Glutathione S-transferase Protein Research Articles

Common structural features in some of the sequentially distant neurotransmitter transporters N-termini

The N-terminal regions of SLC6 transporters are sequentially unrelated, and the majority of such transporters contain only relatively short peptide N-terminal extensions. Currently, it is not clear if a diversity of N-terminal sequences represents diverse functions among the transporters or if there are common functions hidden behind similar, as yet unidentified, structures. Using alignment of amino acid sequences with the hydropathy plot, disorder prediction, and calpain recognition sites, we show that common structural features among the N-termini of some transporters might exist.We previously showed that polymeric neurotransmitter transporter N-termini exhibit very similar profiles of dynamic, time-dependent 465 nm-595 nm-750 nm absorbance metachromasia in the Bradford assay. Here we report that under certain mild denaturing conditions, filamentous aggregation of glutathione S-transferase (GST) protein results in similar near-infrared metachromasia. This effect was eliminated by further GST protein denaturation and solubilization. The results suggest that aggregation of partially denatured GST stabilizes Coomassie dye docking sites, producing a near-infrared absorbance shift similar to that observed in the polymeric unstructured N-termini of transporters.

Molecular and biochemical characterization of a plant-like iota-class glutathione S-transferase from the halotolerant cyanobacterium Halothece sp. PCC7418.

This study identifies a unique glutathione S-transferase (GST) in extremophile using genome, phylogeny, bioinformatics, functional characterization, and RNA sequencing analysis. Five putative GSTs (H0647, H0729, H1478, H3557, and H3594) were identified in Halothece sp. PCC7418. Phylogenetic analysis suggested that H0647, H1478, H0729, H3557, and H3594 are distinct GST classes. Of these, H0729 was classified as an iota-class GST, encoding a high molecular mass GST protein with remarkable features. The protein secondary structure of H0729 revealed the presence of a glutaredoxin (Grx) Cys-Pro-Tyr-Cys (C-P-Y-C) motif that overlaps with the N-terminal domain and harbors a topology similar to the thioredoxin (Trx) fold. Interestingly, recombinant H0729 exhibited a high catalytic efficiency for both glutathione (GSH) and 1-chloro-2, 4-dinitrobenzene (CDNB), with catalytic efficiencies that were 155- and 32-fold higher, respectively compared to recombinant H3557. Lastly, the Halothece gene expression profiles suggested that antioxidant and phase II detoxification encoding genes are crucial in response to salt stress. Iota-class GST was identified in cyanobacteria. This GST exhibited a high catalytic efficiency toward xenobiotic substrates. Our findings shed light a diversified evolution of GST in cyanobacteria and provide functional dynamics of the genes encoding the enzymatic antioxidant and detoxification systems under abiotic stresses.

Biochemical Properties and Immunogenic Epitopes of Echinococcus granulosus Glutathione S-Transferase as a Vaccine Target: In-Silico Study.

The current in silico study was done to determine the primary biochemical features and immunogenic epitopes of Echinococcus granulosus glutathione S-transferase protein as a potential vaccine candidate. Several web tools were employed to predict physico-chemical properties, antigenicity, allergenicity, solubility, post-translational modification (PTM) sites, subcellular localization, signal peptide, transmembrane domain, secondary and tertiary structure followed by refinement and validations. In addition, B-cell epitopes were predicted and were screened using various web servers, while MHC-binding and CTL epitopes were predicted using IEDB and NetCTL servers, respectively. The protein had 219 residues with a molecular weight of 25.55 kDa and alkaline isoelectric pH (7.5). This protein was stable, thermo-tolerant (aliphatic index: 78.04) and hydrophilic (GRAVY: -0.440). The predicted antigenicity scores were low and the protein was nonallergenic in nature. There were no transmembrane domain and signal peptide in the sequence. Moreover, several B-cell, MHC-binding and CTL epitopes were found in the EgGST protein, which could be further used in multi-epitope vaccines. Further studies are needed on the development of vaccines in vivo using EgGST alone or in combination with other antigens in the future.

