Drosophila GSTs display outstanding catalytic efficiencies with the environmental pollutants 2,4,6-trinitrotoluene and 2,4-dinitrotoluene

The nitroaromatic explosive 2,4,6-trinitrotoluene (TNT) and the related 2,4-dinitrotoluene (DNT) aretoxic environmental pollutants. The biotransformation and detoxication of these persistent compoundsin higher organisms are of great significance from a health perspective as well as for the biotechnological challenge of bioremediation of contaminated soil. We demonstrate that different human glutathionetransferases (GSTs) and GSTs from the fruit fly Drosophila melanogaster are catalysts of the biotransformationof TNT and DNT. The human GSTs had significant but modest catalytic activities with both DNT and TNT. However, D. melanogaster GSTE6 and GSTE7 displayed outstanding high activities withboth substrates

Potent inhibitors of equine steroid isomerase EcaGST A3-3.

Equine glutathione transferase A3-3 (EcaGST A3-3) belongs to the superfamily of detoxication enzymes found in all higher organisms. However, it is also the most efficient steroid double-bond isomerase known in mammals. Equus ferus caballus shares the steroidogenic pathway with Homo sapiens, which makes the horse a suitable animal model for investigations of human steroidogenesis. Inhibition of the enzyme has potential for treatment of steroid-hormone-dependent disorders. Screening of a library of FDA-approved drugs identified 16 out of 1040 compounds, which at 10 μM concentration afforded at least 50% inhibition of EcaGST A3-3. The most potent inhibitors, anthralin, sennoside A, tannic acid, and ethacrynic acid, were characterized by IC50 values in the submicromolar range when assayed with the natural substrate Δ5-androstene-3,17-dione

β-actin regulates a heterochromatin landscape essential for optimal induction of neuronal programs during direct reprograming.

During neuronal development, β-actin serves an important role in growth cone mediated axon guidance. Consistent with this notion, in vivo ablation of the β-actin gene leads to abnormalities in the nervous system. However, whether β-actin is involved in the regulation of neuronal gene programs is not known. In this study, we directly reprogramed β-actin+/+ WT, β-actin+/- HET and β-actin-/- KO mouse embryonic fibroblast (MEFs) into chemically induced neurons (CiNeurons). Using RNA-seq analysis, we profiled the transcriptome changes among the CiNeurons. We discovered that induction of neuronal gene programs was impaired in KO CiNeurons in comparison to WT ones, whereas HET CiNeurons showed an intermediate levels of induction. ChIP-seq analysis of heterochromatin markers demonstrated that the impaired expression of neuronal gene programs correlated with the elevated H3K9 and H3K27 methylation levels at gene loci in β-actin deficient MEFs, which is linked to the loss of chromatin association of the BAF complex ATPase subunit Brg1. Together, our study shows that heterochromatin alteration in β-actin null MEFs impedes the induction of neuronal gene programs during direct reprograming. These findings are in line with the notion that H3K9Me3-based heterochromatin forms a major epigenetic barrier during cell fate change

Effect of ITCs on egg laying of fruit flies.

<p>Actin-Gal4 flies crossed to wild-type control (<i>w¹¹¹⁸</i>) or to UAS-GSTE7 flies (GSTE7) were mated for 3–5 days and the females separated and then kept for 7 days on standard control, 0.15 mM PEITC, or 1 mM allyl-ITC supplemented food. They were transferred to fresh vials each day and the number of eggs in the vials counted. Ten vials (approximately 10 flies/vial) with control (<i>w¹¹¹⁸</i>) or GSTE7 flies were used and the number of eggs divided by the number of flies. The graph shows the number of eggs laid per fly, and error bars represent standard error of the mean. * indicates P<0.05, two-tailed unpaired Student's t-test.</p

Overexpression of the GSTE7 protein in fruit flies (<i>Drosophila melanogaster</i>).

<p>(A) Scheme outlining the binary Gal4-UAS system <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110103#pone.0110103-Brand1" target="_blank">[10]</a> used to induce ubiquitous overexpression. The coding sequence of the <i>Drosophila</i> GSTE7 gene was inserted in the pUAST attB vector <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110103#pone.0110103-Bischof1" target="_blank">[9]</a> containing an upstream activating sequence (UAS) and a downstream attB crossover site. The plasmid was introduced by injection into fly embryos carrying an attP landing site on chromosome 3 and inserted into the chromosome by way of ΦC31-mediated integration. (B) Flies overexpressing the GSTE7 transgene were obtained by crossing the UAS-GSTE7 flies with a strain bearing the Actin-Gal4 driver, which induces ubiquitous expression. (C–F) Embryonal expression of <i>D. melanogaster</i> GSTE7 detected by <i>in situ</i> mRNA hybridization. Fly embryos were stained with a digoxigenin-labeled probe recognizing GSTE7 by whole-mount <i>in situ</i> hybridization, and are oriented with anterior to the left and dorsal up. (C) Noninduced pre-cellular embryo showing endogenous GSTE7 mRNA, which is maternally contributed and therefore distributed ubiquitously in the embryo. (D) Stage 13 embryo showing GSTE7 expression lower than in the early embryo, but with pronounced staining in the midgut. In the presence of the Actin-Gal4 driver, the transgenic tissues displayed high ubiquitous expression of GSTE7 in both pre-cellular (E) and stage 13 (F) embryos.</p

Assay conditions for the specific activity determination with alternative substrates.

<p>All measurements were performed in 0.1 M sodium phosphate buffer, pH 6.5 at 30°C. The stock solutions for Isothiocyanates were prepared in acetonitrile (2% final concentration in the assay), and that for CDNB in ethanol (5% final concentration in the assay).</p><p>Assay conditions for the specific activity determination with alternative substrates.</p

Survival of adult flies on 0.25 mM PEITC added to standard fly food.

<p>Actin-Gal4 flies crossed to wild-type control (<i>w¹¹¹⁸</i>) or to UAS-GSTE7 flies (GSTE7) were allowed to mate and then transferred to fly food supplemented with PEITC. Twelve vials with 10 flies each (GSTE7) or 10 vials with 10 flies each (<i>w¹¹¹⁸</i>) were used and the number of surviving flies in each vial scored. Females (A) and males (B) were kept in separate vials. Error bars represent standard error of the mean. * indicates P<0.05, two-tailed unpaired Student's t-test.</p

The Multifaceted Role of Glutathione S-Transferases in Health and Disease

In humans, the cytosolic glutathione S-transferase (GST) family of proteins is encoded by 16 genes presented in seven different classes. GSTs exhibit remarkable structural similarity with some overlapping functionalities. As a primary function, GSTs play a putative role in Phase II metabolism by protecting living cells against a wide variety of toxic molecules by conjugating them with the tripeptide glutathione. This conjugation reaction is extended to forming redox sensitive post-translational modifications on proteins: S-glutathionylation. Apart from these catalytic functions, specific GSTs are involved in the regulation of stress-induced signaling pathways that govern cell proliferation and apoptosis. Recently, studies on the effects of GST genetic polymorphisms on COVID-19 disease development revealed that the individuals with higher numbers of risk-associated genotypes showed higher risk of COVID-19 prevalence and severity. Furthermore, overexpression of GSTs in many tumors is frequently associated with drug resistance phenotypes. These functional properties make these proteins promising targets for therapeutics, and a number of GST inhibitors have progressed in clinical trials for the treatment of cancer and other diseases

Specific activities of purified GSTE7 with alternative substrates.

<p>The data are means ± SD of 3 replicate measurements and the background activities were corrected by using the same concentration of solvent without enzyme.</p><p>Specific activities of purified GSTE7 with alternative substrates.</p