Shifting substrate specificity of human glutathione transferase (from class Pi to class alpha) by a single point mutation

Substrate selectivity, among glutathione transferase (GST) isoenzymes, appears to be determined by a few residues. As part of study to determine which residues are class-specific determinants, Tyr 108 (an important residue of the class Pi) has been changed to a valine, the structural equivalent of a class Alpha enzyme. Using a panel of selected substrates, "diagnostic" for either class Pi or Alpha, it is shown here that this single mutation significantly alters the catalytic properties of the class Pi enzyme and shifts the substrate specificity of the enzyme toward that of the class Alpha enzyme.     

Solution structure of glutathione bound to human glutathione transferase P1-1: comparison of NMR measurements with the crystal structure

The conformation of the bound glutathione (GSH) in the active site of the human glutathione transferase P1-1 (EC 2.5.1.18) has been studied by transferred NOE measurements and compared with those obtained by X-ray diffraction data. Two-dimensional TRNOESY and TRROESY experiments have been performed under fast-exchange conditions. The family of GSH conformers, compatible with TRNOE distance constraints, shows a backbone structure very similar to the crystal model. Interesting differences have been found in the side chain regions. After restrained energy minimization of a representative NMR conformer in the active site, the sulfur atom is not found in hydrogen-bonding distance of the hydroxyl group of Tyr 7. This situation is similar to the one observed in an "atypical" crystal complex grown at low pH and low temperature. The NMR conformers display also a poorly defined structure of the glutamyl moiety, and the presence of an unexpected intermolecular NOE could indicate a different interaction of this substrate portion with the G-site. The NMR data seem to provide a snapshot of GSH in a precomplex where the GSH glutamyl end is bound in a different fashion. The existence of this precomplex is supported by pre-steady-state kinetic experiments [Caccuri, A. M., Lo Bello, M., Nuccetelli, M., Nicotra, M., Rossi, P., Antonini, G., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3028-3034] and preliminary time-resolved fluorescence data.     

A homology model for the human theta-class glutathione transferase T1-1

A manual threading approach is used to model the human glutathione transferase T1-1 based on the coordinates of the related Theta class enzyme T2-2. The low level of sequence identity (about 20%), found in the C-terminal extension in conjunction with a relative deletion of about five residues makes this a challenging modeling problem. The C-terminal extension contributes to the active site of the molecule and is thus of particular interest for understanding the molecular mechanism of the enzyme. Manual docking of known substrates and non-substrates has implicated potential candidates for the T1-1 catalytic residues involved in the dehalogenation and epoxide-ring opening activities. These include the conserved Theta class residues Arg 107, Trp 115, and the conserved GSTT1 subclass residue His 176. Also, the residue at position 234 is implicated in the modulation of T1-1 activity with different substrates between species.     

Characterization of the human class Mu glutathione S-transferase gene cluster and the GSTM1 deletion

A partial physical map has been constructed of the human class Mu glutathione S-transferase genes on chromosome 1p13.3. The glutathione S-transferase genes in this cluster are spaced about 20 kilobase pairs (kb) apart, and arranged as 5'-GSTM4-GSTM2-GSTM1-GSTM5-3'. This map has been used to localize the end points of the polymorphic GSTM1 deletion. The left repeated region is 5 kb downstream from the 3'-end of the GSTM2 gene and 5 kb upstream from the beginning of the GSTM1 gene; the right repeated region is 5 kb downstream from the 3'-end of the GSTM1 and 10 kb upstream from the 5'-end of the GSTM5 gene. The GSTM1-0 deletion produces a novel 7.4-kb HindIII fragment with the loss of 10.3- and 11.4-kb HindIII fragments. The same novel fragment was seen in 13 unrelated individuals (20 null alleles), suggesting that most GSTM1-0 deletions involve recombinations between the same two regions. We have cloned and sequenced the deletion junction that is produced at the GSTM1-null locus; the 5'- and 3'-flanking regions are more than 99% identical to each other and to the deletion junction sequence over 2.3 kb. Because of the high sequence identity between the left repeat, right repeat, and deletion junction regions, the crossing over cannot be localized within the 2.3-kb region. The 2.3-kb repeated region contains a reverse class IV Alu repetitive element near one end of the repeat.     