Environmental-related doses of afidopyropen induced toxicity effects in earthworms (Eisenia fetida)

Afidopyropen has high activity against pests. However, it poses potential risks to the soil ecology after entering the environment. The toxicity of afidopyropen to earthworms (Eisenia fetida) was studied for the first time in this study. The results showed that afidopyropen had low level of acute toxicity to E. fetida. Under the stimulation of chronic toxicity, the increase of reactive oxygen species (ROS) level activated the antioxidant and detoxification system, which led to the increase of superoxide dismutase (SOD) and glutathione S-transferase (GST) activities. Lipid peroxidation and DNA damage were characterized by the increase of malondialdehyde (MDA) and 8-hydroxy-2’-deoxyguanosine (8-OHdG) contents. Meanwhile, the functional genes SOD, CAT, GST, heat shock protein 70 (HSP70), transcriptionally controlled tumor protein (TCTP), and annetocin (ANN) played a synergistic role in antioxidant defense. However, the comprehensive toxicity of high concentration still increased on the 28th day. In addition, strong histopathological damage in the body wall and intestine was observed, accompanied by weight loss, which indicated that afidopyropen inhibited the growth of E. fetida. The molecular docking revealed that afidopyrene combined with the surface structure of SOD and GST proteins, which made SOD and GST become sensitive biomarkers reflecting the toxicity of afidopyropen to E. fetida. Summing up, afidopyropen destroys the homeostasis of E. fetida through chronic toxic. These results provide theoretical data for evaluating the environmental risk of afidopyropen to soil ecosystem.

Glutathione S-transferase activity facilitates rice tolerance to the barnyard grass root exudate DIMBOA

BackgroundIn paddy fields, the noxious weed barnyard grass secretes 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) to interfere with rice growth. Rice is unable to synthesize DIMBOA. Rice cultivars with high or low levels of allelopathy may respond differently to DIMBOA.ResultsIn this study, we found that low concentrations of DIMBOA (≤ 0.06 mM) promoted seedling growth in allelopathic rice PI312777, while DIMBOA (≤ 0.08 mM) had no significant influence on the nonallelopathic rice Lemont. DIMBOA treatment caused changes in the expression of a large number of glutathione S-transferase (GST) proteins, which resulting in enrichment of the glutathione metabolic pathway. This pathway facilitates plant detoxification of heterologous substances. The basal levels of GST activity in Lemont were significantly higher than those in PI312777, while GST activity in PI312777 was slightly induced by increasing DIMBOA concentrations. Overexpression of GST genes (Os09g0367700 and Os01g0949800) in these two cultivars enhanced rice resistance to DIMBOA.ConclusionsTaken together, our results indicated that different rice accessions with different levels of allelopathy have variable tolerance to DIMBOA. Lemont had higher GST activity, which helped it tolerate DIMBOA, while PI312777 had lower GST activity that was more inducible. The enhancement of GST expression facilitates rice tolerance to DIMBOA toxins from barnyard grass root exudates.

Manganese- and zinc-coordinated interaction of Schistosoma japonicum glutathione S-transferase with neurotransmitter transporters GlyT1 and GAT3 in vitro

Glutathione S-transferases (GSTs) are a family of multifunctional isoenzymes involved in the neutralization of toxic compounds, drug resistance and several other cellular functions. The glutathione S-transferase enzyme of Schistosoma japonicum (SjGST-26) plays a role in human schistosomiasis and is also a frequently used fusion partner in mammalian and bacterial expression and pull-down systems. GSTs seem not to be naturally associated with metal ions. Exceptionally, in vitro, metal binding sites have been previously described in some schistosome GSTs; however, their possible physiological role is unclear. Molecules of several neurotransmitter transporters also contain a regulatory zinc binding site, which affects their transport cycle. Here we show that among several metals, manganese and zinc are able to induce a specific protein interaction of SjGST-26 with the glycine transporter GlyT1 and the GABA transporter GAT3 in vitro. The results suggest that metal-binding sites on SjGST-26 and neurotransmitter transporters might function in metal-coordinated interactions with other metalloproteins. Our results additionally indicate that the presence of metal ions in SjGST-26-based GST protein pull-down assays may lead to a false-positive interaction if the potential interacting target is the metalloprotein.

