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Philip G. Board - One of the best experts on this subject based on the ideXlab platform.
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a role for Glutathione Transferase omega 1 gsto1 1 in the glutathionylation cycle
Journal of Biological Chemistry, 2013Co-Authors: Deepthi Menon, Philip G. BoardAbstract:Abstract The glutathionylation of intracellular protein thiols can protect against irreversible oxidation and can act as a redox switch regulating metabolic pathways. In this study we discovered that the Omega class Glutathione Transferase GSTO1-1 plays a significant role in the glutathionylation cycle. The catalytic activity of GSTO1-1 was determined in vitro by assaying the deglutathionylation of a synthetic peptide by tryptophan fluorescence quenching and in T47-D epithelial breast cancer cells by both immunoblotting and the direct determination of total glutathionylation. Mutating the active site cysteine residue (Cys-32) ablated the deglutathionylating activity of GSTO1-1. Furthermore, we demonstrate that the expression of GSTO1-1 in T47-D cells that are devoid of endogenous GSTO1-1 resulted in a 50% reduction in total glutathionylation levels. Mass spectrometry and immunoprecipitation identified β-actin as a protein that is specifically deglutathionylated by GSTO1-1 in T47-D cells. In contrast to the deglutathionylation activity, we also found that GSTO1-1 is associated with the rapid glutathionylation of cellular proteins when the cells are exposed to S-nitrosoGlutathione. The common A140D genetic polymorphism in GSTO1 was found to have significant effects on the kinetics of both the deglutathionylation and glutathionylation reactions. Genetic variation in GSTO1-1 has been associated with a range of diseases, and the discovery that a frequent GSTO1-1 polymorphism affects glutathionylation cycle reactions reveals a common mechanism where it can act on multiple proteins and pathways.
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the inhibitory Glutathione Transferase m2 2 binding site is located in divergent region 3 of the cardiac ryanodine receptor
Biochemical Pharmacology, 2012Co-Authors: Ruwani Punyakanthi Hewawasam, Angela F Dulhunty, Yamuna Karunasekara, Marco G Casarotto, Philip G. BoardAbstract:Abstract The muscle-specific Glutathione Transferase GSTM2-2 modulates the activity of ryanodine receptor (RyR) calcium release channels: it inhibits the activity of cardiac RyR (RyR2) channels with high affinity and activates skeletal RyR (RyR1) channels with low affinity. The C terminal domain of GSTM2-2 (GSTM2C) alone physically binds to RyR2 and inhibits its activity, but it does not bind to RyR1. We have now used yeast two-hybrid analysis, chemical cross-linking, intrinsic tryptophan fluorescence and Ca2+ release studies to determine that the binding site for GSTM2C is in divergent region 3 (D3) of RyR2. The D3 region encompasses residues 1855–1890 in RyR2. Specific mutagenesis shows the binding primarily involves electrostatic interactions with residues K1875, K1886, R1887 and K1889, all residues that are present in RyR2, but not in RyR1. The significant sequence differences between the D3 regions of RyR2 and RyR1 explain why GSTM2-2 specifically inhibits RyR2. This specific inhibition of RyR2 could modulate Ca cycling and be useful for the treatment of heart failure. RyR2 inhibition during diastole may improve filling of the SR with Ca2+ and improve contractility.
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the structure of the c terminal helical bundle in Glutathione Transferase m2 2 determines its ability to inhibit the cardiac ryanodine receptor
Biochemical Pharmacology, 2010Co-Authors: Ruwani Punyakanthi Hewawasam, Angela F Dulhunty, Marco G Casarotto, Philip G. BoardAbstract:Abstract Ca 2+ release from the sarcoplasmic reticulum through cardiac ryanodine receptors (RyR2) is essential for heart function and is inhibited by the carboxy terminal domain of Glutathione Transferase M2-2 (GSTM2-C) and derivative fragments containing helix 6. Since a peptide encoding helix 6 alone does not fold into a helix and does not inhibit RyR2 Ca 2+ release, the importance of the structure of helix 6 and its role in stabilizing GSTM2-C was tested by inserting potentially destabilizing mutations into this helical segment. GSTM2-C preparations with D156A or L163A mutations were so insoluble that the protein could not be purified. Proteins with F157A and Y260A substitutions were soluble, but had lost their capacity to inhibit both RyR2 Ca 2+ release from vesicles and RyR2 channels in bilayers. Circular dichroism studies indicated that these mutated proteins retained their helical secondary structure, although changes in their endogenous tryptophan fluorescence indicated that the F157A and Y160A mutations caused changes in their folding. The single channel studies were conducted with 2 mM ATP and 10 μM Ca 2+ in the cytoplasmic solution, mimicking concentrations in the cytosol of cardiac myocytes. Wild type GSTM2-C inhibited RyR2 only at a potential of +40 mV, which may develop during Ca 2+ efflux, but not at −40 mV. Together, the results indicate that the structure of helix 6 in the C-terminal fold is critical to the inhibitory action of GSTM2-2 and suggest that therapeutics mimicking this structure may reduce excess Ca 2+ release during diastole, which can lead to fatal arrhythmia.
