| 1. |
- Hubert, Shawna M., et al.
(författare)
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Conservation of Glutathione Transferase mRNA and Protein Sequences Similar to Human and Horse Alpha Class GST A3-3 across Dog, Goat, and Opossum Species
- 2023
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Ingår i: Biomolecules. - 2218-273X. ; 13:9
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Ismail, Aram, et al.
(författare)
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Characterization of Dog Glutathione Transferase P1-1, an Enzyme Relevant to Veterinary Medicine
- 2021
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 22:8
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Tidskriftsartikel (refereegranskat)abstract
- Glutathione transferases (GSTs) form a family of detoxication enzymes instrumental in the inactivation and elimination of electrophilic mutagenic and carcinogenic compounds. The Pi class GST P1-1 is present in most tissues and is commonly overexpressed in neoplastic cells. GST P1-1 in the dog, Canis lupus familiaris, has merits as a marker for tumors and as a target for enzyme-activated prodrugs. We produced the canine enzyme CluGST P1-1 by heterologous bacterial expression and verified its cross-reactivity with antihuman-GST P1-1 antibodies. The catalytic activity with alternative substrates of biological significance was determined, and the most active substrate found was benzyl isothiocyanate. Among established GST inhibitors, Cibacron Blue showed positive cooperativity with an IC50 value of 43 nM. Dog GST P1-1 catalyzes activation of the prodrug Telcyta, but the activity is significantly lower than that of the human homolog.
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| 3. |
- Ismail, Aram, et al.
(författare)
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Human GST P1-1 Redesigned for Enhanced Catalytic Activity with the Anticancer Prodrug Telcyta and Improved Thermostability
- 2024
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Ingår i: Cancers. - 2072-6694. ; 16:4
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Tidskriftsartikel (refereegranskat)abstract
- Protein engineering can be used to tailor enzymes for medical purposes, including antibody-directed enzyme prodrug therapy (ADEPT), which can act as a tumor-targeted alternative to conventional chemotherapy for cancer. In ADEPT, the antibody serves as a vector, delivering a drug-activating enzyme selectively to the tumor site. Glutathione transferases (GSTs) are a family of naturally occurring detoxication enzymes, and the finding that some of them are overexpressed in tumors has been exploited to develop GST-activated prodrugs. The prodrug Telcyta is activated by GST P1-1, which is the GST most commonly elevated in cancer cells, implying that tumors overexpressing GST P1-1 should be particularly vulnerable to Telcyta. Promising antitumor activity has been noted in clinical trials, but the wildtype enzyme has modest activity with Telcyta, and further functional improvement would enhance its usefulness for ADEPT. We utilized protein engineering to construct human GST P1-1 gene variants in the search for enzymes with enhanced activity with Telcyta. The variant Y109H displayed a 2.9-fold higher enzyme activity compared to the wild-type GST P1-1. However, increased catalytic potency was accompanied by decreased thermal stability of the Y109H enzyme, losing 99% of its activity in 8 min at 50 °C. Thermal stability was restored by four additional mutations simultaneously introduced without loss of the enhanced activity with Telcyta. The mutation Q85R was identified as an important contributor to the regained thermostability. These results represent a first step towards a functional ADEPT application for Telcyta.
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| 4. |
- Ismail, Aram, et al.
(författare)
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Marmoset glutathione transferases with ketosteroid isomerase activity
- 2021
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Ingår i: Biochemistry and Biophysics Reports. - : Elsevier BV. - 2405-5808. ; 27
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Tidskriftsartikel (refereegranskat)abstract
- The common marmoset Callithrix jacchus encodes two glutathione transferase (GST) enzymes with ketosteroid double-bond isomerase activity. The most active enzyme is CjaGST A3-3 showing a specific activity with 5-androsten-3,17-dione (Delta(5)-AD) of 62.1 +/- 1.8 mu mol min(-1) mg(-1), and a k(cat) value of 261 +/- 49 s(-1). The second ketostemid isomerase CjaGST A1-1 has a 30-fold lower specific activity with Delta(5)-AD and a 37-fold lower k(cat) value. Thus, the marmoset CjaGST A3-3 would be the main contributor to the biosynthesis of the steroid hormones testosterone and progesterone, like the human ortholog HsaGST A3-3. Two residues differ in the H-site of the 91.4% sequence identical CjaGST A1-1 and CjaGST A3-3, and modeling of the structures suggests that the bulky phenyl ring of Phe111 in CjaGST A1-1 causes steric hindrance in the binding of the steroid substrate. Tributyltin acetate (IC50 =0.16 +/- 0.004 mu M) and ethacrynic acid (IC50 =3.3 +/- 0.2 mu M) were found to be potent inhibitors of CjaGST A3-3, as previously demonstrated with the human and equine orthologs.
