GSTA1 Research Articles

Microcavity-assisted cloning (MAC) of hard-to-clone HepG2 cell lines: cloning made easy

Cloning is a key molecular biology procedure for obtaining a genetically homogenous population of organisms or cell lines. It requires the expansion of new cell populations starting from single genetically modified cells. Despite the technical progress, cloning of many cell lines remains difficult. Cloning often fails either due to the strenuous conditions associated with manipulating cells or because many cells don’t tolerate a single-cell state. Here we describe a new cloning method utilizing low adhesion microcavity plates. This new technique, named microcavity-assisted cloning (MAC) was developed to clone difficult-to-clone HepG2 cells. The clones were produced following CRISPR/Cas9 knockout of the GSTA1 gene by a random distribution of 200, 400, and 800 cells into 550 microcavities of a 24-well low adhesion plate originally designed for the culture of spheroids. The knockout of GSTA1 was verified at the protein level using Western blotting. The advantages of the MAC method are its low cost, ease of the procedure, and the possibility of scaling up the throughput and automatization.

Glutathione S-Transferase α4 Alleviates Hyperlipidemia-Induced Vascular Neointimal Hyperplasia in Arteriovenous Grafts via Inhibiting Endoplasmic Reticulum Stress.

Neointimal hyperplasia causes the failure of coronary artery bypass grafting. Our previous studies have found that endothelial dysfunction is 1 candidate for triggering neointimal hyperplasia, but which factors are involved in this process is unclear. Glutathione S-transferase α4 (GSTA4) plays an important role in metabolizing 4-hydroxynonenal (4-HNE), a highly reactive lipid peroxidation product, which causes endothelial dysfunction or death. Here, we investigated the role of GSTA4 in neointima formation after arteriovenous grafts (AVGs) with or without high-fat diet (HFD). Compared with normal diet, HFD caused endothelial dysfunction and increased neointima formation, concomitantly accompanied by downregulated expression of GSTA4 at the mRNA and protein levels. In vitro, overexpression of GSTA4 attenuated 4-HNE-induced endothelial dysfunction and knockdown of GSTA4 aggravated endothelial dysfunction. Furthermore, silencing GSTA4 expression facilitated the activation of 4-HNE-induced endoplasmic reticulum stress and inhibition of endoplasmic reticulum stress pathway alleviated 4-HNE-induced endothelial dysfunction. In addition, compared with wild-type mice, mice with knockout of endothelial-specific GSTA4 (GSTA4 endothelial cell KO) exhibited exacerbated vascular endothelial dysfunction and increased neointima formation caused by HFD. Together, these results demonstrate the critical role of GSTA4 in protecting the function of endothelial cells and in alleviating hyperlipidemia-induced vascular neointimal hyperplasia in arteriovenous grafts.

Regulatory Effects of 198-bp Structural Variants in the GSTA2 Promoter Region on Adipogenesis in Chickens.

Molecular breeding accelerates animal breeding and improves efficiency by utilizing genetic mutations. Structural variations (SVs), a significant source of genetic mutations, have a greater impact on phenotypic variation than SNPs. Understanding SV functional mechanisms and obtaining precise information are crucial for molecular breeding. In this study, association analysis revealed significant correlations between 198-bp SVs in the GSTA2 promoter region and abdominal fat weight, intramuscular fat content, and subcutaneous fat thickness in chickens. High expression of GSTA2 in adipose tissue was positively correlated with the abdominal fat percentage, and different genotypes of GSTA2 exhibited varied expression patterns in the liver. The 198-bp SVs regulate GSTA2 expression by binding to different transcription factors. Overexpression of GSTA2 promoted preadipocyte proliferation and differentiation, while interference had the opposite effect. Mechanistically, the 198-bp fragment contains binding sites for transcription factors such as C/EBPα that regulate GSTA2 expression and fat synthesis. These SVs are significantly associated with chicken fat traits, positively influencing preadipocyte development by regulating cell proliferation and differentiation. Our work provides compelling evidence for the use of 198-bp SVs in the GSTA2 promoter region as molecular markers for poultry breeding and offers new insights into the pivotal role of the GSTA2 gene in fat generation.

A Monocarbonyl Curcuminoid Derivative Inhibits the Activity of Human Glutathione Transferase A4-4 and Chemosensitizes Glioblastoma Cells to Temozolomide.

