Association between glutathione S-transferase M1 and T1 and the risk for coronary heart disease (CHD)

Deficiency of Glutathione S-transferases (GST) M1 and T1 are associated with chronic diseases (e.g. lung cancer, MS) and could be one factor for the risk for CHD.We conducted a pros-pective case-control study in 93 pts. with angiographically proven CHD and 161 controls matched for age ±2y and gender (resulting in n=91 pairs, of which 18 were female). Genes coding for functional GST M1 and T1 were analysed acoording to previously published methods. The association between GST M1, T1 was tested using Fisher's exact test; logistic regression analysis was performed to control for HDL-cholesterol, diabetes smoking, diabetes, hypertension. 41% of cases were smokers, 25% had diabetes and 68% hypertension, corresponding figures for controls were 31%, 13% and 33%. Mean HDL-cholesterol levels were comparable (pts: 46±14 mg/dl, controls: 43± 19 mg/dl). There was no overall significant correlation between functional GST T1 and M1 genotypes and CHD, however, there seems to be an association between GST M1, HDL-cholesterol and CHD. Larger studies are needed to verify these data.

The purine salvage enzyme hypoxanthine guanine xanthine phosphoribosyl transferase is a major target antigen for cell-mediated immunity to malaria

Although there is good evidence that immunity to the blood stages of malaria parasites can be mediated by different effector components of the adaptive immune system, target antigens for a principal component, effector CD4(+) T cells, have never been defined. We generated CD4+ T cell lines to fractions of native antigens from the blood stages of the rodent parasite, Plasmodium yoelii, and identified fraction-specific T cells that had a Th1 phenotype (producing IL-2, IFN-gamma, and tumor necrosis factor-a, but not IL-4, after antigenic stimulation). These T cells could inhibit parasite growth in recipient severe combined immunodeficient mice. N-terminal sequencing of the fraction showed identity with hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT). Recombinant HGXPRT from the human malaria parasite, Plasmodium falciparum, activated the T cells in vitro, and immunization of normal mice with recombinant HGXPRT reduced parasite growth rates in all mice after challenge.

Crystal structures of fusion proteins with large-affinity tags

The fusion of a protein of interest to a large-affinity tag, such as the maltose-binding protein (MBP), thioredoxin (TRX), or glutathione-S-transferase (GST), can be advantageous in terms of increased expression, enhanced solubility, protection from proteolysis, improved folding, and protein purification via affinity chromatography. Unfortunately, crystal growth is hindered by the conformational heterogeneity induced by the fusion tag, requiring that the tag is removed by a potentially problematic cleavage step. The first three crystal structures of fusion proteins with large-affinity tags have been reported recently. All three structures used a novel strategy to rigidly fuse the protein of interest to MBP via a short three- to five-amino acid spacer. This strategy has the potential to aid structure determination of proteins that present particular experimental challenges and are not conducive to more conventional crystallization strategies (e.g., membrane proteins). Structural genomics initiatives may also benefit from this approach as a way to crystallize problematic proteins of significant interest.

Case-only study of interactions between genetic polymorphisms of GSTM1, P1, T1 and Z1 and smoking in Parkinson's disease

Current opinion contends that complex interactions between genetic and environmental factors play a role in the etiology of Parkinson's disease (PD). Cigarette smoking is thought to reduce risk of PD, and emerging evidence suggests that genetic factors may modulate smoking's effect. We used a case-only design, an approach not previously used to study gene-environment interactions in PD, specifically to study interactions between glutathione-S-transferase (GST) gene polymorphisms and smoking in relation to PD. Four-hundred PD cases (age at onset: 60.0 +/- 10.7 years) were genotyped for common polymorphisms in GSTM1, PI, T1 and Z1 using well-established methods. Smoking exposure data were collected in face-to-face interviews. The independence of the studied GST genotypes and smoking exposure was confirmed by studying 402 healthy, aged individuals. No differences were observed in the distributions of GSTM1, T1 or Z1 polymorphisms between ever-smoked and never-smoked PD cases using logistic regression (all P > 0.43). However, GSTP1 *C haplotypes were over-represented among PD cases who ever smoked (odds ratio for interaction (ORi) = 2.00 (95% Cl: 1.11-3.60, P = 0.03)). Analysis revealed that ORi between smoking and the GSTP1-114Val carrier status increased with increasing smoking dose (P = 0.02 for trend). These data suggest that one or more GSTP1 polymorphisms may interact with cigarette smoking to influence the risk for PD. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

