| 1. |
- Ahmad, Shabbir, et al.
(författare)
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Trimeric microsomal glutathione transferase 2 displays one third of the sites reactivity
- 2015
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Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics. - : Elsevier BV. - 1570-9639 .- 1878-1454. ; 1854:1010 Pt A, s. 1365-1371
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Tidskriftsartikel (refereegranskat)abstract
- Human microsomal glutathione transferase 2 (MGST2) is a trimeric integral membrane protein that belongs to the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) family. The mammalian MAPEG family consists of six members where four have been structurally determined. MGST2 activates glutathione to form a thiolate that is crucial for GSH peroxidase activity and GSH conjugation reactions with electrophilic substrates, such as 1-chloro-2,4-dinitrobenzene (CDNB). Several studies have shown that MGST2 is able to catalyze a GSH conjugation reaction with the epoxide LTA(4) forming the pro-inflammatory LTC4. Unlike its closest homologue leukotriene C-4 synthase (LTC4S), MGST2 appears to activate its substrate GSH using only one of the three potential active sites [Ahmad S, et al. (2013) Biochemistry. 52, 1755-1764]. In order to demonstrate and detail the mechanism of one-third of the sites reactivity of MGST2, we have determined the enzyme oligomeric state, by Blue native PAGE and Differential Scanning Calorimetry, as well as the stoichiometty of substrate and substrate analog inhibitor binding to MGST2, using equilibrium dialysis and Isothermal Titration Calorimetry, respectively. Global simulations were used to fit kinetic data to determine the catalytic mechanism of MGST2 with GSH and CDNB (1-chloro-2,4-dinitrobenzene) as substrates. The best fit was observed with 1/3 of the sites catalysis as compared with a simulation where all three sites were active. In contrast to LTC4S, MGST2 displays a 1/3 the sites reactivity, a mechanism shared with the more distant family member MGST1 and recently suggested also for microsomal prostaglandin E synthase-1.
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| 2. |
- Aslam, Muhammad
(författare)
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The fruit fly Drosophila melanogaster GSTE6 and E7; characterization, immobilization and transgenic overexpression
- 2014
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Glutathione transferases (GSTs) are multifunctional enzymes that are universally distributed in most eukaryotes and prokaryotes. They play a pivotal role in the metabolism and detoxication of numerous endogenous and exogenous electrophiles by conjugating them with ubiquitous tripeptide glutathione. In this study we have immobilized two heterologously expressed and purified Epsilon-class enzymes, GSTE6 and GSTE7, from of Drosophila melanogaster on nanoporous alumina membranes. The membranes were derivatized with 3-aminopropyl-triethoxysilane and the amino groups were activated with carbonyldiimidazole to allow coupling of the enzymes. 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 enzymes in solution. The stability of the immobilized enzymes was virtually identical to that for the enzymes in solution and no leakage of enzyme from the matrix could be observed.Additionally, we have investigated the catalytic activities of GSTE7 with organic isothiocyanates (ITCs). These reactive compounds are strong electrophilic molecules produced in plants by the hydrolysis of glucosinolates and exert toxicity in biological tissues. Our in vitro studies, showed high catalytic activity of GSTE7 towards ITCs. We have then explored the in vivo effect of phenethyl isothiocyanate (PEITC) and allyl isothiocyanate (AITC) in transgenic fruit flies overexpressing GSTE7. A concentration of 0.25 mM PEITC in standard fly food was shown to be toxic to flies and significantly shortened the lifespan. We noticed that overexpression of GSTE7 could protect females from the initial acute toxic effects, but had no positive effect on long term exposure. The effect on males seems to be the opposite to that of females, where a higher mortality was seen in fly males overexpressing GST E7 after one week of exposure. On the other hand 1mM concentration of AITC showed no toxic effects, but dramatically reduced the oviposition activity of wild-type flies in comparison to the transgenic flies.
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| 3. |
- Bresell, Anders, et al.
