• Mass Spectrometry Letters
• /
• v.3 no.1
• /
• pp.10-14
• /
• 2012

Artificially oxidized cysteine residues in peroxiredoxin 6 (Prx6) were detected by electrospray interface capillary liquid chromatography-linear ion trap mass spectrometry after the preparation of two-dimensional gel electrophoresis (2D-GE). We used Prx6 as a model protein because it possesses only two cysteine residues at the 47th and 91st positions. The spot of Prx6 on 2D-GE undergoes a basic (isoelectric point, pI 6.6) to acidic (pI 6.2) shift by exposure to peroxide due to selective overoxidation of the active-site cysteine Cys-47 but not of Cys-91. However, we detected a tryptic peptide containing cysteine sulfonic acid at the 47th position from the basic spot and a peptide containing both oxidized Cys-47 and oxidized Cys-91 from the acidic spot of Prx6 after the separation by 2D-GE. We prepared two types of oxidized Prx6s: carrying oxidized Cys-47 (single oxidized Prx6), and other carrying both oxidized Cys-47 and Cys-91 (double oxidized Prx6). Using these oxidized Prx6s, the single oxidized Prx6 and double oxidized Prx6 migrated to pIs at 6.2 and 5.9, respectively. These results suggest that oxidized Cys-47 from the basic spot and oxidized Cys-91 from the acidic spot are generated by artificial oxidation during sample handling processes after isoelectric focusing of 2D-GE. Therefore, it is important to make sure of the origin of cysteine oxidation, if it is physiological or artificial, when an oxidized cysteine residue(s) is identified.

• Parasites, Hosts and Diseases
• /
• v.39 no.2
• /
• pp.133-141
• /
• 2001

A cDNA of 1.1 kb comprising the gene encoding the peroxiredoxin of Toxo-plasma gondii(TgPrx) has been cloned. The open reading frame of 591 Up was translated into a protein of 196 amino acids with a molecular mass of 25 kDa. Conserved 2 cysteine domains of Phe-Val-Cys-Pro and Glu-Val-Cys-Pro indicated TgPrx belonged to 2-Cys Prx families. TgPrx showed the highest homology with that of Arabidopsis thaliana by 53.9% followed by Entamoeba histolytica with 39.5% by the amino acid sequence alignment. Polyclonal antibody against recombinant TgPrx detected 25 kDa band in T. gondii without binding to host cell proteins TgPrx was located in the cytoplasm of T. gondii extracellularly or intracellularly by immunofluorescence assay. The expression of TgPrx was increased as early as 30 min after the treatment with artemisinin in the intracellular stage, while no changes in those of host Prx I and TgSOD. This result implies that TgPrx may function as an antioxidant protecting the cell from the attack of reactive oxygen intermediates. It is also suggested that TgPrx is a possible target of chemotherapy.

• Genomics & Informatics
• /
• v.3 no.2
• /
• pp.55-60
• /
• 2005

Peroxiredoxins (Prx's) are a superfamily of peroxidases that are ubiquitous in all super-kingdoms. Previous biochemical and structural studies have suggested that Prx's could be divided into five subfamilies (1-Cys, Typical 2-Cys, Atypical 2-Cys C-, L- and R- types). In this work, we have developed a set of regular expression patterns describing subfamily-specific spatial constraints of the key catalytic residues. Using these patterns, 1,016 Prx's available in public databases were classified into the five subfamilies. Our method performed well for most of the types except for Atypical 2 Cys R type.

• BMB Reports
• /
• v.50 no.8
• /
• pp.391-392
• /
• 2017

Overexpression of mammalian 2-Cys peroxiredoxin (Prx) enzymes is observed in most cancer tissues. Nevertheless, their specific roles in colorectal cancer (CRC) progression has yet to be fully elucidated. Here, a novel molecular mechanism by which PrxII/Tankyrase (TNKS) interaction mediates survival of adenomatous polyposis coli (APC)-mutant CRC cells was explored. In mice with an inactivating APC mutation, a model of spontaneous intestinal tumorigenesis, deletion of PrxII reduced intestinal adenomatous polyposis and thereby increased survival. In APC-mutant human CRC cells, PrxII depletion hindered PARP-dependent Axin1 degradation through TNKS inactivation. $H_2O_2-sensitive$ Cys residues in the zinc-binding domain of TNKS1 was found to be crucial for PARsylation activity. Mechanistically, direct binding of PrxII to ARC4/5 domains of TNKS conferred vital redox protection against oxidative inactivation. As a proof-of-concept experiment, a chemical compound targeting PrxII inhibited the growth of tumors xenografted with APC-mutation-positive CRC cells. Collectively, the results provide evidence revealing a novel redox mechanism for regulating TNKS activity such that physical interaction between PrxII and TNKS promoted survival of APC-mutant colorectal cancer cells by PrxII-dependent antioxidant shielding.

• Molecules and Cells
• /
• v.39 no.1
• /
• 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.