A conformationally sensitive GHR [growth hormone (GH) receptor] antibody: Impact on GH signaling and GHR proteolysis

The GH receptor (GHR) mediates metabolic and somatogenic actions of GH. Its extracellular domain (ECD; residues 1-246) has two subdomains, each with seven beta strands organized into two antiparallel beta sheets, connected by a short hinge region. Most of the ECD residues involved in GH binding reside in subdomain 1, whereas subdomain 2 harbors a dimerization interface between GHR dimers that alters conformation in response to GH. A regulated GHR metalloprotease cleavage site is in the membrane-proximal stem region of subdomain 2. We have identified a monoclonal anti-ECD antibody, anti-GHR(ext-mAb), which recognizes the rabbit and human GHRs by immunoprecipitation, but less so after GH treatment. By immunoblotting and immunoprecipitation, anti-GHR(ext-mAb) recognized a glutathione-S-transferase (GST) fusion incorporating subdomain 2, but not one including subdomain 1. In transient transfection experiments, anti-GHR(ext-mAb) failed to recognize by immunoprecipitation a previously characterized dimerization interface mutant GHR that is incompetent for signaling. In signaling experiments, brief pretreatment of GH-responsive human fibrosarcoma cells with anti-GHR(ext-mAb) dramatically inhibited GH-induced Janus kinase 2 and signal transducer and activator of transcription 5 tyrosine phosphorylation and prevented GH-induced GHR disulfide linkage (a reflection of GH-induced conformational changes). In contrast, anti-GHR(ext-mAb) only partially inhibited radiolabeled GH binding, suggesting its effects on signaling were not simply via inhibition of binding. Furthermore, anti-GHR(ext-mAb) prevented phorbol ester-stimulated GHR proteolysis, but GHR cleavage site mutants were normally recognized by the antibody, indicating that the stem region cleavage site is not a direct epitope. A Fab fragment of anti-GHR(ext-mAb) inhibited GH-induced GHR disulfide linkage and signaling, as well as phorbol ester-induced GHR proteolysis, in a fashion similar to the intact antibody. Thus, our findings suggest that anti-GHR(ext-mAb) has promise as a GH antagonist and as a tool in studies of conformational changes required for GHR activation.

Functional expression and characterization of Eehinococcus granulosus thioredoxin peroxidase suggests a role in protection against oxidative damage

A full-length cDNA sequence coding for Echinococcus granulosus thioredoxin peroxidase (EgTPx) was isolated from a sheep strain protoscolex cDNA library by immunoscreening using a pool of sera from mice infected with oncospheres. EgTPx expressed as a fusion protein with glutathione S-transferase (GST) exhibited significant thiol-dependent peroxidase activity that protected plasmid DNA from damage by metal-catalyzed oxidation (MCO) in vitro. Furthermore, the suggested antioxidant role for EgTPx was reinforced in an in vivo assay, whereby its expression in BL21 bacterial cells markedly increased the tolerance and survival of the cells to high concentrations of H2O2 compared with controls. Immunolocalization studies revealed that EgTPx was specifically expressed in all tissues of the protoscolex and brood capsules. Higher intensity of labelling was detected in many, but not all, calcareous corpuscle cells in protoscoleces. The purified recombinant EgTPx protein was used to screen sera from heavily infected mice and patients with confirmed hydatid infection. Only a portion of the sera reacted positively with the EgTPx-GST fusion protein in Western blots, suggesting that EgTPx may form antibody-antigen complexes or that responses to the EgTPx antigen may be immunologically regulated. Recombinant EgTPx may prove useful for the screening of specific inhibitors that could serve as new drugs for treatment of hydatid disease. Moreover, given that TPx from different parasitic phyla were phylogenetically distant from host TPx molecules, the development of antiparasite TPx inhibitors that do not react with host TPx might be feasible. (C) 2003 Elsevier B.V. All rights reserved.