Structure and organization of the human alpha class glutathione S-transferase genes and related pseudogenes

We have isolated and characterized genomic DNA encoding several human Alpha class glutathione S-transferase genes and pseudogenes. All the genes are composed of seven exons with boundaries identical to those of the Alpha class genes in rats. The GSTA1 gene is approximately 12 kb in length and is closely flanked by other Alpha class gene sequences. The complete sequence of the 1.7-kb intergenic region between exon 7 of an upstream pseudogene and exon 1 of the GSTA1 gene has been determined. An additional gene that encodes an uncharacterized Alpha class glutathione S-transferase has been identified. The protein derived from this gene would have 19 amino acid substitutions compared with the GSTA1 isoenzyme. Several pseudogenes with single-base and/or complete exon deletions have been identified, but no reverse-transcribed pseudogenes have been detected. The occurrence of multiple genes and pseudogenes on a single fragment of cloned genomic DNA and the prior identification of a single chromosomal region (6p12) of hybridization (Board and Webb, 1987, Proc. Natl. Acad. Sci. USA 84:2377-2381) suggest that all the Alpha class genes are members of a closely linked gene family that has evolved by duplication and gene conversion events.     

Stereoselective conjugation of prostaglandin A2 and prostaglandin J2 with glutathione, catalyzed by the human glutathione S-transferases A1-1, A2-2, M1a-1a, and P1-1

Prostaglandins containing an alpha,beta-unsaturated keto group, such as prostaglandin A2 (PGA2) and prostaglandin J2 (PGJ2), inhibit cell proliferation. These cyclopentenone prostaglandins may be conjugated with GSH chemically or enzymatically via glutathione S-transferases, and this has been suggested to result in inhibition of the antiproliferative mode of action. In the present study, the role of the major human GSTs in the conjugation of PGA2 and PGJ2 with GSH was investigated with purified enzymes, i.e., the Alpha-class enzymes GST A1-1 and GST A2-2, the Mu-class enzyme GST M1a-1a, and the Pi-class enzyme GST P1-1. The GSH conjugates were separated from the parent compound by HPLC and identified by fast atom bombardment mass spectrometry and 1H-NMR. Two GSH conjugates were found for both PGA2 and PGJ2, the R- and S-GSH conjugates of both prostaglandins. Incubation experiments with PGA2 and PGJ2 (70-600 microM) clearly showed the role of individual GSTs in the conjugation of PGA2 and PGJ2. Compared to the chemical reaction, enzyme activities towards PGA2 were up to 5.4 times as high (GSTA1-1) at the lowest concentration (70 microM), while at the highest concentration (600 microM) enzyme activities were up to 3.0 times as high (GST P1-1). For PGJ2, enzyme activities were up to 4.3 (GSTM1a-1a, 70 microM) and up to 3.1 (GSTM1a-1a, 600 microM) times as high. As expected, similar amounts of the R- and S-conjugates of both prostaglandins were found in the chemical reaction. Striking stereoselectivities in conjugating activities were observed for GST A1-1 and GST P1-1. GST A1-1 favors the formation of the R-GSH conjugates of both prostaglandins. GST P1-1 showed a clear selectivity with regard to the formation of the S-GSH conjugate of PGA2. However, this selectivity was not found for the formation of the S-GSH conjugate of PGJ2. GSTM1a-1a showed no stereoselectivity with regard to the GSH conjugation of both PGA2 and PGJ2. GSTA2-2 only showed some minor formation of the R-GSH conjugate of PGJ2. The possible implications of the observed stereoselectivity on the effects of PGA2 and PGJ2 are discussed.     

