Il-Han Kim

Thioredoxin-linked "Thiol Peroxidase" from Periplasmic Space of Escherichia coli

Three different molecular masses (24, 22, and 20 kDa) of antioxidant proteins were purified in Escherichia coli. These proteins exhibited the preventive effects against the inactivation of glutamine synthetase activity and the cleavage of DNA by a metal-catalyzed oxidation system capable of generating reactive oxygen species. Their antioxidant activities were supported by a thiol-reducing equivalent such as dithiothreitol. Analysis of the amino-terminal amino acid sequences and the immunoblots between 24- and 22-kDa proteins indicates that the 24-kDa protein is an intact form of the 22-kDa protein that was previously identified 22-kDa subunit (AhpC) of E. coli alkyl hydroperoxide reductase (AhpC/AhpF). We isolated and sequenced an E. coli genomic DNA fragment that encodes 20-kDa protein. Comparison of the deduced amino acid sequence of the 20-kDa protein with that of AhpC revealed no sequence homology. A search of a data bank showed that the 20-kDa protein is a new type of antioxidant enzyme. The synthesis of this novel 20-kDa protein was increased in response to oxygen stress during growth. The 20-kDa protein resides mainly in the periplasmic space of E. coli, whereas the 24-kDa AhpC resides mainly in the matrix. The 20-kDa protein was functionally linked to the thioredoxin as an in vivo thiol-regenerating system and exerted a peroxidase activity. This 20-kDa protein is thus named “thiol peroxidase,” which could act as an antioxidant enzyme removing peroxides or H2O2 within the catalase- and peroxidase-deficient periplasmic space of E. coli.

Preferential overexpression of glutaredoxin3 in human colon and lung carcinoma

Glutaredoxin (Glrx) uses the reducing power of glutathione to maintain and regulate the cellular redox state. Substantial evidence indicates that the alteration of cellular redox status is a critical factor involved in cell growth and death and results in tumorigenesis. We investigated levels of expression of all Glrx genes in a variety of cancers using a real-time polymerase chain reaction (RT-PCR). Among members of the Glrx, family, Glrx3 (PICOT: PKC-interacting cousin of thioredoxin) was preferentially induced in lung (55.3+/-30.1-fold induction) and colon (50.2+/-28.8-fold induction) cancer compared to their normal tissues (lung>or=colon>breast>ovary>bladder>prostate>thyroid>lymphoma>liver>or=kidney cancers). By contrast, the magnitude of induction folds in other cancer tissues was ranged from 0.83 to 4.0. Moreover, the induction folds of Glrx3 mRNA in colon and lung cancer tissues were significantly higher when compared to those of all thioredoxin (Trx) and peroxiredoxin (Prx) members. Western blot analysis of different and paired cancer tissues revealed the consistent and preferential expression of Glrx3 in lung and colon cancers. Taken together, these results suggest that Glrx3 could take a pivotal role in colon and lung cancer cells during the tumorigenesis.

Msn2p/Msn4p Act as a Key Transcriptional Activator of Yeast Cytoplasmic Thiol Peroxidase II

