Glutathione S-transferase Substrates Research Articles

Molecular mechanisms of sulforaphane in Alzheimer’s disease: insights from an in-silico study

This study was to identify the molecular pathways that may explain sulforaphane’s Alzheimer’s disease (AD) benefits using multiple advanced in silico approaches. We found that sulforaphane regulates 45 targets, including TNF, INS, and BCL2. Therefore, it may help treat AD by reducing neuroinflammation, insulin resistance, and apoptosis. The important relationships were co-expression and pathways. 45 targets were linked to the midbrain, metabolite interconversion enzymes, 14q23.3 and 1q31.1 chromosomes, and modified residues. “Amyloid precursor protein catabolic process”, “regulation of apoptotic signaling pathway”, and “positive regulation of nitric oxide biosynthetic process” were the main pathways, while NFKB1, SP1, RELA, hsa-miR-17-5p, hsa-miR-16-5p, and hsa-miR-26b-5p were transcription factors and miRNAs implicated in sulforaphane In AD treatment, miRNA sponges, dexibuprofen, and sulforaphane may be effective. Furthermore, its unique physicochemical, pharmacokinetic, and biological qualities make sulforaphane an effective AD treatment, including efficient gastrointestinal absorption, drug-like properties, absence of CYP450 enzyme inhibition, not being a substrate for P-glycoprotein, ability to cross the blood–brain barrier, glutathione S-transferase substrate, immunostimulant effects, and antagonistic neurotransmitter effects. Sulforaphane is a promising compound for AD management. Further work is needed to elucidate its therapeutic effects based on our findings, including genes, miRNAs, molecular pathways, and transcription factors.

Improving reactivity of naphthalimide-based GST probe by imparting TPP cation: Development and application for live cell imaging

Glutathione S-transferases (GSTs) are a superfamily of multifunctional enzymes comprising multiple classes and subtypes. This paper describes the synthesis and characterization of TPPBN-1, a naphthalimide derivative conjugated with a triphenylphosphonium (TPP) cation. When 4-bromonaphthalimide (BrNaph), a previously characterized GST substrate, was conjugated to a TPP cation, the conjugate showed increased reactivity towards most alpha- and mu-class GSTs, particularly the GSTA2 subtype, compared to the parent compound, but hardly towards Pi-class GSTs. Using this probe with enhanced reactivity, the enzymatic activity of endogenous GSTA1/2 in HepG2 cells was visualized by confocal fluorescence microscopy. The results demonstrated that modification with TPP cations, which are often used as tags for targeting mitochondria, can be used to enhance the reactivity of probes for specific GST subtypes.

Targeting redox metabolism of the maize-Azospirillum brasilense interaction exposed to arsenic-affected groundwater.

Arsenic in groundwater constitutes an agronomic problem due to its potential accumulation in the food chain. Among the agro-sustainable tools to reduce metal(oid)s toxicity, the use of plant growth-promoting bacteria (PGPB) becomes important. For that, and based on previous results in which significant differences of As translocation were observed when inoculating maize plants with Az39 or CD Azospirillum strains, we decided to decipher the redox metabolism changes and the antioxidant system response of maize plants inoculated when exposed to a realistic arsenate (AsV ) dose. Results showed that AsV caused morphological changes in the root exodermis. Photosynthetic pigments decreased only in CD inoculated plants, while oxidative stress evidence was detected throughout the plant, regardless of the assayed strain. The antioxidant response was strain-differential since only CD inoculated plants showed an increase in superoxide dismutase, glutathione S-transferase (GST), and glutathione reductase (GR) activities while other enzymes showed the same behavior irrespective of the inoculated strain. Gene expression assays reported that only GST23 transcript level was upregulated by arsenate, regardless of the inoculated strain. AsV diminished the glutathione (GSH) content of roots inoculated with the Az39 strain, and CD inoculated plants showed a decrease of oxidized GSH (GSSG) levels. We suggest a model in which the antioxidant response of the maize-diazotrophs system is modulated by the strain and that GSH plays a central role acting mainly as a substrate for GST. These findings generate knowledge for a suitable PGPB selection, and its scaling to an effective bioinoculant formulation for maize crops exposed to adverse environmental conditions.

