Inhibition effects of pesticides on glutathione‐S‐transferase enzyme activity of Van Lake fish liver

Glutathione‐S‐transferases (GSTs) have a function in xenobiotic metabolism. They are a significant multifunctional family with a wide variety of catalytic activities. In the current study, we determined in vitro inhibition effects of 2,4‐dichlorophenoxyacetic acid dimethylamine salt (2,4‐D DMA), haloxyfop‐P‐methyl, glyphosate isopropylamine, dichlorvos, and λ‐cyhalothrin on purified GST. For this purpose, GST were purified from Van Lake fish (Chalcalburnus tarichii Pallas) liver with 29.25 EU mg^?1 specific activity and 10.76% yield using GSH–agarose affinity chromatographic method. The pesticides were tested at various concentrations on in vitro GST activity. K_i constants were calculated as 0.17 ± 0.01, 0.25 ± 0.05, 3.72 ± 0.32, 0.42 ± 0.06, and 0.025 ± 0.004 mM, for 2,4‐D DMA, haloxyfop‐P‐methyl, glyphosate isopropylamine, dichlorvos, and λ‐cyhalothrin, respectively. λ‐Cyhalothrin showed a better inhibitory effect compared to the other pesticides. The inhibition mechanisms of λ‐cyhalothrin were competitive, while the other pesticides were noncompetitive.     

Structures of yeast glutathione‐S‐transferase Gtt2 reveal a new catalytic type of GST family

Glutathione‐S‐transferases (GSTs) are ubiquitous detoxification enzymes that catalyse the conjugation of electrophilic substrates to glutathione. Here, we present the crystal structures of Gtt2, a GST of Saccharomyces cerevisiae, in apo and two ligand‐bound forms, at 2.23 Å, 2.20 Å and 2.10 Å, respectively. Although Gtt2 has the overall structure of a GST, the absence of the classic catalytic essential residues—tyrosine, serine and cysteine—distinguishes it from all other cytosolic GSTs of known structure. Site‐directed mutagenesis in combination with activity assays showed that instead of the classic catalytic residues, a water molecule stabilized by Ser129 and His123 acts as the deprotonator of the glutathione sulphur atom. Furthermore, only glycine and alanine are allowed at the amino‐terminus of helix‐α1 because of stereo‐hindrance. Taken together, these results show that yeast Gtt2 is a novel atypical type of cytosolic GST.     

Identification and characterization of the zebrafish glutathione S‐transferase Pi‐1

Zebrafish has in recent years emerged as a popular vertebrate model for use in pharmacological and toxicological studies. While there have been sporadic studies on the zebrafish glutathione S‐transferases (GSTs), the zebrafish GST gene superfamily still awaits to be fully elucidated. We report here the identification of 15 zebrafish cytosolic GST genes in NCBI GenBank database and the expression, purification, and enzymatic characterization of the zebrafish cytosolic GST Pi‐1 (GSTP1). The cDNA encoding the zebrafish GSTP1 was cloned from a 3‐month‐old female zebrafish, expressed in Eschelichia coli host cells, and purified. Purified GSTP1 displayed glutathione‐conjugating activity toward 1‐chloro‐2,4‐dinitrobenzene as a representative substrate. The enzymatic characteristics of the zebrafish GSTP1, including pH‐dependency, effects of metal cations, and kinetic parameters, were studied. Moreover, the expression of zebrafish GSTP1 at different developmental stages during embryogenesis, throughout larval development, onto maturity was examined.     

c‐FLIP is involved in erythropoietin‐mediated protection of erythroid‐differentiated cells from TNF‐α‐induced apoptosis

