Molecular analysis of the pathway for the synthesis of thiol tripeptides in the model legume Lotus japonicus

The thiol tripeptides, glutathione (GSH) and homoglutathione (hGSH), perform multiple roles in legumes, including protection against toxicity of free radicals and heavy metals. The three genes involved in the synthesis of GSH and hGSH in the model legume, Lotus japonicus, have been fully characterized and appear to be present as single copies in the genome. The gamma-glutamylcysteine synthetase (gammaecs) gene was mapped on the long arm of chromosome 4 (70.0 centimorgans [cM]) and consists of 15 exons, whereas the glutathione synthetase (gshs) and homoglutathione synthetase (hgshs) genes were mapped on the long arm of chromosome 1 (81.3 cM) and found to be arranged in tandem, with a separation of approximately 8 kb. Both genes consist of 12 exons of exactly the same size (except exon 1, which is similar). Two types of transcripts were detected for the gshs gene, which putatively encode proteins localized in the plastids and cytosol. Promoter regions contain cis-acting regulatory elements that may be involved in the plant's response to light, hormones, and stress. Determination of transcript levels, enzyme activities, and thiol contents in nodules, roots, and leaves revealed that gammaecs and hgshs are expressed in all three plant organs, whereas gshs is significantly functional only in nodules. This strongly suggests an important role of GSH in the rhizobia-legume symbiosis.

Regulation of the gene encoding glutathione S-transferase M1 (GSTM1) by the Myb oncoprotein

The identification of Myb 'target' genes will not only aid in the understanding of how overexpression of Myb, or expression of activated forms of Myb, leads to cellular transformation but will also shed light on its role in normal cells. Using a combination of an estrogen-regulated Myb-transformed cell line (ERMYB) and PCR-based subtractive hybridization, we have identified the gene (GSTM1) encoding the detoxification enzyme glutathione S-transferase M1 as being transcriptionally upregulated by Myb. Functional analysis of the GSTM1 promoter using reporter assays indicated that both the DNA binding and transactivation domains of Myb were required for transcriptional activation. Mutational analysis of consensus Myb-binding sites (MBS) in the promoter and electrophoretic mobility gel shift analysis indicated that one of the three potential MBS can bind Myb protein, and is the primary site involved in the regulation of this promoter by Myb.

Polymorphisms at the glutathione S-transferase GSTM1, GSTT1 and GSTP1 loci: risk of ovarian cancer by histological subtype

The phase II glutathione S-transferases (GSTs) GSTT1, GSTM1 and GSTP1 catalyse glutathione-mediated reduction of exogenous and endogenous electrophiles. These GSTs have broad and overlapping substrate specificities and it has been hypothesized that allelic variants associated with less effective detoxification of potential carcinogens may confer an increased susceptibility to cancer. To assess the role of GST gene variants in ovarian cancer development, we screened 285 epithelial ovarian cancer cases and 299 unaffected controls for the GSTT1 deletion (null) variant, the GSTM1 deletion (null) variant and the GSTP1 codon 104 A-->G Ile-->Val amino acid substitution variant, The frequencies of the GSTT1, GSTM1 and GSTP1 polymorphic variants did not vary with tumour behaviour (low malignant potential or invasive) or p53 immunohistochemical status. There was a suggestion that ovarian cancers of the endometrioid or clear cell histological subtype had a higher frequency of the GSTT1 and GSTM1 deletion genotype than other histological subgroups. The GSTT1, GSTM1 and GSTP1 genotype distributions did not differ significantly between unaffected controls and ovarian cancer cases (overall or invasive cancers only). However, the GSTM1 null genotype was associated with increased risk of endometrioid/clear cell invasive cancer [age-adjusted OR (95% CI) = 2.04 (1.01-4.09), P = 0.05], suggesting that deletion of GSTM1 may increase the risk of ovarian cancer of these histological subtypes specifically. This marginally significant finding will require verification by independent studies.