We observed that the transcription ofSaccharomyces cerevisiae cytoplasmic thiol peroxidase type II (cTPx II) (YDR453C) is regulated in response to various stresses (e.g. oxidative stress, carbon starvation, and heat-shock). It has been suggested that both transcription-activating proteins, Yap1p and Skn7p, regulate the transcription of cTPx II upon exposure to oxidative stress. However, a dramatic loss of transcriptional response to various stresses in yeast mutant strains lacking both Msn2p and Msn4p suggests that the transcription factors act as a principal transcriptional activator. In addition to two Yap1p response elements (YREs), TTACTAA and TTAGTAA, the presence of two stress response elements (STREs) (CCCCT) in the upstream sequence of cTPx II also suggests that Msn2p/Msn4p could control stress-induced expression of cTPx II. Analysis of the transcriptional activity of site-directed mutagenesis of the putative STREs (STRE1 and STRE2) and YREs (TRE1 and YRE2) in terms of the activity of a lacZ reporter gene under control of the cTPx II promoter indicates that STRE2 acts as a principal binding element essential for transactivation of the cTPx II promoter. The transcriptional activity of thecTPx II promoter was exponentially increased after postdiauxic growth. The transcriptional activity of the cTPx II promoter is greatly increased by rapamycin. Deletion ofTor1, Tor2, Ras1, andRas2 resulted in a considerable induction when compared with their parent strains, suggesting that the transcription ofcTPx II is under negative control of the Ras/cAMP and target of rapamycin signaling pathways. Taken together, these results suggest that cTPx II is a target of Msn2p/Msn4p transcription factors under negative control of the Ras-protein kinase A and target of rapamycin signaling pathways. Furthermore, the accumulation of cTPx II upon exposure to oxidative stress and during the postdiauxic shift suggests an important antioxidant role in stationary phase yeast cells.

Crystal structure of the C107S/C112S mutant of yeast nuclear 2‐Cys peroxiredoxin

Introduction. Peroxiredoxins (Prxs) are a superfamily of antioxidant enzymes, which are abundant in several isoforms in all kingdoms. Prxs catalyze the reduction of deleterious substances such as hydrogen peroxide (H2O2), alkyl hydroperoxides, and peroxynitrites by utilizing the thiol group of the “peroxidatic” cysteine (CP), which is conserved within the N-terminal region. Some eukaryotic Prxs also act as regulators of H2O2-mediated signal transduction. All Prxs belonging to the thioredoxin-fold superfamily share the same peroxidatic active-site structure. During a catalytic cycle, the CP residue is oxidized by peroxides to a cysteine sulfenic acid (CP-SOH) intermediate. Prxs are classified into 1-Cys and 2-Cys type based on the occurrence of the “resolving” Cys (CR) residue. The 1-Cys Prxs do not contain a CR residue, and the CP-SOH is recycled by glutathionylation mediated by glutathione S-transferase , followed by spontaneous reduction of the enzyme with glutathione. In 2-Cys Prxs, the CP-SOH and CR-SH react to form a stable disulfide, which is then reduced by oxidoreductases such as thioredoxin, tryparedoxin, AhpD, or AhpF. The 2-Cys Prxs have been further subdivided into “typical” and “atypical” types, depending on the position of the CR residue. In typical 2-Cys Prxs (hereafter referred to as T2-Cys Prxs), the CR residue is located within the C-terminal arm of another subunit of a homodimer. In contrast, the CR residue in an atypical 2-Cys Prx resides within the same subunit. The atypical 2-Cys Prxs have also been further subdivided into “L,” “C,” and “R” type subfamilies (hereafter referred to as L-, C-, and R2-Cys Prxs, respectively), depending on the spatial location of the CR residue. 6 Therefore, from a mechanistic point of view, there are five unique Prx subfamilies in total. To date, five distinct Prxs have been identified in the yeast Saccharomyces cerevisiae. They include three thiol peroxidases (cTPx I, II, and III) localized in the cytoplasm, one (nTPx) in the nucleus and one (mTPx) in the mitochondria. cTPx I, II, and III are T2-Cys Prxs, while mTPx is a 1-Cys Prx. nTPx is a member of the C2-Cys Prxs that contains a CxxxxC motif. Bacterial homologues of such Prxs are frequently referred to as the bacterioferritin comigratory proteins (BCP) and their plant homologues are named as PrxQ. These Prxs are least characterized among the Prx subfamilies and information regarding their structure is not yet available. In this study, we have determined the crystal structure of a truncated mutant of nTPx in which both the catalytic residues of Cys107 and Cys112 were replaced with serine. This mutant protein was gradually and spontaneously degraded by the freezing and thawing process until 56 amino acid residues were cleaved off from its N-terminal. nTPx has nuclear targeting sequences but the cleavage site has not yet been determined. The truncated mutant nTPx (hereafter referred to as tmTPx) may correspond to a physiologically mature nTPx. The present structure of a C2-Cys Prx makes it possible to compare the 3D structures of all the five Prx subfamilies.