Human glutathione transferase A4-4 (hGSTA4-4) displays high catalytic efficiency towards 4-hydroxyalkenals and other cytotoxic and mutagenic products of radical reactions and lipid peroxidation. Its role as a target for the chemosensitization of cancer cells has not been investigated so far. In this study, the inhibitory potency of twelve selected natural products and ten monocarbonyl curcumin derivatives against hGSTA4-4 was studied. Among natural products, ellagic acid turned out to be the strongest inhibitor with an IC50 value of 0.44 ± 0.01 μM. Kinetic analysis using glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) as variable substrates showed that ellagic acid behaved as a competitive inhibitor towards both GSH and CDNB, with Ki values of 0.39 ± 0.02 and 0.63 ± 0.03 μM, respectively. Among the curcumin derivatives studied, three proved to be the most potent inhibitors, in the order DM151 > DM101 > DM100, with IC50 values of 2.4 ± 0.1 μM, 12.7 ± 1.1 μΜ and 16.9 ± 0.4 μΜ, respectively. Further kinetic inhibition analysis of the most active derivative, DM151, demonstrated that this compound is a mixed inhibitor towards CDNB with inhibition constants of Ki = 4.1 ± 0.5 μM and Ki' = 0.536 ± 0.034 μM, while it is a competitive inhibitor towards GSH with a Ki = 0.98 ± 0.11 μM. Molecular docking studies were performed to interpret the differences in binding of ellagic acid and curcumin derivatives to hGSTA4-4. The in silico measured docking scores were consistent with the obtained experimental data. Hydrogen bonds appear to be the main contributors to the specific binding of monocarbonyl curcumin derivatives, while π-π stacking interactions play a key role in the enzyme-ellagic acid interaction. In vitro cytotoxicity assessment of the worst (DM148) and the best (DM151) inhibitors was performed against glioblastoma cell lines U-251 MG and U-87 MG. The results revealed that DM151 displays considerably higher cytotoxicity against both glioblastoma cell lines, while the glioblastoma cytotoxicity of DM148 was very limited. Furthermore, low and non-toxic doses of DM151 sensitized U-251 MG cells to the first-line glioblastoma chemotherapeutic temozolomide (TMZ), allowing us to propose for the first time that hGSTA4-4 inhibitors may be attractive therapeutic partners for TMZ to optimize its clinical effect in glioblastoma chemotherapy.

Nrf2 pathway activation promotes the expression of genes related to glutathione metabolism in alcohol-exposed astrocytes.

Oxidative and antioxidant pathways play essential roles in the development of alcohol-induced brain injury. The Nrf2 pathway is an endogenous antioxidant response pathway, but there has been little research on the role of Nrf2 in alcohol-related diseases. Thus, we examined the effects of alcohol and an Nrf2 agonist (TBHQ) on astrocyte function, mRNA expression, and metabolite content to further explore the protective mechanisms of Nrf2 agonists in astrocytes following alcohol exposure. CTX TNA2 astrocytes were cultured with alcohol and TBHQ and then subjected to transcriptome sequencing, LC-MS/MS analysis, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and malondialdehyde (MDA) and superoxide dismutase (SOD) activity assays. Alcohol exposure significantly increased malondialdehyde (MDA) levels while decreasing superoxide dismutase (SOD) levels in astrocytes. Treatment with TBHQ effectively reversed these effects, demonstrating its protective role against oxidative stress induced by alcohol. Transcriptome sequencing and qRT-PCR analysis revealed that TBHQ specifically upregulates genes involved in glutathione metabolism, including a notable increase in the expression of the glutathione S-transferase A5 (GSTA5) gene, which was suppressed by alcohol exposure. Additionally, metabolomic analysis showed that TBHQ regulates key components of ether lipid metabolism in alcohol-exposed astrocytes, with significant reductions in the levels of lysophosphatidylcholine (18:0) (LysoPC (18:0)) and 2-acetyl-1-alkyl-sn-glycero-3-phosphocholine, both of which are critical markers in the ether lipid metabolic pathway. The findings underscore the role of TBHQ as an Nrf2 agonist in mitigating alcohol-induced oxidative damage in astrocytes by modulating glutathione metabolism and ether lipid metabolism. The regulation of GSTA5 gene expression emerges as a key mechanism through which Nrf2 agonists confer neuroprotection against oxidative stress and lipid oxidation. These insights pave the way for potential therapeutic strategies targeting the Nrf2 pathway to protect astrocytes from alcohol-induced damage.

