• BMB Reports
• /
• 제33권3호
• /
• pp.234-241
• /
• 2000

A new type of the human TSA homologous gene was cloned from a HeLa cell cDNA and characterized. The gene product consists of 161 amino acids with a molecular mass of 16,900. The TSA homologous protein, as a new 6th member of the human TSA (hTSA VI), exerted a thioldependent peroxidase activity with the use of thioredoxin system as a physiological electron donor. The values of $V_{max}/K_m$ of hTSA VI for $H_2O_2$ and t-butyl hydroperoxide (t-BOOH) were calculated as $5.53{\times}10^{-2}$ and $3.70{\times}10^{-2}$, respectively. This implies that hTSA VI is a peroxidase, which reduces $H_2O_2$ and t-BOOH. The mutation of $Cys^{47}$ to serine resulted in a complete loss of the peroxidase activity. This suggests that $Cys^{47}$ acts as a primary site of catalysis. The analysis of the tryptic digest derived from hTSA VI revealed that the $Cys^{47}$ exists as a free thiol form. Taken together, these results suggest that the TSA homologous protein is a new type of the human family, which exerts thioredoxin-linked peroxidase activity toward $H_2O_2$ and alkyl hydroperoxide.

• Molecules and Cells
• /
• 제22권1호
• /
• pp.113-118
• /
• 2006

Thioredoxin reductase (TrxR), a component of the redox control system involving thioredoxin (Trx), is implicated in defense against oxidative stress, control of cell growth and proliferation, and regulation of apoptosis. In the present study a stable transfectant was made by introducing the vector pcDNA3.0 harboring the fission yeast TrxR gene into COS-7 African green monkey kidney fibroblast cells. The exogenous TrxR gene led to an increase in TrxR activity of up to 3.2-fold but did not affect glutathione (GSH) content, or glutaredoxin and caspase-3 activities. Levels of reactive oxygen species (ROS), but not those of nitric oxide (NO), were reduced. Conversely, 1-chloro-2,4-dinitrobezene (CDNB), an irreversible inhibitor of mammalian TrxR, enhanced ROS levels in the COS-7 cells. After treatment with hydrogen peroxide, the level of intracellular ROS was lower in the transfectants than in the vector control cells. These results confirm that TrxR is a crucial determinant of the level of cellular ROS during oxidative stress as well as in the normal state.

• BMB Reports
• /
• 제33권2호
• /
• pp.166-171
• /
• 2000

Thioredoxin is a multifunctional protein that is ubiquitous in microorganisms, animals and plants. Previously, the expression of the Escherichia coli thioredoxin gene (trxA) was found to be negatively regulated by cAMP. In the present study, the effect of temperature on the expression of the E. coli trxA gene was investigated. In order to examine the temperature effect, the fusion plasmid pCL70 that harbors the E. coli trxA P1P2 promoter was used. The other two fusion plasmids, pJH3 and pMH521 that were constructed in different vectors which harbor the E. coli trxA P2 promoter, were also used. When the E. coli strain MC1061/pCL70 was grown in a rich medium at $25^{\circ}C$, $34^{\circ}C$ and $42^{\circ}C$, the cells grown at $42^{\circ}C$ gave the highest $\beta$-galactosidase activity. The E. coli MC1061/pJH3 and MC1061/pMG521 cells showed increased $\beta$-galactosidase activity after the shift of the culture temperature to $42^{\circ}C$. The wild-type trxA gene of the E. coli MC1061 cells produced much higher thioredoxin activity at the higher temperature. These results support the conclusion that the E. coli trxA gene is regulated in a temperature-dependent manner. Especially the expression from its P2 promoter appeared to be sensitive to temperature.

• Molecules and Cells
• /
• 제38권2호
• /
• pp.187-194
• /
• 2015

Thioredoxin (TRX) is a disulfide reductase present ubiquitously in all taxa and plays an important role as a regulator of cellular redox state. Recently, a redox-independent, chaperone function has also been reported for some thioredoxins. We previously identified nodulin-35, the subunit of soybean uricase, as an interacting target of a cytosolic soybean thioredoxin, GmTRX. Here we report the further characterization of the interaction, which turns out to be independent of the disulfide reductase function and results in the co-localization of GmTRX and nodulin-35 in peroxisomes, suggesting a possible function of GmTRX in peroxisomes. In addition, the chaperone function of GmTRX was demonstrated in in vitro molecular chaperone activity assays including the thermal denaturation assay and malate dehydrogenase aggregation assay. Our results demonstrate that the target of GmTRX is not only confined to the nodulin-35, but many other peroxisomal proteins, including catalase (AtCAT), transthyretin-like protein 1 (AtTTL1), and acyl-coenzyme A oxidase 4 (AtACX4), also interact with the GmTRX. Together with an increased uricase activity of nodulin-35 and reduced ROS accumulation observed in the presence of GmTRX in our results, especially under heat shock and oxidative stress conditions, it appears that GmTRX represents a novel thioredoxin that is co-localized to the peroxisomes, possibly providing functional integrity to peroxisomal proteins.

