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Studies on Human and Drosophila melanogaster Glutathione Transferases of Biomedical and Biotechnological Interest
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0001-6258-1443
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Glutathione transferases (GSTs, EC.2.5.1.18) are multifunctional enzymes that are universally distributed in all cellular life forms. They play important roles in metabolism and detoxication of endogenously produced toxic compounds and xenobiotics. GSTs have gained considerable interest over the years for biomedical and biotechnological applications due to their involvement in the conjugation of glutathione (GSH) to a vast array of chemical species. Additionally, the emergence of non-detoxifying functions of GSTs has further increased their biological significance. The present work encompasses four scientific studies aimed at investigating human as well as fruit fly Drosophila melanogaster GSTs.

Paper I presents the immobilization of GSTs on nanoporous alumina membranes. Kinetic analyses with 1-chloro-2,4-dinitrobenzene followed by specificity screening with alternative substrates showed a good correlation between the data obtained from immobilized enzymes and the enzymes in solution. Furthermore, immobilization showed no adverse effects on the stability of the enzymes. Paper II presents inhibition studies of human hematopoietic prostaglandin D2 synthase (HPGDS), a promising therapeutic target for anti-allergic and anti-inflammatory drugs. Our screening results with an FDA-approved drug library revealed a number of effective inhibitors of HPGDS with IC50 values in the low micromolar range. Paper III concerns the toxicity of organic isothiocyanates (ITCs) that showed high catalytic activities with GSTE7 in vitro. The in vivo results showed that phenethyl isothiocyanate (PEITC) and allyl isothiocyanate in millimolar dietary concentrations conferred toxicity to the adult fruit flies leading to death or shortened life-span. The transgenic female flies overexpressing GSTE7 showed increased tolerance against PEITC toxicity compared to the wild-type. However, the effect was opposite in male flies overexpressing GSTE7 after one week exposure. Notably, the transgene enhanced the oviposition activity of flies with and without ITCs exposure. Paper IV highlights Drosophila GSTs as efficient catalysts of the environmental pollutant and explosive 2,4,6-trinitrotoluene and the related 2,4-dinitrotoluene degradation. This result suggests the potential of GST transgenes in plants for biotransformation and phytoremediation of these persistent environmental pollutants. 

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University , 2016. , 43 p.
Keyword [en]
glutathione transferases, detoxication, hpgds inhibition, immobilization, isothiocyanates, drosophila GSTs, environmental pollutants
National Category
Chemical Sciences Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-126723ISBN: 978-91-7649-349-6 (print)OAI: oai:DiVA.org:su-126723DiVA: diva2:902998
Public defence
2016-03-18, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2016-02-24 Created: 2016-02-12 Last updated: 2017-02-23Bibliographically approved
List of papers
1. Glutathione transferases immobilized on nanoporous alumina: Flow system kinetics, screening, and stability
Open this publication in new window or tab >>Glutathione transferases immobilized on nanoporous alumina: Flow system kinetics, screening, and stability
Show others...
2014 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 446, 59-63 p.Article in journal (Refereed) Published
Abstract [en]

The previously uncharacterized Drosophila melanogaster Epsilon-class glutathione transferases E6 and E7 were immobilized on nanoporous alumina. The nanoporous anodized alumina membranes were derivatized with 3-aminopropyl-triethoxysilane, and the amino groups were activated with carbonyldiimidazole to allow coupling of the enzymes via c-amino groups. Kinetic analyses of the immobilized enzymes were carried out in a circulating flow system using CDNB (1-chloro-2,4-dinitrobenzene) as substrate, followed by specificity screening with alternative substrates. A good correlation was observed between the substrate screening data for immobilized enzyme and corresponding data for the enzyme in solution. A limited kinetic study was also carried out on immobilized human GST S1-1 (also known as hematopoietic prostaglandin D synthase). The stability of the immobilized enzymes was virtually identical to that of enzymes in solution, and no leakage of enzyme from the matrix could be observed.

Keyword
Immobilization, Glutathione transferase, Kinetics, Screening, Enzyme reactor, Drosophila
National Category
Biochemistry and Molecular Biology Analytical Chemistry
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-100853 (URN)10.1016/j.ab.2013.10.004 (DOI)000329949500010 ()
Note

AuthorCount:5;

Available from: 2014-02-21 Created: 2014-02-17 Last updated: 2017-12-05Bibliographically approved
2. Identification of new inhibitors for human hematopoietic prostaglandin D-2 synthase among FDA-approved drugs and other compounds
Open this publication in new window or tab >>Identification of new inhibitors for human hematopoietic prostaglandin D-2 synthase among FDA-approved drugs and other compounds
2015 (English)In: Chemico-Biological Interactions, ISSN 0009-2797, E-ISSN 1872-7786, Vol. 229, 91-99 p.Article in journal (Refereed) Published
Abstract [en]