Involvement of ArcA and Fnr in Expression of Escherichia coli Thiol Peroxidase Gene

To explore the oxygen response regulators involved in thiol peroxidase gene (tpx) expression in Escherichia coli, we constructed a single‐copy tpx‐lacZ operon fusion and monitored tpx‐lacZ expression in various genetic backgrounds. Expression of the tpx‐lacZ fusion was increased 4‐fold by aerobic growth. Anaerobic expression of tpx‐lacZ in either Delta arcA or Delta fnr strains was 2.5‐fold depressed compared with that of the wild‐type strain. The results of immunoblotting experiments also demonstrated that ArcA and Fnr regulatory proteins repressed thiol peroxidase gene expression during anaerobic growth. Inspection of the tpx promoter region revealed putative binding sites for ArcA and Fnr. It thus appears that ArcA and Fnr function as repressors by blocking the binding of RNA polymerase to the tpx promoter in E. coli under anaerobic growth conditions.

Thioredoxin 1 as a serum marker for ovarian cancer and its use in combination with CA125 for improving the sensitivity of ovarian cancer diagnoses

Abstract The serum levels of Trx1 in patients with ovarian cancer were significantly higher than those in normal persons and patients with non-cancer inflammatory diseases. The level of Trx1 increased with the Figo stage. Ovarian cancer patients who were determined to be negative for CA125, were observed to have serum Trx1 levels as high as those of CA125-positive patients. In addition, patients with non-cancer inflammatory diseases had lower plasma Trx1 1 levels than did controls, showing that Trx1 allows clear distinctions between ovarian cancer and these non-cancer diseases. Combinational analysis of CA125 with Trx1 for the detection of ovarian cancer suggests that the diagnostic capacity of CA125 alone for the early detection of ovarian cancer, especially regarding sensitivity, is significantly improved by its combination with Trx1. Taken together, we conclude that serum Trx1 is useful for the early diagnosis of ovarian cancer.

A glutaredoxin-fused thiol peroxidase acts as an important player in hydrogen peroxide detoxification in late-phased growth of Anabaena sp. PCC7120.

The Anabaena sp. genome contains an open reading frame with homology to a novel hybrid form of thiol peroxidase, fused with a glutaredoxin domain. The gene was expressed in Escherichia coli. The purified hybrid protein exerted the highest peroxidase activity toward H2O2 using an electron from a reduced form of glutathione. The calculated kcat and kcat/K(m) values for H2O2 are 48.2 s(-1) and 3.29 x 10(6) M(-1) s(-1), respectively. Immunoblot analyses of the heterocystic proteins showed that the level of the protein in the heterocyst is comparable to that in the vegetative cell. All oxidants tested significantly elevated the mRNA and protein levels. The transcript slightly increased during the exponential growth phase, following which it increased steeply. Also, the levels of transcript were significantly increased in response to N2 starvation, carbon starvation, and light elimination. Taken together, the present data reveal for the first time that the glutathione-dependent thiol peroxidase is an adaptive strategy in Anabaena sp. that efficiently combats H2O2 that are produced during later phase of vegetative and heterocystic growth.