Flexibility of helix 2 in the human glutathione transferase P1-1. time-resolved fluorescence spectroscopy

Time-resolved fluorescence spectroscopy and site-directed mutagenesis have been used to probe the flexibility of alpha-helix 2 (residues 35-46) in the apo structure of the human glutathione transferase P1-1 (EC 2.5.1.18) as well as in the binary complex with the natural substrate glutathione. Trp-38, which resides on helix 2, has been exploited as an intrinsic fluorescent probe of the dynamics of this region. A Trp-28 mutant enzyme was studied in which the second tryptophan of glutathione transferase P1-1 is replaced by histidine. Time-resolved fluorescence data indicate that, in the absence of glutathione, the apoenzyme exists in at least two different families of conformational states. The first one (38% of the total population) corresponds to a number of slightly different conformations of helix 2, in which Trp-38 resides in a polar environment showing an average emission wavelength of 350 nm. The second one (62% of the total population) displays an emission centered at 320 nm, thus suggesting a quite apolar environment near Trp-38. The interconversion between these two conformations is much slower than 1 ns. In the presence of saturating glutathione concentrations, the equilibrium is shifted toward the apolar component, which is now 83% of the total population. The polar conformers, on the other hand, do not change their average decay lifetime, but the distribution becomes wider, indicating a slightly increased rigidity. These data suggest a central role of conformational transitions in the binding mechanism, and are consistent with NMR data (Nicotra, M., Paci, M., Sette, M., Oakley, A. J., Parker, M. W., Lo Bello, M., Caccuri, A. M., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3020-3027) and pre-steady state kinetic experiments (Caccuri, A. M., Lo Bello, M., Nuccetelli, M., Nicotra, M., Rossi, P., Antonini, G., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3028-3034) indicating the existence of a pre-complex in which GSH is not firmly bound to the active site.     

Optimized heterologous expression of the polymorphic human glutathione transferase M1-1 based on silent mutations in the corresponding cDNA

An expression clone for large-scale production of the polymorphic human glutathione transferase (GST) M1-1 has been developed. Heterologous expression in Escherichia coli afforded a yield of 100 mg of GST M1-1 per 3 liters of culture medium, corresponding to an approximately 10-fold increased yield compared to the parental expression construct. Overproduction of the enzyme was dependent on the codon usage in the 5' region of the DNA sequence encoding glutathione transferase M1-1. High-level expression clones were generated by a combination of random silent mutations in selected wobble positions in the coding sequence and immunoselection of clones from the library of random mutants. The strategy used is generally applicable for the production of recombinant proteins provided that a suitable selection procedure is available for identifying the desired mutants.     

Solution structure of the carboxyl terminus of a human class Mu glutathione S-transferase: NMR assignment strategies in large proteins

Strategies to obtain the NMR assignments for the HN, N, CO, Calpha and Cbeta resonance frequencies for the human class mu glutathione-S-transferase GSTM2-2 are reported. These assignments were obtained with deuterated protein using a combination of scalar and dipolar connectivities and various specific labeling schemes. The large size of this protein (55 kDa, homodimer) necessitated the development of a novel pulse sequence and specific labeling strategies. These aided in the identification of residue type and were essential components in determining sequence specific assignments. These assignments were utilized in this study to characterize the structure and dynamics of the carboxy-terminal residues in the unliganded protein. Previous crystallographic studies of this enzyme in complex with glutathione suggested that this region may be disordered, and that this disorder may be essential for catalysis. Furthermore, in the related class alpha protein extensive changes in conformation of the C terminus are observed upon ligand binding. On the basis of the results presented here, the time-averaged conformation of the carboxyl terminus of unliganded GSTM2-2 is similar to that seen in the crystal structure. NOE patterns and 1H-15N heteronuclear nuclear Overhauser enhancements suggest that this region of the enzyme does not undergo motion on a rapid time scale.     

Mutations of Gly to Ala in human glutathione transferase P1-1 affect helix 2 (G-site) and induce positive cooperativity in the binding of glutathione

Previous kinetic studies on human glutathione transferase P1-1 have indicated that the motions of an irregular alpha-helix (helix 2) lining the glutathione (GSH) binding site are viscosity dependent and may modulate the affinity of GSH binding. The effect of single amino acid residue substitutions (Gly to Ala) in this region is investigated here by site-directed mutagenesis. Three mutants (Gly41Ala, Gly50Ala and Gly41Ala/Gly50Ala) were overexpressed in Escherichia coli, purified, and characterized by kinetic, structural, and spectroscopic studies. All these mutant enzymes show kcat values similar to that of the wild-type enzyme, while the [S]0.5 for GSH increases about eight-fold in the Gly41Ala mutant and more than 100-fold in the Gly41Ala/Gly50Ala double mutant. This change in affinity towards GSH is accompanied by an induced positive cooperativity as reflected by Hill coefficients of 1.4 (Gly41Ala) and 1.7 (Gly41Ala/Gly50Ala) upon substrate binding. Taken together, these data suggest that the region around helix 2 is markedly altered leading to the observed intersubunit communication. Molecular modeling of the Gly41Ala/Gly50Ala mutant and of the inactive oxidized form of the native enzyme provides a structural explanation of our results.     