Proteomics associated with coronary high-risk plaque features on optical coherence tomography

Abstract Introduction Cardiovascular disease is the major cause of death worldwide. The physiological status of the cardiovascular system is reflected in plasma proteins and a newly developed technique based on proximity extension assays has enabled the analysis of a broad range of protein biomarkers associated with cardiovascular disease. However, biomarkers for coronary high-risk plaques, including lipid-rich plaques, macrophages, cholesterol crystals, and microvessels, have not yet been found. Purpose This study aimed to identify proteins specific for coronary high-risk plaques. Methods We analyzed 57 patients (71.1 ± 10.6 years, male: 68.4%) who underwent intracoronary optical coherence tomography and provided blood samples for proteomic analysis. Forty-seven patients (86.0%) had lipid-rich plaques, 46 patients (80.7%) had macrophages, 16 patients (28.1%) had cholesterol crystals, and 27 patients (47.4%) had microvessels. A total of 1,470 plasma proteins were analyzed using the Olink® Explore 1,536 Reagent Kit and P2 100 cycle reagent kit on a NextSeq 2000 sequencer. Results In patients with lipid-rich plaques, the protein expressions of Interstitial collagenase (MMP1), Wiskott-Aldrich syndrome protein family member 1 (WASF1), and Protein phosphatase 1 regulatory subunit 12A (PPP1R12RA) were significantly increased, while the expressions of Fc receptor-like protein 3 (FCRL3), Folate receptor gamma (FOLR3), Cobalamin binding intrinsic factor (CBLIF), Glutathione S-transferase A1 (GSTA1), and Keratin, type I cytoskeletal 18 (KRT18) were significantly decreased (Figure A). In patients with macrophages, the protein expressions of MMP1, WASF1, PPP1R12RA, and Scavenger receptor cysteine-rich domain-containing group B protein (SSC4D) were significantly increased, while the expressions of Fatty acid-binding protein, intestinal (FABP2), FOLR3, CBLIF, GSTA1, and Fibroblast growth factor 21 (FGF21) were significantly decreased (Figure B). In patients with cholesterol crystals, the protein expressions of FOLR3, Secreted frizzled-related protein 1 (SFRP1), and Anterior gradient protein 2 homolog (AGR2) were significantly decreased (Figure C). In patients with microvessels, the expression of FOLR3 was significantly decreased (Figure D). Conclusion These proteins might be new biomarkers in patients with coronary high-risk plaques.

Conservation of Glutathione Transferase mRNA and Protein Sequences Similar to Human and Horse Alpha Class GST A3-3 across Dog, Goat, and Opossum Species.

The glutathione transferase A3-3 (GST A3-3) homodimeric enzyme is the most efficient enzyme that catalyzes isomerization of the precursors of testosterone, estradiol, and progesterone in the gonads of humans and horses. However, the presence of GST A3-3 orthologs with equally high ketosteroid isomerase activity has not been verified in other mammalian species, even though pig and cattle homologs have been cloned and studied. Identifying GSTA3 genes is a challenge because of multiple GSTA gene duplications (e.g., 12 in the human genome); consequently, the GSTA3 gene is not annotated in most genomes. To improve our understanding of GSTA3 gene products and their functions across diverse mammalian species, we cloned homologs of the horse and human GSTA3 mRNAs from the testes of a dog, goat, and gray short-tailed opossum, the genomes of which all currently lack GSTA3 gene annotations. The resultant novel GSTA3 mRNA and inferred protein sequences had a high level of conservation with human GSTA3 mRNA and protein sequences (≥70% and ≥64% identities, respectively). Sequence conservation was also apparent for the 12 residues of the "H-site" in the 222 amino acid GSTA3 protein that is known to interact with the steroid substrates. Modeling predicted that the dog GSTA3-3 may be a more active ketosteroid isomerase than the corresponding goat or opossum enzymes. However, expression of the GSTA3 gene was higher in liver than in other dog tissue. Our results improve understanding of the active sites of mammalian GST A3-3 enzymes, inhibitors of which might be useful for reducing steroidogenesis for medical purposes, such as fertility control or treatment of steroid-dependent diseases.

Direct observation of negative cooperativity in a detoxification enzyme at the atomic level by Electron Paramagnetic Resonance spectroscopy and simulation.

The catalytic activity of human glutathione S-transferase A1-1 (hGSTA1-1), a homodimeric detoxification enzyme, is dependent on the conformational dynamics of a key C-terminal helix α9 in each monomer. However, the structural details of how the two monomers interact upon binding of substrates is not well understood and the structure of the ligand-free state of the hGSTA1-1 homodimer has not been resolved. Here, we used a combination of electron paramagnetic resonance (EPR)distance measurements and weighted ensemble (WE) simulations to characterize the conformational ensemble of the ligand-free state at the atomic level. EPR measurements reveal a broad distance distribution between a pair of Cu(II) labels in the ligand-free state that gradually shifts and narrows as a function of increasing ligand concentration. These shifts suggest changes in the relative positioning of the two α9 helices upon ligand binding. WE simulations generated unbiased pathways for the seconds-timescale transition between alternate states of the enzyme, leading to the generation of atomically detailed structures of the ligand-free state. Notably, the simulations provide direct observations of negative cooperativity between the monomers of hGSTA1-1, which involve the mutually exclusive docking of α9 in each monomer as a lid over the active site. We identify key interactions between residues that lead to this negative cooperativity. Negative cooperativity may be essential for interaction of hGSTA1-1 with a wide variety of toxic substrates and their subsequent neutralization. More broadly, this work demonstrates the power of integrating EPR distances with WE rare-events sampling strategy to gain mechanistic information on protein function at the atomic level.