• Tuberculosis and Respiratory Diseases
• /
• 제47권5호
• /
• pp.650-659
• /
• 1999

목 적: 산소를 이용하는 세포들은 반응성 산소기(reactive oxygen species, ROS)로 부터 자신을 보호하는 기전을 가지고 있는데, thioredoxin system으로부터 전자를 받아 과산화물을 물로 환원시키는 생화학적 성질을 가진 효모 단백질을 발견하여 이를 thioredoxin peroxidase(TPX)로 명명하였다. 이는 효모 뿐 아니라 체내 여러 장기에 고루 분포되어 있으며, 특허 종양세포에서 유리되는 반응생 산소기를 제거함으로써 종양세포의 발생(initiation), 성장(promotion) 및 전이(metastasis)를 억제하는 역할을 할 것으로 알려져 있으며, 폐암, 유방암 등의 종양세포내에서의 발현이 증가되는 것으로 보고되었다. 한편, 폐암의 치료에 있어서 방사선 치료는 폐암의 국소적 치료 및 증상 경감을 위해 중요한 위치를 차지하고 있으나, 폐의 방사선 치료에 있어 가장 중요한 장애요인은 치료후 발생하는 폐실질의 섬유화로서 방사선 조사후의 산소유리가(free radical) 손상 등에 의한 것으로 알려져 있으며, 이런 섬유화에 관여하는 인자 및 방어기전에 대한 연구는 미흡한 실정이다. 이에 저자 등은 방사선 조사 후에 백서의 폐조직에서 thioredoxin peroxidase의 아형 및 catalase등이 방사선 조사로 인한 폐손상의 방어기전에 어떤 역할을 하는지 알아보기 위하여 다음과 같은 연구를 시행하였다. 대상 및 방법: 백서 18마리를 각각 3마리씩 6개 군으로 나누었고, 그 중 15마리에 900cGY와 방사선을 조사하였다. 방사선 조사를 받은 백서를 조사직후, 1주후, 2주후, 3주후 및 6주후에 희생시켜 폐조직을 얻었고, 이들 폐조직과 대조군 3마리의 폐조직을 H&E 염색하여 폐섬유화의 정도를 관찰함과 동시에 TPX 아형 및 catalase에 대한 항체로 Western blot analysis를 시행하여 각각의 단백질의 발현정도를 비교하였다. 결 과: 대조군과 비교하여 볼 때 폐조직에서 H&E 염색상 방사선 조사후 시기별로 폐섬유화의 정도가 점차로 진행됨을 볼 수 있었으나, thioredoxin peroxidase 5가지 아형(TPX-A & B, HS22, MER5, HORF-06) 모두 방사선 조사전과 조사후, 시간경과에 따른 면역반응대(immunoreactive band)의 차이를 보이지는 않았다. Catalase 역시 면역반응대의 차이를 보이지 않았다. 결 론: 체내 여러장기, 특히 폐장에 많이 분포하고, 폐암세포에 의한 반응성 산소기(reactive oxygen species)에 의하여 발현이 증가되는 것으로 알려진 thioredoxin peroxidase가 방사선 조사후에 발생한 백서의 폐섬유화에서는 그 발현의 정도가 변하지 않는 것으로 미루어 방사선으로 인한 폐손상의 복원과정에는 관여하지 않는 것으로 생각된다.

• BMB Reports
• /
• 제31권6호
• /
• pp.554-559
• /
• 1998

Activities of glutathione- and thioredoxin-related enzymes and phenylpropanoid-synthesizing enzymes were measured and compared in the developing leaves of Arabidopsis thaliana. Phenylalanine ammonia-lyase activity is maximal in the leaves of 2-wk-grown Arabidopsis. Tyrosine ammonia-lyase activity is maximal in the leaves of 3-wk-grown and 4-wk-grown Arabidopsis. Activity of thioitransferase, an enzyme involved in the reduction of various disulfide compounds, is higher in younger leaves than in older ones. A similar pattern was obtained in the activity of thioredoxin, a small protein known as a cofactor of ribonucleotide reductase and a regulator of photosynthesis. Activity of glutathione reductase is also higher in the younger leaves. Malate debydrogenase activity remains relatively constant during the development of Arabidopsis leaves. The results offer preliminary information for further approach to elucidate the mechanism of growth-dependent variations of these enzymes.