Characterization of two alkyl hydroperoxide reductase C homologs alkyl hydroperoxide reductase C_H1 and alkyl hydroperoxide reductase C_H2 in Bacillus subtilis

AIM To identify alkyl hydroperoxide reductase subunit C (AhpC) homologs in Bacillus subtilis (B. subtilis) and to characterize their structural and biochemical properties. AhpC is responsible for the detoxification of reactive oxygen species in bacteria. METHODS Two AhpC homologs (AhpC_H1 and AhpC_H2) were identified by searching the B. subtilis database; these were then cloned and expressed in Escherichia coli. AhpC mutants carrying substitutions of catalytically important Cys residues (C37S, C47S, C166S, C37/47S, C37/166S, C47/166S, and C37/47/166S for AhpC_H1; C52S, C169S, and C52/169S for AhpC_H2) were obtained by site-directed mutagenesis and purified, and their structure-function relationship was analyzed. The B. subtilis ahpC genes were disrupted by the short flanking homology method, and the phenotypes of the resulting AhpC-deficient bacteria were examined. RESULTS Comparative characterization of AhpC homologs indicates that AhpC_H1 contains an extra C37, which forms a disulfide bond with the peroxidatic C47, and behaves like an atypical 2-Cys AhpC, while AhpC_H2 functions like a typical 2-Cys AhpC. Tryptic digestion analysis demonstrated the presence of intramolecular Cys37-Cys47 linkage, which could be reduced by thioredoxin, resulting in the association of the dimer into higher-molecular-mass complexes. Peroxidase activity analysis of Cys→Ser mutants indicated that three Cys residues were involved in the catalysis. AhpC_H1 was resistant to inactivation by peroxide substrates, but had lower activity at physiological H2O2 concentrations compared to AhpC_H2, suggesting that in B. subtilis, the enzymes may be physiologically functional at different substrate concentrations. The exposure to organic peroxides induced AhpC_H1 expression, while AhpC_H1-deficient mutants exhibited growth retardation in the stationary phase, suggesting the role of AhpC_H1 as an antioxidant scavenger of lipid hydroperoxides and a stress-response factor in B. subtilis. CONCLUSION AhpC_H1, a novel atypical 2-Cys AhpC, is functionally distinct from AhpC_H2, a typical 2-Cys AhpC.

Interaction between Saccharomyces cerevisiae glutaredoxin 5 and SPT10 and their in vivo functions

Glutaredoxin 5 (Grx5) is a monothiol member of the Grx family that comprises two dithiol and three monothiol members. Using a yeast two-hybrid system, we isolated a Grx5-binding protein, SPT10, which has been previously suggested to act as a global transcriptional regulator of specific histone genes. We find that among the five members of the Grx family and two members of the thioredoxin (Trx) family (Trx1 and Trx2), Grx5 alone interacts with SPT10 via an intermolecular disulfide linkage between Cys60 of Grx5 and Cys385 of SPT10. To evaluate the physiological function of the Grx5/SPT10 interaction, we investigated the phenotypes of three null mutant strains (Grx5Δ, SPT10Δ, and Grx5ΔSPT10Δ). Taken together, the results show that all of these phenotypes are probably a consequence of the disruption of the interaction between Grx5 and SPT10. From this study, we suggest an interaction between Grx5 and SPT10 via intermolecular disulfide linkage and propose a model for a role of Grx5 in the regulation of protein expression under the control of SPT10.

Crystallization and preliminary X-ray analysis of a truncated mutant of yeast nuclear thiol peroxidase, a novel atypical 2-Cys peroxiredoxin

Saccharomyces cerevisiae nTPx is a thioredoxin-dependent thiol peroxidase that is localized in the nucleus. nTPx belongs to the C-type atypical 2-Cys peroxiredoxin family members, which are frequently called BCPs or PrxQs. A double mutant (C107S/C112S) of nTPx overexpressed in Escherichia coli was spontaneously degraded upon freezing and thawing and its truncated form (residues 57-215; MW = 17837 Da) was crystallized with PEG 3350 and mercury(II) acetate as precipitants using the hanging-drop vapour-diffusion method. Diffraction data were collected to 1.8 A resolution using X-ray synchrotron radiation. The crystals belong to the trigonal space group P3(2), with unit-cell parameters a = b = 37.54, c = 83.26 A. The asymmetric unit contains one molecule of truncated mutant nTPx, with a corresponding VM of 1.91 A3 Da(-1) and a solvent content of 35.5%.