Characterising the Binding Specificities of the Subunits Associated with the KMT2/Set1 Histone Lysine Methyltransferase

KMT2/Set1 is the catalytic subunit of the complex of proteins associated with Set1 (COMPASS) that is responsible for the methylation of lysine 4 of histone H3 (H3K4) in Saccharomyces cerevisiae. Whereas monomethylated H3K4 (H3K4me1) is found throughout the genome, di- (H3K4me2) and tri- (H3K4me3) methylated H3K4 are enriched at specific loci, which correlates with the promoter and 5′-ends of actively transcribed genes in the case of H3K4me3. The COMPASS subunits contain a number of domains that are conserved in homologous complexes in higher eukaryotes and are reported to interact with modified histones. However, the exact organization of these subunits and their role within the complex have not been elucidated. In this study we showed that: (1) subunits Swd1 and Swd3 form a stable heterodimer that dissociates upon binding to a modified H3K4me2 tail peptide, suggesting a regulatory role in COMPASS; (2) the affinity of the subunit Spp1 for modified histone H3 substrates is much higher than that of Swd1 and Swd3; (3) Spp1 has a preference for H3K4me2/3 methylation state; and (4) Spp1 contains a high-affinity DNA-binding domain in the previously uncharacterised C-terminal region. These data allow us to suggest a mechanism for the regulation of COMPASS activity at an actively transcribed gene.     

Ultrastructural localization of 28 kDa glutathione S-transferase in adult Clonorchis sinensis

Glutathione S-transferase (28GST) with molecular mass of 28 kDa is an antioxidant enzyme abundant in Clonorchis sinensis. In adult C. sinensis, 28GST was localized in tegumental syncytium, cytons, parenchyma, and sperm tails examined by immunoelectron microscopy. C. sinensis 28GST was earlier found to neutralize bioreactive compounds and to be rich in eggs. Accordingly, it is suggested that 28GST plays important roles in phase II defense system and physiological roles in worm fecundity of C. sinensis.     

Malondialdehyde, Glutathione, and Nitric Oxide Levels in Toxoplasma gondii Seropositive Patients

The aim of this study was to investigate the difference in the serum malondialdehyde (MDA), glutathione (GSH), and nitric oxide (NO) levels between normal and T. gondii-infected patients. To this end, MDA, GSH, and NO levels in the sera of 37 seropositive patients and 40 participants in the control group were evaluated. In Toxoplasma ELISA, IgG results of the patient group were 1,013.0 ± 543.8 in optical density (mean ± SD). A statistically significant difference was found between patients and the control group in terms of MDA, GSH, and NO levels. A decrease in GSH activity was detected, while MDA and NO levels increased significantly. Consequently, it is suggested that the use of antioxidant vitamins in addition to a parasite treatment shall prove useful. The high infection vs control ratio of MDA and NO levels probably suggests the occurrence as a mechanism of tissue damage in cases of chronic toxoplasmosis. Moreover, it is recommended that the patient levels of MDA, GSH, and NO should be evaluated in toxoplasmosis.     

Toxicological and biochemical analysis of the susceptibility of sylvatic Triatoma infestans from the Andean Valley of Bolivia to organophosphate insecticide

To increase our knowledge of the natural susceptibility of Triatoma infestans to an organophosphate insecticide, we performed toxicological and biochemical studies on three sylvatic populations from Bolivia and two populations from domestic dwellings from Bolivia and Argentina. Fifty-per-cent lethal doses (LD50) were determined based on the topical application of fenitrothion on first instar nymphs and mortality was assessed at 24 h. Both type of populations exhibited LD50ratios significantly higher than 1 with a range of the values (1.42-2.47); the maximum value were found in a sylvatic (-S) population, Veinte de Octubre-S. Samples were biochemically analysed using a glutathione S-transferase activity assay. The highest significant activity was obtained for Veinte de Octubre-S and the lowest activity was obtained for the reference population (102.69 and 54.23 pmol per minute per mg of protein respectively). Two out of the three sylvatic populations (Veinte de Octubre-S and Kirus Mayu-S) exhibited significantly higher glutathione S-transferase activity than that of the reference population. Based on this analysis of the natural susceptibility of this organism to organophosphate insecticides, continental and focal surveys of organophosphate susceptibility should be conducted to evaluate the evolution and distribution of this phenomenon.     