The TNF‐α (tumour necrosis factor) affects a wide range of biological activities, such as cell proliferation and apoptosis. Cell life or death responses to this cytokine might depend on cell conditions. This study focused on the modulation of factors that would affect the sensitivity of erythroid‐differentiated cells to TNF‐α. Hemin‐differentiated K562 cells showed higher sensitivity to TNF‐induced apoptosis than undifferentiated cells. At the same time, hemin‐induced erythroid differentiation reduced c‐FLIP (cellular FLICE‐inhibitory protein) expression. However, this negative effect was prevented by prior treatment with Epo (erythropoietin), which allowed the cell line to maintain c‐FLIP levels. On the other hand, erythroid‐differentiated UT‐7 cells – dependent on Epo for survival – showed resistance to TNF‐α pro‐apoptotic action. Only after the inhibition of PI3K (phosphatidylinositol‐3 kinase)‐mediated pathways, which was accompanied by negative c‐FLIP modulation and increased erythroid differentiation, were UT‐7 cells sensitive to TNF‐α‐triggered apoptosis. In summary, erythroid differentiation might deregulate the balance between growth promotion and death signals induced by TNF‐α, depending on cell type and environmental conditions. The role of c‐FLIP seemed to be critical in the protection of erythroid‐differentiated cells from apoptosis or in the determination of their sensitivity to TNF‐mediated programmed cell death. Epo, which for the first time was found to be involved in the prevention of c‐FLIP down‐regulation, proved to have an anti‐apoptotic effect against the pro‐inflammatory factor. The identification of signals related to cell life/death switching would have significant implications in the control of proliferative diseases and would contribute to the understanding of mechanisms underlying the anaemia associated with inflammatory processes.     

LINE‐1 hypomethylation induced by reactive oxygen species is mediated via depletion of S‐adenosylmethionine

Whether long interspersed nuclear element‐1 (LINE‐1) hypomethylation induced by reactive oxygen species (ROS) was mediated through the depletion of S‐adenosylmethionine (SAM) was investigated. Bladder cancer (UM‐UC‐3 and TCCSUP) and human kidney (HK‐2) cell lines were exposed to 20 μM H₂O₂ for 72 h to induce oxidative stress. Level of LINE‐1 methylation, SAM and homocysteine (Hcy) was measured in the H₂O₂‐exposed cells. Effects of α‐tocopheryl acetate (TA), N‐acetylcysteine (NAC), methionine, SAM and folic acid on oxidative stress and LINE‐1 methylation in the H₂O₂‐treated cells were explored. Viabilities of cells treated with H₂O₂ were not significantly changed. Intracellular ROS production and protein carbonyl content were significantly increased, but LINE‐1 methylation was significantly decreased in the H₂O₂‐treated cells. LINE‐1 methylation was restored by TA, NAC, methionine, SAM and folic acid. SAM level in H₂O₂‐treated cells was significantly decreased, while total glutathione was significantly increased. SAM level in H₂O₂‐treated cells was restored by NAC, methionine, SAM and folic acid; while, total glutathione level was normalized by TA and NAC. Hcy was significantly decreased in the H₂O₂‐treated cells and subsequently restored by NAC. In conclusion, in bladder cancer and normal kidney cells exposed to H₂O₂, SAM and Hcy were decreased, but total glutathione was increased. Treatments with antioxidants (TA and NAC) and one‐carbon metabolites (SAM, methionine and folic acid) restored these changes. This pioneer finding suggests that exposure of cells to ROS activates glutathione synthesis via the transsulfuration pathway leading to deficiency of Hcy, which consequently causes SAM depletion and eventual hypomethylation of LINE‐1. Copyright © 2015 John Wiley & Sons, Ltd.     

