Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Comparison of the NSP4 amino acid sequences from 31 strains of mammalian rotaviruses revealed the presence of four distinct NSP4 alleles; i.e., the Wa, KUN, AU-1, and EW alleles. The EW allele consists only of NSP4s from murine rotavirus strains and is divergent from other NSP4 alleles from the evolutionary perspective. There have been conflicting reports regarding the enterotoxigenic activity of NSP4 in the mouse model system; heterologous simian and porcine rotavirus NSP4s function as an enterotoxin in mice, while a homologous EC NSP4 does not play a dominant role as an enterotoxin in the cystic fibrosis conductance regulator knockout mice. To further examine the enterotoxigenic activity of NSP4, we expressed in Escherichia coli a recombinant protein consisting of glutathione S-transferase and amino acid residues 86-175 of the EW NSP4. We found that this fusion protein caused diarrhea in the majority (8/14) of 5- to 6-day-old CD1 mice. This study confirmed and extended that group A rotavirus NSP4s were able to induce diarrhea in neonatal mice and had an enterotoxigenic activity.
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PMID:Diarrhea induction by rotavirus NSP4 in the homologous mouse model system. 1050 18

The cystic fibrosis transmembrane conductance regulator (CFTR) is known to function as a regulated chloride channel and, when genetically impaired, to cause the disease cystic fibrosis. The novel studies reported here were undertaken to gain greater molecular insight into possible interactions among CFTR's soluble domains, which include two nucleotide binding domains (NBF1 and NBF2) and a regulatory domain (R). The NBF1+R and NBF2 regions of CFTR were highly expressed in Escherichia coli, purified to near homogeneity under denaturing conditions, and refolded. Both refolded proteins bound TNP-ATP and TNP-ADP, which could be readily replaced with ATP. Four different approaches were then used to determine whether the NBF1+R and NBF2 proteins interact. First, the purified NBF2 protein was labeled near its C-terminus with a fluorescent probe, 7-diethyl amino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM). Addition of the unlabeled NBF1+R to the CPM-labeled NBF2 caused a red-shift in lambda(max) of the CPM fluorescence, consistent with a direct interaction between the two proteins. Second, when the NBF1+R protein, the NBF2 protein, and a mixture of the two proteins were folded separately and analyzed by molecular sieve chomatography, the mixture was found to elute prior to either NBF1+R or NBF2. Third, na-tive-PAGE gel studies revealed that the mixture of the NBF1+R and NBF2 domains migrated as a single band with an R(F) value between that of NBF1+R and NBF2. Fourth, trypsin digestion of a mixture of the NBF1+R and NBF2 proteins occurred at a slower rate than that for the individual proteins. Finally, studies were carried out to determine whether an NBF1+R/NBF2 interaction could be demonstrated after expressing one of the two proteins in soluble, native form, thus avoiding the inclusion body, denaturation, and renaturation approach. Specifically, the NBF1+R protein was overexpressed in E. coli in fusion with glutathione-S-transferase near a thrombin cleavage site. Following binding of the GST-(NBF1+R) fusion protein to a GST Sepharose affinity column, added NBF2 was shown to bind and then to coelute with NBF1+R upon addition of glutathione or thrombin. Collectively, these experiments demonstrate that CFTR's NBF1+R region and its NBF2 domain, after folding separately as distinct units, have a strong propensity to interact and that this interaction is stable in the absence of added nucleotides or exogenously induced phosphorylation. These findings, together with the additional observation that the NBF1+R/NBF2 interaction induces a change in the C-terminus of NBF2, which resides within the C-terminal region of CFTR, may have important implications not only for the function of CFTR per se, but its interaction with other proteins.
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PMID:Cystic fibrosis transmembrane conductance regulator: the purified NBF1+R protein interacts with the purified NBF2 domain to form a stable NBF1+R/NBF2 complex while inducing a conformational change transmitted to the C-terminal region. 1068 44