The Detection of Circulating Antigen Glutathione S-Transferase in Sheep Infected with Fasciola hepatica with Double-Antibody Sandwich Signal Amplification Enzyme-Linked Immunosorbent Assay

Fasciolosis is a global zoonotic parasitic disease caused by F. hepatica infection that is particularly harmful to cattle and sheep. A biotin–streptavidin signal amplification ELISA (streptavidin-ELISA/SA-ELISA) based on circulating antigens can allow for the early detection of F. hepatica-infected animals and is suitable for batch detection. It is considered to be a better means of detecting F. hepatica infection than traditional detection methods. In this study, using the serum of sheep artificially infected with F. hepatica, the cDNA expression library of F. hepatica was screened, 17 immunodominant antigen genes of F. hepatica were obtained, and glutathione s-transferase (GST) was selected as the candidate detection antigen. Firstly, the GST cDNA sequence was amplified from F. hepatica, followed by the preparation of recombinant protein GST (rFhGST). Then, monoclonal and polyclonal antibodies against rFhGST were prepared using the GST protein. Afterward, the immunolocalization of the target protein in the worm was observed via confocal microscopy, and it was found that the GST protein was localized in the uterus, intestinal tract, and body surface of F. hepatica. Finally, a double-antibody sandwich SA-ELISA based on the detection of circulating antigens was established. There was no cross-reaction with positive sera infected with Dicrocoelium lanceatum (D. lanceatum), Haemonchus contortus (H. contortus), Neospora caninum (N. caninum), or Schistosoma japonicum (S. japonicum). Forty serum and fecal samples from the same batch of sheep in Nong’an County, Changchun City, Jilin Province, China were analyzed using the established detection method and fecal detection method. The positive rate of the SA-ELISA was 17.5%, and the positive rate of the fecal detection method was 15%. The detection results of this method were 100% consistent with commercial ELISA kits. A total of 152 sheep serum samples were tested in Nong’an County, Changchun City, Jilin Province, and the positive rate was 5.92%. This study laid the foundation for the development of serological detection preparations for F. hepatica infection based on the detection of circulating antigens.

Computational biology of antibody epitope, tunnels and pores analysis of protein glutathione S-transferase P, and quantum mechanics

Different tissues of various plants contain allelochemicals such as phenolics, flavonoids, etc., which exhibit antioxidants and protect the plants from harmful infections. The widespread group of plant allelochemicals in the ecosystem is phenolic compounds. They are substances composed of an aromatic hydrocarbon group and a hydroxyl group. The 3-Hydroxyflavone skeleton of flavonol has a phenolic and a hydroxyl substitution. A comparison of experimental and calculated data of FT-IR and Raman was studied for the vibrational assessment of these allelochemicals. PES scan and molecular geometry analysis are done for the conformation of the 3-Hydroxyflavone ligand. 3-Hydroxyflavone is docked with the three proteins of Homo sapiens such as Prothrombin with 622 amino acids synthesized in the liver, human neutrophils found within intracellular granules with 467 amino acids, Glutathione S-transferase P is produced from exogenous xenobiotics with 210 amino acids. The active site residues by using the Prothrombin (1A2C), Neutrophil collagenase (1A86), Glutathione S-transferase P(18 GS) protein with ligand 3-Hydroxyflavone, fair binding affinity was found for the Glutathione S-transferase P (18 GS). The MOLE online server web interface's ability to see and analyze tunnels and pores allows for simple, online interaction with bio-macromolecule investigation. The automatic transmembrane channel calculation on the MOLE web generates the quickest list of ligands for transport analysis and tunnel identification. Pore-forming proteins (PFPs) are recognized as crucial agents in immunity and infection. They target membranes by opening channels through them. ElliPro is thought to be a potentially effective method for identifying antibody epitopes in protein antigens. Molecular dynamics result the general time-dependent structural deviation/degree of similarity among the structures that the trajectory records. The epitope technique sought to examine the effectiveness of its web tool on linear and discontinuous epitopes known from the structures of antibodies of 18 GS with 3-Hydroxyflavone complexes and find effective scores.

