Thermosensitive phenotype of transgenic mice overproducing human glutathione peroxidases.

Exposure of humans and other mammals to hyperthermic conditions elicits many physiological responses to stress in various tissues leading to profound injuries, which eventually result in death. It has been suggested that hyperthermia may increase oxidative stress in tissues to form reactive oxygen species harmful to cellular functions. By using transgenic mice with human antioxidant genes, we demonstrate that the overproduction of glutathione peroxidase (GP, both extracellular and intracellular) leads to a thermosensitive phenotype, whereas the overproduction of Cu,Zn-superoxide dismutase has no effect on the thermosensitivity of transgenic mice. Induction of HSP70 in brain, lung, and muscle in GP transgenic mice at elevated temperature was significantly inhibited in comparison to normal animals. Measurement of peroxide production in regions normally displaying induction of HSP70 under hyperthermia revealed high levels of peroxides in normal mice and low levels in GP transgenic mice. There was also a significant difference between normal and intracellular GP transgenic mice in level of prostaglandin E2 in hypothalamus and cerebellum. These data suggest direct participation of peroxides in induction of cytoprotective proteins (HSP70) and cellular mechanisms regulating body temperature. GP transgenic mice provide a model for studying thermoregulation and processes involving actions of hydroxy and lipid peroxides in mammals.

Forced evolution of glutathione S-transferase to create a more efficient drug detoxication enzyme.

Glutathione S-transferases (EC 2.5.1.18) in mammalian cells catalyze the conjugation, and thus, the detoxication of a structurally diverse group of electrophilic environmental carcinogens and alkylating drugs, including the antineoplastic nitrogen mustards. We proposed that structural alteration of the nonspecific electrophile-binding site would produce mutant enzymes with increased efficiency for detoxication of a single drug and that these mutants could serve as useful somatic transgenes to protect healthy human cells against single alkylating agents used in cancer chemotherapy protocols. Random mutagenesis of three regions (residues 9-14, 102-112, and 210-220), which together compose the glutathione S-transferase electrophile-binding site, followed by selection of Escherichia coli expressing the enzyme library with the nitrogen mustard mechlorethamine (20-500 microM), yielded mutant enzymes that showed significant improvement in catalytic efficiency for mechlorethamine conjugation (up to 15-fold increase in kcat and up to 6-fold increase in kcat/Km) and that confer up to 31-fold resistance, which is 9-fold greater drug resistance than that conferred by the wild-type enzyme. The results suggest a general strategy for modification of drug- and carcinogen-metabolizing enzymes to achieve desired resistance in both prokaryotic and eukaryotic plant and animal cells.

Cloning of an organic solvent-resistance gene in Escherichia coli: the unexpected role of alkylhydroperoxide reductase.

Although bacterial strain able to grow in the presence of organic solvents have been isolated, little is known about the mechanism of their resistance. In the present study, 1,2,3,4-tetrahydronaphthalene (tetralin), a solvent with potential applications in industrial biocatalysis, was used to select a resistant mutant of Escherichia coli. The resultant mutant strain was tested for resistance to a wide range of solvents of varying hydrophobicities and was found to be resistant not only to tetralin itself but also to cyclohexane, propylbenzene, and 1,2-dihydronaphthalene. A recombinant library from mutant DNA was used to clone the resistance gene. The sequence of the cloned locus was determined and found to match the sequence of the previously described alkylhydroperoxide reductase operon ahpCF. The mutation was localized to a substitution of valine for glycine at position 142 in the coding region of ahpC, which is the gene encoding the catalytic subunit of the enzyme. The ahpC mutant was found to have an activity that was three times that of the wild type in reducing tetralin hydroperoxide to 1,2,3,4-tetrahydro-1-naphthol. We conclude that the toxicity of such solvents as tetralin is caused by the formation of toxic hydroperoxides in the cell. The ahpC mutation increases the activity of the enzyme toward hydrophobic hydroperoxides, thereby conferring resistance. The ahpC mutant was sensitive to the more hydrophilic solvents xylene and toluene, suggesting that there are additional mechanisms of solvent toxicity. Mutants resistant to a mixture of xylene and tetralin were isolated from the ahpC mutant but not from the wild-type strain.