• Molecules and Cells
• /
• 제39권1호
• /
• pp.65-71
• /
• 2016

A challenge in the redox field is the elucidation of the molecular mechanisms, by which $H_2O_2$ mediates signal transduction in cells. This is relevant since redox pathways are disturbed in some pathologies. The transcription factor OxyR is the $H_2O_2$ sensor in bacteria, whereas Cys-based peroxidases are involved in the perception of this oxidant in eukaryotic cells. Three possible mechanisms may be involved in $H_2O_2$ signaling that are not mutually exclusive. In the simplest pathway, $H_2O_2$ signals through direct oxidation of the signaling protein, such as a phosphatase or a transcription factor. Although signaling proteins are frequently observed in the oxidized state in biological systems, in most cases their direct oxidation by $H_2O_2$ is too slow ($10^1M^{-1}s^{-1}$ range) to outcompete Cys-based peroxidases and glutathione. In some particular cellular compartments (such as vicinity of NADPH oxidases), it is possible that a signaling protein faces extremely high $H_2O_2$ concentrations, making the direct oxidation feasible. Alternatively, high $H_2O_2$ levels can hyperoxidize peroxiredoxins leading to local building up of $H_2O_2$ that then could oxidize a signaling protein (floodgate hypothesis). In a second model, $H_2O_2$ oxidizes Cys-based peroxidases that then through thiol-disulfide reshuffling would transmit the oxidized equivalents to the signaling protein. The third model of signaling is centered on the reducing substrate of Cys-based peroxidases that in most cases is thioredoxin. Is this model, peroxiredoxins would signal by modulating the thioredoxin redox status. More kinetic data is required to allow the identification of the complex network of thiol switches.

• BMB Reports
• /
• 제34권3호
• /
• pp.278-283
• /
• 2001

Antioxidant and redox enzyme activities are known to be involved in the cellular responses to various stresses. Their variations were observed according to the growth cycle of the fission yeast Schizosaccharomyces pombe. Peroxidase activity appeared to be notably higher in the early exponential phase than in the mid-exponential and stationary phases. However, catalase activity showed a variation pattern resembling the growth curve. Glutathione S-transferase activity was higher in the early exponential and late stationary phases. Activities of the two redox enzymes, thioredoxin and thioltransferase (glutaredoxin), were high in the stationary phase. However, their activities appeared to increase from the early exponential to mid-exponential phase. Total glutathione content had a varying pattern similar to that of thioredoxin and thioltransferase. However, its content in the early exponential phase was high. These results propose that antioxidant and redox enzymes tested are also involved in the mechanism of cell growth.

• BMB Reports
• /
• 제38권5호
• /
• pp.550-554
• /
• 2005

YKR049C is a mitochondrial protein in Saccharomyces cerevisiae that is conserved among yeast species, including Candida albicans. However, no biological function for YKR049C has been ascribed based on its primary sequence information. In the present study, NMR spectroscopy was used to determine the putative biological function of YKR049C based on its solution structure. YKR049C shows a well-defined thioredoxin fold with a unique insertion of helices between two $\beta$-strands. The central $\beta$-sheet divides the protein into two parts; a unique face and a conserved face. The 'unique face' is located between ${\beta}2$ and ${\beta}3$. Interestingly, the sequences most conserved among YKR049C families are found on this 'unique face', which incorporates L109 to E114. The side chains of these conserved residues interact with residues on the helical region with a stretch of hydrophobic surface. A putative active site composed by two short helices and a single Cys97 was also well observed. Our findings suggest that YKR049C is a redox protein with a thioredoxin fold containing a single active cysteine.

• Archives of Pharmacal Research
• /
• 제28권9호
• /
• pp.1065-1072
• /
• 2005

The nascent thyroglobulin (Tg) multimer molecule, which is generated during the initial fate of Tg in ER, undergoes the rapid reductive depolymerization. In an attempt to determine the depolymerization process, various types of Tg multimers, which were generated from deoxy­cholate-treated/reduced Tg, partially unfolded Tg or partially unfolded/reduced Tg, were subjected to various GSH (reduced glutathione) reducing systems using protein disulfide isomerase (PDI), glutathione reductase (GR), glutaredoxin or thioredoxin reductase. The Tg multimers generated from deoxycholate-treated/reduced Tg were depolymerized readily by the PDI/GSH system, which is consistent with the reductase activity of PDI. The PDI/GSH-induced depolymerization of the Tg multimers, which were generated from either partially unfolded Tg or partially unfolded/reduced Tg, required the simultaneous inclusion of glutathione reductase, which is capable of reducing glutathionylated mixed disulfide (PSSG). This suggests that PSSG was generated during the Tg multimerization stage or its depolymerization stage. In particular, the thioredoxin/thioredoxin reductase system or glutaredoxin system was also effective in depolymerizing the Tg multimers generated from the unfolded Tg. Overall, under the net GSH condition, the depolymerization of Tg multimers might be mediated by PDI, which is assisted by other reductive enzymes, and the mechanism for depolymerizing the Tg multimers differs according to the type of Tg multimer containing different degrees and types of disulfide linkages.