MRNA level of antioxidant genes and activity of NADPH-generating enzymes in rotenone-induced parkinsonism in rats

Aim. To analyze the mRNA level of genes encoding antioxidant enzymes and the transcription factors Nrf2 and Foxo1 regulating their expression and the activity of glucose-6-phosphate dehydrogenase (G6PDH) and NADPdependent isocitrate dehydrogenase (NADP-IDH) and assess the correlation between these parameters, oxidative status, and motor coordination parameters in rats with rotenone-induced parkinsonism.Materials and methods. The study was performed on male Wistar rats aged 4–6 months and weighing 200–250 g. Parkinsonism was modeled by subcutaneous administration of rotenone for 10 days at a dose of 2.5 mg / kg. To confirm the development of the pathology, motor coordination tests and histological staining of the cerebral cortex and striatum with hematoxylin and eosin were used. The oxidative status was analyzed based on the levels of conjugated dienes, carbonyl amino acid residues in proteins, and α-tocopherol. The enzyme activity was studied spectrophotometrically by the formation of NADPH. Real-time PCR was used to analyze the level of gene mRNA.Results. During the study, an increase in serum and brain concentrations of conjugated dienes, carbonyl amino acid residues, and α-tocopherol was observed in the experimental group of rats compared to the controls. It could be associated with the redistribution of this compound between tissues during pathology development. The animals with experimental parkinsonism, in addition, were characterized by a decrease in the mRNA level of the Sod1, Gpx1, Gsr, Gsta2, Nfe2l2, and Foxo1 genes, as well as the activity of G6PDH and NADP-IDH. In the rats with experimental parkinsonism, a negative correlation of NADPH-IDH activity in the brain with serum α-tocopherol level and a positive correlation with Gpx1 and Foxo1 mRNA levels in the striatum were found. The level of oxidatively modified proteins in the brain of the animals with PD was negatively correlated with the concentration of Gsta2 mRNA in the striatum, while the specific activity of G6PDH in the serum was characterized by the positive relationship with grip strength.Conclusion. The data obtained indicate that the inhibition of transcription of the genes encoding antioxidant enzymes and regulatory factors Nrf2 and Foxo1 contributed significantly to the development of oxidative stress in PD. A decrease in the activity of G6PDH and NADP-IDH led to a decrease in the availability of NADPH, which is a limiting factor in the functioning of the glutathione antioxidant system. Obviously, the inhibition of G6PDH and NADP-IDH was also an important pathogenic factor in the progression of the pathology. Along with a decrease in the content of antioxidant gene mRNA in the brain tissues, the level of α-tocopherol increased in the rats with parkinsonism, which could be the result of an imbalance in the functioning of antioxidant system.

Lansoprazole protects hepatic cells against cisplatin-induced oxidative stress through the p38 MAPK/ARE/Nrf2 pathway.

Lansoprazole, a proton pump inhibitor, can exert antioxidant effects through the induction of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, independently of the inhibition of acid secretion in the gastrointestinal tract. Lansoprazole has been reported to provide hepatoprotection in a drug-induced hepatitis animal model through the Nrf2/heme oxygenase-1 (HO1) pathway. We sought to investigate the molecular mechanism of cytoprotection by lansoprazole. An in vitro experimental model was conducted using cultured rat hepatic cells treated with lansoprazole to analyze the expression levels of Nrf2 and its downstream genes, the activity of Nrf2 using luciferase reporter assays, cisplatin-induced cytotoxicity, and signaling pathways involved in Nrf2 activation. Lansoprazole treatment of rat liver epithelial RL34 cells induced transactivation of Nrf2 and the expression of the Nrf2-dependent antioxidant genes encoding HO1, NAD(P)H quinone oxidoreductase-1, and glutathione S-transferase A2. Furthermore, cycloheximide chase experiments revealed that lansoprazole prolongs the half-life of the Nrf2 protein. Notably, cell viability was significantly increased by lansoprazole treatment in a cisplatin-induced cytotoxicity model. Moreover, the siRNA knockdown of Nrf2 fully abolished the cytoprotective effect of lansoprazole, whereas the inhibition of HO1 by tin-mesoporphyrin only partially abolished this. Finally, lansoprazole promoted the phosphorylation of p38 mitogen-activated protein kinase (MAPK) but not that of the extracellular signal-regulated kinase or the c-Jun N-terminal kinase. Using SB203580, a specific inhibitor for p38 MAPK, the lansoprazole-induced Nrf2/antioxidant response elements pathway activation and cytoprotective effects were shown to be exclusively p38 MAPK dependent. Lansoprazole was shown by these results to exert a cytoprotective effect on liver epithelial cells against the cisplatin-induced cytotoxicity through the p38 MAPK signaling pathway. This could have potential applications for the prevention and treatment of oxidative injury in the liver.