Residues 417-830 of the cystic fibrosis transmembrane conductance regulator (CFTR) were expressed as a glutathione-S-transferase fusion protein. This fusion protein, NBD1/R/GST, contains the regulatory and first nucleotide binding domains of CFTR. NBD1/R/GST hydrolyzed ATP with a K(M) (60 microM) and V(max) (330 nmol/min/mg) that differed from those reported for CFTR and for a peptide containing CFTR residues 433-589. The ATPase inhibitor profile of NBD1/R/GST indicates that CFTR resembles P-glycoprotein with respect to the NBD1 ATPase catalytic mechanism. ATP hydrolysis by NBD1/R/GST was unaffected by genistein, glybenclamide, and other agents known to affect CFTR's chloride channel function, suggesting that these agents do not act by directly influencing the ATPase function of NBD1. The disease-causing mutation, G551D, reduced ATP hydrolysis by NBD1/R/GST by increasing the K(M) for ATP fourfold. This suggests that when G551D occurs in patients with cystic fibrosis, it affects CFTR function by reducing the affinity of NBD1 for ATP.
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PMID:ATP hydrolysis by a CFTR domain: pharmacology and effects of G551D mutation. 1079 28

Adenylate kinase (AK; ATP:AMP phosphotransferase, EC 2.7.4.3) is a ubiquitous enzyme that contributes to the homeostasis of adenine nucleotides in eukaryotic and prokaryotic cells. AK catalyzes the reversible reaction Mg. ATP + AMP <--> Mg. ADP + ADP. In this study we show that AK secreted by the pathogenic strains of Pseudomonas aeruginosa appears to play an important role in macrophage cell death. We purified and characterized AK from the growth medium of a cystic fibrosis isolate strain of P. aeruginosa 8821 and hyperproduced it as a fusion protein with glutathione S-transferase. We demonstrated enhanced macrophage cell death in the presence of both the secreted and recombinant purified AK and its substrates AMP plus ATP or ADP. These data suggested that AK converts its substrates to a mixture of AMP, ADP, and ATP, which are potentially more cytotoxic than ATP alone. In addition, we observed increased macrophage killing in the presence of AK and ATP alone. Since the presence of ATPase activity on the macrophages was confirmed in the present work, external macrophage-effluxed ATP is converted to ADP, which in turn can be transformed by AK into a cytotoxic mixture of three adenine nucleotides. Evidence is presented in this study that secreted AK was detected in macrophages during infection with P. aeruginosa. Thus, the possible role of secreted AK as a virulence factor is in producing and keeping an intact pool of toxic mixtures of AMP, ADP, and ATP, which allows P. aeruginosa to exert its full virulence.
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PMID:Adenylate kinase as a virulence factor of Pseudomonas aeruginosa. 1134 42

Liver disease in patients with cystic fibrosis (CF) is inconstant and has not yet been clearly related to any specific risk factor. While the expression of cystic fibrosis transmembrane conductance regulator (CFTR) is restricted to the biliary epithelium in the liver, recent findings indicate that CFTR modulates reduced glutathione (GSH) transport and that CFTR dysfunction creates an imbalance in the antioxidant defense. Among liver detoxifying enzymes, the glutathione S-transferases (GSTs) play a key role in the protection against oxidative stress. Because oxidative injury contributes to the development of liver disease, we hypothesized that 2 members of the GST superfamily, GSTM1 and GSTP1, which are expressed in the biliary epithelium, could influence the hepatic status in patients with CF. The potential impact of GSTM1 and GSTP1 gene polymorphisms was assessed in 106 children with CF (mean age, 11.5 years). Based on polymerase chain reaction/restriction fragment length polymorphism analysis, we found that the frequency of GSTP1-Ile(105)/Ile(105) genotype was significantly higher in patients with CF with liver disease than in those without (P <.03). Among the youngest patients, aged 6 years, GSTP1-Ile(105)/Ile(105) genotype was associated with a 8-fold increase in the risk of liver disease compared with other GSTP1 genotypes (P =.002). No association between the GSTM1 genotype and liver status was documented. In conclusion, GSTP1-Ile(105)-encoding allele contributes to hepatic dysfunction in CF. Identification of this polymorphism may have prognostic value and prompt early treatment in patients with CF with an increased risk of liver disease.
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PMID:Liver disease in pediatric patients with cystic fibrosis is associated with glutathione S-transferase P1 polymorphism. 1260 71