Distinctive structure of the human GSTM3 gene-inverted orientation relative to the mu class glutathione transferase gene cluster

The goals of this study were 1) to compare the effects of transforming growth factor-beta (TGF-beta) and parathyroid hormone-related protein (PTHrP) on mouse blastocyst attachment and outgrowth in vitro, 2) to determine whether TGF-beta acts through a mechanism involving PTHrP, 3) to examine effects of PTHrP on preimplantation mouse embryo development, and 4) to determine the pattern of expression of PTHrP protein in the uterus of the mouse during early gestation. In the first set of experiments, hatched blastocysts were placed in fibronectin-coated wells. Cultures were treated with PTHrP or TGF-beta1 and assessed at 24, 48, and 72 h for attachment and surface area of blastocyst outgrowth. Results showed that both PTHrP and TGF-beta1 increased blastocyst outgrowth significantly. A PTHrP-neutralizing antibody blocked the stimulatory effect of both PTHrP and TGF-beta1, suggesting that TGF-beta1 acts to increase endogenous production of PTHrP by the blastocyst. Immunoassay of conditioned medium from blastocysts treated with either TGF-beta1 or PTHrP 1-34 confirmed a 3- to 4-fold increase in levels of PTHrP 1-141. In the second series of experiments, pronuclear zygotes were cultured in various concentrations of PTHrP for 96 h. Blastocysts then were subjected to differential fluorescent staining of inner cell mass and trophectoderm cells. Treatment of mouse embryos with the various concentrations of PTHrP altered neither the number developing to the blastocyst stage nor the number of inner cell mass or trophectoderm cells in the resulting blastocysts. In the third experiment, pregnant mice were killed at Days 3, 4, 5, 6, and 7 of gestation, and uterine horns were processed for immunohistochemistry. Uterine sections were stained with antibodies to PTHrP, desmin, and laminin. On Days 3, 4, and 5, uterine luminal and glandular epithelial cells stained intensely for PTHrP, while stromal cells were negative. By Days 6 and 7, decidualized stromal cells stained positively for PTHrP, desmin, and laminin. These results support the hypothesis that TGF-beta and PTHrP play an important role in the process of implantation.     

Conformational changes in the crystal structure of rat glutathione transferase M1-1 with global substitution of 3-fluorotyrosine for tyrosine

Brain tumors are the most common solid neoplasms in childhood. This article reviews the current classification, clinical presentations, diagnostic procedures, and principles of treatment of pediatric brain tumors. The specific presentation, treatment, and prognosis of the most common, individual types of pediatric brain tumors are discussed.     

Evaluation of the effects of a specific alpha 2-adrenoceptor antagonist, atipamezole, on alpha 1- and alpha 2-adrenoceptor subtype binding, brain neurochemistry and behaviour in comparison with yohimbine