No contribution of GSTM1 and GSTT1 null genotypes to the risk of neutropenia due to benzene exposure in Southeastern Brazil

Exposure to benzene has been associated with haematological diseases such as neutropenia (NEB) and acute myeloid leukaemia (AML). We tested whether the null genotypes of the GSTM1 and GSTT1 genes, involved in benzene inactivation, altered the risk for NEB in southeastern Brazil. Genomic DNA from 55 NEB patients and 330 controls was analysed by multiplex-polymerase chain reaction. The frequency of the GSTM1, GSTT1 and combined null genotypes was similar in patients and controls (GSTM1, 27.3% vs. 38.8%, p = 0.16; GSTT1, 25.5% vs. 19.7%, p = 0.24; GSTM1/GSTT1, 12.7% vs. 6.7%, p = 0.26; respectively). The distribution of genotype classes in NEB patients was similar to normal controls, suggesting that GSTM1 and GSTT1 null genotypes make no specific contribution to the risk of NEB. As the GSTM1 and GSTT1 null genotypes were previously associated with increased risk for AML in Brazil and elsewhere, we hypothesise that different thresholds of chemical exposure relative to distinct GSTM1 and GSTT1 genotypes may determine whether AML or NEB manifests in benzene exposed individuals from southeastern Brazil. Although indicative, our results still require support by prospective and large scale epidemiological studies, with rigorous assessment of daily chemical exposures and control of the possible contribution of other polymorphic genes involved in benzene metabolism.     

Physicochemical consequences of the perdeuteriation of glutathione S-transferase from S. japonicum

Glutathione S:-transferase (GST) from Schistosoma japonicum has been prepared in both normal protiated (pGST) and fully deuteriated (dGST) form by recombinant DNA technology. Electrospray mass spectrometry showed that the level of deuteriation in dGST was 96% and was homogeneous across the sample. This result is attributed to the use of a deuterium-tolerant host Escherichia coli strain in the preparation of the protein. 10 heteroatom-bound deuteriums (in addition to the carbon-bound deuteriums) were resistant to exchange when dGST was incubated in protiated buffer. The physicochemical and biological properties of the two proteins were compared. dGST was relatively less stable to heat denaturation and to proteolytic cleavage than was pGST. The midpoint transition temperature for pGST was 54.9 degrees C, whereas that for dGST was 51.0 degrees C. Static light-scattering measurements revealed that the association behavior of dGST is also different from that of pGST. The perdeuteriated enzyme shows a tendency to associate into dimers of the fundamental dimer. This is in contrast with results that have been obtained for other perdeuteriated proteins in which perdeuteriation has been shown to promote dissociation of aggregates. dGST showed a similar K(m) to pGST; similar results had been obtained previously with bacterial alkaline phosphatase. However, whereas the alkaline phosphatase showed a reduced rate of catalysis on deuteriation, dGST exhibited a slightly higher rate of catalysis than pGST. It is clear that the bulk substitution of deuterium for protium has significant effects on the properties of proteins. Until many more examples have been studied, it will be difficult to predict these effects for any given protein. Nevertheless, deuteriation represents an intriguing method of preparing functional analogs of recombinant proteins.     

Possible function of two insect phospholipid-hydroperoxide glutathione peroxidases

We compared the functional properties of two insect members of the phospholipid hydroperoxide glutathione peroxidases (PHGPx) family, VLP1, a major component of virus-like particles from the hymenopteran endoparasitoid Venturia canescens and its closest Drosophila relative, one of the putative PHGPx-proteins predicted from the Berkeley Drosophila genome sequence project. Recombinant Drosophila PHGPx shows enzymatic activity towards a number of PHGPx substrates, while the recombinant PHGPx-like domain of VLP1 lacks a functionally relevant cysteine and enzyme activity. A possible function of a non-enzymatic extracellular PHGPx-like protein is discussed.     

The thioredoxin specificity of Drosophila GPx: a paradigm for a peroxiredoxin-like mechanism of many glutathione peroxidases

Some members of the glutathione peroxidase (GPx) family have been reported to accept thioredoxin as reducing substrate. However, the selenocysteine-containing ones oxidise thioredoxin (Trx), if at all, at extremely slow rates. In contrast, the Cys homolog of Drosophila melanogaster exhibits a clear preference for Trx, the net forward rate constant, k'(+2), for reduction by Trx being 1.5x10(6) M(-1) s(-1), but only 5.4 M(-1) s(-1) for glutathione. Like other CysGPxs with thioredoxin peroxidase activity, Drosophila melanogaster (Dm)GPx oxidized by H(2)O(2) contained an intra-molecular disulfide bridge between the active-site cysteine (C45; C(P)) and C91. Site-directed mutagenesis of C91 in DmGPx abrogated Trx peroxidase activity, but increased the rate constant for glutathione by two orders of magnitude. In contrast, a replacement of C74 by Ser or Ala only marginally affected activity and specificity of DmGPx. Furthermore, LC-MS/MS analysis of oxidized DmGPx exposed to a reduced Trx C35S mutant yielded a dead-end intermediate containing a disulfide between Trx C32 and DmGPx C91. Thus, the catalytic mechanism of DmGPx, unlike that of selenocysteine (Sec)GPxs, involves formation of an internal disulfide that is pivotal to the interaction with Trx. Hereby C91, like the analogous second cysteine in 2-cysteine peroxiredoxins, adopts the role of a "resolving" cysteine (C(R)). Molecular modeling and homology considerations based on 450 GPxs suggest peculiar features to determine Trx specificity: (i) a non-aligned second Cys within the fourth helix that acts as C(R); (ii) deletions of the subunit interfaces typical of tetrameric GPxs leading to flexibility of the C(R)-containing loop. Based of these characteristics, most of the non-mammalian CysGPxs, in functional terms, are thioredoxin peroxidases.     