Role of glutathione in the export of compounds from cells by the multidrug-resistance-associated protein.

Multidrug-resistance-associated protein (MRP) is a plasma membrane glycoprotein that can confer multidrug resistance (MDR) by lowering intracellular drug concentration. Here we demonstrate that depletion of intracellular glutathione by DL-buthionine (S,R)-sulfoximine results in a complete reversal of resistance to doxorubicin, daunorubicin, vincristine, and VP-16 in lung carcinoma cells transfected with a MRP cDNA expression vector. Glutathione depletion had less effect on MDR in cells transfected with MDR1 cDNA encoding P-glycoprotein and did not increase the passive uptake of daunorubicin by cells, indicating that the decrease of MRP-mediated MDR was not due to nonspecific membrane damage. Glutathione depletion resulted in a decreased efflux of daunorubicin from MRP-transfected cells, but not from MDR1-transfected cells, suggesting that glutathione is specifically required for the export of drugs from cells by MRP. We also show that MRP increases the export of glutathione from the cell and this increased export is further elevated in the presence of arsenite. Our results support the hypothesis that MRP functions as a glutathione S-conjugate carrier.

A potassium channel beta subunit related to the aldo-keto reductase superfamily is encoded by the Drosophila hyperkinetic locus.

Genetic and physiological studies of the Drosophila Hyperkinetic (Hk) mutant revealed defects in the function or regulation of K+ channels encoded by the Shaker (Sh) locus. The Hk polypeptide, determined from analysis of cDNA clones, is a homologue of mammalian K+ channel beta subunits (Kv beta). Coexpression of Hk with Sh in Xenopus oocytes increases current amplitudes and changes the voltage dependence and kinetics of activation and inactivation, consistent with predicted functions of Hk in vivo. Sequence alignments show that Hk, together with mammalian Kv beta, represents an additional branch of the aldo-keto reductase superfamily. These results are relevant to understanding the function and evolutionary origin of Kv beta.

Coenzyme Q reductase from liver plasma membrane: purification and role in trans-plasma-membrane electron transport.

A specific requirement for coenzyme Q in the maintenance of trans-plasma-membrane redox activity is demonstrated. Extraction of coenzyme Q from membranes resulted in inhibition of NADH-ascorbate free radical reductase (trans electron transport), and addition of coenzyme Q10 restored the activity. NADH-cytochrome c oxidoreductase (cis electron transport) did not respond to the coenzyme Q status. Quinone analogs inhibited trans-plasma-membrane redox activity, and the inhibition was reversed by coenzyme Q. A 34-kDa coenzyme Q reductase (p34) has been purified from pig-liver plasma membranes. The isolated enzyme was sensitive to quinone-site inhibitors. p34 catalyzed the NADH-dependent reduction of coenzyme Q10 after reconstitution in phospholipid liposomes. When plasma membranes were supplemented with extra p34, NADH-ascorbate free radical reductase was activated but NADH-cytochrome c oxidoreductase was not. These results support the involvement of p34 as a source of electrons for the trans-plasma-membrane redox system oxidizing NADH and support coenzyme Q as an intermediate electron carrier between NADH and the external acceptor ascorbate free radical.

Crystallization and preliminary x-ray studies of NADPH-cytochrome P450 reductase.

NADPH-cytochrome P450 reductase (CPR; NADPH:ferrihemoprotein reductase, EC 1.6.2.4) catalyzes the transfer of electrons to all known microsomal cytochromes P450. CPR is unique in that it is one of only two mammalian enzymes known to contain both flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), the other being the various isoforms of nitric oxide synthase. Similarities in amino acid sequence and in functional domain arrangement with other key flavoproteins, including nitric oxide synthase, make CPR an excellent prototype for studies of interactions between two flavin cofactors. We have obtained diffraction-quality crystals of rat liver CPR, expressed in Escherichia coli and solubilized by limited proteolysis with trypsin. The crystals were grown in Hepes buffer (pH 7.0), containing polyethylene glycol 4500 and NaCl. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 103.3 A, b = 116.1 A, and c = 120.4 A. If we assume that there are two molecules of the 72-kDa CPR polypeptide per asymmetric unit, the calculated value of Vm is 2.54 A3/Da.

