| 1. |
- Mazari, Aslam M. A., et al.
(författare)
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Overexpression of Glutathione Transferase E7 in Drosophila Differentially Impacts Toxicity of Organic Isothiocyanates in Males and Females
- 2014
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:10
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
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| 3. |
- Zhang, Wei
(författare)
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Directed Evolution of Glutathione Transferases with Altered Substrate Selectivity Profiles : A Laboratory Evolution Study Shedding Light on the Multidimensional Nature of Epistasis
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Directed evolution is generally regarded as a useful approach in protein engineering. By subjecting members of a mutant library to the power of Darwinian evolution, desired protein properties are obtained. Numerous reports have appeared in the literature showing the success of tailoring proteins for various applications by this method. Is it a one-way track that protein practitioners can only learn from nature to enable more efficient protein engineering? A structure-and-mechanism-based approach, supplemented with the use of reduced amino acid alphabets, was proposed as a general means for semi-rational enzyme engineering. Using human GST A2-2*E, the most active human enzyme in the bioactivation of azathioprine, as a parental enzyme to test this approach, a L107G/L108D/F222H triple-point mutant of GST A2-2*E (thereafter designated as GDH) was discovered with 70-fold increased activity, approaching the upper limit of specific activity of the GST scaffold. The approach was further experimentally verified to be more successful than intuitively choosing active-site residues in proximity to the bound substrate for the improvement of enzyme performance. By constructing all intermediates along all putative mutational paths leading from GST A2-2*E to mutant GDH and assaying them with nine alternative substrates, the fitness landscapes were found to be “rugged” in differential fashions in substrate-activity space. The multidimensional fitness landscapes stemming from functional promiscuity can lead to alternative outcomes with enzymes optimized for other features than the selectable markers that were relevant at the origin of the evolutionary process. The results in this thesis suggest that in this manner an evolutionary response to changing environmental conditions can readily be mounted. In summary, the thesis demonstrates the attractive features of the structure-and-mechanism-based semi-rational directed evolution approach for optimizing enzyme performance. Moreover, the results gained from the studies show that laboratory evolution may refine our understanding of evolutionary process in nature.
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| 4. |
- Zuo, Shusheng, 1968-
(författare)
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Quantitation, Purification and Reconstitution of the Red Blood Cell Glucose Transporter GLUT1
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The human glucose transporter GLUT1 facilitates glucose to be accumulated on the other side of the cell membrane. The functional state of GLUT1 is uncertain due to diversity of the reports. In this thesis, the activity of red blood cell GLUT1 was extensively studied to further characterize this protein.The human red blood cell membranes were stripped to become vesicles with low-ionic alkaline solution in the presence or absence of dithioerithritol. The supernatant of partially solubilized membrane vesicles provided approximately 65% of the vesicle proteins. GLUT1 purified from this supernatant showed a little high-affinity cytochalasin B binding activity. On the other hand, the vesicles stripped with dithioerythritol provided mostly monomeric GLUT1 and those without dithioerythritol provided monomeric and oligomeric GLUT1. MALDI-ToF-MS detected variant GLUT1 fragments between the two preparations. Residual endogenous phospholipids per GLUT1 also showed difference. However, the equilibrium exchange of glucose was retained for both GLUT1 preparations. Cytochalasin B-binding activity of GLUT1 in streptoavidin-biotin-immobilized red blood cells showed that both dissociation constant and binding sites per GLUT1 fell between those of wheat germ lectin-immobilized red blood cells with or without polylysine coating, which indicated the switching of two cytochalasin B-binding states of GLUT1. It is concluded that GLUT1 in red blood cells contains approximately two equal portions, monomeric with high-affinity cytochalasin B-binding activity and oligomeric without high-affinity cytochalasin B-binding activity. In the partial solubilization of the membrane vesicles, GLUT1 which does not have high-affinity cytochalasin B-binding activity is pooled. This might provide a resolution to select oligomerically and functionally different GLUT1 for crystallization.In addition a modified micro-Bradford assay with CaPE precipitation was developed to achieve a routine quantitation method for membrane proteins and the effects of cholesterol and PEG(5000)-DSPE on reconstituted GLUT1 were preliminarily determined.
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| 5. |
- Bolelli, K., et al.