A class C radical S‐adenosylmethionine methyltransferase synthesizes 8‐methylmenaquinone

The membranous quinone/quinol pool is essential to the majority of life forms and has been widely used as an important biomarker in microbial taxonomy. In the anaerobic world, the most important quinones are menaquinone (MK) and a methylated form of MK, designated methylmenaquinone (MMK), which is anticipated to serve specifically in low‐potential electron transport chains involved in anaerobic respiration. However, it has remained unclear how MMK is generated. Here, we show that a novel enzyme homologous to class C radical SAM methyltransferases (RSMTs) synthesizes MMK using MK as substrate. Such enzymes, termed either MenK or MqnK, are present in MMK‐producing bacteria (and some archaea) that possess either the classical MK biosynthesis pathway (Men) or the futalosine pathway (Mqn). An mqnK deletion mutant of the model Epsilonproteobacterium Wolinella succinogenes was unable to produce MMK₆ but its formation was restored upon genomic complementation using either the native mqnK gene or menK from the human gut bacterium Adlercreutzia equolifaciens or Shewanella oneidensis. Moreover, any of the menK genes enabled Escherichia coli cells to produce MMK₈ and a methylated form of 2‐demethylmenaquinone₈ (DMK₈). The results expand the knowledge on quinone synthesis and demonstrate an unprecedented function for a class C RSMT‐type enzyme in primary cell metabolism.     

Glutathione S‐transferase π complexes with and stimulates Na+,K+‐ATPase

Glutathione S‐transferase (GST) was found to complex with the Na⁺,K⁺‐ATPase as shown by binding assay using quartz crystal microbalance. The complexation was obstructed by the addition of antiserum to the α‐subunit of the Na⁺,K⁺‐ATPase, suggesting the specificity of complexation between GST and the Na⁺,K⁺‐ATPase. Co‐immunoprecipitation experiments, using the anti‐α‐subunit antiserum to precipitate the GST‐Na⁺,K⁺‐ATPase complex and then using antibodies specific to an isoform of GST to identify the co‐precipitated proteins, revealed that GSTπ was complexed with the Na⁺,K⁺‐ATPase. GST stimulated the Na⁺,K⁺‐ATPase activity up to 1.4‐fold. The level of stimulation exhibited a saturable dose–response relationship with the amount of GST added, although the level of stimulation varied depending on the content of GSTπ in the lots of GST received from supplier. The stimulation was also obtained when recombinant GSTπ was used, confirming the results. When GST was treated with reduced glutathione, GST activity was greatly stimulated, whereas the level of stimulation of the Na⁺,K⁺‐ATPase activity was similar to that when untreated GST was added. When GST was treated with H₂O₂, GST activity was greatly diminished while the stimulation of the Na⁺,K⁺‐ATPase activity was preserved. The results suggest that GSTπ complexes with the Na⁺,K⁺‐ATPase and stimulates the latter independent of its GST activity. Copyright © 2012 John Wiley & Sons, Ltd.     

Nucleocytoplasmic trafficking is essential for BAK1‐ and BKK1‐mediated cell‐death control

BRI1‐ASSOCIATED KINASE 1 (BAK1) was initially identified as a co‐receptor of the brassinosteroid (BR) receptor BRI1. Genetic analyses also revealed that BAK1 and its closest homolog BAK1‐LIKE 1 (BKK1) regulate a BR‐independent cell‐death control pathway. The double null mutant bak1 bkk1 displays a salicylic acid‐ and light‐dependent cell‐death phenotype even without pathogen invasion. Molecular mechanisms of the spontaneous cell death mediated by BAK1 and BKK1 remain unknown. Here we report our identification of a suppressor of bak1 bkk1 (sbb1–1). Genetic analyses indicated that cell‐death symptoms in a weak double mutant, bak1–3 bkk1–1, were completely suppressed by the loss‐of‐function mutation in SBB1, which encodes a nucleoporin (NUP) 85‐like protein. Genetic analyses also demonstrated that individually knocking out three other nucleoporin genes from the SBB1‐located sub‐complex was also able to rescue the cell‐death phenotype of bak1–3 bkk1–1. In addition, a DEAD‐box RNA helicase, DRH1, was identified in the same protein complex as SBB1 via a proteomic approach. The drh1 mutation also rescues the cell‐death symptoms of bak1–3 bkk1–1. Further analyses indicated that export of poly(A)⁺ RNA was greatly blocked in the nup and drh1 mutants, resulting in accumulation of significant levels of mRNAs in the nuclei. Over‐expression of a bacterial NahG gene to inactivate salicylic acid also rescues the cell‐death phenotype of bak1–3 bkk1–1. Mutants suppressing cell‐death symptoms always showed greatly reduced salicylic acid contents. These results suggest that nucleocytoplasmic trafficking, especially of molecules directly or indirectly involved in endogenous salicylic acid accumulation, is critical in BAK1‐ and BKK1‐mediated cell‐death control.     