(författare)
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Bioinformatic and enzymatic characterization of the MAPEG superfamily
- 2005
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 272:7, s. 1688-1703
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Tidskriftsartikel (refereegranskat)abstract
- The membrane associated proteins in eicosanoid and glutathione metabolism (MAPEG) superfamily includes structurally related membrane proteins with diverse functions of widespread origin. A total of 136 proteins belonging to the MAPEG superfamily were found in database and genome screenings. The members were found in prokaryotes and eukaryotes, but not in any archaeal organism. Multiple sequence alignments and calculations of evolutionary trees revealed a clear subdivision of the eukaryotic MAPEG members, corresponding to the six families of microsomal glutathione transferases (MGST) 1, 2 and 3, leukotriene C4 synthase (LTC4), 5-lipoxygenase activating protein (FLAP), and prostaglandin E synthase. Prokaryotes contain at least two distinct potential ancestral subfamilies, of which one is unique, whereas the other most closely resembles enzymes that belong to the MGST2/FLAP/LTC4 synthase families. The insect members are most similar to MGST1/prostaglandin E synthase. With the new data available, we observe that fish enzymes are present in all six families, showing an early origin for MAPEG family differentiation. Thus, the evolutionary origins and relationships of the MAPEG superfamily can be defined, including distinct sequence patterns characteristic for each of the subfamilies. We have further investigated and functionally characterized representative gene products from Escherichia coli, Synechocystis sp., Arabidopsis thaliana and Drosophila melanogaster, and the fish liver enzyme, purified from pike (Esox lucius). Protein overexpression and enzyme activity analysis demonstrated that all proteins catalyzed the conjugation of 1-chloro-2,4-dinitrobenzene with reduced glutathione. The E. coli protein displayed glutathione transferase activity of 0.11 µmol·min−1·mg−1 in the membrane fraction from bacteria overexpressing the protein. Partial purification of the Synechocystis sp. protein yielded an enzyme of the expected molecular mass and an N-terminal amino acid sequence that was at least 50% pure, with a specific activity towards 1-chloro-2,4-dinitrobenzene of 11 µmol·min−1·mg−1. Yeast microsomes expressing the Arabidopsis enzyme showed an activity of 0.02 µmol·min−1·mg−1, whereas the Drosophila enzyme expressed in E. coli was highly active at 3.6 µmol·min−1·mg−1. The purified pike enzyme is the most active MGST described so far with a specific activity of 285 µmol·min−1·mg−1. Drosophila and pike enzymes also displayed glutathione peroxidase activity towards cumene hydroperoxide (0.4 and 2.2 µmol·min−1·mg−1, respectively). Glutathione transferase activity can thus be regarded as a common denominator for a majority of MAPEG members throughout the kingdoms of life whereas glutathione peroxidase activity occurs in representatives from the MGST1, 2 and 3 and PGES subfamilies.
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| 4. |
- Busenlehner, Laura S., et al.
(författare)
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Location of substrate binding sites within the integral membrane protein microsomal glutathione transferase-1
- 2007
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 46:10, s. 2812-2822
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Tidskriftsartikel (refereegranskat)abstract
- Microsomal glutathione transferase-1 (MGST1) is a trimeric, membrane-bound enzyme with both glutathione (GSH) transferase and hydroperoxidase activities. As a member of the MAPEG superfamily, MGST1 aids in the detoxication of numerous xenobiotic substrates and in cellular protection from oxidative stress through the GSH-dependent reduction of phospholipid hydroperoxides. However, little is known about the location of the different substrate binding sites, including whether the transferase and peroxidase activities overlap structurally. Although molecular density attributed to GSH has been observed in the 3.2 A resolution electron crystallographic structure of MGST1, the electrophilic and phospholipid hydroperoxide substrate binding sites remain elusive. Amide H-D exchange kinetics and H-D ligand footprinting experiments indicate that GSH and hydrophobic substrates bind within similar, but distinct, regions of MGST1. Site-directed mutagenesis, guided by the H-D exchange results, demonstrates that specific residues within the GSH footprint effect transferase activity toward 1-chloro-2,4-dinitrobenzene. In addition, cytosolic residues surrounding the chemical stress sensor C49 but not modeled in the crystal structure appear to play an important role in the formation of the binding site for hydrophobic substrates. Although the fatty acid/phospholipid binding site structurally overlaps that for GSH, it does not appear to be localized to the same region as other hydrophobic substrates. Finally, H-D exchange mass spectrometry reveals a specific conformational transition that may mediate substrate binding and/or product release. Such structural changes in MGST1 are essential for activation of the enzyme and are important for its biological function.
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| 5. |
- Cebula, Marcus, et al.
(författare)
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Catalytic Conversion of Lipophilic Substrates by Phase constrained Enzymes in the Aqueous or in the Membrane Phase
- 2016
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Both soluble and membrane-bound enzymes can catalyze the conversion of lipophilic substrates. The precise substrate access path, with regard to phase, has however, until now relied on conjecture from enzyme structural data only (certainly giving credible and valuable hypotheses). Alternative methods have been missing. To obtain the first experimental evidence directly determining the access paths (of lipophilic substrates) to phase constrained enzymes we here describe the application of a BODIPY-derived substrate (PS1). Using this tool, which is not accessible to cytosolic enzymes in the presence of detergent and, by contrast, not accessible to membrane embedded enzymes in the absence of detergent, we demonstrate that cytosolic and microsomal glutathione transferases (GSTs), both catalyzing the activation of PS1, do so only within their respective phases. This approach can serve as a guideline to experimentally validate substrate access paths, a fundamental property of phase restricted enzymes. Examples of other enzyme classes with members in both phases are xenobiotic-metabolizing sulphotransferases/UDP-glucuronosyl transferases or epoxide hydrolases. Since specific GSTs have been suggested to contribute to tumor drug resistance, PS1 can also be utilized as a tool to discriminate between phase constrained members of these enzymes by analyzing samples in the absence and presence of Triton X-100.