Glutathione in Parkinson's disease

Thesis (Ph.D.)--University of Washington, 2016-03

The critical role of tryptophan-116 in the catalytic cycle of dimethylsulfoxide reductase from Rhodobacter capsulatus

In dimethylsulfoxide reductase of Rhodobacter capsulatus tryptophan-116 forms a hydrogen bond with a single oxo ligand bound to the molybdenum ion. Mutation of this residue to phenylalanine affected the UV/visible spectrum of the purified Mo-VI form of dimethylsulfoxide reductase resulting in the loss of the characteristic transition at 720 nm. Results of steady-state kinetic analysis and electrochemical studies suggest that tryptophan 116 plays a critical role in stabilizing the hexacoordinate monooxo Mo-VI form of the enzyme and prevents the formation of a dioxo pentacoordinate Mo-VI species, generated as a consequence of the dissociation of one of the dithiolene ligands of the molybdopterin cofactor from the Mo ion. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

NmlR of Neisseria gonorrhoeae: a novel redox responsive transcription factor from the MerR family

A MerR-like regulator (NmlR -Neisseria merR-like Regulator) identified in the Neisseria gonorrhoeae genome lacks the conserved cysteines known to bind metal ions in characterized proteins of this family. Phylogenetic analysis indicates that NmlR defines a subfamily of MerR-like transcription factors with a distinctive pattern of conserved cysteines within their primary structure. NmlR regulates itself and three other genes in N. gonorrhoeae encoding a glutathione-dependent dehydrogenase (AdhC), a CPx-type ATPase (CopA) and a thioredoxin reductase (TrxB). An nmlR mutant lacked the ability to survive oxidative stress induced by diamide and cumene hydroperoxide. It also had > 50-fold lower NADH-S-nitrosoglutathione oxidoreductase activity consistent with a role for AdhC in protection against nitric oxide stress. The upstream sequences of the NmlR regulated genes contained typical MerR-like operator/promoter arrangements consisting of a dyad symmetry located between the -35 and -10 elements of the target genes. The NmlR target operator/promoters were cloned into a beta-galactosidase reporter system and promoter activity was repressed by the introduction of NmlR in trans. Promoter activity was activated by NmlR in the presence of diamide. Under metal depleted conditions NmlR did not repress P-AdhC (or P-CopA) promoter activity, but this was reversed in the presence of Zn(II), indicating repression was Zn(II)-dependent. Analysis of mutated promoters lacking the dyad symmetry revealed constitutive promoter activity which was independent of NmlR. Gel shift assays further confirmed that NmlR bound to the target promoters possessing the dyad symmetry. Site-directed mutagenesis of the four NmlR cysteine residues revealed that they were essential for activation of gene expression by NmlR.

Polymorphisms in glutathione S-transferases and non-melanoma skin cancer risk in Australian renal transplant recipients

Caucasian renal transplant recipients from Queensland, Australia have the highest non-melanoma skin cancer (NMSC) risk worldwide. Although ultraviolet light (UVR) exposure is critical, genetic factors also appear important. We and others have shown that polymorphism in the glutathione S-transferases (GST) is associated with NMSC in UK recipients. However, the effect of high UVR exposure and differences in immunosuppressive regimen on these associations is unknown. In this study, we examined allelism in GSTM1, GSTM3, GSTT1 and GSTP1 in 361 Queensland renal transplant recipients. Data on squamous (SCC) and basal cell carcinoma (BCC), UVR/tobacco exposure and genotype were obtained. Associations with both NMSC risk and numbers were examined using logistic and negative binomial regression, respectively. In the total group, GSTM1 AB [P = 0.049, rate ratio (RR) = 0.23] and GSTM3 AA (P = 0.015, RR = 0.50) were associated with fewer SCC. Recipients were then stratified by prednisolone dose (less than or equal to7 versus >7 mg/day). In the low-dose group, GSTT1 null (P = 0.006, RR = 0.20) and GSTP1 Val/Val (P = 0.021, RR = 0.20) were associated with SCC numbers. In contrast, in the high-dose group, GSTM1 AB (P = 0.009, RR = 0.05), GSTM3 AB (P = 0.042, RR = 2.29) and BB (P = 0.014, RR = 5.31) and GSTP1 Val/Val (P = 0.036, RR = 2.98) were associated with SCC numbers. GSTM1 AB (P = 0.016) and GSTP1 Val/Val (P = 0.046) were also associated with fewer BCC in this group. GSTP1 associations were strongest in recipients with lower UVR/tobacco exposure. The data confirm our UK findings, suggesting that protection against UVR-induced oxidative stress is important in NMSC development in recipients, but that this effect depends on the immunosuppressant regimen.