Defects in MAP1S‐mediated autophagy cause reduction in mouse lifespans especially when fibronectin is overexpressed

Autophagy is a cellular process that executes the turnover of dysfunctional organelles and misfolded or abnormally aggregated proteins. Microtubule‐associated protein MAP1S interacts with autophagy marker LC3 and positively regulates autophagy flux. LC3 binds with fibronectinmRNA and facilitates its translation. The synthesized fibronectin protein is exported to cell surface to initiate the assembly of fibronectin extracellular matrix. Fibronectin is degraded in lysosomes after it is engulfed into cytosol via endocytosis. Here, we show that defects in MAP1S‐mediated autophagy trigger oxidative stress, sinusoidal dilation, and lifespan reduction. Overexpression of LC3 in wild‐type mice increases the levels of fibronectin and γ‐H₂AX, a marker of DNA double‐strand breakage. LC3‐induced fibronectin is efficiently degraded in lysosomes to maintain a balance of fibronectin levels in wild‐type mice so that the mice live a normal term of lifespan. In the LC3 transgenic mice with MAP1S deleted, LC3 enhances the synthesis of fibronectin but the MAP1S depletion causes an impairment of the lysosomal degradation of fibronectin. The accumulation of fibronectin protein promotes liver fibrosis, induces an accumulation of cell population at the G0/G1 stage, and further intensifies oxidative stress and sinusoidal dilatation. The LC3‐induced overexpression of fibronectin imposes stresses on MAP1S‐deficient mice and dramatically reduces their lifespans. Therefore, MAP1S‐mediated autophagy plays an important role in maintaining mouse lifespan especially in the presence of extra amount of fibronectin.     

Identification and expression analysis of components involved in rice Xa21‐mediated disease resistance signalling

Rice Xa21 gene encodes a receptor‐like kinase that confers broad‐spectrum resistance against Xanthomonas oryzae pv. oryzae (Xoo). Recently, a number of genes involved in the Xa21‐mediated disease resistance pathway have been identified. Based on our previous data and the literature, we chose 16 candidate proteins and made corresponding antibodies. Using Western blotting, we systematically investigated the expression profile of the proteins in Xa21‐mediated disease resistance response. We found nine proteins with altered expression. We further compared their expression in resistance, susceptible and mock responses, and found that GST expression was up‐regulated during the resistance process, indicating GST is a positive regulator in resistance responses. ATPsB expression was down‐regulated during both the resistance and susceptible response processes, although it was higher in the resistance response than that in the susceptible response. The total amount of MYB, GAPDH, CatB, Trx and NB‐ARC proteins was lower in the resistance than in the susceptible response, but their abundance per unit bacteria in the resistance response was still higher than in the susceptible response, suggesting that these proteins might be positive regulators in the resistance response. In addition, expression of another ERF was induced by inoculation with bacterial blight pathogen, and expression of Zf‐LSD1 was activated by wounding stress alone. Interestingly, most proteins showed similar altered expression patterns in the resistance and susceptible responses, but differed to some extents, implying that both responses might share common molecular mechanisms. This study revealed evidence of resistance‐related protein expression, providing a foundation for better understanding of their functions.     