The aim of the study was to evaluate serum a-glutathione S-transferase (s-GSTA) levels in patients with cystic fibrosis (CF) and to compare s-GSTA with other liver function tests and with a hepatic ultrasound scan (US). The cytosolic enzyme, alpha-glutathione S-transferase is predominantly found in the liver and is distributed uniformly in the liver tissue. In our study s-GSTA levels were measured in 37 CF patients aged 1 to 28 years (mean age 10.4 years, 24 males). The control group consisted of 27 patients aged 2 to 17 years (mean age 8.5 years, 18 males). The presence of hepatobiliary abnormalities was assessed by clinical examination, ultrasound scan, s-GSTA, and conventional liver enzymes: alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST) and gama-glutamyl transferase (GMT). The calculated 5-95 % range of s-GSTA for the control group was 0.098-2.54 microg/l, for the CF group 0.43-9.76 microg/l. Mean s-GSTA level in the control group was 1.55 microg/l (S.D.=1.57), and 2.05 micro/l (S.D.=2.60) in the CF group. In the group of CF patients, the serum levels were significantly higher than in the control group (P<0.01). No significant correlation existed in the CF group between s-GSTA and conventional liver tests (ALT, AST, ALP and GMT). Four patients in the CF group had hepatobiliary abnormalities detectable by conventional liver tests, s-GSTA and US. Four patients had abnormal s-GSTA, while conventional liver tests and US were normal. One other patient had abnormal hepatic US, but normal standard liver tests and s-GSTA. The study has suggested that a raised s-GSTA level might be a marker of possible pathological changes of the hepatobiliar system in CF patients. Serum GSTA seems to be a more sensitive marker than transaminases for the monitoring of hepatocellular integrity and as an early predictor of hepatic damage.
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PMID:Serum alpha-glutathione S-transferase as a sensitive marker of hepatocellular damage in patients with cystic fibrosis. 1279 Jul 69

The unphosphorylated regulatory (R) domain of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is often viewed as an inhibitor that is released by phosphorylation. To test this notion, we studied domain interactions using CFTR channels assembled from three polypeptides. Nucleotides encoding the R domain (aa 635-836) were replaced with an internal ribosome entry sequence so that amino- and carboxyl-terminal half-molecules would be translated from the same mRNA transcript. Although only core glycosylation was detected on SplitDeltaR, biotinylation, immunostaining, and functional studies clearly demonstrated its trafficking to the plasma membrane. SplitDeltaR generated a constitutive halide permeability, which became responsive to cAMP when the missing R domain was coexpressed. Each half-molecule was co-precipitated by antibody against the other half. Contrary to expectations, GST-R domain was pulled down only if prephosphorylated by protein kinase A, and coexpressed R domain was precipitated with SplitDeltaR much more efficiently when cells were stimulated with cAMP. These results indicate that phosphorylation regulates CFTR by promoting association of the R domain with other domains rather than by causing its dissociation from an inhibitory site.
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PMID:Phosphorylation of CFTR by PKA promotes binding of the regulatory domain. 1600 Oct 79

It is known that only 10-20% of smokers develop COPD, implying that apart from environmental features, additional factors such as genetic variability contribute to smoke susceptibility. This proposal is in compatibility with the "Dutch Hypothesis", formulated in the early 60's. Alpha-1-antitrypsin gene was implicated in the pathogenesis of COPD, especially the homozygous state of z allele. Since then many other genes have stepped forward as possible contributors to COPD development. In the present review we attempt to summarize the majority of these, including the genes of matrix metalloproteinases and their inhibitors, elastin, serpine2, tumor necrosis factor - a, transforming growth factor beta, a variety of interleukins and their receptors and antagonists, high affinity IgE receptor , human calcium-activated chloride channel 1, heme oxygenase, vascular endothelial growth factor, microsomal epoxide hydrolase, glutathione S-transferase, cytochrome P45O, superoxide dismutase, vitamin D binding protein, beta2-adrenergic receptor, Toll like receptor, human B defensins, mucins, cystic fibrosis transmembrane regulator, surfactant protein and Nuclear Factor E2 Related Factor 2.
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PMID:Genetics of chronic obstructive pulmonary disease, beyond a1-antitrypsin deficiency. 1707 33