Assay of the enzymes of glutathione biosynthesis

This article is dedicated to the late long-time Editor-in-Chief of Analytical Biochemistry, William Jakoby. As a graduate student, I remember reading many articles in Analytical Biochemistry and Methods in Enzymology, both volumes that Bill edited. I first met him as a graduate student presenting at the American Society of Biochemistry (and Molecular Biology) meetings. My Ph.D. advisor, Alton Meister, would bring over well-known biochemists and introduce me as Dr. Anderson, leaving me a bit tongue-tied being that I was still actually a humble graduate student! I next met Bill at my first Analytical Biochemistry Executive Editors meeting in San Diego when he was Editor-in-Chief Emeritus; I felt honored to be on the same board with him and serving the journal to which he had brought to prominence. His eyes were piercing and he was so sharp; his knowledge was both broad and deep. Since much of the large body of Bill's research was on glutathione S-transferases, my article focuses on the assay of the enzymes that synthesize glutathione, a substrate for glutathione S-transferases.

Glutathione S-transferase activity in aquatic macrophytes and halophytes and biotransformation potential for biocides.

Glutathione S-transferase (GST) participates in the biotransformation of many xenobiotics including biocides. Its activity in plants is generally associated with their phytoremediation capabilities. Biocides have been used in agriculture and antifouling paints and they represent risks for the aquatic environment. The present study aimed to: (1) evaluate the basal GST activity in roots, stems, and leaves from thirteen plants (eleven aquatic macrophytes and two halophytes) collected at South Brazil wetlands; (2) estimate the biotransformation potential of Nothoscordum gracile for five biocides using competitive kinetic assays with 1-chloro-2,4-dinitrobenzene (CDNB), a typical GST substrate. The N. gracile, Spartina alterniflora and Cakile maritima presented the highest GST activities among the tested plants. The Lineweaver-Burk plot obtained from the GST competitive kinetic assays confirmed that the biocides chlorothalonil, 4,5-dichloro-N-octyl-3(2H)-isothiazolone (DCOIT), dichlofluanid, and diuron, but not irgarol, compete with the substrate CDNB for GST. Chlorothalonil and DCOIT showed the lowest IC20 values (11.1 and 10.6μM, respectively), followed by dichlofluanid (38.6μM) and diuron (353.1μM). The inhibition of GST-CDNB activity by 100nM biocide was higher for chlorothalonil, DCOIT, and dichlofluanid (46.5, 49.0, and 45.1%, respectively) than for diuron (6.5%) and irgarol (2.2%). The present study indicates plant species that have significant GST activity and could be potentially used for phytoremediation. The competitive kinetic tests suggest that among the five biocides that were tested, chlorothalonil, DCOIT, and dichlofluanid are probably preferred for biotransformation via GST in plant.

Structure of the Complete Dimeric Human GDAP1 Core Domain Provides Insights into Ligand Binding and Clustering of Disease Mutations.

Charcot-Marie-Tooth disease (CMT) is one of the most common inherited neurological disorders. Despite the common involvement of ganglioside-induced differentiation-associated protein 1 (GDAP1) in CMT, the protein structure and function, as well as the pathogenic mechanisms, remain unclear. We determined the crystal structure of the complete human GDAP1 core domain, which shows a novel mode of dimerization within the glutathione S-transferase (GST) family. The long GDAP1-specific insertion forms an extended helix and a flexible loop. GDAP1 is catalytically inactive toward classical GST substrates. Through metabolite screening, we identified a ligand for GDAP1, the fatty acid hexadecanedioic acid, which is relevant for mitochondrial membrane permeability and Ca2+ homeostasis. The fatty acid binds to a pocket next to a CMT-linked residue cluster, increases protein stability, and induces changes in protein conformation and oligomerization. The closest homologue of GDAP1, GDAP1L1, is monomeric in its full-length form. Our results highlight the uniqueness of GDAP1 within the GST family and point toward allosteric mechanisms in regulating GDAP1 oligomeric state and function.