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| 5. |
- Lindström, Helena, 1961-
(författare)
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Exploring the steroidogenic activity of glutathione transferases across species
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Glutathione transferases (GSTs) comprise a superfamily of enzymes prominently involved in detoxication. However, some GSTs have developed alternative functions. Thus, a member of the Alpha class GSTs in tissues of Homo sapiens (humans), Sus scrofa (pigs) and ruminants is involved in biosynthesis of steroid hormones, catalyzing a double-bond isomerization reaction as the last step of synthesis of Δ4-pregnene-3,20-dione (progesterone) and the obligatory step in the synthesis of the last precursor of testosterone, Δ4-androstenene-3,17-dione. Steroids regulate several vital aspects of life such as for example glucose homeostasis, inflammation, immunosuppression, blood pressure, reproduction and pregnancy.The human GST A3-3 was the most efficient steroid double-bond isomerase known so far in mammals. Our work extends discoveries of GSTs that act in the steroidogenic pathways in large mammals to Equus ferus caballus (horse). The kinetic profile of EcaGST A3-3 reveals a catalytic efficiency higher than that of the human enzyme making EcaGST A3-3 the most efficient steroid double-bond isomerase known today in mammals.In contrast to the rodents, Equus ferus caballus shares the steroidogenic pathway with Homo sapiens, which makes it a more suitable model for human steroidogenesis than the murine one. Inhibition of EcaGST A3-3 might help treat endocrine disorders. We screened a library of 1040 FDA-approved compounds for novel inhibitors of EcaGST A3-3 and made a further characterization of the most potent inhibitors.To extend the search for steroidogenic GSTs to other mammals, we probed the degree of GST A3-3 amino acid sequence conservation in Homo sapiens, Equus ferus caballus, Canis lupus familiaris (dog), Capra hircus (goat) and Monodelphis domestica (gray short-tailed opossum). We generated expression vectors containing homologous DNA from these species to facilitate further evaluation of the activity of these GSTs in mammals.We continued to expand the research to insects by investigating the steroidogenic activity of GSTE14 in Drosophila melanogaster (fruit fly), where this enzyme has been shown to be implicated in molting.Our work has provided insights into the role of GSTs in steroidogenesis in mammals and insects, further accentuating the functional versatility of GSTs. We have provided an initial step for the development of potential treatments of steroidogenic disorders as well as tools for further investigation of activity of these GSTs in mammals.
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| 6. |
- Mannervik, Bengt, et al.
(författare)
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Glutathione Transferases as Efficient Ketosteroid Isomerases
- 2021
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 8
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Forskningsöversikt (refereegranskat)abstract
- In addition to their well-established role in detoxication, glutathione transferases (GSTs) have other biological functions. We are focusing on the ketosteroid isomerase activity, which appears to contribute to steroid hormone biosynthesis in mammalian tissues. A highly efficient GST A3-3 is present in some, but not all, mammals. The alpha class enzyme GST A3-3 in humans and the horse shows the highest catalytic efficiency with kcat/Km values of approximately 107 M−1s−1, ranking close to the most active enzymes known. The expression of GST A3-3 in steroidogenic tissues suggests that the enzyme has evolved to support the activity of 3β-hydroxysteroid dehydrogenase, which catalyzes the formation of 5-androsten-3,17-dione and 5-pregnen-3,20-dione that are substrates for the double-bond isomerization catalyzed by GST A3-3. The dehydrogenase also catalyzes the isomerization, but its kcat of approximately 1 s−1 is 200-fold lower than the kcat values of human and equine GST A3-3. Inhibition of GST A3-3 in progesterone-producing human cells suppress the formation of the hormone. Glutathione serves as a coenzyme contributing a thiolate as a base in the isomerase mechanism, which also involves the active-site Tyr9 and Arg15. These conserved residues are necessary but not sufficient for the ketosteroid isomerase activity. A proper assortment of H-site residues is crucial to efficient catalysis by forming the cavity binding the hydrophobic substrate. It remains to elucidate why some mammals, such as rats and mice, lack GSTs with the prominent ketosteroid isomerase activity found in certain other species. Remarkably, the fruit fly Drosophila melanogaster, expresses a GSTE14 with notable steroid isomerase activity, even though Ser14 has evolved as the active-site residue corresponding to Tyr9 in the mammalian alpha class.
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| 7. |
- Musdal, Yaman, et al.
(författare)
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Potent GST Ketosteroid Isomerase Activity Relevant to Ecdysteroidogenesis in the Malaria Vector Anopheles gambiae
- 2023
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Ingår i: Biomolecules. - 2218-273X. ; 13:6
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Tidskriftsartikel (refereegranskat)abstract
- Nobo is a glutathione transferase (GST) crucially contributing to ecdysteroid biosynthesis in insects of the orders Diptera and Lepidoptera. Ecdysone is a vital steroid hormone in insects, which governs larval molting and metamorphosis, and the suppression of its synthesis has potential as a novel approach to insect growth regulation and combatting vectors of disease. In general, GSTs catalyze detoxication, whereas the specific function of Nobo in ecdysteroidogenesis is unknown. We report that Nobo from the malaria-spreading mosquito Anopheles gambiae is a highly efficient ketosteroid isomerase catalyzing double-bond isomerization in the steroids 5-androsten-3,17-dione and 5-pregnen-3,20-dione. These mammalian ketosteroids are unknown in mosquitoes, but the discovered prominent catalytic activity of these compounds suggests that the unknown Nobo substrate in insects has a ketosteroid functionality. Aminoacid residue Asp111 in Nobo is essential for activity with the steroids, but not for conventional GST substrates. Further characterization of Nobo may guide the development of new insecticides to prevent malaria.