The Protective Potential eff ect of Metformin during Acetaminophen Hepatotoxicity through Nrf2 Activation

Acetaminophen (N-acetyl-para-aminophenol [APAP]) is the most commonly used medication for the relief of pain and fever around the world. Although APAP is safe and eff ective at a therapeutic level, acute overdose causes hepatotoxicity and severe liver damage. The hepatotoxicity induced by APAP is a persistent global problem that results in hepatotoxicity cases; acute liver failure and even death worldwide. This toxicity is characterized by extensive oxidant stress which consequently causes hepatocyte cell death. On the other hand, scientifi c studies have proven that MET shows hepatoprotective eff ect against hepatotoxicity induced by APAP through numerous mechanisms, e.g., antioxidant activity that alleviate the hepatotoxicity by activating Nrf2 pathway. The activation of Nrf2 pathway is anticipated to protect cells from oxidative stress that forms during hepatotoxicity. The benefi cial of using MET to protect against hepatotoxicity after APAP has been performed in an in-vitro experiment using Hep2G cell culture. However, up to our knowledge non, an in-vivo study (experimental animal) has been used to approve the benefi t of MET. Justifi cation: Nrf2 pathway activation by MET is one of the most important issues that need to be explained and followed up in laboratory animals since it has been previously studied in-vitro. Our study focuses on studying Nrf2 pathway in-vivo, since the results of in-vivo study are more relevant and similar to that of human beings. Aim of the study: To examine the possible stimulant eff ect of MET on Nrf2 pathway in experimental animals and its role in protecting the liver from oxidative stress that formed during APAP-induced hepatotoxicity. Methodology: Twenty-four wistar rats were divided randomly into four groups (six rats/Grp). Grp1; normal saline orally, Grp2; toxic dose of APAP (1000 mg/kg) orally; Grp3; 200 mg/kg of MET ip after 1-hour of APAP (1000 mg/kg) orally, Grp 4; 200 mg/kg MET ip for 24 hours. Then sera from experimental animals were collected for subsequent assessments of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) enzyme activities. Liver tissue was harvested to detect the expression of keap1 which is the negative regulator of Nrf2, and HO-1 and GST A1, which are related to Nrf2 pathway, by western blotting technique. Results: The results were showed a signifi cant elevation in ALT and AST activities in APAP treated group while MET normalized these biomarkers (AST and ALT). Western blotting assay showed that keap1 expression increased in APAP-treated animals while MET showed a signifi cant decrement in keap1 expression. The expression of antioxidant proteins HO-1 and GST A1 are decreased signifi cantly in APAP-treated animals while increased signifi cantly by MET treatment. Conclusion: The present results demonstrated that MET has a hepatoprotective eff ect in experimental animals against hepatotoxicity induced by APAP through activating Nrf2 pathway.

Tandem mass tag-based quantitative proteomic profiling identifies candidate serum biomarkers of drug-induced liver injury in humans

Diagnosis of drug-induced liver injury (DILI) and its distinction from other liver diseases are significant challenges in drug development and clinical practice. Here, we identify, confirm, and replicate the biomarker performance characteristics of candidate proteins in patients with DILI at onset (DO; n = 133) and follow-up (n = 120), acute non-DILI at onset (NDO; n = 63) and follow-up (n = 42), and healthy volunteers (HV; n = 104). Area under the receiver operating characteristic curve (AUC) for cytoplasmic aconitate hydratase, argininosuccinate synthase, carbamoylphosphate synthase, fumarylacetoacetase, fructose-1,6-bisphosphatase 1 (FBP1) across cohorts achieved near complete separation (range: 0.94–0.99) of DO and HV. In addition, we show that FBP1, alone or in combination with glutathione S-transferase A1 and leukocyte cell-derived chemotaxin 2, could potentially assist in clinical diagnosis by distinguishing NDO from DO (AUC range: 0.65–0.78), but further technical and clinical validation of these candidate biomarkers is needed.