In the present study we evaluated the alpha 1- and alpha 2-adrenoceptor subtype binding, central alpha 2-adrenoceptor antagonist potency, as well as effects on brain neurochemistry and behavioural pharmacology of two alpha 2-adrenoceptor antagonists, atipamezole and yohimbine. Atipamezole had higher selectivity for alpha 2- vs. alpha 1-adrenoceptors than yohimbine regardless of the subtypes studied. Both compounds had comparable affinity for the alpha 2A-, alpha 2C- and alpha 2B-adrenoceptors, but yohimbine had significantly lower affinity for the alpha 2D-subtype. This may account for the fact that significantly higher doses of yohimbine than atipamezole were needed for reversal of alpha 2-agonist (medetomidine)-induced effects in rats (mydriasis) and mice (sedation and hypothermia). The effect on central monoaminergic activity was estimated by measuring the concentrations of transmitters and their main metabolites in whole brain homogenate. At equally effective alpha 2-antagonising doses in the rat mydriasis model, both drugs stimulated central noradrenaline turnover (as reflected by increase in metabolite levels) to the same extent. Atipamezole increased dopaminergic activity only slightly, whereas yohimbine elevated central dopamine but decreased central 5-hydroxytryptamine turnover rates. In behavioural tests, atipamezole (0.1-10 mg/kg) did not affect motor activity but stimulated food rewarded operant (FR-10) responding (0.03-3 mg/kg) whereas yohimbine both stimulated (1 mg/kg) and decreased (> or = 3 mg/kg) behaviour in a narrow dose range in these tests. In the staircase test, both antagonists increased neophobia, but in the two compartment test only yohimbine (> or = 3 mg/kg) decreased exploratory behaviour. The dissimilar effects of the antagonists on neurochemistry and behaviour are thought to be caused by non alpha 2-adrenoceptor properties of yohimbine. In conclusion, the alpha 2-antagonist atipamezole blocked all alpha 2-adrenoceptor subtypes at low doses, stimulated central noradrenergic activity and had only slight effects on behaviour under familiar conditions, but increased neophobia. The low affinity for the alpha 2D-adrenoceptor combined with its unspecific effects complicates the use of yohimbine as pharmacological tool to study alpha 2-adrenoceptor physiology and pharmacology.     

Distribution of alpha 1A, alpha 1B and alpha 1E voltage-dependent calcium channel subunits in the human hippocampus and parahippocampal gyrus

The distribution of voltage-dependent calcium channel subunits in the central nervous system may provide information about the function of these channels. The present study examined the distribution of three alpha-1 subunits, alpha 1A, alpha 1B and alpha 1E, in the normal human hippocampal formation and parahippocampal gyrus using the techniques of in situ hybridization and immunocytochemistry. All three subunit mRNAs appeared to be similarly localized, with high levels of expression in the dentate granule and CA pyramidal layer. At the protein level, alpha 1A, alpha 1B and alpha 1E subunits were differentially localized. In general, alpha 1A-immunoreactivity was most intense in cell bodies and dendritic processes, including dentate granule cells, CA3 pyramidal cells and entorhinal cortex pre-alpha and pri-alpha cells. The alpha 1B antibody exhibited relatively weak staining of cell bodies but stronger staining of neuropil, especially in certain regions of high synaptic density such as the polymorphic layer of the dentate gyrus and the stratum lucidum and radiatum of the CA regions. The alpha 1E staining pattern shared features in common with both alpha 1A and alpha 1B, with strong immunoreactivity in dentate granule, CA3 pyramidal and entorhinal cortex pri-alpha cells, as well as staining of the CA3 stratum lucidum. These findings suggest regions in which particular subunits may be involved in synaptic communication. For example, comparison of alpha 1B and alpha 1E staining in the CA3 stratum lucidum with calbindin-immuno-reactivity suggested that these two calcium channels subunits may be localized presynaptically in mossy fibre terminals and therefore may be involved in neurotransmitter release from these terminals.     

Species differences in the glutathione transferase GSTT1-1 activity towards the model substrates methyl chloride and dichloromethane in liver and kidney

Glutathione transferase (GST) GSTT1-1 is involved in the biotransformation of several chemicals widely used in industry, such as butadiene and dichloro methane DCM. The polymorphic hGSTT1-1 may well play a role in the development of kidney tumours after high and long-term occupational exposure against trichloroethylene. Although several studies have investigated the association of this polymorphism with malignant diseases little is known about its enzyme activity in potential extrahepatic target tissues. The known theta-specific substrates methyl chloride (MC) dichloromethane and 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) were used to assay GSTT1-1 activity in liver and kidney of rats, mice, hamsters and humans differentiating the three phenotypes (non-conjugators, low conjugators, high conjugators) seen in humans. In addition GSTT1-1 activity towards MC and DCM was determined in human erythrocytes. No GSTT1-1 activity was found in any tissue of non-conjugators (NC). In all organs high conjugators (HC) showed twofold higher activity towards MC and DCM than low conjugators (LC). The activity in human samples towards EPNP was too close to the detection limit to differentiate between the three conjugator phenotypes. GSTT1-1 activity towards MC was two to seven-times higher in liver cytosol than in kidney cytosol. The relation for MC between species was identical in both organs: mouse > HC > rat > LC > hamster > NC. In rats, mice and hamsters GSTT1-1 activity in liver cytosol towards DCM was also two to seven-times higher than in the kidney cytosol. In humans this activity was twice as high in kidney cytosol than in liver cytosol. The relation between species was mouse > rat > HC > LC > hamster > NC for liver, but mouse > HC > LC/rat > hamster/NC for kidney cytosol. The importance to heed the specific environment at potential target sites in risk assessment is emphasized by these results.     

