substrates, and has thus allowed for pseudo structure based drug design.
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Ho, Ian-ian, i 何欣欣. "Does Ras/MEK signaling stimulate the expression of thioredoxin reductase?" Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2007. http://hub.hku.hk/bib/B45011217.
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Floen, Miranda J. "Thioredoxin-1| Identification of redox substrates and response to hyperoxia". Thesis, University of South Dakota, 2016. http://pqdtopen.proquest.com/#viewpdf?dispub=10132866.
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Bronchopulmonary dysplasia (BPD) is a serious respiratory complication for the preterm newborn characterized clinically by prolonged respiratory distress and histologically by alveolar simplification and decreased pulmonary vasculature. The development of BPD is well linked to oxidative stress suffered by the newborn as a result of a preterm fetal-neonatal transition, supplemental oxygen, infection, increased inflammation, and mechanical ventilation. Damage suffered by oxidative stress may be through direct mechanisms or through alteration of redox¬sensitive pathways involved in cell death, cell survival, differentiation, and proliferation. Redox¬sensitive modifications regulating protein function and redox-sensitive pathways have mainly been ascribed to oxidative modification of cysteine thiols. As their modification is critical for protein function, maintenance of the thiol redox status is crucial. Thioredoxin-1 (Trx1) functions in maintenance of thiol redox homeostasis, and its redox activity is intimately linked to antioxidant, cytoprotection, proliferation responses, and cytoprotection. While Trx1 targets of redox regulation have been identified, we hypothesize that additional protein may be redox regulated and that Trx1 target profiles may change during oxidative stress. Therefore a novel immunoprecipitation approach, identified as the substrate trap approach, was developed to identify Trx1 targets. The following demonstrates the use of the substrate trap approach for identification of Trx1 redox targets and further application of the approach to identify alterations in target profiles in response to oxidative stress. Use of nuclear targeted substrate trap was successfully employed to enrich from nuclear Trx1 targets. As a final component the characterization of the Trx1 system in mouse from late embryonic development through the first week of life animals were exposed to room air or hyperoxia (model of BPD). Characterization indicates impairment of the Trx1 system in response to hyperoxic injury. As Trx1 is known to regulate proliferation, cell death, survival, differentiation pathways, impairment of the Trx1 system during early neonatal development may potentiate hyperoxic injury and alterations in lung development. Better understanding of Trx1 interactions occur through the substrate trap in a physiological model of BPD will help elucidate redox-signaling pathways involved in BPD pathogenesis.
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Tan, Aiguo. "Thioredoxin-1 attenuates indomethacin-induced gastric mucosal injury in mice". Kyoto University, 2008. http://hdl.handle.net/2433/135862.
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Hanschmann, Eva-Maria [Verfasser]. "Thioredoxin family proteins in physiology and disease / Eva-Maria Hanschmann". Marburg : Universitätsbibliothek Marburg, 2011. http://d-nb.info/1016617615/34.
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Zurek, Mark [Verfasser]. "Regulation der kardialen Myofibroblastendifferenzierung – Rolle von Thioredoxin-1 / Mark Zurek". Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2020. http://d-nb.info/1212238664/34.
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Dai, Shaodong. "Structural and functional studies of NADPH and ferredoxin dependent thioredoxin reductases /". Uppsala : Swedish Univ. of Agricultural Sciences (Sveriges lantbruksuniv.), 1998. http://epsilon.slu.se/avh/1998/91-576-5480-8.gif.
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Attarian, Rodgoun. "Detoxification of glutathione and nitrosoglutathione by thioredoxin system of Mycobacterium tuberculosis". Thesis, University of British Columbia, 2009. http://hdl.handle.net/2429/11175.
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Tuberculosis is the leading cause of mortality due to a single pathogenic infection. Its etiological agent, Mycobacterium tuberculosis infects, resides and multiplies within human alveolar macrophages. It is exposed to reactive oxygen intermediates and reactive nitrogen intermediates (RNI) such as nitric oxide (NO) produced within phagosomes and granulomas against invading pathogens. Therefore, proliferation of M. tuberculosis within the host depends on its strategies to counteract the onslaught of these intermediates. One example is recruitment of the thioredoxin system as one of the most proficient pathways for protection against oxidative stress, since it dominates peroxide detoxification pathways.