NMR studies of exchange between intra- and extracellular glutathione in human erythrocytes

Glutathione is the main source of intracellular antioxidant protection in the human erythrocyte and its redox status has frequently been used as a measure of oxidative stress. Extracellular glutathione has been shown to enhance intracellular reduced glutathione levels in some cell types. However, there are conflicting reports in the literature and it remains unclear as to whether erythrocytes can utilise extracellular glutathione to enhance the intracellular free glutathione pool. We have resolved this issue using a C-13-NMR approach. The novel use of L-gamma-glutamyl-L-cysteinyl-[2-C-13] glycine allowed the intra- and extracellular glutathione pools to be distinguished unequivocally, enabling the direct and non-invasive observation over time of the glutathione redox status in both compartments. The intracellular glutathione redox status was measured using H-1 spin-echo NMR, while C-13[H-1-decoupled] NMR experiments were used to measure the extracellular status. Extracellular glutathione was not oxidised in the incubations, and did not affect the intracellular glutathione redox status. Extracellular glutathione also did not affect erythrocyte glucose metabolism, as measured from the lactate-to-pyruvate ratio. The results reported here refute the previously attractive hypothesis that, in glucose-starved erythrocytes, extracellular GSH can increase intracellular GSH concentrations by releasing bound glutathione from mixed disulfides with membrane proteins.

Cytochrome P450-mediated metabolism of haloperidol and reduced haloperidol to pyridinium metabolites

Haloperidol ( HP) has been reported to undergo cytochrome P450 (P450)-mediated metabolism to potentially neurotoxic pyridinium metabolites; however, the chemical pathways and specific enzymes involved in these reactions remain to be identified. The aims of the current study were to (i) fully identify the cytochrome P450 enzymes capable of metabolizing HP to the pyridinium metabolite, 4-(4-chlorophenyl)- 1-(4-fluorophenyl)-4-oxobutylpyridinium (HPP+), and reduced HP (RHP) to 4-(4-chlorophenyl)- 1-(4-fluorophenyl)-4-hydroxybutylpyridinium (RHPP+); and (ii) determine whether 4-(4-chlorophenyl)- 1-(4-fluorophenyl)-4-oxobutyl-1,2,3,6-tetrahydropyridine (HPTP) and 4-(4-chlorophenyl)1-( 4-fluorophenyl)-4-hydroxybutyl-1,2,3,6-tetrahydropyridine (RHPTP) were metabolic intermediates in these pathways. In vitro studies were conducted using human liver microsomal preparations and recombinant human cytochrome P450 enzymes (P450s 1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19 2D6, 2E1, 3A4, 3A5, and 3A7) expressed in bicistronic format with human NADPH cytochrome P450 reductase in Escherichia coli membranes. Pyridinium formation from HP and RHP was highly correlated across liver preparations, suggesting the same enzyme or enzymes were responsible for both reactions. Cytochrome P450s 3A4, 3A5, and 3A7 were the only recombinant enzymes which demonstrated significant catalytic activity under optimized conditions, although trace levels of activity could be catalyzed by NADPHP450 reductase alone. NADPH-P450 reductase-mediated activity was inhibited by reduced glutathione but not catalase or superoxide dismutase, suggesting O-2-dependent oxidation. No evidence was obtained to support the contention that HPTP and RHPTP are intermediates in these pathways. K-m values for HPP+ (34 +/- 5 mu M) and RHPP+ (64 +/- 4 mu M) formation by recombinant P450 3A4 agreed well with those obtained with human liver microsomes, consistent with P450 3A4 being the major catalyst of pyridinium metabolite formation in human liver.