A bi-functional fluorescent probe for visualized and rapid natural drug screening via GSTs activity monitoring

Glutathione S-transferase (GSTs), as an important target for drug screening, is closely associated with physiological and pathological process regulation. Hence, effective monitoring of GSTs activity is benificial for GSTs-induced diseases diagnosis and therapeutic drugs discovery. Resorufin-typed probes with high sensitivity, excellent selectivity, and low cytotoxicity have become a powerful tool for target monitoring. In this study, a bi-functional fluorescent probe, resorufin-typed probe (RP) with resorufin as a fluorophore was designed as the GSTs substrate and drug screening tool. GSTs can trigger significant fluorescence signal enhancement of RP by catalyzing the combination of glutathione (GSH) and its recognition site in RP. Meaningfully, the change of fluorescence signal positively correlated with GSTs activity can be used to monitor the regulatory effect of natural drugs on GSTs. Based on the axis relation of “drug-GSTs activity-fluorescence signal”, this powerful probe was successfully employed for visual and rapid screening GSTs-targeted natural compounds in vitro by monitoring the change of GSTs activity. Natural compound 1, 2 and 7 (C1, C2 and C7) were screened as the GSTs inhibitors, and C7 was helpful for enhancing the sensitivity of cisplatin in cancer cells. Overall, the designed natural drug screening strategy based on bi-functional fluorescence probe demonstrated its hopeful application to drug rapid discovery.

Systematic characterization of glutathione S-transferases in common marmosets

The common marmoset is an important primate species used in drug metabolism studies. However, glutathione S-transferases (GSTs), essential drug-metabolizing enzymes involved in the conjugation of various endogenous and exogenous substrates, have not been identified or characterized in this species. In this study, 20 GSTs [including 3 microsomal GSTs (MGSTs)] were identified and characterized in marmosets. Marmoset GSTs had amino acid sequences highly identical (86–99%) to human GSTs, except for GSTA4L, which had lower identities (59–62%) with human GSTAs. Phylogenetic analysis revealed that marmoset GSTs were closely clustered with their human counterparts. Marmoset GSTs had gene and genomic structures generally similar to their human counterparts, with some differences in GSTA, GSTM, and GSTT clusters. Marmoset GST mRNAs exhibited distinct tissue expression patterns: GSTA1, GSTA3, GSTA4L, GSTK1, GSTT1, GSTZ1, and MGST1 mRNAs were expressed most abundantly in liver. Other GST mRNAs were expressed most abundantly in small intestine, lung, brain, or kidney. Expression of GSTT4 and GSTT4L mRNAs was detected only in testis. Among all 20 marmoset GST mRNAs, the most abundant mRNAs were GSTA1 mRNA in liver, small intestine, and kidney; GSTM3 mRNA in testis; and MSGT3 mRNA in brain and lung. All 20 GSTs mediated the conjugation of GST substrates 1-chloro-2,4-dinitrobenzene; 1,2-epoxy-3-(p-nitrophenoxy)propane; styrene 7,8-oxide; and/or 1-iodohexane, but with different activity levels. Kinetic analyses showed that marmoset GSTM2/GSTM5 and GSTM5/GSTT1 effectively conjugated styrene 7,8-oxide and 1-iodohexane, respectively, with the highest affinity. These results suggest that the 20 newly identified marmoset GSTs were functional drug-metabolizing enzymes able to conjugate typical GST substrates.

An omega-class glutathione S-transferase in the brown planthopper Nilaparvata lugens exhibits glutathione transferase and dehydroascorbate reductase activities.

A complementary DNA that encodes an omega-class glutathione S-transferase (GST) of the brown planthopper, Nilaparvata lugens (nlGSTO), was isolated by reverse transcriptase polymerase chain reaction. A recombinant protein (nlGSTO) was obtained via overexpression in the Escherichia coli cells and purified. nlGSTO catalyzes the biotransformation of glutathione with 1-chloro-2,4-dinitrobenzene, a general substrate for GST, as well as with dehydroascorbate to synthesize ascorbate. Mutation experiments revealed that putative substrate-binding sites, including Phe28, Cys29, Phe30, Arg176, and Lue225, were important for glutathione transferase and dehydroascorbate reductase activities. As ascorbate is a reducing agent, nlGSTO may participate in antioxidant resistance.