A burst of NO within macrophages parallels the production of glutathione (GSH) to protect the host against NO toxicity. The macrophage GSH pool reduces NO to S-nitrosoglutathione (GSNO). Both glutathione disulphide (GSSG) and GSNO possess mycobactericidal activities in vitro.
This thesis is focused on characterizing the role of M. tuberculosis thioredoxin system in detoxification of antimycobacterial compounds produced within the host such as GSSG and GSNO, due to the intrinsic capacity of the system to universally reduce disulfide bonds and reduce GSNO in humans. By performing NADPH oxidation assays and HPLC analysis we demonstrate that M. tuberculosis thioredoxin redox cascade is a general reduction system able to efficiently reduce the low molecular weight disulfides GSSG and MSSM, and dissimilate GSNO.
We also investigated the cellular pathways in which thioredoxin of M. tuberculosis participates. Here, we present an analysis of the thioredoxin-linked M. tuberculosis proteome by using a substrate trapping assay and mass spectrometry. We have identified eleven proteins associated with TrxC, implicating the involvement of thioredoxin in distinct cellular processes in this pathogen.
The findings described in this thesis elucidate a novel function for the thioredoxin system of M. tuberculosis. We demonstrate that this system serves as a detoxification pathway against mycobactericidal compounds such as GSSG and GSNO. Overall, the data presented here establishes that M. tuberculosis thioredoxin has pleiotropic roles and is involved in a spectrum of processes from metabolic pathways to gene expression and signal transduction.
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Petry, Sebastian Friedrich [Verfasser]. "Thioredoxin family proteins in the db/db mouse / Sebastian Friedrich Petry". Gießen : Universitätsbibliothek, 2015. http://d-nb.info/1077438826/34.
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Findlay, Victoria Jane. "The role of thioredoxin peroxidases in the yeast oxidative stress response". Thesis, University of Newcastle Upon Tyne, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.391954.
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Susanti, Dwi. "Sulfite reductase and thioredoxin in oxidative stress responses of methanogenic archaea". Diss., Virginia Tech, 2013. http://hdl.handle.net/10919/51423.
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Methanogens are a group of microorganisms that utilize simple compounds such as H2 + CO2, acetate and methanol for the production of methane, an end-product of their metabolism. These obligate anaerobes belonging to the archaeal domain inhabit diverse anoxic environments such as rice paddy fields, human guts, rumen of ruminants, and hydrothermal vents. In these habitats, methanogens are often exposed to O2 and previous studies have shown that many methanogens are able to tolerate O2 exposure. Hence, methanogens must have developed survival strategies to be able to live under oxidative stress conditions. The anaerobic species that lived on Earth during the early oxygenation event were first to face oxidative stress. Presumably some of the strategies employed by extant methanogens for combating oxidative stress were developed on early Earth. Our laboratory is interested in studying the mechanism underlying the oxygen tolerance and oxidative stress responses in methanogenic archaea, which are obligate anaerobe. Our research concerns two aspects of oxidative stress. (i) Responses toward extracellular toxic species such as SO32-, that forms as a result of reactions of O2 with reduced compounds in the environment. These species are mostly seen in anaerobic environments upon O2 exposure due to the abundance of reduced components therein. (ii) Responses toward intracellular toxic species such as superoxide and hydrogen peroxide that are generated upon entry of O2 and subsequent reaction of O2 with reduced component inside the cell. Aerobic microorganisms experience the second problem. Since a large number of microorganisms of Earth are anaerobes and the oxidative defense mechanisms of anaerobes are relatively less studied, the research in our laboratory has focused on this area. My thesis research covers two studies that fall in the above-mentioned two focus areas.