Natural Perylenequinone Compounds as Potent Inhibitors of Schistosoma mansoni Glutathione S-Transferase.

The existing treatment strategy for Schistosomiasis centers on praziquantel, a single drug, but its effectiveness is limited due to resistance and lack of preventive benefits. Thus, there is an urgent need for novel antischistosomal agents. Schistosoma glutathione S-transferase (GST) is an essential parasite enzyme, with a high potential for targeted drug discovery. In this study, we conducted a screening of compounds possessing antihelminth properties, focusing on their interaction with the Schistosoma mansoni glutathione S-transferase (SmGST) protein. We demonstrated the unique nature of SmGST in comparison to human GST. Evolutionary analysis indicated its close relationship with other parasitic worms, setting it apart from free-living worms such as C. elegans. Through an assessment of binding pockets and subsequent protein-ligand docking, we identified Scutiaquinone A and Scutiaquinone B, both naturally derived Perylenequinones, as robust binders to SmGST. These compounds have exhibited effectiveness against similar parasites and offer promising potential as antischistosomal agents.

Physiological, Proteomic, and Resin Yield-Related Genes Expression Analysis Provides Insights into the Mechanisms Regulating Resin Yield in Masson Pine.

Masson pine (Pinus massoniana Lamb.) is an important resin-producing conifer species in China. Resin yield is a highly heritable trait and varies greatly among different genotypes. However, the mechanisms regulating the resin yield of masson pine remain largely unknown. In this study, physiological, proteomic, and gene expression analysis was performed on xylem tissues of masson pine with high and low resin yield. Physiological investigation showed that the activity of terpene synthase, as well as the contents of soluble sugar, jasmonic acid (JA), methyl jasmonate (MeJA), gibberellins (GA1, GA4, GA9, GA19, and GA20), indole-3-acetic acid (IAA), and abscisic acid (ABA) were significantly increased in the high yielder, whereas sucrose and salicylic acid (SA) were significantly decreased compared with the low one. A total of 2984 differentially expressed proteins (DEPs) were identified in four groups, which were mainly enriched in the biosynthesis of secondary metabolites, protein processing in the endoplasmic reticulum, carbohydrate metabolism, phytohormone biosynthesis, glutathione metabolism, and plant-pathogen interaction. Integrated physiological and proteomic analysis revealed that carbohydrate metabolism, terpenoid biosynthesis, resistance to stress, as well as JA and GA biosynthesis and signaling, play key roles in regulating resin yield. A series of proteins associated with resin yield, e.g., terpene synthase proteins (TPSs), ATP-binding cassette transporters (ABCs), glutathione S-transferase proteins (GSTs), and heat shock proteins (HSPs), were identified. Resin yield-related gene expression was also associated with resin yield. Our study unveils the implicated molecular mechanisms regulating resin yield and is of pivotal significance to breeding strategies of high resin-yielding masson pine cultivars.

Decreased Klotho Expression Causes Accelerated Decline of Male Fecundity through Oxidative Injury in Murine Testis.

Oxidative stress is the etiology for 30-80% of male patients affected by infertility, which is a major health problem worldwide. Klotho protein is an aging suppressor that functions as a humoral factor modulating various cellular processes including antioxidation and anti-inflammation, and its dysregulation leads to human pathologies. Male mice lacking Klotho are sterile, and decreased Klotho levels in the serum are observed in men suffering from infertility with lower sperm counts. However, the mechanism by which Klotho maintains healthy male fertility remains unclear. Klotho haplodeficiency (Kl+/-) accelerates fertility reduction by impairing sperm quality and spermatogenesis in Kl+/- mice. Testicular proteomic analysis revealed that loss of Klotho predominantly disturbed oxidation and the glutathione-related pathway. We further focused on the glutathione-S-transferase (GST) family which counteracts oxidative stress in most cell types and closely relates with fertility. Several GST proteins, including GSTP1, GSTO2, and GSTK1, were significantly downregulated, which subsequently resulted in increased levels of the lipid peroxidation product 4-hydroxynonenal and apoptosis in murine testis with low or no expression of Klotho. Taken together, the loss of one Kl allele accelerates male fecundity loss because diminished antioxidant capability induces oxidative injury in mice. This is the first study that highlights a connection between Klotho and GST proteins.