4-Bromo-1,8-naphthalimide derivatives as fluorogenic substrates for live cell imaging of glutathione S-transferase (GST) activity

Fluorogenic substrates are used to visualize the activity of cancer-associated enzymes and to interpret biological events. Certain types of glutathione S-transferase (GST), such as Pi class GST (referred to as GSTP1), are more highly expressed in a wide variety of human cancer tissues compared to their corresponding normal tissues. Pi class GST is thus a cancer cell molecular marker and potential target for overcoming resistance to chemotherapy. Here, we report that 4-bromo-1,8-naphthalimide (BrNaph) is a practical fluorogenic GST substrate. We have found that HE-BrNaph, an N-hydroxyethyl derivative, shows remarkable fluorescence enhancement upon GST-catalyzed SNAr replacement of the bromo group with a glutathionyl group. This substitution was highly selective and occurred only in the presence of GSH/GSTs; no non-enzymatic reaction was observed. We demonstrated that HE-BrNaph allows visualization of GST activity in living cells and enables to distinguish cancer cells from normal cells. Further, various N-substitutions in BrNaph retain susceptibility to enzymatic activity and isozyme selectivity, suggesting the applicability of BrNaph derivatives. Thus, BrNaph and its derivatives are GST substrates useful for fluorescence imaging and the intracellular detection of GSTP1 activity in living cells.

Glutathione‐S‐Transferases Represent a Novel Pathway Contributing to the Metabolic Clearance of the Anti‐Cancer Agent and Aromatase Inhibitor, Exemestane

Breast Cancer is the second leading cause of cancer death among women in the USA, with estrogen‐receptor positive (ER+) tumors accounting for 75% of breast cancers in postmenopausal women. Endocrine therapy targeting the ER pathway is a major treatment modality for ER+ patients. Exemestane (EXE) is a third generation aromatase inhibitor which blocks the aromatase enzyme in the final step of estrogen biosynthesis. Studies examining the long‐term use of EXE as a chemopreventive agent demonstrated a reduction in breast cancer incidence by more than 65%. EXE represents an improvement in breast cancer therapy, but inter‐individual variability exists in overall patient response and adverse events that may be attributed to variation in EXE metabolism. Preliminary studies have demonstrated that cysteine conjugates of EXE and its active metabolite 17β‐dihydro‐EXE (17β‐DHE) comprise 77% of total EXE metabolites in the urine of subjects taking EXE. The initial step in cysteine conjugate formation is glutathione (GSH) conjugation in a reaction catalyzed by the glutathione‐S‐transferase (GST) family of the enzymes involved in the metabolism of a variety of exogenous and endogenous substances. The goal of the present study was to identify hepatic GSTs active in the metabolism of EXE and 17β‐DHE. In a screening of the Human Protein Atlas, a total of 12 cytosolic and 3 microsomal GSTs were found to be hepatically expressed. Commercially available cytosolic GSTs were purchased, and their activity was verified with a common GST substrate, 1‐chloro‐2.4‐dinitrobenzene (CDNB). Additionally, GSTT1, GSTO1, GSTZ1, GSTA1, GSTM3, and GSTP1 were produced as recombinant histidine‐tagged proteins using an E‐coli expression system and found to be 97–99% pure by silver staining. Microsomal GSTs were cloned with a V5 epitope tag, overexpressed in the HEK293 cell line, and verified by Western Blot analysis using an anti‐V5 antibody. The 12 cytosolic and 3 microsomal GSTs were screened by incubating GSH with EXE or 17β‐DHE (0.125mM) using 0.5 μg of pure recombinant protein or 20 μg of microsomal protein. GS‐conjugates of EXE and DHE were detected using ultra‐performance liquid chromatography combined with tandem mass spectrometry (UPLC‐MS/MS). Results from the individual EXE‐GSH conjugation assays indicate that the cytosolic enzymes GSTA1, GSTM3, GSTP1 and GSTT1 are active against EXE and marginally active against 17β‐DHE, with GSTA1 exhibiting the highest relative activity. The three screened microsomal MGST1, MGST2 and MGST3 all exhibit activity against both EXE and 17β‐DHE, with MGST2 exhibiting the highest activity against 17β‐DHE. These data suggest that both cytosolic and microsomal GSTs are responsible for EXE‐GS and 17β‐DHE‐GS conjugate formation in vitro and that GSH conjugation by GSTs may be an important metabolic pathway contributing to excretion and elimination of EXE in people.Support or Funding InformationThis work is supported by grants from NIH, National Cancer Institute (grant R01‐ CA164366; to P. Lazarus) and the Health Sciences and Services Authority of Spokane, Washington (grant WSU002292 to College of Pharmacy and Pharmaceutical Sciences, Washington State University)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Selective Disruption of Mitochondrial Thiol Redox State in Cells and In Vivo