Identification of alternative splicing events related to fatty liver formation in duck using full-length transcripts

BackgroundNon-alcoholic fatty liver disease (NAFLD) is one of most common diseases in the world. Recently, alternative splicing (AS) has been reported to play a key role in NAFLD processes in mammals. Ducks can quickly form fatty liver similar to human NAFLD after overfeeding and restore to normal liver in a short time, suggesting that ducks are an excellent model to unravel molecular mechanisms of lipid metabolism for NAFLD. However, how alternative splicing events (ASEs) affect the fatty liver process in ducks is still unclear.ResultsHere we identify 126,277 unique transcripts in liver tissue from an overfed duck (77,237 total transcripts) and its sibling control (69,618 total transcripts). We combined these full-length transcripts with Illumina RNA-seq data from five pairs of overfed ducks and control individuals. Full-length transcript sequencing provided us with structural information of transcripts and Illumina RNA-seq data reveals the expressional profile of each transcript. We found, among these unique transcripts, 30,618 were lncRNAs and 1,744 transcripts including 155 lncRNAs and 1,589 coding transcripts showed significantly differential expression in liver tissues between overfed ducks and control individuals. We also detected 27,317 ASEs and 142 of them showed significant relative abundance changes in ducks under different feeding conditions. Full-length transcript profiles together with Illumina RNA-seq data demonstrated that 10 genes involving in lipid metabolism had ASEs with significantly differential abundance in normally fed (control) and overfed ducks. Among these genes, protein products of five genes (CYP4F22, BTN, GSTA2, ADH5, and DHRS2 genes) were changed by ASEs.ConclusionsThis study presents an example of how to identify ASEs related to important biological processes, such as fatty liver formation, using full-length transcripts alongside Illumina RNA-seq data. Based on these data, we screened out ASEs of lipid-metabolism related genes which might respond to overfeeding. Our future ability to explore the function of genes showing AS differences between overfed ducks and their sibling controls, using genetic manipulations and co-evolutionary studies, will certainly extend our knowledge of genes related to the non-pathogenic fatty liver process.

A Key Role in Catalysis and Enzyme Thermostability of a Conserved Helix H5 Motif of Human Glutathione Transferase A1-1.

Glutathione transferases (GSTs) are promiscuous enzymes whose main function is the detoxification of electrophilic compounds. These enzymes are characterized by structural modularity that underpins their exploitation as dynamic scaffolds for engineering enzyme variants, with customized catalytic and structural properties. In the present work, multiple sequence alignment of the alpha class GSTs allowed the identification of three conserved residues (E137, K141, and S142) at α-helix 5 (H5). A motif-directed redesign of the human glutathione transferase A1-1 (hGSTA1-1) was performed through site-directed mutagenesis at these sites, creating two single- and two double-point mutants (E137H, K141H, K141H/S142H, and E137H/K141H). The results showed that all the enzyme variants displayed enhanced catalytic activity compared to the wild-type enzyme hGSTA1-1, while the double mutant hGSTA1-K141H/S142H also showed improved thermal stability. X-ray crystallographic analysis revealed the molecular basis of the effects of double mutations on enzyme stability and catalysis. The biochemical and structural analysis presented here will contribute to a deeper understanding of the structure and function of alpha class GSTs.

Reversibility and Low Commitment to Forward Catalysis in the Conjugation of Lipid Alkenals by Glutathione Transferase A4-4.

High concentrations of electrophilic lipid alkenals formed during oxidative stress are implicated in cytotoxicity and disease. However, low concentrations of alkenals are required to induce antioxidative stress responses. An established clearance pathway for lipid alkenals includes conjugation to glutathione (GSH) via Michael addition, which is catalyzed mainly by glutathione transferase isoform A4 (GSTA4-4). Based on the ability of GSTs to catalyze hydrolysis or retro-Michael addition of GSH conjugates, and the antioxidant function of low concentrations of lipid alkenals, we hypothesize that GSTA4-4 contributes a homeostatic role in lipid metabolism. Enzymatic kinetic parameters for retro-Michael addition with trans-2-Nonenal (NE) reveal the chemical competence of GSTA4-4 in this putative role. The forward GSTA4-4-catalyzed Michael addition occurs with the rapid exchange of the C2 proton of NE in D2O as observed by NMR. The isotope exchange was completely dependent on the presence of GSH. The overall commitment to catalysis, or the ratio of first order kcat,f for 'forward' Michael addition to the first order kcat,ex for H/D exchange is remarkably low, approximately 3:1. This behavior is consistent with the possibility that GSTA4-4 is a regulatory enzyme that contributes to steady-state levels of lipid alkenals, rather than a strict 'one way' detoxication enzyme.