Glutathione and glutathione metabolizing enzymes in yeasts

Total glutathione content, glutathione peroxidase, glutathione transferase and glutathione reductase activities have been measured in 12 species of yeasts. All the strains tested contained glutathione, though in different amounts, as well as the above mentioned enzymes. To discriminate between the selenium-dependent and the selenium-independent form, glutathione peroxidase activity has been measured with both H₂O₂ and cumene hydroperoxide. Rhodotorula glutinis appeared to be the only strain in which the selenium-dependent form was not found, but this yeast exhibited the highest level of selenium-independent glutathione peroxidase activity as compared to the other strains.     

Crystallographic survey of active sites of an unclassified glutathione transferase from Bombyx mori

Background

Glutathione transferase (GST) catalyzes a major step in the xenobiotic detoxification pathway. We previously identified a novel, unclassified GST that is upregulated in an insecticide-resistant silkworm (Bombyx mori) upon insecticide exposure. Here, we sought to further characterize this GST, bmGSTu, by solving and refining its crystal structure and identifying its catalytic residues.

Methods

The structure of wild-type bmGSTu was determined with a resolution of 2.1 Å by synchrotron radiation and molecular modeling. Potential catalytic residues were mutated to alanine by means of site-directed mutagenesis, and kinetic data determined for wild-type and mutated bmGSTu.

Results

We found that bmGSTu occurred as a dimer, and that, like other GSTs, each subunit displayed a G-site and an H-site in the active center. Bound glutathione could be localized at the G-site. Kinetic data of the mutated forms of bmGSTu show that Val55, Glu67, and Ser68 in the G-site are important for catalysis. Furthermore, the H-site showed some unique features.

Conclusions

This is the first study to our knowledge to elucidate the molecular conformation of this B. mori GST. Our results indicate that residues Val55, Glu67, and Ser68, as well as Tyr7 and Ser12, in the glutathione-binding region of bmGSTu are critical for catalytic function.

General Significance

Our results, together with our previous finding that bmGSTu was preferentially induced in an insecticide-resistant strain, support the idea that bmGSTu functions in the transformation of exogenous chemical agents. Furthermore, the unique features observed in bmGSTu may shed light on mechanisms of insecticide resistance.     

Oxidative Stress in Vivax Malaria

Malaria is still a leading cause of morbidity and mortality. The increase in lipid peroxidation reported in malaria infection and antioxidant status may be a useful marker of oxidative stress during malaria infection. The aim of this study was to investigate the role of antioxidant enzymes against toxic reactive oxygen species in patients infected with Plasmodium vivax and healthy controls. Malondialdehyde levels, superoxide dismutase, and glutathione peroxidase activities were determined in 91 P. vivax patients and compared with 52 controls. Malondialdehyde levels, superoxide dismutase, and glutathione peroxidase activities were 8.07±2.29 nM/ml, 2.69±0.33 U/ml, and 49.6±3.2 U/g Hb in the patient group and 2.72±0.50 nM/ml, 3.71±0.47 U/ml, and 62.3±4.3 U/g Hb in the control group, respectively. Malondialdehyde levels were found statistically significant in patients with vivax malaria higher than in healthy controls (P<0.001). On the other hand, superoxide dismutase and glutathione peroxidase activities were found to be significantly lower in vivax malaria patients than in controls (P<0.05). There was an increase in oxidative stress in vivax malaria. The results suggested that antioxidant defense mechanisms may play an important role in the pathogenesis of P. vivax.     