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| 6. |
- Chaudhry, Qasim Ali, et al.
(författare)
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Influence of Biological Cell Geometry on Reaction and Diffusion Simulation
- 2012
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Rapport (övrigt vetenskapligt/konstnärligt)abstract
- Mathematical modeling of reaction-diffusion system in a biological cellis an important and difficult task, especially when the chemical compoundsare lipophilic. The difficulty level increases, when we take into account theheterogeneity of the cell, and the variation of cellular architecture. Mathematicalmodeling of reaction-diffusion systems in spherical cell geometryhas earlier been performed by us. In the present paper, we have workedwith non-spherical cell geometry, because the cellular geometry can play animportant role for drug diffusion in the cell. Homogenization techniques,which were earlier applied in the case of a spherical cell model, have beenused for the numerical treatment of the model. This technique considerablyreduces the complexity of the model. To further reduce the complexity ofthe model, a simplified model was also developed. The key idea of this simplifiedmodel has been advocated in Virtual Cell, where PDEs are used forthe extracellular domain, cytoplasm and nucleus, whereas the plasma andnuclear membranes have been taken away, and replaced by membrane flux,using Fick’s Law of diffusion. The numerical results of the non-sphericalcell model have been compared with the results of the spherical cell model,where the numerical results of spherical cell model have already been validatedagainst in vitro cell experimental results. From the numerical results,we conclude that the plasma and nuclear membranes can be protective reservoirsof significance. The numerical results of the simplified model werecompared against the numerical results of our detailed model, revealing theimportance of detailed modeling of membranes in our model.
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| 7. |
- Chaudhry, Qasim Ali, et al.
(författare)
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Mathematical Modeling of Reaction and Diffusion Systems in a Cell Including Surface Reactions on the Cytoplasmic Membranes
- 2012
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Rapport (övrigt vetenskapligt/konstnärligt)abstract
- Benzo[a]pyrene (BP) is a toxic polycyclic aromatic hydrocarbon (PAH) whichis found in our environment. These BPs are metabolized to benzo[a]pyrene diol(BPD) by enzymes bound to the cytoplasmic membranes e.g. members of thecytochrome P450 protein family and epoxide hydrolyses. BPDs are further metabolizedto two stereochemical variants of Benzo[a]pyrene diol expoxide (BPDE) bythe cytochrome P450 family of proteins. These are the two steps of metabolismcategorized as Phase I. In Phase II, BPDEs are further metabolized by soluble enzymesin the cytoplasm e.g. members of the glutathione transferase protein familyto GSH conjugates. BPDE can also diffuse into the cellular nucleus and reactwith DNA forming mutagenic DNA adducts. The formation of GSH conjugatesand DNA adducts, was earlier studied by us by developing a mathematical modeldescribing the intracellular reaction and diffusion of lipophilic PAHs taking intoaccount the partitioning phenomenon (Dreij K et al. PLoS One 6(8), 2011). In thispaper part of Phase I metabolism i.e formation of BPDE metabolites, will be addedto the model, thus enhancing the previous model. These cytochrome P450 reactionstake place on the intracellular membranes, and are modeled as a membranesurface reaction within the cytoplasm using the standard process of adsorption anddesorption. The effective equations are derived using iterative homogenization forthe numerical treatment of the cytoplasm including surface effects. The numericalresults of some of the species have been qualitatively verified against in vitroresults found in the literature.
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| 8. |
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| 9. |
- Chaudhry, Qasim Ali, 1982-, et al.
(författare)
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On the numerical approximation of drug diffusion in complex cell geometry
- 2009
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Ingår i: Proceedings of the 6th International Conference on Frontiers of Information Technology, FIT '09. - Abbottabad : ACM. - 9781605586427
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The mathematical modeling of a mammalian cell is a very tedious work due to its very complex geometry. Especially, taking into account the spatial distribution and the inclusion of lipophilic toxic compounds greatly increases its complexity. The nonhomogeneity and the different cellular architecture of the cell certainly affect the diffusion of these compounds. The complexity of the whole system can be reduced by a homogenization technique. To see the effect of these compounds on different cell architectures, we have implemented a mathematical model. The work has been done in 2-dimensional space. The simulation results have been qualitatively verified using compartmental modeling approach. This work can be extended with a more complex reaction-diffusion system and to 3-dimensional space as well. Copyright 2009 ACM.
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| 10. |
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