• Journal of Life Science
• /
• v.24 no.12
• /
• pp.1291-1300
• /
• 2014

Peroxiredoxins (Prxs) are a ubiquitous family of antioxidant enzymes that participate in a variety of biological processes, including $H_2O_2$-mediated signal transduction, molecular chaperoning, and mitochondrial function. In this study, we isolated and characterized a Prx 2 cDNA from abalone (Haliotis discus hannai). The abalone Prx 2 cDNA encoded a 199-amino acid polypeptide that belongs to a class of typical 2-Cys Prxs that contain peroxidatic and resolving cysteines. The deduced abalone Prx 2 protein showed strong homology (64-99%) with Prx 2 proteins from other species, including mollusk, fish, amphibians, and mammals, and it was most closely related to disk abalone (H. discus discus) Prx 2. Abalone Prx 2 mRNA was ubiquitously detected in tested tissues, and its expression was comparatively high in the mantle, gills, liver, foot, and digestive duct. The expression level of abalone Prx 2 mRNA was 106.7-fold, 51.9-fold, and 437.8-fold higher, respectively, in the gills, digestive duct, and liver than in the muscles. The expression level of abalone Prx 2 mRNA in the liver peaked at 6 hr postinfection with Vibrio parahemolyticus and decreased at 12 hr postinfection. The expression level of abalone Prx 2 mRNA in hemocytes was drastically increased at 1 hr postinfection with V. parahemolyticus. These results suggest that abalone Prx 2 is conserved through evolution and that it may play a role similar to that of its mammalian counterpart.

• Molecules and Cells
• /
• v.39 no.1
• /
• pp.31-39
• /
• 2016

The peroxiredoxins (Prxs) constitute a very large and highly conserved family of thiol-based peroxidases that has been discovered only very recently. We consider here these enzymes through the angle of their discovery, and of some features of their molecular and physiological functions, focusing on complex phenotypes of the gene mutations of the 2-Cys Prxs subtype in yeast. As scavengers of the low levels of $H_2O_2$ and as $H_2O_2$ receptors and transducers, 2-Cys Prxs have been highly instrumental to understand the biological impact of $H_2O_2$, and in particular its signaling function. 2-Cys Prxs can also become potent chaperone holdases, and unveiling the in vivo relevance of this function, which is still not established, should further increase our knowledge of the biological impact and toxicity of $H_2O_2$. The diverse molecular functions of 2-Cys Prx explain the often-hard task of relating them to peroxiredoxin genes phenotypes, which underscores the pleiotropic physiological role of these enzymes and complex biologic impact of $H_2O_2$.

• Journal of Plant Biotechnology
• /
• v.36 no.1
• /
• pp.75-80
• /
• 2009

In order to develop transgenic sweetpotato plants [Ipomoea batatas (L.) Lam. cv. Yulmi] with enhanced tolerance to oxidative stress, we constructed transformation vectors expressing 2-Cys peroxiredoxin (Prx) gene under the control of the stress-inducible SWPA2 or enhanced 35S promoter (named as SP or EP). Transgenic sweetpotato plants were attempted to generate from embryogenic calli using an Agrobacterium-mediated transformation system. Embryogenic calli gave rise to somatic embryos and then converted into plantlets on MS medium containing 100 mg/L kanamycin. Transgenic plants were regenerated in the same medium. Southern blot analysis confirmed that the Prx gene was inserted into the genome of the plants. To further study we selected the transgenic plant lines with enhanced tolerance against methyl viologen (MV). When sweetpotato leaf discs were subjected to methyl MV at $20{\mu}M$, transgenic plants showed about 40% higher tolerance than non-transgenic or empty vector-transformed plants.

• Molecules and Cells
• /
• v.39 no.8
• /
• pp.594-602
• /
• 2016

Alkyl hydroperoxide reductase subunit C from Pseudomonas aeruginosa PAO1 (PaAhpC) is a member of the 2-Cys peroxiredoxin family. Here, we examined the peroxidase and molecular chaperone functions of PaAhpC using a site-directed mutagenesis approach by substitution of Ser and Thr residues with Cys at positions 78 and 105 located between two catalytic cysteines. Substitution of Ser with Cys at position 78 enhanced the chaperone activity of the mutant (S78C-PaAhpC) by approximately 9-fold compared with that of the wild-type protein (WT-PaAhpC). This increased activity may have been associated with the proportionate increase in the high-molecular-weight (HMW) fraction and enhanced hydrophobicity of S78C-PaAhpC. Homology modeling revealed that mutation of $Ser^{78}$ to $Cys^{78}$ resulted in a more compact decameric structure than that observed in WT-PaAhpC and decreased the atomic distance between the two neighboring sulfur atoms of $Cys^{78}$ in the dimer-dimer interface of S78C-PaAhpC, which could be responsible for the enhanced hydrophobic interaction at the dimer-dimer interface. Furthermore, complementation assays showed that S78C-PaAhpC exhibited greatly improved the heat tolerance, resulting in enhanced1 survival under thermal stress. Thus, addition of Cys at position 78 in PaAhpC modulated the functional shifting of this protein from a peroxidase to a chaperone.

• Proceedings of the Korean Society of Sericultural Science Conference
• /
• 2003.10a
• /
• pp.130-133
• /
• 2003

Peroxiredoxins are a family of antioxidant proteins ubiquitously found in all living organisms. A type of peroxidase enzyme, named thioredoxin peroxidase (TPx), that reduces $H_2O$$_2$ with the use of electrons from thioredoxin and contains two essential cysteines was identified in a wide variety of organisms ranging from prokaryotes to mammals. TPx homologs, termed peroxiredoxin (Prx), have also been identified and include several proteins, designated 1-Cys Prx, that contain only one conserved cysteine. (omitted)