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| 8. |
- Schwartz, Mathieu, et al.
(författare)
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Interactions Between Odorants and Glutathione Transferases in the Human Olfactory Cleft
- 2020
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Ingår i: Chemical Senses. - : Oxford University Press (OUP). - 0379-864X .- 1464-3553. ; 45:8, s. 645-654
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Tidskriftsartikel (refereegranskat)abstract
- Xenobiotic metabolizing enzymes and other proteins, including odorant-binding proteins located in the nasal epithelium and mucus, participate in a series of processes modulating the concentration of odorants in the environment of olfactory receptors (ORs) and finely impact odor perception. These enzymes and transporters are thought to participate in odorant degradation or transport. Odorant biotransformation results in 1) changes in the odorant quantity up to their clearance and the termination of signaling and 2) the formation of new odorant stimuli (metabolites). Enzymes, such as cytochrome P450 and glutathione transferases (GSTs), have been proposed to participate in odorant clearance in insects and mammals as odorant metabolizing enzymes. This study aims to explore the function of GSTs in human olfaction. Using immunohistochemical methods, GSTs were found to be localized in human tissues surrounding the olfactory epithelium. Then, the activity of 2 members of the GST family toward odorants was measured using heterologously expressed enzymes. The interactions/reactions with odorants were further characterized using a combination of enzymatic techniques. Furthermore, the structure of the complex between human GSTA1 and the glutathione conjugate of an odorant was determined by X-ray crystallography. Our results strongly suggest the role of human GSTs in the modulation of odorant availability to ORs in the peripheral olfactory process.
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| 9. |
- Segura-Aguilar, Juan, et al.
(författare)
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A Preclinical Model for Parkinson's Disease Based on Transcriptional Gene Activation via KEAP1/NRF2 to Develop New Antioxidant Therapies
- 2023
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Ingår i: Antioxidants. - : MDPI AG. - 2076-3921. ; 12:3
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Forskningsöversikt (refereegranskat)abstract
- Investigations of the effect of antioxidants on idiopathic Parkinson’s disease have been unsuccessful because the preclinical models used to propose these clinical studies do not accurately represent the neurodegenerative process of the disease. Treatment with certain exogenous neurotoxins induces massive and extremely rapid degeneration; for example, MPTP causes severe Parkinsonism in just three days, while the degenerative process of idiopathic Parkinson´s disease proceeds over many years. The endogenous neurotoxin aminochrome seems to be a good alternative target since it is formed in the nigrostriatal system neurons where the degenerative process occurs. Aminochrome induces all the mechanisms reported to be involved in the degenerative processes of idiopathic Parkinson’s disease. The presence of neuromelanin-containing dopaminergic neurons in the postmortem brain of healthy elderly people suggests that neuromelanin synthesis is a normal and harmless process despite the fact that it requires oxidation of dopamine to three ortho-quinones that are potentially toxic, especially aminochrome. The apparent contradiction that neuromelanin synthesis is harmless, despite its formation via neurotoxic ortho-quinones, can be explained by the protective roles of DT-diaphorase and glutathione transferase GSTM2-2 as well as the neuroprotective role of astrocytes secreting exosomes loaded with GSTM2-2. Increasing the expression of DT-diaphorase and GSTM2-2 may be a therapeutic goal to prevent the degeneration of new neuromelanin-containing dopaminergic neurons. Several phytochemicals that induce DT-diaphorase have been discovered and, therefore, an interesting question is whether these phytochemical KEAP1/NRF2 activators can inhibit or decrease aminochrome-induced neurotoxicity.
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| 10. |
- Segura-Aguilar, Juan, et al.
(författare)
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Astrocytes protect dopaminergic neurons against aminochrome neurotoxicity
- 2022
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Ingår i: Neural Regeneration Research. - : Medknow. - 1673-5374 .- 1876-7958. ; 17:9, s. 1861-1866
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Forskningsöversikt (refereegranskat)abstract
- Astrocytes protect neurons by modulating neuronal function and survival. Astrocytes support neurons in several ways. They provide energy through the astrocyte-neuron lactate shuttle, protect neurons from excitotoxicity, and internalize neuronal lipid droplets to degrade fatty acids for neuronal metabolic and synaptic support, as well as by their high capacity for glutamate uptake and the conversion of glutamate to glutamine. A recent reported astrocyte system for protection of dopamine neurons against the neurotoxic products of dopamine, such as aminochrome and other o-quinones, were generated under neuromelanin synthesis by oxidizing dopamine catechol structure. Astrocytes secrete glutathione transferase M2-2 through exosomes that transport this enzyme into dopaminergic neurons to protect these neurons against aminochrome neurotoxicity. The role of this new astrocyte protective mechanism in Parkinson´s disease is discussed.
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