Adipocyte-specific FXR-deficiency protects adipose tissue from oxidative stress and insulin resistance and improves glucose homeostasis

ObjectiveObesity is associated with metabolic dysfunction of white adipose tissue (WAT). Activated adipocytes secrete pro-inflammatory cytokines resulting in the recruitment of pro-inflammatory macrophages, which contribute to WAT insulin resistance. The bile acid (BA)-activated nuclear Farnesoid X Receptor (FXR) controls systemic glucose and lipid metabolism. Here, we studied the role of FXR in adipose tissue function. MethodsWe first investigated the immune phenotype of epididymal WAT (eWAT) from high fat diet (HFD)-fed whole-body FXR-deficient (FXR−/−) mice by flow cytometry and gene expression analysis. We then generated adipocyte-specific FXR-deficient (Ad-FXR−/−) mice and analyzed systemic and eWAT metabolism and immune phenotype upon HFD feeding. Transcriptomic analysis was done on mature eWAT adipocytes from HFD-fed Ad-FXR−/− mice. ResultseWAT from HFD-fed whole-body FXR−/− and Ad-FXR−/− mice displayed decreased pro-inflammatory macrophage infiltration and inflammation. Ad-FXR−/− mice showed lower blood glucose concentrations, improved systemic glucose tolerance and WAT insulin sensitivity and oxidative stress. Transcriptomic analysis identified Gsta4, a modulator of oxidative stress in WAT, as the most upregulated gene in Ad-FXR−/− mouse adipocytes. Finally, chromatin immunoprecipitation analysis showed that FXR binds the Gsta4 gene promoter. ConclusionsThese results indicate a role for the adipocyte FXR-GSTA4 axis in controlling HFD-induced inflammation and systemic glucose homeostasis.

Differentially Expressed Genes and Signalling Pathways Regulated by High Selenium Involved in Antioxidant and Immune Functions of Goats Based on Transcriptome Sequencing.

The objective of this study is to observe the effect of high selenium on the antioxidant and immune functions of growing goats based on transcriptome sequencing. Eighteen goats were randomly divided into three groups: (1) the control (CON) group was fed a basal diet, and (2) the treatment 1 group (LS) and treatment 2 group (HS) were fed a basal diet with 2.4 and 4.8 mg/kg selenium-yeast (SY), respectively. The results indicate that HS treatment significantly (p < 0.05) increased the apparent digestibility of either extract and significantly increased (p < 0.05) total antioxidant capacity, whereas it significantly (p < 0.05) decreased plasma aspartate aminotransferase and malondialdehyde relative to the control group. The LS treatment had significantly (p < 0.05) increased glutathione S-transferase and catalase compared to CON. A total of 532 differentially expressed genes (DEGs) between the CON and HS were obtained using transcriptome sequencing. Kyoto Encyclopedia of Genes and Genomes analysis identified upregulated (p < 0.05) DEGs mainly related to vascular smooth muscle contraction, alpha-linolenic acid metabolism, biosynthesis of unsaturated fatty acids, the VEGF signalling pathway, and proteoglycans in cancer; downregulated (p < 0.05) DEGs mainly related to the NOD-like receptor signalling pathway, influenza A, cytokine-cytokine receptor interaction, haematopoietic cell lineage, and African trypanosomiasis. Ontology analyses of the top genes show that the identified DEGs are mainly involved in the regulation of granulocyte macrophage colony-stimulating factor production for biological processes, the external side of the plasma membrane for cellular components, and carbohydrate derivative binding for molecular functions. Seven genes are considered potential candidate genes for regulating antioxidant activity, including selenoprotein W, 1, glutathione peroxidase 1, glutathione S-transferase A1, tumour necrosis factor, tumour necrosis factor superfamily member 10, tumour necrosis factor superfamily member 8, and tumour necrosis factor superfamily member 13b. The experimental observations indicate that dietary supplementation with 4.8 mg/kg SY can enhance antioxidant and immune functions by improving muscle immunity, reducing the concentrations of inflammatory molecules, and modulating antioxidant and inflammatory signalling pathways in growing goats.

An integrated targeted metabolomics and network pharmacology approach to exploring the mechanism of ellagic acid against sleep deprivation-induced memory impairment and anxiety.