(författare)
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Synthesis and activity mechanism of some novel 2-substituted benzothiazoles as hGSTP1-1 enzyme inhibitors
- 2017
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Ingår i: SAR and QSAR in environmental research (Print). - 1062-936X .- 1029-046X. ; 28:11, s. 927-940
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Tidskriftsartikel (refereegranskat)abstract
- Human GSTP1-1 is one of the most important proteins, which overexpresses in a large number of human tumours and is involved in the development of resistance to several anticancer drugs. So, it has become an important target in cancer treatment. In this study, 12 benzothiazole derivatives were synthesized and screened for their in vitro inhibitory activity for hGSTP1-1. Among these compounds, two of them (compounds #2 and #5) have been found to be the leads when compared with the reference drug etoposide. In order to analyse the structure-activity relationships (SARs) and to investigate the binding side interactions of the observed lead compounds, a HipHop pharmacophore model was generated and the molecular docking studies were performed by using CDocker method. In conclusion, it is observed that the lead compounds #2 and #5 possessed inhibitory activity on the hGSTP1-1 by binding to the H-site as a substrate in which the para position of the phenyl ring of the benzamide moiety on the benzothiazole ring is important. Substitution at this position with a hydrophobic group that reduces the electron density at the phenyl ring is required for the interaction with the H side active residue Tyr108.
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| 6. |
- Dourado, Daniel F. A. R., et al.
(författare)
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Isomerization of Delta(5)-Androstene-3,17-dione into Delta(4)-Androstene-3, 17-dione Catalyzed by Human Glutathione Transferase A3-3 : A Computational Study Identifies a Dual Role for Glutathione
- 2014
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Ingår i: Journal of Physical Chemistry A. - : American Chemical Society (ACS). - 1089-5639 .- 1520-5215. ; 118:31, s. 5790-5800
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Tidskriftsartikel (refereegranskat)abstract
- Glutathione transferases (GSTs) are important enzymes in the metabolism of electrophilic xenobiotic and endobiotic toxic compounds. In addition, human GST A3-3 also catalyzes the double bond isomerization of Delta 5-androstene-3,17-dione (Delta(5)-AD) and Delta(5)-pregnene-3,20-dione (Delta(5)-PD), which are the immediate precursors of testosterone and progesterone. In fact, GST A3-3 is the most efficient human enzyme known to exist in the catalysis of these reactions. In this work, we have used density functional theory (DFT) calculations to propose a refined mechanism for the isomerization of Delta(5)-AD catalyzed by GST A3-3. In this mechanism the glutathione (GSH) thiol and Tyr9 catalyze the proton transfer from the Delta(5)-AD C4 atom to the Delta(5)-AD C6 atom, with a rate limiting activation energy of 15.8 kcal.mol(-1). GSH has a dual function, because it is also responsible for stabilizing the negative charge that is formed in the 03 atom of the enolate intermediate. The catalytic role of Tyr9 depends on significant conformational rearrangements of its side chain. Neither of these contributions to catalysis has been observed before. Residues Phe10, Leul11, Ala 208, and Ala 216 complete the list of the important catalytic residues. The mechanism detailed here is based on the GST A3-3:GSH:Delta(4)-AD crystal structure and is consistent with all available experimental data.
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| 7. |
- Zhang, Wei, et al.
(författare)
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Evolution of the active site of human glutathione transferase A2-2 for enhanced activity with dietary isothiocyanates
- 2015
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Ingår i: Biochimica et Biophysica Acta - General Subjects. - : Elsevier BV. - 0304-4165 .- 1872-8006. ; 1850:4, s. 742-749
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Tidskriftsartikel (refereegranskat)abstract
- Background: Organic isothiocyanates (ITCs) are produced by plants, in which they are released from glucosinolates by myrosinase. ITCs are generally toxic and serve as a chemical defense against herbivorous insects and against infections by microorganisms. In mammalian tissues subtoxic concentrations of ITCs can provide protective effects against cancer and other diseases partially by induction of glutathione transferases (GSTs) and other detoxication enzymes. Thus, human consumption of edible plants rich in ITCs is presumed to provide health benefits. ITCs react with intracellular glutathione to form dithiocarbamates, catalyzed by GSTs. Formation of glutathione conjugates is central to the biotransformation of ITCs and leads to a route for their excretion. Clearly, the emergence of ITC conjugating activity in GSTs is essential from the biological and evolutionary perspective. Methods: In the present investigation an active-site-focused mutant library of GST A2-2 has been screened for enzyme variants with enhanced ITC activity. Results: Significantly superior activities were found in 34 of the approximately 2000 mutants analyzed, and the majority of the superior GSTs featured His and Gly residues in one of the three active-site positions subjected to mutagenesis. Conclusions: We explored the propensity of GSTs to obtain altered substrate selectivity and moreover, identified a specific pattern of mutagenesis in GST for enhanced PEITC detoxification, which may play an important role in the evolution of adaptive responses in organisms subjected to ITCs. General significance: The facile acquisition of enhanced ITC activity demonstrates that this important detoxication function can be promoted by numerous evolutionary trajectories in sequence space.