A sensitive and specific assay for glutathione with potential application to glutathione disulphide, using high-performance liquid chromatography-tandem mass spectrometry

We have utilised the combination of sensitivity and specificity afforded by coupling high-performance liquid chromatography (HPLC) to a tandem mass spectrometer (MS-MS) to produce an assay which is suitable for assaying glutathione (GSH) concentrations in liver tissue. The sensitivity suggests it may also be suitable for extrahepatic tissues, The method has been validated for GSH using mouse liver samples and also allows the assay of GSSG. The stability of GSH under conditions relevant to the assay has been determined. A 20-mul amount of a diluted methanol extract of tissue is injected with detection limits of 0.2 pmol for GSH and 2 pmol for GSSG. The HPLC uses an Altima C-18 (150X4.6 mm, 5 mum) column at 35 degreesC. Chromatography utilises a linear gradient from 0 to 10% methanol in 0.1% formic acid over 5 min, with a final isocratic stage holding at 10% methanol for 5 min. Total flow rate is 0.8 ml/min. The transition from the M+H ion (308.1 m/z for GSH, and 613.3 m/z for GSSG) to the 162.0 m/z (GSH) and 355.3 m/z (GSSG) fragments are monitored. (C) 2001 Elsevier Science B.V. All rights reserved.

A multifunctional polyketide-peptide synthetase essential for albicidin biosynthesis in Xanthomonas albilineans

Albicidins, a family of potent antibiotics and phytotoxins produced by the sugarcane leaf scald pathogen Xanthomonas albilineans, inhibit DNA replication in bacteria and plastids. A gene located by Tn5-tagging was confirmed by complementation to participate in albicidin biosynthesis. The gene (xabB) encodes a large protein (predicted Mr 525695), with a modular architecture indicative of a multifunctional polyketide synthase (PKS) linked to a non-ribosomal peptide synthetase (NRPS). At 4801 amino acids in length, XabB is the largest reported PKS–NRPS. Twelve catalytic domains in this multifunctional enzyme are arranged in the order N terminus–acyl-CoA ligase (AL)–acyl carrier protein (ACP)–ß-ketoacyl synthase (KS)–ß-ketoacyl reductase (KR)–ACP–ACP–KS–peptidyl carrier protein (PCP)–condensation (C)–adenylation–PCP–C. The modular architecture of XabB indicates likely steps in albicidin biosynthesis and approaches to enhance antibiotic yield. The novel pattern of domains, in comparison with known PKS–NRPS enzymes for antibiotic production, also contributes to the knowledge base for rational design of enzymes producing novel antibiotics.

Expression of asparagine synthetase in response to carbohydrate supply in model callus cultures and shoot tips of asparagus (Asparagus officinalis L.)

Sugar uptake and metabolism were studied in callus cultures and shoot tips of asparagus. Asparagus callus cultures were used to model senescence in shoot tips. Callus cultures absorbed glucose from a nutrient medium, and accumulated sucrose, glucose and fructose. This uptake of glucose by the callus cultures down-regulated expression of asparagine synthetase and beta -galactosidase transcripts that otherwise accumulated when sugar was withheld. When 80 mm-long asparagus shoots were excised from growing plants and placed in 2% and 8% sucrose solutions, endogenous concentrations of sucrose, glucose, fructose, UDPglucose, and glucose-6-phosphate declined in the 30mm-long meristematic tip regions. At the same time, asparagine and asparagine synthetase gene transcripts began to accumulate in these tips. When 10 mm-long asparagus shoot tips were placed on glucose- or fructose-containing agar, the tips accumulated sucrose, glucose and fructose, and asparagine accumulation and expression of asparagine synthetase were marginally reduced. We concluded that in callus cultures, asparagine synthetase expression was sugar regulated, but that sugar regulation was not as pronounced in asparagus shoot tips. This may be due in part to slower rates of sugar uptake into shoot tips and in part to compartmentation of sugars in the tips. We suggest that callus cultures are not a suitable model for metabolic studies in asparagus shoot tips.