In 2005-2007 our laboratory discovered that certain methanogens use an unusual sulfite reductase, named F420-dependent sulfite reductase (Fsr), for the detoxification of SO32- that is produced outside the cell from a reaction between oxygen and sulfide. This reaction occurred during early oxygenation of Earth and continues to occur in deep-sea hydrothermal vents. Fsr, a flavoprotein, carries out a 6-electron reduction of SO32- to S2-. It is a chimeric protein where N- and C-terminal halves (Fsr-N and Fsr-C) are homologs of F420H2 dehydrogenase and dissimilatory sulfite reductase (Dsr), respectively. We hypothesized that Fsr was developed in a methanogen from pre-existing parts. To begin testing this hypothesis we have carried out bioinformatics analyses of methanogen genomes and found that both Fsr-N homologs and Fsr-C homologs are abundant in methanogens. We called the Fsr-C homolog dissimilatory sulfite reductase-like protein (Dsr-LP). Thus, Fsr was likely assembled from freestanding Fsr-N homologs and Dsr-like proteins (Dsr-LP) in methanogens. During the course of this study, we also identified two new putative F420H2-dependent enzymes, namely F420H2-dependent glutamate synthase and assimilatory sulfite reductase.
Another aspect of my research concerns the reactivation of proteins that are deactivated by the entry of oxygen inside the cell. Here I focused specifically on the role of thioredoxin (Trx) in methanogens. Trx, a small redox regulatory protein, is ubiquitous in all living cells. In bacteria and eukarya, Trx regulates a wide variety of cellular processes including cell divison, biosynthesis and oxidative stress response. Though some Trxs of methanogens have been structurally and biochemically characterized, their physiological roles in these organisms are unknown. Our bioinformatics analysis suggested that Trx is ubiquitous in methanogens and the pattern of its distribution in various phylogenetic classes paralleled the respective evolutionary histories and metabolic versatilities. Using a proteomics approach, we have identified 155 Trx targets in a hyperthermophilic phylogenetically deeply-rooted methanogen, Methanocaldococcus jannaschii. Our analysis of two of these targets employing biochemical assays suggested that Trx is needed for reactivation of oxidatively deactivated enzymes in M. jannaschii. To our knowledge, this is the first report on the role of Trx in an organism from the archaeal domain.
During the course of our work on methanogen Trxs, we investigated the evolutionary histories of different Trx systems that are composed of Trxs and cognate Trx reductases. In collaboration with other laboratories, we conducted bioinformatics analysis for the distribution of one of such systems, ferredoxin-dependent thioredoxin reductase (FTR), in all organisms. We found that FTR was most likely originated in the phylogenetically deeply-rooted microaerophilic bacteria where it regulates CO2 fixation via the reverse citric acid cycle.
Ph. D.
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Kawasaki, Kimio. "Helicobacter felis-induced gastritis was suppressed in mice overexpressing thioredoxin-1". Kyoto University, 2005. http://hdl.handle.net/2433/144494.
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Okubo, Kenichi. "Amelioration of ischemia-reperfusion injury by human thioredoxin in rabbit lung". Kyoto University, 1997. http://hdl.handle.net/2433/202158.
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Andres, Allen Mariano. "Metabolic role of thioredoxin-interacting protein in facilitating the fasting response". Diss., [La Jolla] : [San Diego] : University of California, San Diego ; San Diego State University, 2009. http://wwwlib.umi.com/cr/ucsd/fullcit?p3369671.
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Thesis (Ph. D.)--University of California, San Diego and San Diego State University, 2009.Title from first page of PDF file (viewed September 15, 2009). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 99-119).
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Karlenius, Therese Christina. "Regulation of the Thioredoxin System under Hypoxia and Different Oxygen Conditions". Thesis, Griffith University, 2011. http://hdl.handle.net/10072/365526.