SummaryMitochondrial glutathione (GSH) and thioredoxin (Trx) systems function independently of the rest of the cell. While maintenance of mitochondrial thiol redox state is thought vital for cell survival, this was not testable due to the difficulty of manipulating the organelle's thiol systems independently of those in other cell compartments. To overcome this constraint we modified the glutathione S-transferase substrate and Trx reductase (TrxR) inhibitor, 1-chloro-2,4-dinitrobenzene (CDNB) by conjugation to the mitochondria-targeting triphenylphosphonium cation. The result, MitoCDNB, is taken up by mitochondria where it selectively depletes the mitochondrial GSH pool, catalyzed by glutathione S-transferases, and directly inhibits mitochondrial TrxR2 and peroxiredoxin 3, a peroxidase. Importantly, MitoCDNB inactivates mitochondrial thiol redox homeostasis in isolated cells and in vivo, without affecting that of the cytosol. Consequently, MitoCDNB enables assessment of the biomedical importance of mitochondrial thiol homeostasis in reactive oxygen species production, organelle dynamics, redox signaling, and cell death in cells and in vivo.

Role of glutathione‐S‐transferases in the metabolism of the anti‐cancer agent and aromatase inhibitor, exemestane

Breast cancer is the most commonly diagnosed cancer in women, with approximately 1 in 8 women in the U.S. developing breast cancer over their lifetime. The majority of breast cancers diagnosed each year are estrogen receptor positive (ER+). Drugs targeting the ER pathway are a major treatment modality for ER+ patients and are additionally prescribed for the prevention of breast cancer. Exemestane (EXE) is an inhibitor which blocks the enzyme aromatase in the final step of estrogen biosynthesis. Studies examining the long‐term use of EXE as a chemopreventative agent demonstrated a reduction in breast cancer incidence by more than 65%. However, response and toxicity varied widely among individuals, which may be attributed to genetic polymorphisms in enzymes that metabolize EXE. Data from our lab demonstrate that a cysteine conjugates of EXE and its active metabolite 17β‐dihydro‐EXE (17β‐DHE) make up 77% of the total metabolite in urine. Cysteine conjugates are formed from metabolism of glutathione (GSH) conjugates in a reaction catalyzed by the glutathione‐S‐transferases (GST) family of the enzymes. The goal of the present study was to identify hepatic GSTs active in the metabolism of EXE and 17β‐DHE and to determine kinetic parameters for the identified enzymes. In a screening of the Protein Atlas, a total of 12 cytosolic and 3 microsomal GSTs were found to be hepatically expressed. EXE‐GSH conjugation activity in human liver samples was screened by incubating GSH with EXE or 17β‐DHE (0.125–1.0 mM) in cytosol (HLC) or microsomes (HLM) prepared from human livers. GSH conjugates were formed for both cellular fractions, indicating that both cytosolic and microsomal GST enzymes are active against EXE and 17β‐DHE. GSH conjugates were detected by UPLC‐MS and quantified using synthesized EXE‐GS and 17β‐DHE‐GS standards. The purity and identity of both standards and internal standards were confirmed using NMR and UPLC‐MS and found to be > 95% pure. Individual recombinant human GSTs were then screened to determine which enzyme is primarily responsible for the metabolism of EXE. Commercially available cytosolic GSTs were purchased, and their activity was verified with a common GST substrate, 1‐chloro‐2.4‐dinitrobenzene. Results from the individual EXE‐GSH conjugation assays indicate the cytosolic enzymes GSTA1, GSTM3 and GSTP1 are active against EXE and 17β‐DHE; studies of microsomal GSTs are on‐going. These data suggest that several GSTs, both cytosolic and microsomal, may be important in the phase II conjugation of EXE and its major active metabolite, 17β‐DHE.Support or Funding InformationThis study is supported by National Institutes of Health (NIH) (Grant R01‐ES025460 and R01‐CA164366) and the Health Sciences and Services Authority of SpokaneThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Molecular cloning and functional characterization of theta class glutathione S-transferase from Apostichopus japonicus