The Skp1‐like protein SSK1 is required for cross‐pollen compatibility in S‐RNase‐based self‐incompatibility

The self‐incompatibility (SI) response occurs widely in flowering plants as a means of preventing self‐fertilization. In these self/non‐self discrimination systems, plant pistils reject self or genetically related pollen. In the Solanaceae, Plantaginaceae and Rosaceae, pistil‐secreted S‐RNases enter the pollen tube and function as cytotoxins to specifically arrest self‐pollen tube growth. Recent studies have revealed that the S‐locus F‐box (SLF) protein controls the pollen expression of SI in these families. However, the precise role of SLF remains largely unknown. Here we report that PhSSK1 (Petunia hybrida SLF‐interacting Skp1‐like1), an equivalent of AhSSK1 of Antirrhinum hispanicum, is expressed specifically in pollen and acts as an adaptor in an SCF(Skp1‐Cullin1‐F‐box)^SLF complex, indicating that this pollen‐specific SSK1‐SLF interaction occurs in both Petunia and Antirrhinum, two species from the Solanaceae and Plantaginaceae, respectively. Substantial reduction of PhSSK1 in pollen reduced cross‐pollen compatibility (CPC) in the S‐RNase‐based SI response, suggesting that the pollen S determinant contributes to inhibiting rather than protecting the S‐RNase activity, at least in solanaceous plants. Furthermore, our results provide an example that a specific Skp1‐like protein other than the known conserved ones can be recruited into a canonical SCF complex as an adaptor.     

Evaluation of chalcones as inhibitors of glutathione S‐transferase

Glutathione S‐transferases (GSTs) are the superfamily of multifunctional detoxification isoenzymes and play an important role in cellular signaling. In the present study, potential inhibition effects of chalcones were tested against human GST. For this purpose, GST was purified from human erythrocytes with 5.381 EU?mg^?1 specific activity and 51.95% yield using a GSH–agarose affinity chromatographic method. The effects of chalcones on in vitro GST activity were tested at various concentrations. K_i constants of chalcones were found in the range of 7.76–41.93 μM. According to the results, 4‐fluorochalcone showed a better inhibitory effect compared with the other compounds. The inhibition mechanisms of 2'‐hydroxy‐4‐methoxychalcone and 4‐methoxychalcone were noncompetitive, whereas the inhibition mechanisms of 4'‐ hydroxychalcone, 4‐ fluorochalcone, and 4,4'‐ diflurochalcone were competitive.     

Pollen S–locus F–box proteins of Petunia involved in S–RNase‐based self‐incompatibility are themselves subject to ubiquitin‐mediated degradation

Many flowering plants show self‐incompatibility, an intra‐specific reproductive barrier by which pistils reject self‐pollen to prevent inbreeding and accept non‐self pollen to promote out‐crossing. In Petunia, the polymorphic S–locus determines self/non‐self recognition. The locus contains a gene encoding an S–RNase, which controls pistil specificity, and multiple S‐locus F‐box (SLF) genes that collectively control pollen specificity. Each SLF is a component of an SCF (Skp1/Cullin/F‐box) complex that is responsible for mediating degradation of non‐self S‐RNase(s), with which the SLF interacts, via the ubiquitin–26S proteasome pathway. A complete set of SLFs is required to detoxify all non‐self S‐RNases to allow cross‐compatible pollination. Here, we show that SLF1 of Petunia inflata is itself subject to degradation via the ubiquitin–26S proteasome pathway, and identify an 18 amino acid sequence in the C‐terminal region of S₂‐SLF1 (SLF1 of S₂ haplotype) that contains a degradation motif. Seven of the 18 amino acids are conserved among all 17 SLF proteins of S₂ haplotype and S₃ haplotype involved in pollen specificity, suggesting that all SLF proteins are probably subject to similar degradation. Deleting the 18 amino acid sequence from S₂‐SLF1 stabilized the protein but abolished its function in self‐incompatibility, suggesting that dynamic cycling of SLF proteins is an integral part of their function in self‐incompatibility.