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The thioredoxin system is one of the key antioxidant systems in the cell and is crucial for cell survival. It is comprised of thioredoxin and thioredoxin reductase and plays important roles in maintaining the redox homeostasis within the cell. The thioredoxin system is upregulated under conditions of oxidative stress to help re-establish the redox environment. This induced expression is mainly regulated through the action of regulatory elements present in their promoters, especially the antioxidant responsive element (ARE) via binding of the Nrf-2 transcription factor. The regulation of the thioredoxin system in response to a decrease in oxygen tension (hypoxic stress) is still not well known, although it is generally accepted as being up-regulated during hypoxia. The thioredoxin system is highly expressed in cancer cells, especially in more aggressive and therapeutic resistant cancers. The oxygen supply to a tumor is unstable due to abnormal vascular growth and this leads to a tumor oxygen environment that is constantly switching between hypoxic and oxidative stress, caused by the re-oxygenation step. Therefore, an understanding of how the expression of thioredoxin system is regulated in response to the different oxygen conditions occurring in cancers may aid in elucidating the link between the thioredoxin system and cancer progression.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Sciences
Science, Environment, Engineering and Technology
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Bhatia, Maneet. "Inhibition of the Thioredoxin System: Regulation by the Cancer Cell Environment". Thesis, Griffith University, 2016. http://hdl.handle.net/10072/367262.
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The oxygen environment in tumors is not static and involves constant cycling between hypoxic and re-oxygenation phases, a phenomenon known as intermittent hypoxia. Hypoxic and redox pathways are upregulated in response to intermittent hypoxia. The thioredoxin system, comprised of thioredoxin and thioredoxin reductase, is one of the main antioxidant systems, while hypoxia inducible factor 1 (HIF1) is the major hypoxia responsive system. High levels of both the thioredoxin system proteins and HIF1α have been correlated with extremely aggressive and highly metastatic tumors. Both these systems have also been linked to development of resistance against anti-cancer therapies. Moreover, HIF1α is indirectly redox regulated by thioredoxin, suggesting a potential cross-talk between the two systems, which becomes more apparent under intermittent hypoxia. Therefore, an understanding of these two systems under different oxygen conditions occurring in cancers may aid in the designing more effective therapeutics.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Natural Sciences
Science, Environment, Engineering and Technology
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Wang, Sicong. "Investigating cellular responses after inhibition of the thioredoxin system in lymphoma". Thesis, Griffith University, 2022. http://hdl.handle.net/10072/417233.
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Lymphoma is a haematological cancer that develops in the lymphatic system. Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphoma (NHL) are the two main lymphoma subtypes. HL is commonly diagnosed in young people and in adults over the age of 55. NHL is a more aggressive subtype than HL, and it accounts for approximately 90% of all lymphoma cases. Despite the development of different chemotherapy regimens for lymphoma treatment, 40-50% of lymphoma patients fail to achieve long-term survival rates because some patients do not respond to chemotherapy or they become resistant to the treatment. Additionally, lymphoma patients also suffer from the side effects of chemotherapy. Therefore, improved chemotherapies are desired, including the identification and investigation of new targets. In this study, three lymphoma cell lines (KMH2, SUDHL2, and SUDHL4) representing three different subtypes were used. This thesis focused on evaluating the efficacy of inhibiting the thioredoxin system in lymphoma cells using [Au(d2pype)2]Cl, assessing the cellular response after inhibiting TrxR, and evaluating its potential role as part of a combination treatment strategy in lymphoma.
Antioxidant systems, especially the thioredoxin (Trx) system, are known to provide cancer cells with a survival advantage, with thioredoxin reductase (TrxR) recently suggested as a potential anticancer target. In this project, overexpression of antioxidant system genes, including the Trx system, was observed in lymphoma patient samples. Furthermore, [Au(d2pype)2]Cl could significantly inhibit cell proliferation in three lymphoma cell lines by targeting TrxR as a specific TrxR inhibitor. Since the glutathione (GSH) system compensates for some functions of the Trx system, the GSH system was investigated in the lymphoma cell lines after the [Au(d2pype)2]Cl treatment. It was found that the activity of the Gpx selenoprotein was also inhibited by [Au(d2pype)2]Cl. In addition, the NHL cell lines were shown to be more sensitive to [Au(d2pype)]Cl than the HL cell line. Further investigation indicated that the KMH2 cell line (HL) had higher GSH levels than SUDHL2 and SUDHL4 cell lines (NHL), which may be a reason why KMH2 cells were not as sensitive to [Au(d2pype)2]Cl treatment.