Glutathione S-transferases (GSTs) are the superfamily of multifunctional detoxification isoenzymes and play crucial roles in innate immunity. In the present study, a theta class GST homology was identified from A. japonicus (designated as AjGST-θ) by RACE approaches. The full-length cDNA of AjGST-θ was of 1013 bp encoded a cytosolic protein of 231 amino acids residues. Structural analysis revealed that AjGST-θ processed the characteristic N-terminal GSH-binding site (G-site) and the C-terminal hydrophobic substrate binding site (H-site). Multiple sequence alignment and phylogenetic analysis together supported that AjGST-θ belonged to a new member of theta class GST protein subfamily. Spatial expression analysis revealed that AjGST-θ was ubiquitously expressed in all examined tissues with the larger magnitude in intestine. The Vibrio splendidus challenge in vivo and LPS stimulation in vitro could both significantly up-regulate the mRNA expression of AjGST-θ when compared with control group. The recombinant protein was expressed in Escherichia coli and the purified AjGST-θ showed high activity with GST substrate. Meantime, disc diffusion assay showed that recombinant AjGST-θ protein could markedly improve bacterial growth under Cumene hydroperoxide exposure. More importantly, the recombinant AjGST-θ could effectively prevent primary coelomocytes apoptosis after LPS exposure. Our present findings suggested that AjGST-θ might play significantly roles in the modulation of immune response and protect cells from pathogens infection in A. japonicus.

LcGST4 is an anthocyanin-related glutathione S-transferase gene in Litchi chinensis Sonn.

A novel LcGST4 was identified and characterized from Litchi chinensis . Expression and functional analysis demonstrated that it might function in anthocyanin accumulation in litchi. Glutathione S-transferases (GSTs) have been defined as detoxification enzymes for their ability to recognize reactive electrophilic xenobiotic molecules as well as endogenous secondary metabolites. Anthocyanins are among the few endogenous substrates of GSTs for vacuolar accumulation. The gene encoding a GST protein that is involved in anthocyanin sequestration from Litchi chinensis Sonn. has not been reported. Here, LcGST4, an anthocyanin-related GST, was identified and characterized. Phylogenetic analysis showed that LcGST4 was clustered with other known anthocyanin-related GSTs in the same clade. Expression analysis revealed that the expression pattern of LcGST4 was strongly correlated with anthocyanin accumulation in litchi. ABA- and light-responsive elements were found in the LcGST4 promoter, which is in agreement with the result that the expression of LcGST4 was induced by both ABA and debagging treatment. A GST activity assay in vitro verified that the LcGST4 protein shared universal activity with the GST family. Functional complementation of an Arabidopsis mutant tt19 demonstrated that LcGST4 might function in anthocyanin accumulation in litchi. Dual luciferase assay revealed that the expression of LcGST4 was activated by LcMYB1, a key R2R3-MYB transcription factor that regulates anthocyanin biosynthesis in litchi.