Genes encoding cytochrome P450 monooxygenases and glutathione S-transferases associated with herbicide resistance evolved before the origin of land plants.

Cytochrome P450 (CYP) monooxygenases and glutathione S-transferases (GST) are enzymes that catalyse chemical modifications of a range of organic compounds. Herbicide resistance has been associated with higher levels of CYP and GST gene expression in some herbicide-resistant weed populations compared to sensitive populations of the same species. By comparing the protein sequences of 9 representative species of the Archaeplastida-the lineage which includes red algae, glaucophyte algae, chlorophyte algae, and streptophytes-and generating phylogenetic trees, we identified the CYP and GST proteins that existed in the common ancestor of the Archaeplastida. All CYP clans and all but one land plant GST classes present in land plants evolved before the divergence of streptophyte algae and land plants from their last common ancestor. We also demonstrate that there are more genes encoding CYP and GST proteins in land plants than in algae. The larger numbers of genes among land plants largely results from gene duplications in CYP clans 71, 72, and 85 and in the GST phi and tau classes [1,2]. Enzymes that either metabolise herbicides or confer herbicide resistance belong to CYP clans 71 and 72 and the GST phi and tau classes. Most CYP proteins that have been shown to confer herbicide resistance are members of the CYP81 family from clan 71. These results demonstrate that the clan and class diversity in extant plant CYP and GST proteins had evolved before the divergence of land plants and streptophyte algae from a last common ancestor estimated to be between 515 and 474 million years ago. Then, early in embryophyte evolution during the Palaeozoic, gene duplication in four of the twelve CYP clans, and in two of the fourteen GST classes, led to the large numbers of CYP and GST proteins found in extant land plants. It is among the genes of CYP clans 71 and 72 and GST classes phi and tau that alleles conferring herbicide resistance evolved in the last fifty years.

Enzymatic Investigation of Spongospora subterranea Zoospore Attachment to Roots of Potato Cultivars Resistant or Susceptible to Powdery Scab Disease.

For potato crops, host resistance is currently the most effective and sustainable tool to manage diseases caused by the plasmodiophorid Spongospora subterranea. Arguably, zoospore root attachment is the most critical phase of infection; however, the underlying mechanisms remain unknown. This study investigated the potential role of root-surface cell-wall polysaccharides and proteins in cultivars resistant/susceptible to zoospore attachment. We first compared the effects of enzymatic removal of root cell-wall proteins, N-linked glycans and polysaccharides on S. subterranea attachment. Subsequent analysis of peptides released by trypsin shaving (TS) of root segments identified 262 proteins that were differentially abundant between cultivars. These were enriched in root-surface-derived peptides but also included intracellular proteins, e.g., proteins associated with glutathione metabolism and lignin biosynthesis, which were more abundant in the resistant cultivar. Comparison with whole-root proteomic analysis of the same cultivars identified 226 proteins specific to the TS dataset, of which 188 were significantly different. Among these, the pathogen-defence-related cell-wall protein stem 28 kDa glycoprotein and two major latex proteins were significantly less abundant in the resistant cultivar. A further major latex protein was reduced in the resistant cultivar in both the TS and whole-root datasets. In contrast, three glutathione S-transferase proteins were more abundant in the resistant cultivar (TS-specific), while the protein glucan endo-1,3-beta-glucosidase was increased in both datasets. These results imply a particular role for major latex proteins and glucan endo-1,3-beta-glucosidase in regulating zoospore binding to potato roots and susceptibility to S. subterranea.