As a neuroprotective agent, ellagic acid (EA) is extremely beneficial. Our previous study found that EA can alleviate sleep deprivation (SD)-induced abnormal behaviors, although the mechanisms underlying this protective effect have not yet been fully elucidated. An integrated network pharmacology and targeted metabolomics approach was utilized in this study to investigate the mechanism of EA against SD-induced memory impairment and anxiety. Behavioral tests were conducted on mice after 72 h of SD. Hematoxylin and eosin staining and nissl staining were then carried out. Integration of network pharmacology and targeted metabolomics was performed. Eventually, the putative targets were further verified using molecular docking analyses and immunoblotting assays. The present study findings confirmed that EA ameliorated the behavioral abnormalities induced by SD and prevented histopathological and morphological damage to hippocampal neurons. Through multivariate analysis, clear clustering was obtained among different groups, and potential biomarkers were identified. Four key targets, catechol-O-methyltransferase (COMT), cytochrome P450 1B1 (CYP1B1), glutathione S-transferase A2 (GSTA2), and glutathione S-transferase P1 (GSTP1), as well as the related potential metabolites and metabolic pathways, were determined by further integrated analysis. Meanwhile, in-silico studies revealed that EA is well located inside the binding site of CYP1B1 and COMT. The experimental results further demonstrated that EA significantly reduced the increased expression of CYP1B1 and COMT caused by SD. The findings of this study extended our understanding of the underlying mechanisms by which EA treats SD-induced memory impairment and anxiety, and suggested a novel approach to address the increased health risks associated with sleep loss.

Long-Term Sleep Deprivation-Induced Myocardial Remodeling and Mitochondrial Dysfunction in Mice Were Attenuated by Lipoic Acid and N-Acetylcysteine.

The impact of long-term sleep deprivation on the heart and its underlying mechanisms are poorly understood. The present study aimed to investigate the impact of chronic sleep deprivation (CSD) on the heart and mitochondrial function and explore an effective drug for treating CSD-induced heart dysfunction. We used a modified method to induce CSD in mice; lipoic acid (LA) and N-acetylcysteine (NAC) were used to treat CSD mice. Echocardiography, hematoxylin-eosin (H&E) staining, Sirius red staining, and immunohistochemistry were used to determine heart function and cardiac fibrosis. The serum levels of brain natriuretic peptide (BNP), superoxide Dismutase (SOD), micro malondialdehyde (MDA), and glutathione (GSH) were measured to determine cardiovascular and oxidative stress-related damage. Transmission electron microscopy was used to investigate mitochondrial damage. RNA-seq and Western blotting were used to explore related pathways. We found that the left ventricular ejection fraction (LVEF) and fraction shortening (LVFS) values were significantly decreased and myocardial hypertrophy was induced, accompanied by damaged mitochondria, elevated reactive oxygen species (ROS), and reduced SOD levels. RNA-sequence analysis of the heart tissue showed that various differentially expressed genes in the metabolic pathway were enriched. Sirtuin 1 (Sirt1) and Glutathione S-transferase A3 (Gsta3) may be responsible for CSD-induced heart and mitochondrial dysfunction. Pharmacological inhibition of ROS by treating CSD mice with LA and NAC effectively reduced heart damage and mitochondrial dysfunction by regulating Sirt1 and Gsta3 expression. Our data contribute to understanding the pathways of CSD-induced heart dysfunction, and pharmacological targeting to ROS may represent a strategy to prevent CSD-induced heart damage.

Phase I Metabolism of Pterostilbene, a Dietary Resveratrol Derivative: Metabolite Identification, Species Differences, Isozyme Contribution, and Further Bioactivation

Pterostilbene (PTE), a dietary derivative of resveratrol, displayed pleiotropic health-promoting activities. This study aimed to explore the metabolic profiles and species differences of the phase I metabolism of PTE and to investigate subsequent detoxification after PTE bioactivation. PTE was found to be biotransformed to two pharmacologically active metabolites, pinostilbene and 3'-hydroxypterostilbene, in vivo and in vitro with substantial species differences. Human CYP1A2 was proved to be mainly responsible for the demethylation and 3'-hydroxylation of PTE, with its contribution to a demethylation of 94.5% and to a 3'-hydroxylation of 97.9%. An in vitro glutathione trapping experiment revealed the presence of an ortho-quinone intermediate formed by further oxidation of 3'-hydroxypterostilbene. Human glutathione S-transferase isoforms A2, T1, and A1 inactivated the ortho-quinone intermediate by catalyzing glutathione conjugation, implicating a potential protective pathway against PTE bioactivation-derived toxicity. Overall, this study provided a comprehensive view of PTE phase I metabolism and facilitated its further development as a promising nutraceutical.