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| 8. |
- Cuevas, Carlos, et al.
(författare)
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Glutathione Transferase-M2-2 Secreted from Glioblastoma Cell Protects SH-SY5Y Cells from Aminochrome Neurotoxicity
- 2015
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Ingår i: Neurotoxicity research. - : Springer Science and Business Media LLC. - 1029-8428 .- 1476-3524. ; 27:3, s. 217-228
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Tidskriftsartikel (refereegranskat)abstract
- U373MG cells are able to take up aminochrome that induces glutathione transferase M2-2 (GSTM2) expression in a concentration-dependent manner where 100 A mu M aminochrome increases GSTM2 expression by 2.1-fold (P < 0.001) at 3 h. The uptake of H-3-aminochrome into U373MG cells was significantly reduced in the presence of 2 A mu M nomifensine (P < 0.001) 100 A mu M imipramine (P < 0.001) and 50 mM dopamine (P < 0.001). Interestingly, U373MG cells excrete GSTM2 into the conditioned medium and the excretion was significantly increased (2.7-fold; P < 0.001) when the cells were pretreated with 50 A mu M aminochrome for 3 h. The U373MG-conditioned medium containing GSTM2 protects SH-SY5Y cells incubated with 10 A mu M aminochrome. The significant protection provided by U373MG-conditioned medium in SH-SY5Y cells incubated with aminochrome was dependent on GSTM2 internalization into SH-SY5Y cells as evidenced by (i) uptake of C-14-GSTM2 released from U373MG cells into SH-SY5Y cells, a process inhibited by anti-GSTM2 antiserum; (ii) lack of protection of U373MG-conditioned medium in the presence of anti-GSTM2 antiserum on SH-SY5Y cells treated with aminochrome; and (iii) lack of protection of conditioned medium from U373MGsiGST6 that expresses an siRNA directed against GSTM2 on SH-SY5Y cells treated with aminochrome. In conclusion, our results demonstrated that U373MG cells protect SH-SY5Y cells against aminochrome neurotoxicity by releasing GSTM2 into the conditioned medium and subsequent internalization of GSTM2 into SH-SY5Y cells. These results suggest a new mechanism of protection of dopaminergic neurons mediated by astrocytes by releasing GSTM2 into the intersynaptic space and subsequent internalization into dopaminergic neuron in order to protect these cells against aminochrome neurotoxicity.
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| 9. |
- Govindarajan, Sridhar, et al.
(författare)
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Mapping of Amino Acid Substitutions Conferring Herbicide Resistance in Wheat Glutathione Transferase
- 2015
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Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 4:3, s. 221-227
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Tidskriftsartikel (refereegranskat)abstract
- We have used design of experiments (DOE) and systematic variance to efficiently explore glutathione transferase substrate specificities caused by amino acid substitutions. Amino acid substitutions selected using phylogenetic analysis were synthetically combined using a DOE design to create an information-rich set of gene variants, termed infologs. We used machine learning to identify and quantify protein sequence-function relationships against 14 different substrates The resulting models were quantitative and predictive, serving as a guide for engineering of glutathione transferase activity toward a diverse set of herbicides Predictive quantitative models like those presented here have broad applicability for bioengineering.
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| 10. |
- Mannervik, Bengt
(författare)
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Five Decades with Glutathione and the GSTome
- 2012
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 287:9, s. 6072-6083
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Tidskriftsartikel (refereegranskat)abstract
- Uncle Folke inspired me to become a biochemist by demonstrating electrophoresis experiments on butterfly hemolymph in his kitchen. Glutathione became the subject for my undergraduate project in 1964 and has remained a focal point in my research owing to its multifarious roles in the cell. Since the 1960s, the multiple forms of glutathione transferase (GST), the GSTome, were isolated and characterized, some of which were discovered in our laboratory. Products of oxidative processes were found to be natural GST substrates. Examples of toxic compounds against which particular GSTs provide protection include 4-hydroxynonenal and ortho-quinones, with possible links to the etiology of Alzheimer and Parkinson diseases and other degenerative conditions. The role of thioltransferase and glutathione reductase in the cellular reduction of disulfides and other oxidized forms of thiols was clarified. Glyoxalase I catalyzes still another glutathione-dependent detoxication reaction. The unusual steady-state kinetics of this zinc-containing enzyme initiated model discrimination by regression analysis. Functional properties of the enzymes have been altered by stochastic mutations based on DNA shuffling and rationally tailored by structure-based redesign. We found it useful to represent promiscuous enzymes by vectors or points in multidimensional substrate-activity space and visualize them by multivariate analysis. Adopting the concept molecular quasi-species, we describe clusters of functionally related enzyme variants that may emerge in natural as well as directed evolution.
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