Although the gene expression of the Trx and GSH systems was altered after the [Au(d2pype)2]Cl treatment, it was still unknown whether other signaling pathways might be affected. Hence, RNA-seq was performed to investigate altered gene expression after the inhibition of TrxR using [Au(d2pype)2]Cl. Bioinformatic analysis suggested that the Nrf-2-mediated oxidative stress response was the most affected pathway after TrxR was inhibited using [Au(d2pype)2]Cl in lymphoma cells. Furthermore, cytokine signaling-related genes were strongly linked to the antioxidant genes, as assessed by STRING analysis. Follow-up experiments using RT-qPCR and western blotting analysis confirmed that the IL6/JAK/STAT3 pathway was downregulated after the [Au(d2pype)2]Cl treatment, leading to the downregulation of c-Myc expression in lymphoma cells.
The B-cell receptor (BCR) signaling pathway is critical in lymphoma survival and development, and Bruton’s tyrosine kinase (BTK) is a key component of the BCR signaling pathway. In this project, the overexpression of BTK mRNA and protein was confirmed in lymphoma patients' samples from the TCGA and human protein atlas public databases. It was found that the mRNA expression levels of BTK and NF-κB (a downstream protein of the BCR pathway) were significantly correlated with the expression levels of TrxR in patient samples from the TCGA database. Further investigation indicated that TrxR inhibition using either specific siRNA or [Au(d2pype)2]Cl resulted in a decreased expression of both BTK mRNA and protein levels. In addition, the combination treatment performed by co-targeting TrxR and BTK using [Au(d2pype)2]Cl and ibrutinib showed a synergistic effect on inhibiting lymphoma cell proliferation. Meanwhile, cytoplasmic accumulation of p65 was observed after the combination treatment in DLBCL cells, indicating that the NF-κB pathway was inactive. The combination treatment also stimulated apoptosis in all three lymphoma cell lines. Moreover, the ferroptosis pathway was also activated after the combination treatment in the SUDHL4 cell line.
Over several decades, metal-based compounds such as cisplatin have shown anticancer activity in the clinic. However, side effects or resistance to cisplatin still occur in treated patients. Therefore, alternative metal-based compounds need to be identified and studied to find potential drugs with fewer side effects, including drugs that will be effective in overcoming drug resistance. This study assessed 12 newly synthesized indole-metal-based compounds, including iron-based, cobalt-based, and gold-based. It was found that the indole-gold-based compounds showed the best anti-lymphoma activity via inhibiting selenoproteins TrxR and Gpx, compared to the iron-based and cobalt-based compounds. Further investigation revealed that two of the gold-based compounds, Inac-Au2, and Inac-Au3, decreased the GSH level in the DLBCL cell line and induced the expression of ferroptosis-related genes. In addition, lipid peroxidation was significantly increased, indicating the activation of ferroptosis in the DLBCL cell line after the Inac-Au2 and Inac-Au3 treatment. These results showed the potential of Inac-Au2 and Inac-Au3 as TrxR and Gpx inhibitors in lymphoma treatment, warranting further assessment in animal models.
In conclusion, this thesis demonstrates the effectiveness of [Au(d2pype)2]Cl as a TrxR inhibitor in lymphoma and the validation of the Trx system as a potential target in malignant lymphoma treatment. Understanding the interaction between the Trx system and the BCR signaling pathway may provide a valid co-therapeutic strategy for lymphoma treatment.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environment and Sc
Science, Environment, Engineering and Technology
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Nonn, Larisa. "The role of the mitochondrial thioredoxin-2 system in cell function". Diss., The University of Arizona, 2003. http://hdl.handle.net/10150/289903.