Glutathione Reductase and Thioredoxin Reductase: Novel Antioxidant Enzymes from Plasmodium berghei

Malaria parasites adapt to the oxidative stress during their erythrocytic stages with the help of vital thioredoxin redox system and glutathione redox system. Glutathione reductase and thioredoxin reductase are important enzymes of these redox systems that help parasites to maintain an adequate intracellular redox environment. In the present study, activities of glutathione reductase and thioredoxin reductase were investigated in normal and Plasmodium berghei-infected mice red blood cells and their fractions. Activities of glutathione reductase and thioredoxin reductase in P. berghei-infected host erythrocytes were found to be higher than those in normal host cells. These enzymes were mainly confined to the cytosolic part of cell-free P. berghei. Full characterization and understanding of these enzymes may promise advances in chemotherapy of malaria.     

Biochemical markers of oxidative stress in Perna viridis exposed to mercury and temperature

Oxidative damage and antioxidant properties have been studied in Perna viridis subjected to short-term exposure to Hg along with temperature (72h) and long-term temperature exposures (14 days) as pollution biomarkers. The elevated thiobarbituric acid reactive substances (TBA-RS) levels observed in gills and digestive gland under exposure to Hg, individually and combined with temperature, as also long-term temperature stress have been assigned to the oxidative damage resulting in lipid peroxidation (LPX). Increased activities of antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR) and glutathione-S-transferase (GST) both in gills and digestive glands under long-term exposures to temperatures are more prominent to heat rather than cold stress suggesting activation of physiological mechanism to scavenge the ROS produced during heat stress. Also decreased values of reduced glutathione (GSH) on long exposures to temperature stress indicate utilisation of this antioxidant, either to scavenge oxiradicals or act in combination with other enzymes, was more than its production capacity under heat stress. The results suggest that temperature variation does alter the active oxygen metabolism by modulating antioxidant enzyme activities, which can be used as biomarker to detect sublethal effects of pollution.     

Structure of a Drosophila sigma class glutathione S-transferase reveals a novel active site topography suited for lipid peroxidation products

Insect glutathione-S-transferases (GSTs) are grouped in three classes, I, II and recently III; class I (Delta class) enzymes together with class III members are implicated in conferring resistance to insecticides. Class II (Sigma class) GSTs, however, are poorly characterized and their exact biological function remains elusive. Drosophila glutathione S-transferase-2 (GST-2) (DmGSTS1-1) is a class II enzyme previously found associated specifically with the insect indirect flight muscle. It was recently shown that GST-2 exhibits considerable conjugation activity for 4-hydroxynonenal (4-HNE), a lipid peroxidation product, raising the possibility that it has a major anti-oxidant role in the flight muscle. Here, we report the crystal structure of GST-2 at 1.75A resolution. The GST-2 dimer shows the canonical GST fold with glutathione (GSH) ordered in only one of the two binding sites. While the GSH-binding mode is similar to other GST structures, a distinct orientation of helix alpha6 creates a novel electrophilic substrate-binding site (H-site) topography, largely flat and without a prominent hydrophobic-binding pocket, which characterizes the H-sites of other GSTs. The H-site displays directionality in the distribution of charged/polar and hydrophobic residues creating a binding surface that explains the selectivity for amphipolar peroxidation products, with the polar-binding region formed by residues Y208, Y153 and R145 and the hydrophobic-binding region by residues V57, A59, Y211 and the C-terminal V249. A structure-based model of 4-HNE binding is presented. The model suggest that residues Y208, R145 and possibly Y153 may be key residues involved in catalysis.     

Optimizing expression and purification of an ATP-binding gene gsiA from Escherichia coli k-12 by using GFP fusion

The cloning, expression and purification of the glutathione (sulfur) import system ATP-binding protein (gsiA) was carried out. The coding sequence of Escherichia coli gsiA, which encodes the ATP-binding protein of a glutathione importer, was amplified by PCR, and then inserted into a prokaryotic expression vector pWaldo-GFPe harboring green fluorescent protein (GFP) reporter gene. The resulting recombinant plasmid pWaldo-GFP-GsiA was transformed into various E. coli strains, and expression conditions were optimized. The effect of five E. coli expression strains on the production of the recombinant gsiA protein was evaluated. E. coli BL21 (DE3) was found to be the most productive strain for GsiA-GFP fusion-protein expression, most of which was insoluble fraction. However, results from in-gel and Western blot analysis suggested that expression of recombinant GsiA in Rosetta (DE3) provides an efficient source in soluble form. By using GFP as reporter, the most suitable host strain was conveniently obtained, whereby optimizing conditions for overexpression and purification of the proteins for further functional and structural studies, became, not only less laborious, but also time-saving.     