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Polymorphisms in the human Glutathione Transferase zeta promoter.
Pharmacogenetics and Genomics, 2006Co-Authors: Yu-yan Fang, M. W. Anders, Uliana Kashkarov, Philip G. BoardAbstract:ObjectivesThe zeta-class Glutathione Transferase GSTZ1-1 catalyses the Glutathione-dependent isomerization of maleylacetoacetate to fumarylacetoacetate in the tyrosine catabolic pathway and the biotransformation of α-halo acids, including dichloroacetic acid (DCA). Genetic polymorphisms in the codin
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characterization of the monomethylarsonate reductase and dehydroascorbate reductase activities of omega class Glutathione Transferase variants implications for arsenic metabolism and the age at onset of alzheimer s and parkinson s diseases
Pharmacogenetics and Genomics, 2005Co-Authors: Erica Schmuck, Anneke C. Blackburn, Philip G. Board, Astrid K Whitbread, Natasha Tetlow, Juleen A Cavanaugh, Amir MasoumiAbstract:There are two functional Omega class Glutathione Transferase (GST) genes in humans. GSTO1 is polymorphic with several coding region alleles, including an A140D substitution, a potential deletion of E155 and an E208K substitution. GSTO2 is also polymorphic with an N142D substitution in the coding reg
Bengt Mannervik - One of the best experts on this subject based on the ideXlab platform.
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Optimizing the heterologous expression of Glutathione Transferase.
Methods in Enzymology, 2020Co-Authors: Bengt MannervikAbstract:The heterologous expression of a protein may be enhanced by silent mutations in the coding region of its corresponding DNA. This simple approach has been successfully used for optimized production of a number of Glutathione-linked enzymes. For example, the yield of human Glutathione Transferase M2-2 was elevated by 140-fold in a clone isolated by immunoscreening of a library of plasmids with randomized synonymous codons in the 5'-segment of the region encoding the enzyme.
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The polymorphic human Glutathione Transferase T1-1, the most efficient Glutathione Transferase in the denitrosation and inactivation of the anticancer drug 1,3-bis(2-chloroethyl)-1-nitrosourea.
Biochemical Pharmacology, 2002Co-Authors: Samantha Lien, Anna Karin Larsson, Bengt MannervikAbstract:The polymorphic human Glutathione Transferase T1-1, the most efficient Glutathione Transferase in the denitrosation and inactivation of the anticancer drug 1,3-bis(2-chloroethyl)-1-nitrosourea
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differences in the catalytic efficiencies of allelic variants of Glutathione Transferase p1 1 towards carcinogenic diol epoxides of polycyclic aromatic hydrocarbons
Carcinogenesis, 1998Co-Authors: Kathrin Sundberg, Bengt Mannervik, Gun Stenberg, Anne Sofie Johansson, Mikael Widersten, Albrecht Seidel, Bengt JernstromAbstract:Previous studies have identified allelic variants of the human Glutathione Transferase (GST) Pi gene and showed that the two different encoded proteins with isoleucine (GSTP1-1/I-105) or valine (GSTP1-1/V-105) at position 105, respectively, differ significantly in their catalytic activities with model substrates. Moreover, recent epidemiological studies have demonstrated that individuals differing in the expression of these allelic variants also differ in susceptibility to tumour formation in certain organs, including such in which polycyclic aromatic hydrocarbons (PAH) may be etiological factors. In the present study the catalytic efficiencies (kcat/Km) of these GSTP1-1 variants were determined with a number of stereoisomeric bay-region diol epoxides, known as the ultimate mutagenic and carcinogenic metabolites of PAH, including those from chrysene, benzo[a]pyrene and dibenz[a,h]anthracene. In addition, GSTP1-1 mutants in which amino residue 105 is alanine (GSTP1-1/A-105) or tryptophan (GSTP1-1/W-105) have been constructed and characterized. GSTP1-1/V-105 was found to be more active than GSTP1-1/I-105 in conjugation reactions with the