The Effect of Carnosine on UVA-Induced Changes in Intracellular Signaling of Human Skin Fibroblast Spheroids

Dermis fibroblasts are very sensitive to penetrating UVA radiation and induce photo-damage. To protect skin cells against this environmental damage, there is an urgent need for effective compounds, specifically targeting UVA-induced mitochondrial injury. This study aimed to analyze the effect of carnosine on the proteome of UVA-irradiated human skin fibroblast, cultured in a three-dimensional (3D) biological system recapitulating dermal compartment as a test system to investigate the altered cellular pathways after 48 h and 7 days of culture with or without carnosine treatment. The obtained results indicate that UVA dysregulates Oxidative Phosphorylation, the Fibrosis Signaling Pathway, Glycolysis I and Nrf2-mediated Oxidative Stress Response. Carnosine exercises provide a protective function against the harmful effects of UVA radiation by activating the Nrf2 pathway with the upregulations of some ROS-detoxifying enzymes such as the glutathione S-transferase (GST) protein family. Additionally, carnosine regulates the activation of the Epithelial Adherens Junction and Wound Healing Signaling Pathway by mediating the activation of structural proteins such as vinculin and zyxin as well as fibronectin 1 and collagen type XVIII alpha 1 chain against UVA-induced changes.

Ethanolic Extracts of Datura innoxia Have Promising Acaricidal Activity against Rhipicephalus microplus as It Blocks the Glutathione S-Transferase Activity of the Target Tick.

Rhipicephalus microplus is a major bovine ectoparasite that negatively impacts the cattle industry. The acaricidal activity of Datura innoxia ethanolic plant extract against R. microplus, compared with trichlorfon, was examined using the adult immersion test (AIT), and larval packet test (LPT). In vitro acaricidal activity of the selected plant extract against R. microplus engorged females was evaluated at different concentrations (2.5, 5, 10, 20, and 40 mg/mL), and was the same for AIT and LPT. It was further supported by in silico molecular docking of D. innoxia's 21 phytochemicals against the R. microplus Glutathione S-transferases (RmGST) protein's three-dimensional (3D) structure predicted by the trRosetta server. The modeled 3D structure was then evaluated and confirmed with PROCHECK, ERRAT, and Verify3D online servers. To predict the binding mechanisms of these compounds, molecular docking was performed using Auto dock Vina software, and molecular dynamic (MD) simulations were used to investigate the protein atom's dynamic motion. D. innoxia has a relatively higher inhibitory effect on oviposition (from 9.81% to 45.37%) and total larval mortality (42.33% at 24 h and 93.67% at 48 h) at 40 mg/mL. Moreover, the docking results showed that the chemicals norapoatropine and 7-Hydroxyhyoscyamine have strong interactions with active site residues of the target protein, with a docking score of -7.3 and -7.0 Kcal/mol, respectively. The current work also provided a computational basis for the inhibitors of Glutathione S-transferases that were studied in this research work, and this new knowledge should aid in creating new and effective acaricidal chemicals. Furthermore, this plant extract's acaricide activity and its effect on oviposition and larval mortality were established in this work for the first time, indicating the possible use of this extract in the management of ticks.

Detoxification enzyme activity, reproductive and developmental fitness of abamectin-resistant Bryobia praetiosa (Acari:Tetranychidae)

Abamectin is an important and environmentally friendly antibiotic pesticide, and its resistance has become an area of intense research. In this study, we employed laboratory bioassays, biochemical analyses, and life history analyses to systematically evaluate abamectin resistance, the underlying biochemical mechanisms of resistance, and the reproductive and developmental characteristics of the arthropod pest Bryobia praetiosa. An abamectin-resistant B. praetiosa strain (RR) was obtained by intensive selection of an abamectin-susceptible strain (SS) using abamectin exposure in the laboratory for 24 generations, yielding a resistance index (RI) of 30.11. Comparison of detoxification enzymes suggested that the specific activities of carboxylesterases (CarEs), glutathione S-transferases (GSTs), and mixed-function oxidases (MFOs) in the RR strain were higher than those of the SS strain by 1.13-, 1.75-, and 4.02-fold, respectively. GST and MFOS protein concentrations were also significantly different between RR and SS strains. Further analysis of life history parameters demonstrated that the developmental duration and lifespan of RR strain in addition to egg numbers were markedly decreased relative to SS strain. The net reproductive rate (R0), finite rate of increase (λ), and population doubling time (Dt) of RR strain were significantly lower than those of SS strain. The results indicated that the RR strain was more disadvantageous in developmental and reproductive characteristics than the SS strain. In addition, the relative fitness value of the resistant strain is about half (Rf = 0.49) that of the susceptible strain. In conclusion, increased abamectin resistance in B. praetiosa was associated with a rapid increase in MFOs activity, while GSTs also played a role in abamectin resistance development and RR strain exhibited reproductive disadvantages.