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The hypothesis upon which this research is based is that the mitochondrial thioredoxin-2 system, which consists of mitochondrial thioredoxin-2 (Trx-2), mitochondrial thioredoxin reductase (TrxR-2) and mitochondrial thioredoxin peroxidase (Prdx-3), protects cells against apoptosis and regulates cell growth via mitochondrial redox homeostasis. Trx-2 mRNA was found expressed in a panel of cancer cell lines and Trx-2 protein is localized exclusively to the mitochondria. An alternate splice form of Trx-2 lacking exon 2 was identified that does not yield protein. Forced overexpression of Trx-2 in cell lines by using constitutive and inducible promoter systems increased mRNA levels, but did not yield an increase in Trx-2 protein. The role of Trx-2 in mammalian development was examined by generating a mouse deficient in Trx-2. Massive apoptosis, exencephaly and early embryonic lethality was seen in the Trx-2(-/-) homozygous embryos. Trx-2(-/-) embryonic fibroblasts were not viable under normal growth conditions, but could be rescued with maintenance in hypoxia. Trx-2(-/-) cells were also lacking mature cytochrome c, implicating Trx-2 in cytochrome c biogenesis. The Trx-2(+/-) heterozygous mice, which appear normal, had an increased sensitivity to some forms of oxidative toxicity (eg. acute doxorubicin) compared to the wild-type mice. To investigate the role of Prdx-3, WEHI7.2 cells with overexpression of Prdx-3 were generated by stable transfection. Overexpression of Prdx-3 inhibited cell proliferation, reduced cellular hydrogen peroxide levels and altered the mitochondrial membrane potential. Prdx-3 overexpression protected the cell from various drug-induced and hypoxia-induced apoptosis. Prdx-3 protein degradation was decreased during hypoxia, leading to a longer half-life under hypoxic conditions. Additionally, Prdx-3 protein levels were increased in a RCC4 cells expressing wild-type VHL compared to RCC4 cells with mutant VHL.
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SABELLI, RENATO. "Organ sulfur compounds and interactions with the detoxification and redox system enzymes". Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2010. http://hdl.handle.net/2108/1194.
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Molti composti organici contenenti zolfo (OSCs) ritrovati nell'aglio sono in grado di indurre apoptosi in varie linee tumorali [80]. Recentemente, è stato ritrovato nella fase acquosa dell'aglio bollito il 2-propenil tiosolfato (2-PTS), ed è stato visto essere in grado di indurre apoptosi in più linee tumorali, tramite la produzione di ROS intracellulari [16, 75]. Inoltre, recenti
lavori [38, 39, 75] hanno messo in evidenza il fatto che la tioalchenilazione degli enzimi sia una delle cause dell'effetto citotossico di questi composti. Sulla base di ciò è stato valutato l'effetto citotossico del 2-PTS su una linea di linfoblastoma umano (HuT 78), sulla quale è stata monitorata l'attività e l'espressione di alcuni enzimi, che è stato visto essere coinvolti metabolismo degli OSCs, come la Rodanesi (TST), la Glutatione-S-transferasi (GST), la Tioredossina (Trx), la Tioredossina reduttasi (Trd). Tutti questi enzimi sono importantissimi per la vitalità cellulare, ed una loro disfunzione causa gravi patologie [7] ed è visto essere associata anche all'induzione apoptotica [2,
54, 76]. Nei nostri esperimenti è emersa la capacità del 2-PTS di ossidare tutte le proteine in esame attraverso un meccanismo di tioalchenilazione, tranne che per la Trx, di residui di cisteina essenziali per la loro attività enzimatica.
E' stata, inoltre, monitorata la reazione tra il 2-PTS ed un'altra molecola importante, il glutatione (GSH). Il 2-PTS è in grado di reagire con il GSH in condizioni fisiologiche, producendo l' S-allil-mercaptoglutatione (GSSP), il quale dopo essere stato caratterizzato è stato utilizzato per studi molecolari e cellulari. Gli studi molecolari evidenziano che il GSSP è un inibitore competitivo della GSTM1-1, ed ha una Ki di circa 0.1 mM. Gli studi cellulari sulla linea HuT 78, invece, mostrano un'inibizione della proliferazione cellulare, associata ad un blocco della fase G1/S del ciclo cellulare, e un'attivazione della MAPKinasi p38. E' stata inoltre osservata la capacità del GSSP di incrementare la concentrazione intracellulare di Doxorubicina, aumentando così l'effetto citotossico del chemioterapico, nel caso si utilizzasse un co-trattamento di GSSP e Doxo, rispetto al solo trattamento con una concentrazione non tossica di Doxo. L'aumento dell'effetto della Doxorubicina è accompagnato con un aumento dell'espressione della CDKN1A, p21.