Detoxification of optically active bay- and fjord-region polycyclic aromatic hydrocarbon dihydrodiol epoxides by human glutathione transferase P1-1 expressed in Chinese hamster V79 cells

Dihydrodiol epoxides (DEs) are important carcinogenic metabolites of polycyclic aromatic hydrocarbons (PAHs). The metabolic formation of four stereoisomeric DEs (a pair of optically active diastereomers termed as syn- and anti-form) is possible. Glutathione tranferases (GSTs) have been demonstrated to catalyze the detoxification of DEs. Purified GSTs display remarkable differences in catalytic efficiencies towards bay- and fjord-region DEs along with a high degree of regio- and stereoselectivity. Here we determined to which extent heterologously expressed human GSTP1-1, a major GST isoform in lung, affects the mutagenicity of stereoisomeric bay-region DEs of benzo[a]pyrene in Chinese hamster V79 cells. To evaluate the influence of sterical crowding in the substrate on the activity of GSTP-1, the study was extended to the strongly mutagenic fjord-region (-)-anti-DEs of benzo[c]phenanthrene and dibenzo[a,l]pyrene. GSTP1-1,reduced preferentially the mutagenicity (studied at the hprt locus) of (+)-anti and (+)-syn-DEs of benzo[a]pyrene (by 66 and 67%) as compared with the corresponding (-)-anti- and (-)-syn-enantiomers (by 15 and 13%). These results are in line with previous studies on the enantioselectivity of purified GSTP1-1 towards the DE isomers of benzo[a]pyrene and benzo[c]phenanthrene showing that enantiomers with (R)-configuration at the benzylic oxiranyl carbon are better substrates than those with (S)-configuration. Interestingly, the (-)-anti-DEs of benzo[c]phenanthrene and dibenzo[a,l]pyrene were efficiently detoxified by GSTP-1-1 in the constructed cell line (reduction of mutagenicity by 66 and 64%). This study demonstrates that differences in the caalytic activity seen for purified GST towards individual mutagens do not necessarily reflect the detoxification of DEs by the same enzyme in a living cell and provides further evidence that specific human GSTs play a role in the detoxification of DEs of PAHs.     

Aromatic DNA adducts in foundry workers in relation to exposure, life style and CYP1A1 and glutathione transferase M1 genotype

Levels of aromatic DNA adducts in foundry workers and controls were followed at four annual samplings. During this time exposure to polycyclic aromatic hydrocarbons (PAH) decreased and the level of DNA adducts decreased accordingly. In the total group exposure was related to the level of adducts. Adduct levels correlated with urinary 1-hydroxypyrene (LOGU1OH), air benzo[a]pyrene, weekly working hours and daily cigarette consumption. In a multivariate model 1-hydroxypyrene had a consistent effect. Neither glutathione transferase M1 (GSTM1) nor cytochrome P450 1A1 (CYP1A1) genotypes had clear effects. Yet the individuals lacking GSTM1 had a stronger effect of LOGU1OH and some effect by other sources of PAH, such as charcoal broiled food, although all these variables were not significant in the multivariate model. The rare individuals with a CYP1A1 polymorphism MspI containing an amino acid change at isoleucine had an increased level of adducts. The results showed that the postlabelling method used was able to detect an increase in aromatic DNA adducts in leukocytes when exposure to benzo[a]pyrene in air was approximately 5 ng/m3. At such low levels smoking and charcoal broiled food may be important contributors to adducts.