亚硒酸钠对小麦幼苗中谷胱甘肽过氧化物酶和谷胱甘肽转硫酶活性以及谷胱甘肽含量的影响

用1.0 mg·L-1的亚硒酸钠根施小麦幼苗,测定亚硒酸钠对谷胱甘肽过氧化物酶和谷胱甘肽转硫酶活性以及还原性谷胱甘肽含量的结果表明,外源亚硒酸钠对麦苗地上部的谷胱甘肽过氧化物酶和谷胱甘肽转硫酶活性均有诱导作用,使麦苗体内的谷胱甘肽含量水平增加.     

Oxygen stress: a regulator of apoptosis in yeast.

Oxygen radicals are important components of metazoan apoptosis. We have found that apoptosis can be induced in the yeast Saccharomyces cerevisiae by depletion of glutathione or by low external doses of H2O2. Cycloheximide prevents apoptotic death revealing active participation of the cell. Yeast can also be triggered into apoptosis by a mutation in CDC48 or by expression of mammalian bax. In both cases, we show oxygen radicals to accumulate in the cell, whereas radical depletion or hypoxia prevents apoptosis. These results suggest that the generation of oxygen radicals is a key event in the ancestral apoptotic pathway and offer an explanation for the mechanism of bax-induced apoptosis in the absence of any established apoptotic gene in yeast.     

Distribution of glutathione transferases in Gram-positive bacteria and Archaea

Glutathione transferases (GSTs) have been widely studied in Gram-negative bacteria and the structure and function of several representatives have been elucidated. Conversely, limited information is available about the occurrence, classification and functional features of GSTs both in Gram-positive bacteria and in Archaea. An analysis of 305 fully-sequenced Gram-positive genomes highlights the presence of 49 putative GST genes in the genera of both Firmicutes and Actinobacteria phyla. We also performed an analysis on 81 complete genomes of the Archaea domain. Eleven hits were found in the Halobacteriaceae family of the Euryarchaeota phylum and only one in the Crenarchaeota phylum. A comparison of the identified sequences with well-characterized GSTs belonging to both Gram-negative and eukaryotic GSTs sheds light on their putative function and the evolutionary relationships within the large GST superfamily. This analysis suggests that the identified sequences mainly cluster in the new Xi class, while Beta class GSTs, widely distributed in Gram-negative bacteria, are under-represented in Gram-positive bacteria and absent in Archaea.     

Development of Mechanisms of Protection Against Oxidative Stress in Doxorubicin-Resistant Rat Tumoral Cells in Culture

We have compared some mechanisms involved in the defense against doxorubicin-induced free radical damage in rat hepatoma and glioblastoma cell lines and their doxorubicin-resistant variants presenting an overexpression of the multidrug resistance gene.

Immediate in vivo production of malondialdehyde was minor and was not different in sensitive and resistant cells. Alpha-tocopherol was undetectable in all cell lines. Glutathione levels were not different in sensitive and resistant cells and these levels did not vary upon doxorubicin treatment. Resistant cells exhibited either a 50% decrease (hepatoma) or a 25% increase (glioblastoma) of glutathione-S-transferase activity. Glutathione reductase presented no important change upon acquisition of resistance. In contrast, selenium-dependent glutathione peroxidase activity was consistently 2-6-fold increased in the resistant cells, which suggests a magnification of protection mechanisms against hydroxyle radical formation from H₂O₂ in resistant cells. Depletion of glutathione levels by buthionine sulfoximine sensitized hepatoma resistant cells to doxorubicin, but had no effect on doxorubicin cytotoxicity to glioblastoma cells.     

Development of Mechanisms of Protection Against Oxidative Stress in Doxorubicin-Resistant Rat Tumoral Cells in Culture

We have compared some mechanisms involved in the defense against doxorubicin-induced free radical damage in rat hepatoma and glioblastoma cell lines and their doxorubicin-resistant variants presenting an overexpression of the multidrug resistance gene.

Immediate in vivo production of malondialdehyde was minor and was not different in sensitive and resistant cells. Alpha-tocopherol was undetectable in all cell lines. Glutathione levels were not different in sensitive and resistant cells and these levels did not vary upon doxorubicin treatment. Resistant cells exhibited either a 50% decrease (hepatoma) or a 25% increase (glioblastoma) of glutathione-S-transferase activity. Glutathione reductase presented no important change upon acquisition of resistance. In contrast, selenium-dependent glutathione peroxidase activity was consistently 2-6-fold increased in the resistant cells, which suggests a magnification of protection mechanisms against hydroxyle radical formation from H₂O₂ in resistant cells. Depletion of glutathione levels by buthionine sulfoximine sensitized hepatoma resistant cells to doxorubicin, but had no effect on doxorubicin cytotoxicity to glioblastoma cells.