bulky diol epoxides of PAH, being up to 3-fold as active towards the anti- and syn-diol epoxide enantiomers with R-absolute configuration at the benzylic oxiranyl carbon. Comparing the four enzyme variants, GSTP1-1/A-105 generally demonstrated the highest kcat/Km value and GSTP1-1/W-105 the lowest with the anti-diol epoxides. A close correlation was observed between the volume occupied by the amino acid residue at position 105 and the value of kcat/Km. With the syn-diol epoxides, such a correlation was observed with alanine, valine and isoleucine, whereas tryptophan was associated with increased kcat/Km values. The mutational replacement of isoleucine with alanine or tryptophan at position 105 did not alter the enantio selectivity of the GSTP1-1 variants compared with the naturally occurring allelic variants GSTP1-1/I-105 and GSTP1-1/V-105. Since the amino acid at position 105 forms part of the substrate binding site (H-site) the effect of increasing bulkiness is expected to cause restricted access of the diol epoxide and proper alignment of the two reactants for efficient glutathionyl
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human Glutathione Transferase a4 4 an alpha class enzyme with high catalytic efficiency in the conjugation of 4 hydroxynonenal and other genotoxic products of lipid peroxidation
Biochemical Journal, 1998Co-Authors: Ina Hubatsch, Marianne Ridderstrom, Bengt MannervikAbstract:A sequence encoding a novel Glutathione Transferase, GST A4-4, has been identified in a human fetal brain cDNA library. The protein has been produced in Escherichia coli after optimization of the codon usage for high-level heterologous expression. The dimeric protein has a subunit molecular mass of 25704 Da based on the deduced amino acid composition. Human GST A4-4 is a member of the Alpha class but shows only 53% amino acid sequence identity with the major liver enzyme GST A1-1. High catalytic efficiency with 4-hydroxyalkenals and other cytotoxic and mutagenic products of radical reactions and lipid peroxidation is a significant feature of GST A4-4. The k cat / K m values for 4-hydroxynonenal and 4-hydroxydecenal are > 3×10 6 M -1 ·s -1 , several orders of magnitude higher than the values for conventional GST substrates. 4-Hydroxynonenal and other reactive electrophiles produced by oxidative metabolism have been linked to aging, atherosclerosis, cataract formation, Parkinson9s disease and Alzheimer9s disease, as well as other degenerative human conditions, suggesting that human GST A4-4 fulfills an important protective role and that variations in its expression may have significant pathophysiological consequences.
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Characterization of a marsupial Glutathione Transferase, a class Alpha enzyme from Brown Antechinus (Antechinus stuartii).
FEBS letters, 1997Co-Authors: R. M. Bolton, Jorma T. Ahokas, L Curstedt, E Cederlund, L Hjelmqvist, Bengt Mannervik, Hans JörnvallAbstract:The major form of Glutathione Transferase from the marsupial Antechinus stuartii has been purified and characterized as an Alpha class enzyme (Ast GST A1-1) with distant sequence relationships to other class Alpha sublines, compatible with the early origin of marsupials. Amino acid replacements toward the closest enzyme characterized (chicken, form A3) involve no less than 79 positions (36%). At the active site, as deduced from comparisons with the known tertiary structure of the corresponding human enzyme, over half of the residues (8 of 15) ascribed to substrate binding interactions are exchanged although the general character of that site is conserved, while only 1 of 11 positions ascribed to interactions with GSH is exchanged. Class variability and species variability appear to coincide, with divergent segments centering around positions 33-49, 103-130 and 205-222. The pattern is reminiscent of that in similarly multiple MDR alcohol dehydrogenases. Both these enzyme families involved in cellular defense reactions have diverged considerably.
Angela F Dulhunty - One of the best experts on this subject based on the ideXlab platform.