Genome-Wide Evolutionary Analysis of Putative Non-Specific Herbicide Resistance Genes and Compilation of Core Promoters between Monocots and Dicots.

Herbicides are key weed-control tools, but their repeated use across large areas has favored the evolution of herbicide resistance. Although target-site has been the most prevalent and studied type of resistance, non-target-site resistance (NTSR) is increasing. However, the genetic factors involved in NTSR are widely unknown. In this study, four gene groups encoding putative NTSR enzymes, namely, cytochrome-P450, glutathione-S-transferase (GST), uridine 5′-diphospho-glucuronosyltransferase (UDPGT), and nitronate monooxygenase (NMO) were analyzed. The monocot and dicot gene sequences were downloaded from publicly available databases. Phylogenetic trees revealed that most of the CYP450 resistance-related sequences belong to CYP81 (5), and in GST, most of the resistance sequences belonged to GSTU18 (9) and GSTF6 (8) groups. In addition, the study of upstream promoter sequences of these NTSR genes revealed stress-related cis-regulatory motifs, as well as eight transcription factor binding sites (TFBS) were identified. The discovered TFBS were commonly present in both monocots and dicots, and the identified motifs are known to play key roles in countering abiotic stress. Further, we predicted the 3D structure for the resistant CYP450 and GST protein and identified the substrate recognition site through the homology approach. Our description of putative NTSR enzymes may be used to develop innovative weed control techniques to delay the evolution of NTSR.

Quantitative Succinyl-Proteome Profiling of Turnip (Brassica rapa var. rapa) in Response to Cadmium Stress.

Protein post-translational modification (PTM) is an efficient biological mechanism to regulate protein structure and function, but its role in plant responses to heavy metal stress is poorly understood. The present study performed quantitative succinyl-proteome profiling using liquid chromatography–mass spectrometry analysis to explore the potential roles of lysine succinylation modification in turnip seedlings in response to cadmium (Cd) stress (20 μM) under hydroponic conditions over a short time period (0–8 h). A total of 547 succinylated sites on 256 proteins were identified in the shoots of turnip seedlings. These succinylated proteins participated in various biological processes (e.g., photosynthesis, tricarboxylic acid cycle, amino acid metabolism, and response to stimulation) that occurred in diverse cellular compartments according to the functional classification, subcellular localization, and protein interaction network analysis. Quantitative analysis showed that the intensities of nine succinylation sites on eight proteins were significantly altered (p < 0.05) in turnip shoots after 8 h of Cd stress. These differentially succinylated sites were highly conserved in Brassicaceae species and mostly located in the conserved domains of the proteins. Among them, a downregulated succinylation site (K150) in the glycolate oxidase protein (Gene0282600.1), an upregulated succinylation site (K396) in the catalase 3 protein (Gene0163880.1), and a downregulated succinylation site (K197) in the glutathione S-transferase protein (Gene0315380.1) may have contributed to the altered activity of the corresponding enzymes, which suggests that lysine succinylation affects the Cd detoxification process in turnip by regulating the H2O2 accumulation and glutathione metabolism. These results provide novel insights into understanding Cd response mechanisms in plants and important protein modification information for the molecular-assisted breeding of Brassica varieties with distinct Cd tolerance and accumulation capacities.