Benzene oxide is a substrate for glutathione S-transferases

Benzene is a known human carcinogen which must be activated to benzene oxide (BO) to exert its carcinogenic potential. BO can be detoxified in vivo by reaction with glutathione and excretion in the urine as S-phenylmercapturic acid. This process may be catalyzed by glutathione S-transferases (GSTs), but kinetic data for this reaction have not been published. Therefore, we incubated GSTA1, GSTT1, GSTM1, and GSTP1 with glutathione and BO and quantified the formation of S-phenylglutathione. Kinetic parameters were determined for GSTT1 and GSTP1. At 37 °C, the putative Km and Vmax values for GSTT1 were 420 μM and 450 fmol/s, respectively, while those for GSTP1 were 3600 μM and 3100 fmol/s. GSTA1 and GSTM1 did not exhibit sufficient activity for determination of kinetic parameters. We conclude that GSTT1 is a critical enzyme in the detoxification of BO and that GSTP1 may also play an important role, while GSTA1 and GSTM1 seem to be less important.

Characterization of Glutathione-Homocystine Transhydrogenase as a Novel Isoform of Glutathione S-Transferase from Aspergillus flavipes

Glutathione-homocystine transhydrogenase/oxidoreductase (GHTHase) is an enzyme that catalyzes the reversible oxidation/reduction of reduced glutathione (GSH) and homocystine (Hcy2) to oxidized glutathione and homocysteine (Hcy). GHTHase is implicated in regulation of the metabolism of homocysteine and glutathione. Aspergillus flavipes JF831014 exhibits the highest productivity of GHTHase, using GSH as electron donor and Hcy2 as acceptor. GHTHase yield from A. flavipes has been nutritionally optimized to reach the maximum activity (21.14 U/mg) using GSH (0.4%) combined with Hcy2 (0.01%) and glucose (0.4%), NADH + H (30 mM) at medium initial pH 7.8. The yield of GHTHase was increased about 1.2 times upon starvation of the culture of A. flavipes for two days, as compared to the non-sulfur starved culture. The GHTHase activity was increased by 13.7 fold with total yield of 8.8%. According to denaturing PAGE, GHTHase had 35 kDa, and 75 kDa by non-denaturing PAGE, gel-filtration and DLS analysis, ensuring its homodimeric identity. The enzyme displayed a highest activity at pH 6.5 – 7.6, 40°C, and pH stability within 6.0 – 8.0. GHTHase has higher affinity for GSH (K m = 14.3 mM) and cysteine (K m = 15.1 mM) as hydrogen donor for Hcy2 reduction, other than CDNB for glutathione S-transferase. The variant catalytic response to standard glutathione S-transferase substrates, esteem the unique catalytic properties of GHTHase. The enzyme was significantly inhibited by iodoacetate, hydroxylamine, and propargylglycine, revealing their sulfur active site dependence. Thus, a new isoform of glutathione S-transferase, GHTHase, with unique potency to reduce Hcy2 to soluble Hcy using GSH as hydrogen donor, was discovered. The specificity of GHTHase to oxidize toxic insoluble Hcy2 (cardiovascular disorder risk factor) can be a novel route to attack cardiovascular diseases.

Acute myeloid leukemia cells MOLM-13 and SKM-1 established for resistance by azacytidine are crossresistant to P-glycoprotein substrates

Establishment of the acute myeloid leukemia cells SKM-1 and MOLM-13 for resistance by azacytidine (AzaC) resulted in SKM-1/AzaC and MOLM-13/AzaC cell variants with reduced sensitivity to AzaC. Despite the fact that AzaC is not substrate of P-glycoprotein (P-gp), the adaptation procedure resulted in an induction in P-gp expression/efflux activity that confers crossresistance to P-gp substrates in both resistant cell variants. While the resistance to P-gp substrates in SKM-1/AzaC and MOLM-13/AzaC cells could be reversed by the P-gp inhibitors, resistance to AzaC was insensitive to these inhibitors in both resistant cell variants. In addition, NF-κB and the antiapoptotic protein Bcl-2 were downregulated and the proapoptotic proteins Bax and p53 were upregulated in both resistant cell variants when compared with their sensitive counterparts. Moreover, at least five times the elevation in overall glutathione S-transferase activity was measured with 1-chloro-2, 5-dinitrobenzene as a substrate in the resistant variant of both cell lines. Taken together, the findings of the present study indicate that the treatment of AML cells with AzaC might lead to a drug resistance phenotype that may be associated with cross resistance to P-gp substrates and substrates of glutathione S-transferases.