Interaction of the Hsp90 cochaperone cyclophilin 40 with Hsc70

The high-affinity ligand-binding form of unactivated steroid receptors exists as a multicomponent complex that includes heat shock protein (Hsp)90; one of the immunophilins cyclophilin 40 (CyP40), FKBP51, or FKBP52; and an additional p23 protein component. Assembly of this heterocomplex is mediated by Hsp70 in association with accessory chaperones Hsp40, Hip, and Hop. A conserved structural element incorporating a tetratricopeptide repeat (TPR) domain mediates the interaction of the immunophilins with Hsp90 by accommodating the C-terminal EEVD peptide of the chaperone through a network of electrostatic and hydrophobic interactions. TPR cochaperones recognize the EEVD structural motif common to both Hsp90 and Hsp70 through a highly conserved clamp domain. In the present study, we investigated in vitro the molecular interactions between CyP40 and FKBP52 and other stress-related components involved in steroid receptor assembly, namely Hsp70 and Hop. Using a binding protein-retention assay with CyP40 fused to glutathione S-transferase immobilized on glutathione-agarose, we have identified the constitutively expressed form of Hsp70, heat shock cognate (Hsc)70, as an additional target for CyP40. Deletion mapping studies showed the binding determinants to be similar to those for CyP40-Hsp90 interaction. Furthermore, a mutational analysis of CyP40 clamp domain residues confirmed the importance of this motif in CyP40-Hsc70 interaction. Additional residues thought to mediate binding specificity through hydrophobic interactions were also important for Hsc70 recognition. CyP40 was shown to have a preference for Hsp90 over Hsc70. Surprisingly, FKBP52 was unable to compete with CyP40 for Hsc70 binding, suggesting that FKBP52 discriminates between the TPR cochaperone-binding sites in Hsp90 and Hsp70. Hop, which contains multiple units of the TPR motif, was shown to be a direct competitor with CyP40 for Hsc70 binding. Similar to Hop, CyP40 was shown not to influence the adenosine triphosphatase activity of Hsc70. Our results suggest that CyP40 may have a modulating role in Hsc70 as well as Hsp90 cellular function.

Nedd4-2 functionally interacts with ClC-5 - Involvement in constitutive albumin endocytosis in proximal tubule cells

Constitutive albumin uptake by the proximal tubule is achieved by a receptor-mediated process in which the Cl- channel, ClC-5, plays an obligate role. Here we investigated the functional interaction between ClC-5 and ubiquitin ligases Nedd4 and Nedd4-2 and their role in albumin uptake in opossum kidney proximal tubule (OK) cells. In vivo immunoprecipitation using an anti-HECT antibody demonstrated that ClC-5 bound to ubiquitin ligases, whereas glutathione S-transferase pull-downs confirmed that the C terminus of ClC-5 bound both Nedd4 and Nedd4-2. Nedd4-2 alone was able to alter ClC-5 currents in Xenopus oocytes by decreasing cell surface expression of ClC-5. In OK cells, a physiological concentration of albumin (10 mug/ml) rapidly increased cell surface expression of ClC-5, which was also accompanied by the ubiquitination of ClC-5. Albumin uptake was reduced by inhibiting either the lysosome or proteasome. Total levels of Nedd4-2 and proteasome activity also increased rapidly in response to albumin. Overexpression of ligase defective Nedd4-2 or knockdown of endogenous Nedd4-2 with small interfering RNA resulted in significant decreases in albumin uptake. In contrast, pathophysiological concentrations of albumin (100 and 1000 mug/ml) reduced the levels of ClC-5 and Nedd4-2 and the activity of the proteasome to the levels seen in the absence of albumin. These data demonstrate that normal constitutive uptake of albumin by the proximal tubule requires Nedd4-2, which may act via ubiquitination to shunt ClC-5 into the endocytic pathway.