Genetic variations in NLRP1 and Glutathione S-Transferase are associated with Generalized Vitiligo of Phlegmatic Clinical Phenotypes in Unani Medicine Philosophy of South Indian Population

Background: Generalized vitiligo is a common, multifaceted, polygenic condition in which autoimmune loss of melanocytes results in depigmented skin patches, overlying hair and mucous membranes. NLRP1 has been proposed to be implicated in the susceptibility of a broad variety of autoimmune disorders, including generalized vitiligo (GV). Genetic polymorphisms in the NLRP1 encoding gene (formerly known as NALP1) have previously been found to be linked with GV and there is uncertainty as to their role in the modulation of NLRP1 expression. Oxidative stress is a significant pathogenesis theory for vitiligo. Glutathione S-transferases (GSTs) are enzymes active in the defense of cells against chemical toxicity and stress.This study validates some of the Unani concepts of humors or temperaments (Phenotypes), with regard to Vitiligo, Where vitiligo is regarded as a phlegmatic disease. We selected Vitiligo subjects with Phlegmatic Clinical Phenotype for our study, with an aim to determine its association with the genetic biomarkers- NLRP1, GSTM1 and GSTT1 null genotypes and other biochemical parameters. Methods: The Unani clinicians randomly selected 100 vitiligo patients with a phlegmatic Clinical Phenotype who were attending NRIUMSD for treatment and 100 healthy volunteers belonging to Phlegmatic (Phlegmatic clinical Phenotype). Besides looking at temperaments/ humors as susceptibility factors – we included a genetic factor- NLRP1, GSTM1- and GSTT1-null genotypes to our investigation. We have genotyped the NLRP1, GSTM1- and GSTT1-null genotypes by PCR-RFLP and by Multiplex PCR, GST protein level estimation by ELISA method. Results: NLRP1 rs2670660 polymorphism was shown to be in significant association with GV, with the presence of minor alleles in active GV. We found that the frequencies of GSTM1 null genotype and GSTT1 null genotype in vitiligo patients were significantly high compared to the controls (OR= 1.47, 95% CI=0.765--2.861), (OR = 4.75, 95% CI = 2.131-10.63), respectively. In combination analysis with both genes, the results suggested significant association of vitiligo risk with both GSTM1\GSTT1 null genotypes (OR=4.83, 95% CI=1.523– 15.32).We observed a significant decrease (p&lt;0.001) in GST protein levels. Conclusion: Our findings indicate that NLRP1 rs2670660 polymorphism may be genetic risk factor for susceptibility to GV and the null genotypes of GSTM1 and GSTT1 of both genes increase the risk of the disease. A significant decrease (p&lt;0.001) in GST protein levels appeared to be a key feature in Vitiligo subjects, Therefore, detection of antioxidant enzyme levels can be effective biomarkers for early detection of the disease. We believed that GSTM1-and GSTT1-null genotype polymorphisms were associated with an increased risk of vitiligo. This is the first study of its kind along with Clinical Phenotype as per Unani Philosophy.

Molecular Cloning of a TCHQD Class Glutathione S-Transferase and GST Function in Response to GABA Induction of Melon Seedlings under Root Hypoxic Stress

Glutathione-S-transferase (GST), a versatile enzyme that occurs widely in plants, plays a key role in plant resistance to environmental stresses. Previous results have demonstrated that GST proteins are involved in alleviating root hypoxic injury caused by gamma-aminobutyric acid (GABA); however, the induction mechanism of the GST gene in the melon under root hypoxic stress and its functional mechanisms remain unclear. In this study, based on gene cloning and bioinformatics analysis, GST gene expression and activity and glutathione (GSH) content were assessed under root hypoxic and normoxic conditions with or without GABA. The results showed that the CmGST locus includes an 804 bp gene sequence that encodes 267 amino acids. The sequence was highly similar to those of other plant TCHQD GSTs, and the highest value (94%) corresponded to Cucumis sativus. Real-time PCR results showed that the CmGST gene was induced by root hypoxic stress and GABA, and this induction was accompanied by increased GST activity and GSH content. Root hypoxic stress significantly upregulated CmGST expression in melon roots (0.5–6 d), stems, and leaves (0.5–4 d), and GST activity and GSH content were also significantly increased. Exogenous GABA treatment upregulated CmGST gene expression, GST activity, and GSH content, particularly under root hypoxic conditions. As a result, CmGST expression in GABA-treated roots and leaves at 0.5–4 d and stems at 0.5–6 d was significantly higher than that under root hypoxic stress alone. This study provides evidence that the TCHQD CmGST may play a vital role in how GABA increases melon hypoxia tolerance by upregulating gene expression and improving metabolism.