Characterization of a Highly pH Stable Chi-Class Glutathione S-Transferase from Synechocystis PCC 6803.

Glutathione S-transferases (GSTs) are multifunctional enzymes present in virtually all organisms. Besides having an essential role in cellular detoxification, they also perform various other functions, including responses in stress conditions and signaling. GSTs are highly studied in plants and animals; however, the knowledge regarding GSTs in cyanobacteria seems rudimentary. In this study, we report the characterization of a highly pH stable GST from the model cyanobacterium- Synechocystis PCC 6803. The gene sll0067 was expressed in Escherichia coli (E. coli), and the protein was purified to homogeneity. The expressed protein exists as a homo-dimer, which is composed of about 20 kDa subunit. The results of the steady-state enzyme kinetics displayed protein’s glutathione conjugation activity towards its class specific substrate- isothiocyanate, having the maximal activity with phenethyl isothiocyanate. Contrary to the poor catalytic activity and low specificity towards standard GST substrates such as 1-chloro-2,4-dinitrobenzene by bacterial GSTs, PmGST B1-1 from Proteus mirabilis, and E. coli GST, sll0067 has broad substrate degradation capability like most of the mammalian GST. Moreover, we have shown that cyanobacterial GST sll0067 is catalytically efficient compared to the best mammalian enzymes. The structural stability of GST was studied as a function of pH. The fluorescence and CD spectroscopy in combination with size exclusion chromatography showed a highly stable nature of the protein over a broad pH range from 2.0 to 11.0. To the best of our knowledge, this is the first GST with such a wide range of pH related structural stability. Furthermore, the presence of conserved Proline-53, structural motifs such as N-capping box and hydrophobic staple further aid in the stability and proper folding of cyanobacterial GST- sll0067.

Characterization of glutathione-S-transferases in zebrafish (Danio rerio)

Glutathione-S-transferases (GSTs) are one of the key enzymes that mediate phase II of cellular detoxification. The aim of our study was a comprehensive characterization of GSTs in zebrafish (Danio rerio) as an important vertebrate model species frequently used in environmental research. A detailed phylogenetic analysis of GST superfamily revealed 27 zebrafish gst genes. Further insights into the orthology relationships between human and zebrafish GSTs/Gsts were obtained by the conserved synteny analysis. Expression of gst genes in six tissues (liver, kidney, gills, intestine, brain and gonads) of adult male and female zebrafish was determined using qRT-PCR. Functional characterization was performed on 9 cytosolic Gst enzymes after overexpression in E. coli and subsequent protein purification. Enzyme kinetics was measured for GSH and a series of model substrates. Our data revealed ubiquitously high expression of gstp, gstm (except in liver), gstr1, mgst3a and mgst3b, high expression of gsto2 in gills and ovaries, gsta in intestine and testes, gstt1a in liver, and gstz1 in liver, kidney and brain. All zebrafish Gsts catalyzed the conjugation of GSH to model GST substrates 1-chloro-2,4-dinitrobenzene (CDNB) and monochlorobimane (MCB), apart from Gsto2 and Gstz1 that catalyzed GSH conjugation to dehydroascorbate (DHA) and dichloroacetic acid (DCA), respectively. Affinity toward CDNB varied from 0.28mM (Gstp2) to 3.69mM (Gstm3), while affinity toward MCB was in the range of 5μM (Gstt1a) to 250μM (Gstp1). Affinity toward GSH varied from 0.27mM (Gstz1) to 4.45mM (Gstt1a). Turnover number for CDNB varied from 5.25s−1 (Gstt1a) to 112s−1 (Gstp2). Only Gst Pi enzymes utilized ethacrynic acid (ETA). We suggest that Gstp1, Gstp2, Gstt1a, Gstz1, Gstr1, Mgst3a and Mgst3b have important role in the biotransformation of xenobiotics, while Gst Alpha, Mu, Pi, Zeta and Rho classes are involved in the crucial physiological processes. In summary, this study provides the first comprehensive analysis of GST superfamily in zebrafish, presents new insight into distinct functions of individual Gsts, and offers methodological protocols that can be used for further verification of interaction of environmental contaminants with fish Gsts.