Occupational and non-occupational risk factors in bladder cancer patients in an industrialized area located in former east-Germany

Purpose: Several occupational carcinogens are metabolized by polymorphic enzymes. The distribution of the polymorphic enzymes N-acetyltransferase 2 (NAT2; substrates: aromatic amines), glutathione S-transferase M1 (GSTM1; substrates: e.g., reactive metabolites of polycyclic aromatic hydrocarbons), and glutathione S-transferase T1 (GSTT1; substrates: small molecules with 1 - 2 carbon atoms) were investigated. Material and Methods: At the urological department in Lutherstadt Wittenberg, 136 patients with a histologically proven transitional cell cancer of the urinary bladder were investigated for all occupations performed for more than 6 months. Several occupational and non-occupational risk factors were asked. The genotypes of NAT2, GSTM1, and GSTT1 were determined from leucocyte DNA by PCR. Results: Compared to the general population in Middle Europe, the percentage of GSTT1 negative persons (22.1%) was ordinary; the percentage of slow acetylators (59.6%) was in the upper normal range, while the percentage of GSTM1 negative persons (58.8%) was elevated in the entire group. Shifts in the distribution of the genotypes were observed in subgroups who had been exposed to asbestos (6/6 GSTM1 negative, 5/6 slow acetylators), rubber manufacturing (8/10 GSTM1 negative), and chlorinated solvents (9/15 GSTM1 negative). Conclusions: The overrepresentation of GSTM1 negative bladder cancer patients also in this industrialized area and more pronounced in several occupationally exposed subgroups points to an impact of the GSTM1 negative genotype in bladder carcinogenesis.

Selective modulation of neuronal nicotinic acetylcholine receptor channel subunits by G(o)-protein subunits

protein modulation of neuronal nicotinic acetylcholine receptor ( nAChR) channels in rat intrinsic cardiac ganglia was examined using dialyzed whole-cell and excised membrane patch-recording configurations. Cell dialysis with GTP gamma S increased the agonist affinity of nAChRs, resulting in a potentiation of nicotine-evoked whole-cell currents at low concentrations. ACh- and nicotine-evoked current amplitudes were increased approximately twofold in the presence of GTP gamma S. In inside-out membrane patches, the open probability (NPo) of nAChR-mediated unitary currents was reversibly increased fourfold after bath application of 0.2mM GTP gamma S relative to control but was unchanged in the presence of GDP gamma S. The modulation of nAChR-mediated whole- cell currents was agonist specific; currents evoked by the cholinergic agonists ACh, nicotine, and 1,1-dimethyl-4-phenylpiperazinium iodide, but not cytisine or choline, were potentiated in the presence of GTP gamma S. The direct interaction between G-protein subunits and nAChRs was examined by bath application of either G(o)alpha or G beta gamma subunits to inside-out membrane patches and in glutathione S-transferase pull-down and coimmunoprecipitation experiments. Bath application of 50 nM G beta gamma increased the open probability of ACh- activated single-channel currents fivefold, whereas G(o)alpha( 50 nM) produced no significant increase in NPo. Neuronal nAChR subunits alpha 3-alpha 5 and alpha 2 exhibited a positive interaction with G(o)alpha and G beta gamma, whereas beta 4 and alpha 7 failed to interact with either of the G-protein subunits. These results provide evidence for a direct interaction between nAChR and G-protein subunits, underlying the increased open probability of ACh-activated single-channel currents and potentiation of nAChR-mediated whole-cell currents in parasympathetic neurons of rat intrinsic cardiac ganglia.

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.