Anti-inflammatory properties of azithromycin (AZM) have been proposed as possible mechanisms of clinical beneficial effects in patients with cystic fibrosis (CF). Altered glutathione (GSH) transport in cystic fibrosis transmembrane regulator protein (CFTR)-deficient cells leads to the occurrence of oxidative stress that finally induces glutathione S-transferase (GST) activity. The present investigation was aimed to verify the effects of AZM on GST activity and expression in CF airway cells in vitro and in vivo. AZM exposure significantly decreased GSTT1 and GSTM1 mRNA and protein expression in IB3-1, restoring the levels to those observed in non-CF C38 cells, which also express lower levels of gamma-glutamyltransferase (GGT) activity than IB3-1. In another CF cell line, 2CFSMEo-, AZM produced 45% reduction in GSTT1 and GSTM1 mRNA levels. AZM reduced GST activity by approximately 25% and 40% in IB3-1 and 2CFSMEo- cells, respectively. GSTP1 was similarly expressed in all CF and non-CF cells and was unaffected by AZM. The anti-inflammatory cytokine IL-10 down-modulated GST activity at similar levels, supporting a link between GST inhibition and anti-inflammatory properties of AZM. In bronchoalveolar lavage fluid of CF mice homozygous for the F508 del mutation, GSTM1 protein levels were undetectable after AZM treatment. The association between increased GST expression and activity, together with its reversal by AZM treatment in vitro and in vivo, suggest novel antioxidant properties for this drug. The issue whether decreased GST activity may directly concur to anti-inflammatory properties of AZM or is rather a marker of the oxidative status of CF cells will require additional studies.
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PMID:Effects of azithromycin on glutathione S-transferases in cystic fibrosis airway cells. 1909 86

Cystic fibrosis (CF) is a major genetic disease in Caucasians affecting 1 in 2500 newborns. Hepatobiliary pathology is a major cause of morbidity and mortality in CF second only to pulmonary disease. SULT1E1 activity is significantly elevated, generally 20-30-fold, in hepatocytes of mouse models of CF. SULT1E1 is responsible for the inactivation of beta-estradiol (E2) at physiological concentrations via conjugation with sulfonate. The increase in SULT1E1 activity results in the alteration of E2-regulated protein expression in CF mouse liver. To investigate the mechanism by which the absence of CFTR in human cholangiocytes induces SULT1E1 expression in hepatocytes, a membrane-separated human MMNK-1 cholangiocyte and human HepG2 hepatocyte co-culture system was developed. The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in bile duct cholangiocytes but not hepatocytes, whereas SULT1E1 is expressed in hepatocytes but not cholangiocytes. CFTR expression in MMNK-1 cells was inhibited with siRNA by >90% as determined by immunoblot and immunohistochemical analysis. Control and CFTR-siRNA-MMNK-1 cells were co-cultured with HepG2 cells in a Transwell membrane-separated system. After 8h of co-culture, HepG2 cells were removed from exposure to MMNK-1 cells and placed in fresh medium. After 24-48h, expression of SULT1E1 and selected E2-regulated proteins was analyzed in the HepG2 cells. Results demonstrated that SULT1E1 message and activity were selectively induced in HepG2 cells co-cultured with CFTR-deficient MMNK-1 cells. The expression of E2-regulated proteins (IGF-1, GST-P1 and carbonic anhydrase II) was also altered in response to decreased E2 levels. Thus, the loss of CFTR activity in cholangiocytes stimulates the expression of SULT1E1 in hepatocytes by a paracrine mechanism. SULT1E1 expression in HepG2 cells is inducible by sterol mediated liver-X-receptor (LXR) activation although not by progestins that induce SULT1E1 in the endometrium. SULT1E1 induction in the human cholangiocyte/hepatocyte co-culture system is consistent with and supports the results observed in CF mice. The changes in hepatocyte gene expression affect liver biochemistry and may facilitate the development of CF liver disease.
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PMID:Regulation of hepatic sulfotransferase (SULT) 1E1 expression and effects on estrogenic activity in cystic fibrosis (CF). 1942 40


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