Tutti questi risultati danno molta importanza alla scoperta del ruolo del sistema Trx-Trd-TST come sistema detossificante per gli OSCs, poichè una disregolazione dell'espressione o dell'attività di uno o più componenti del
sistema, come nel caso di alcune patologie [8, 9, 88, 91], potrebbe compromettere la capacità detossificante della cellula, giustificando così la maggiore sensibilità di alcune cellule tumorali al trattamento con gli OSCs, rispetto a quelle non tumorali, o dare luogo allo sviluppo di patologie correlate allo scorretto metabolismo degli OSCs [88, 91]. L'inibizione, inoltre, della GST da parte del 2-PTS e del GSSP da ulteriori indicazioni sul meccanismo d'azione di questo tipo di composti all'interno della cellula.
Infine, la scoperta degli effetti del GSSP da indicazioni sulla possibilità di utilizzare il co-trattamento con chemioterapici (Doxo) e OSCs come possibile approccio metodologico per abbassare le dosi di chemioterapico somministrato.Many Organ Sulfur Compounds (OSCs), founded in garlic, are able to induce apoptosis in various tumoral cell lines [80]. Recently, in the aqueous phase of boiled garlic has been found the 2-propenyl thiosulfate (2-PTS), and it can induce apoptosis in several tumor cell lines, through the intracellular ROS production [16, 75]. Besides, recent works [38, 39, 75] highlight that the enzymes thioalkenylation can be one of the causes of cytotoxic effect of this type of compounds. Since to this observations, has been valuated the cytotoxic effect of 2-PTS on a human lymphoblastoma cell line (HuT 78), monitoring the activity and expression of some enzymes involved in the OSCs metabolism, such as Rhodanese (TST), Glutathione-Stransferase (GST), Thioredoxin (Trx), Thioredoxin reductase (Trd). All these enzyme are very important for the cell vitality, and their dysfunction causes severe diseases [7] and is related to the apoptotic induction [2, 54, 76]. In our experiments we have observed the ability of 2-PTS to oxidase all the proteins examined trough a thioalkenylation mechanism of cysteine residues, except Trx, essential for their enzymatic activity. Has been also observed the reaction between the 2-PTS and another important molecule, the glutathione (GSH). The 2-PTS can react with the GSH at physiological condition, producing S-allyl-mercaptoglutathione (GSSP), which is used, after its characterization, for molecular and cellular studies. Molecular studies indicates that GSSP is a competitive inhibitor of GSTM1-1, with a Ki of about 0,1 mM. Cellular studies on HuT 78 cell line, shows an inhibition of cell proliferation, with a G1/S phase blockage of the cell cycle, and an activation of p38 MAPKinase. It has been also observed the ability of GSSP to increment the intracellular concentration of Doxorubicin, incrementing its cytotoxic effect, in the case of combinate treatment of GSSP and Dox, respect to the only treatment with a non-toxic concentration of Doxo. The increase of the effect of Doxo is attended with an increment of expression of CDKN1A, p21. All these results gives prominence to the discovery of the role of the Trx-Trd-TST system as detoxifying system of the OSCs, because a dysregulation of expression or activity of one o more components of the system, as the case of some pathology [8, 9, 88, 91], could compromise the detoxification skill of the cell, explaining the major sensibility of some tumor cells to the OSCs treatment, or causing the develop of pathology correlated to the incorrect OSCs metabolism [88, 91]. Moreover, the inhibition of GST from 2-PTS and GSSP gives further indications on the intracellular mechanism of action of this type of compounds. In the end, the discover of the GSSP effect give indications on the possibility to use the co-treatment with chemoterapic (Doxo) and OSCs as possible methodological approach to reduce the used chemoterapic dose.