Objective: Hematopoietic prostaglandin D-2 synthase (HPGDS) is a member of the Sigma class glutathione transferases (GSTs) catalyzing the isomerization of prostaglandin H-2 to prostaglandin D-2, a mediator of allergy and inflammation responses. Selective inhibitors of human HPGDS are expected to be of therapeutic importance in relieving symptoms related to allergy and asthma. Hence, a collection of diverse FDA-approved compounds was screened for potential novel applications as inhibitors of HPGDS. Methods: The catalytic activity of purified HPGDS was used for inhibition studies in vitro. Results: Our inhibition studies revealed 23 compounds as effective inhibitors of HPGDS with IC50 values in the low micromolar range. Erythrosine sodium, suramin, tannic acid and sanguinarine sulfate were characterized with IC50 values of 0.2, 0.3, 0.4, and 0.6 mu M, respectively. Kinetic inhibition analysis showed that erythrosine sodium is a nonlinear competitive inhibitor of HPGDS, while suramin, tannic acid and sanguinarine sulfate are linear competitive inhibitors. Conclusion: The results show that certain FDA-approved compounds may have pharmacological effects not previously realized that warrant further consideration in their clinical use.

Keyword
Prostaglandin D-2 synthase, FDA-approved drugs, HPGDS inhibitors, Glutathione transferase S1-1
National Category
Biochemistry and Molecular Biology Pharmacology and Toxicology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-117004 (URN)10.1016/j.cbi.2015.01.014 (DOI)000352050800011 ()25603235 (PubMedID)
Funder
Swedish Cancer SocietySwedish Research Council
Available from: 2015-05-08 Created: 2015-05-05 Last updated: 2017-12-04Bibliographically approved
3. Overexpression of Glutathione Transferase E7 in Drosophila Differentially Impacts Toxicity of Organic Isothiocyanates in Males and Females
Open this publication in new window or tab >>Overexpression of Glutathione Transferase E7 in Drosophila Differentially Impacts Toxicity of Organic Isothiocyanates in Males and Females
2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 10, e110103Article in journal (Refereed) Published
Abstract [en]

Organic isothiocyanates (ITCs) are allelochemicals produced by plants in order to combat insects and other herbivores. The compounds are toxic electrophiles that can be inactivated and conjugated with intracellular glutathione in reactions catalyzed by glutathione transferases (GSTs). The Drosophila melanogaster GSTE7 was heterologously expressed in Escherichia coli and purified for functional studies. The enzyme showed high catalytic activity with various isothiocyanates including phenethyl isothiocyanate (PEITC) and allyl isothiocyanate (AITC), which in millimolar dietary concentrations conferred toxicity to adult D. melanogaster leading to death or a shortened life-span of the flies. In situ hybridization revealed a maternal contribution of GSTE7 transcripts to embryos, and strongest zygotic expression in the digestive tract. Transgenesis involving the GSTE7 gene controlled by an actin promoter produced viable flies expressing the GSTE7 transcript ubiquitously. Transgenic females show a significantly increased survival when subjected to the same PEITC treatment as the wild-type flies. By contrast, transgenic male flies show a significantly lower survival rate. Oviposition activity was enhanced in transgenic flies. The effect was significant in transgenic females reared in the absence of ITCs as well as in the presence of 0.15 mM PEITC or 1 mM AITC. Thus the GSTE7 transgene elicits responses to exposure to ITC allelochemicals which differentially affect life-span and fecundity of male and female flies.

National Category
Biological Sciences Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-109267 (URN)10.1371/journal.pone.0110103 (DOI)000343210800055 ()
Available from: 2014-11-21 Created: 2014-11-17 Last updated: 2017-12-05Bibliographically approved
4. Drosophila GSTs display outstanding catalytic efficiencies with the environmental pollutants 2,4,6-trinitrotoluene and 2,4-dinitrotoluene
Open this publication in new window or tab >>Drosophila GSTs display outstanding catalytic efficiencies with the environmental pollutants 2,4,6-trinitrotoluene and 2,4-dinitrotoluene
2016 (English)In: Biochemistry and Biophysics Reports, ISSN 2405-5808, Vol. 5, 141-145 p.Article in journal (Refereed) Published
Abstract [en]

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

Keyword
Glutathione conjugation, 2, 4, 6-trinitrotoluene detoxication, 2, 4-dinitrotoluene detoxication, Drosophila GSTs, Human GSTs
National Category
Biochemistry and Molecular Biology Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-126346 (URN)10.1016/j.bbrep.2015.12.003 (DOI)
Funder
Swedish Research Council, 2012-5161
Available from: 2016-02-01 Created: 2016-02-01 Last updated: 2016-02-23Bibliographically approved

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