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the inhibitory Glutathione Transferase m2 2 binding site is located in divergent region 3 of the cardiac ryanodine receptor
Biochemical Pharmacology, 2012Co-Authors: Ruwani Punyakanthi Hewawasam, Angela F Dulhunty, Yamuna Karunasekara, Marco G Casarotto, Philip G. BoardAbstract:Abstract The muscle-specific Glutathione Transferase GSTM2-2 modulates the activity of ryanodine receptor (RyR) calcium release channels: it inhibits the activity of cardiac RyR (RyR2) channels with high affinity and activates skeletal RyR (RyR1) channels with low affinity. The C terminal domain of GSTM2-2 (GSTM2C) alone physically binds to RyR2 and inhibits its activity, but it does not bind to RyR1. We have now used yeast two-hybrid analysis, chemical cross-linking, intrinsic tryptophan fluorescence and Ca2+ release studies to determine that the binding site for GSTM2C is in divergent region 3 (D3) of RyR2. The D3 region encompasses residues 1855–1890 in RyR2. Specific mutagenesis shows the binding primarily involves electrostatic interactions with residues K1875, K1886, R1887 and K1889, all residues that are present in RyR2, but not in RyR1. The significant sequence differences between the D3 regions of RyR2 and RyR1 explain why GSTM2-2 specifically inhibits RyR2. This specific inhibition of RyR2 could modulate Ca cycling and be useful for the treatment of heart failure. RyR2 inhibition during diastole may improve filling of the SR with Ca2+ and improve contractility.
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the structure of the c terminal helical bundle in Glutathione Transferase m2 2 determines its ability to inhibit the cardiac ryanodine receptor
Biochemical Pharmacology, 2010Co-Authors: Ruwani Punyakanthi Hewawasam, Angela F Dulhunty, Marco G Casarotto, Philip G. BoardAbstract:Abstract Ca 2+ release from the sarcoplasmic reticulum through cardiac ryanodine receptors (RyR2) is essential for heart function and is inhibited by the carboxy terminal domain of Glutathione Transferase M2-2 (GSTM2-C) and derivative fragments containing helix 6. Since a peptide encoding helix 6 alone does not fold into a helix and does not inhibit RyR2 Ca 2+ release, the importance of the structure of helix 6 and its role in stabilizing GSTM2-C was tested by inserting potentially destabilizing mutations into this helical segment. GSTM2-C preparations with D156A or L163A mutations were so insoluble that the protein could not be purified. Proteins with F157A and Y260A substitutions were soluble, but had lost their capacity to inhibit both RyR2 Ca 2+ release from vesicles and RyR2 channels in bilayers. Circular dichroism studies indicated that these mutated proteins retained their helical secondary structure, although changes in their endogenous tryptophan fluorescence indicated that the F157A and Y160A mutations caused changes in their folding. The single channel studies were conducted with 2 mM ATP and 10 μM Ca 2+ in the cytoplasmic solution, mimicking concentrations in the cytosol of cardiac myocytes. Wild type GSTM2-C inhibited RyR2 only at a potential of +40 mV, which may develop during Ca 2+ efflux, but not at −40 mV. Together, the results indicate that the structure of helix 6 in the C-terminal fold is critical to the inhibitory action of GSTM2-2 and suggest that therapeutics mimicking this structure may reduce excess Ca 2+ release during diastole, which can lead to fatal arrhythmia.
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the Glutathione Transferase structural family includes a nuclear chloride channel and a ryanodine receptor calcium release channel modulator
Journal of Biological Chemistry, 2001Co-Authors: Angela F Dulhunty, Gareth Chelvanayagam, Peter W Gage, Suzanne M Curtis, Philip G. BoardAbstract:Abstract The ubiquitous Glutathione Transferases (GSTs) catalyze Glutathione conjugation to many compounds and have other diverse functions that continue to be discovered. We noticed sequence similarities between Omega class GSTs and a nuclear chloride channel, NCC27 (CLIC1), and show here that NCC27 belongs to the GST structural family. The structural homology prompted us to investigate whether the human Omega class Glutathione Transferase GSTO1–1 forms or modulates ion channels. We find that GSTO1–1 modulates ryanodine receptors (RyR), which are calcium channels in the endoplasmic reticulum of various cells. Cardiac RyR2 activity was inhibited by GSTO1–1, whereas skeletal muscle RyR1 activity was potentiated. An enzymatically active conformation of GSTO1–1 was required for inhibition of RyR2, and mutation of the active site cysteine (Cys-32 → Ala) abolished the inhibitory activity. We propose a novel role for GSTO1–1 in protecting cells containing RyR2 from apoptosis induced by Ca2+ mobilization from intracellular stores.
Ruwani Punyakanthi Hewawasam - One of the best experts on this subject based on the ideXlab platform.