Use of heterologously-expressed cytochomre P450 and glutathione transferase enzymes in toxicity assays

Our groups have had a long-term interest in utilizing bacterial systems in the characterization of bioactivation and detoxication reactions catalyzed by cytochrome P450 (P450) and glutathione transferase (GST) enzymes. Bacterial systems remain the first choice for initial screens with new chemicals and have advantages, including high-throughput capability. Most human P450s of interest in toxicology have been readily expressed in Escherichia coli with only minor sequence modification. These enzymes can be readily purified and used in assays of activation of chemicals. Bicistronic systems have been developed in order to provide the auxiliary NADPH-P450 reductase. Alternative systems involve these enzymes expressed together within bacteria. In one approach, a lac selection system is used with E. coli and has been applied to the characterization of inhibitors of P450s 1A2 and 1131, as well as in basic studies involving random mutagenesis. Another approach utilizes induction of the SOS (umu) response in Salmonella typhimurium, and systems have now been developed with human P450s 1A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4, which have been used to report responses from heterocyclic amines. S. typhimurium his reporter systems have also been used with GSTs, first to demonstrate the role of rat GST 5-5 in the activation of dihalomethanes. These systems have been used to compare these GSTs with regard to activation of dihaloalkanes and potential toxicity. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Distinct cis-elements in the Asparagus officinalis asparagine synthetase promoter respond to carbohydrate and senescence signals

The Asparagus officinalis L. asparagine (Asn) synthetase (AS) promoter was analysed for elements responding to carbohydrate and senescence signals. Transgenic Arabidopsis thaliana L. plants containing deletion constructs of the –1958 bp AS promoter linked to the β-glucuronidase (GUS) reporter gene (AS::GUS) were analysed by measuring GUS specific activity. Inclusion of sucrose (Suc), glucose (Glc) or fructose (Fru) in plant media repressed levels of GUS activity in –1958AS::GUS plants, regardless of the light environment, with increases in GUS found 1 d after incubation on Suc-lacking media. Hexokinase is likely to be involved in the signal pathway, as Suc, Glc, Fru, 2-deoxy-d-glucose and mannose were more effective repressors than 3-O-methylglucose, and the hexokinase inhibitor mannoheptulose reduced repression. Plants containing AS::GUS constructs with deletions that reduced the promoter to less than –405 bp did not show low sugar induction. AS::GUS activity was significantly higher in excised leaves induced to senesce by dark storage for 24 h, compared to fresh leaves, for lines containing at least –640 bp of the AS promoter but not those with –523 bp or smaller promoter fragments. Fusion of the –640 to –523 bp region to a –381AS::GUS construct generated a promoter that retained senescence induction but lacked low sugar induction. Alignment of this region to the 33-bp senescence-related sequence of the Arabidopsis and Brassica napus L. SAG12 promoters identified the sequence TTGCACG as being conserved in all the promoters, and which may be an important senescence-responsive element.

Structure of the N-terminal domain of Escherichia coli glutamine synthetase adenylyltransferase

We report the crystal structure of the N-terminal domain of Escherichia coli adenylyltransferase that catalyzes the reversible nucleotidylation of glutamine synthetase (GS), a key enzyme in nitrogen assimilation. This domain (AT-N440) catalyzes the deadenylylation and subsequent activation of GS. The structure has been divided into three subdomains, two of which bear some similarity to kanamycin nucleotidyltransferase (KNT). However, the orientation of the two domains in AT-N440 differs from that in KNT. The active site of AT-N440 has been identified on the basis of structural comparisons with KNT, DNA polymerase beta, and polyadenylate polymerase. AT-N440 has a cluster of metal binding residues that are conserved in polbeta-like nucleotidyl transferases. The location of residues conserved in all ATase sequences was found to cluster around the active site. Many of these residues are very likely to play a role in catalysis, substrate binding, or effector binding.

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.