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the inhibitory Glutathione Transferase m2 2 binding site is located in divergent region 3 of the cardiac ryanodine receptor
Biochemical Pharmacology, 2012Co-Authors: Ruwani Punyakanthi Hewawasam, Angela F Dulhunty, Yamuna Karunasekara, Marco G Casarotto, Philip G. BoardAbstract:Abstract The muscle-specific Glutathione Transferase GSTM2-2 modulates the activity of ryanodine receptor (RyR) calcium release channels: it inhibits the activity of cardiac RyR (RyR2) channels with high affinity and activates skeletal RyR (RyR1) channels with low affinity. The C terminal domain of GSTM2-2 (GSTM2C) alone physically binds to RyR2 and inhibits its activity, but it does not bind to RyR1. We have now used yeast two-hybrid analysis, chemical cross-linking, intrinsic tryptophan fluorescence and Ca2+ release studies to determine that the binding site for GSTM2C is in divergent region 3 (D3) of RyR2. The D3 region encompasses residues 1855–1890 in RyR2. Specific mutagenesis shows the binding primarily involves electrostatic interactions with residues K1875, K1886, R1887 and K1889, all residues that are present in RyR2, but not in RyR1. The significant sequence differences between the D3 regions of RyR2 and RyR1 explain why GSTM2-2 specifically inhibits RyR2. This specific inhibition of RyR2 could modulate Ca cycling and be useful for the treatment of heart failure. RyR2 inhibition during diastole may improve filling of the SR with Ca2+ and improve contractility.
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the structure of the c terminal helical bundle in Glutathione Transferase m2 2 determines its ability to inhibit the cardiac ryanodine receptor
Biochemical Pharmacology, 2010Co-Authors: Ruwani Punyakanthi Hewawasam, Angela F Dulhunty, Marco G Casarotto, Philip G. BoardAbstract:Abstract Ca 2+ release from the sarcoplasmic reticulum through cardiac ryanodine receptors (RyR2) is essential for heart function and is inhibited by the carboxy terminal domain of Glutathione Transferase M2-2 (GSTM2-C) and derivative fragments containing helix 6. Since a peptide encoding helix 6 alone does not fold into a helix and does not inhibit RyR2 Ca 2+ release, the importance of the structure of helix 6 and its role in stabilizing GSTM2-C was tested by inserting potentially destabilizing mutations into this helical segment. GSTM2-C preparations with D156A or L163A mutations were so insoluble that the protein could not be purified. Proteins with F157A and Y260A substitutions were soluble, but had lost their capacity to inhibit both RyR2 Ca 2+ release from vesicles and RyR2 channels in bilayers. Circular dichroism studies indicated that these mutated proteins retained their helical secondary structure, although changes in their endogenous tryptophan fluorescence indicated that the F157A and Y160A mutations caused changes in their folding. The single channel studies were conducted with 2 mM ATP and 10 μM Ca 2+ in the cytoplasmic solution, mimicking concentrations in the cytosol of cardiac myocytes. Wild type GSTM2-C inhibited RyR2 only at a potential of +40 mV, which may develop during Ca 2+ efflux, but not at −40 mV. Together, the results indicate that the structure of helix 6 in the C-terminal fold is critical to the inhibitory action of GSTM2-2 and suggest that therapeutics mimicking this structure may reduce excess Ca 2+ release during diastole, which can lead to fatal arrhythmia.
Laure Menin - One of the best experts on this subject based on the ideXlab platform.
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a structure based mechanism of cisplatin resistance mediated by Glutathione Transferase p1 1
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Anastasia De Luca, Lorien J Parker, Carlo Rodolfo, Valentina Gabbarini, Nancy C Hancock, Francesca Palone, A P Mazzetti, Laure MeninAbstract:Cisplatin [cis-diamminedichloroplatinum(II) (cis-DDP)] is one of the most successful anticancer agents effective against a wide range of solid tumors. However, its use is restricted by side effects and/or by intrinsic or acquired drug resistance. Here, we probed the role of Glutathione Transferase (GST) P1-1, an antiapoptotic protein often overexpressed in drug-resistant tumors, as a cis-DDP–binding protein. Our results show that cis-DDP is not a substrate for the Glutathione (GSH) Transferase activity of GST P1-1. Instead, GST P1-1 sequesters and inactivates cisplatin with the aid of 2 solvent-accessible cysteines, resulting in protein subunits cross-linking, while maintaining its GSH-conjugation activity. Furthermore, it is well known that GST P1-1 binding to the c-Jun N-terminal kinase (JNK) inhibits JNK phosphorylation, which is required for downstream apoptosis signaling. Thus, in turn, GST P1-1 overexpression and Pt-induced subunit cross-linking could modulate JNK apoptotic signaling, further confirming the role of GST P1-1 as an antiapoptotic protein.