UNIPROT:P05412 - FACTA Search

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Query: UNIPROT:P05412 (c-Jun)
11,453 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe a multipurpose eukaryotic expression vector that incorporates the following features: restriction sites for in-frame insertion of cDNAs of interest between sequences encoding the glutathione-S-transferase (GST) and an oligohistidine element, allowing expression of the corresponding fusion proteins; a phosphorylation site for protein kinase A for in vitro labeling of the fusion protein; a T7 promoter for in vitro transcription and subsequent translation; and signals for single-stranded DNA production in bacteria. We have used this vector to demonstrate the formation in vivo of complexes between the transcription factor ATFa, a member of the family of ATF/CRE binding proteins, and the c-Jun or c-Fos proteins. Such interactions could be detected in crude extracts from cells transfected with vectors expressing the GST-ATFa fusion protein, as well as the c-Jun or c-Fos proteins. Complexes containing both ATFa and either c-Jun or c-Fos were specifically retained on glutathione (GSH)-agarose beads as revealed by immunoblot analyses. We also show that the leucine zipper domain of ATFa is essential for this interaction.
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PMID:Eukaryotic GST fusion vector for the study of protein-protein associations in vivo: application to interaction of ATFa with Jun and Fos. 770 40

The gene GLCLC encodes the catalytic subunit of gamma-glutamylcysteine synthetase (glutamate-cysteine ligase E.C. 6.3.2.2), the rate limiting enzyme for glutathione synthesis. When HepG2 cells were exposed to the serine/threonine phosphatase inhibitor okadaic acid (OA), increased expression of GLCLC was observed, as was the development of resistance to xenobiotic induced GSH depletion. Okadaic acid is known to activate both NF-kappaB and AP-1 activity. Inhibition of NF-kappaB activity by overexpression of an IkappaB alpha transdominant inhibitor or exposure to the protease inhibitor TLCK did not inhibit the OA mediated increase in GLCLC transcripts. Fibroblasts derived from a mouse containing a c-Jun null mutation exhibited diminished AP-1 binding activity, reduced levels of GLCLC message, and a correspondingly low GSH concentration compared to wild type cells. When the null cells, which express Jun B and Jun D, were exposed to OA, AP-1 binding activity increased, as did expression of GLCLC message. These results indicate that AP-1 transcription factors participate in the regulation of glutathione metabolism.
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PMID:Expression of glutathione and gamma-glutamylcysteine synthetase mRNA is Jun dependent. 917 57

In order to study the role played by known and novel genes in growth control and neoplasia, we here compare the pEX and pGEX bacterial expression systems for recombinant oncoprotein production and for generation of specific antisera. The results of five pEX (MS2-c-Fos, MS2-Fra-1, MS2-JunD, bgal-c-Jun and bgal-JunB) and two pGEX [glutathione S-transferase (GSH)-JE/MCP-1 and GST-JunD] fusion-protein productions are presented. Higher (15-43-fold) yields are obtained with the pEX system, but only the pGEX system allows separation of the protein of interest from the fusion moiety by digestion with specific proteases. The degree of fusion-protein purification, as assessed by SDS/PAGE, is similar for both systems. Proteins produced by both systems were successfully used in the generation of specific antisera. The choice between the pEX and pGEX systems is dependent upon the specific recombinant protein produced.
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PMID:Use of pEX and pGEX bacterial heterologous protein expression systems for recombinant oncoprotein production and antisera generation. 919 74

Thioredoxin (Trx) is a small ubiquitous dithiol protein which together with the FAD-containing enzyme thioredoxin reductase (TR) and NADPH (the Trx system) is a hydrogen donor for ribonucleotide reductase essential for DNA synthesis and a general protein disulfide reductase involved in redox regulation. Selenite, selenodiglutathione (GS-Se-SG) and selenocystine are efficiently reduced by thioredoxins and also directly by NADPH and mammalian TR but not by the E. coli enzyme. Incubation of selenite or GS-Se-SG with the Trx system or with mammalian TR results in a rapid formation of selenide, which by redox cycling with oxygen may cause a large non-stoichiometric oxidation of NADPH. Selenocystine is efficiently reduced into two molecules of the selenol amino acid selenocysteine by mammalian TR with a K(m)-value (6 mumol.L-1) and a high turnover number (kappa cat 3200 min-1) almost identical to the natural substrate Trx-S2. TR also directly reduces lipid hydroperoxides and this peroxidase reaction is strongly stimulated by the presence of catalytic amounts of free selenocysteine. Glutaredoxin (Grx) which catalyzes GSH-dependent disulfide reduction also via a redox-active disulfide and Trx are both efficient electron donors to the human plasma glutathione peroxidase providing a mechanism by which human plasma glutathione peroxidase may reduce hydroperoxides in an environment almost free from glutathione. Selenate is reduced by Grx and Trx in the presence of GSH. The DNA-binding of the transcription factor AP-1 is strongly inhibited by GS-Se-SG and selenite. Furthermore, selenide formed by TR-mediated reduction of selenite and GS-Se-SG inhibits lipoxygenase and changes the electron spin resonance spectrum of the active site iron. Mammalian TR with two subunits of 57 kDa has recently been cloned and shown to be homologous to glutathione reductase. The rat enzyme contains a selenocysteine residue in a unique Cterminal position and a conserved SECIS sequence directing insertion of the selenocysteine. The discovery of selenocysteine in mammalian TR may explain the broad substrate specificity of the enzyme and the requirement of selenium for cell proliferation.
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PMID:Selenium and the thioredoxin and glutaredoxin systems. 931 20

The synthesis of glutathione (GSH) and its conjugation to xenobiotics are essential for detoxification in liver cells. To understand how cellular levels of GSH are balanced in response to environmental stress, we cloned two cell lines, HLE/BSO1-1 and HLE/BSO1-2, from human hepatic HLE/WT cells resistant to buthionine sulfoximine (BSO), an irreversible inhibitor of gamma-glutamylcysteine synthetase (GCS). HLE/BSO1-1 and HLE/BSO1-2 showed 35- and 40-fold higher resistance respectively, than the wild type to BSO. In the absence of BSO, cellular levels of GSH were 3.0-fold higher, whereas levels of Pi class glutathione thiol transferase, GSTP1, were 2-fold lower, in the subclones than in the wild type cells. GCS heavy subunit (GCSh) mRNA level were 2.5-fold higher in HLE/BSO1-1 and HLE/BSO1-2 as compared with HLE/WT. Sequences between -315 and -241 base pairs of the 5' region, which contain an AP1 site, were shown to be responsible for the enhanced expression of GCSh in HLE/BSO1-1 cells. The expression of a dominant-negative mutant of c-Jun was found to inhibit the AP1-dependent GCSh promoter activity in HLE/WT and HLE/BSO1-1. Both protein level of c-Jun and binding activity of AP-1 were increased in both HLE/BSO1-1 and HLE/BSO1-2 cells. The up-regulation of GCSh gene appeared to be due to enhanced GCSh promoter acting through AP-1 activation in BSO-resistant hepatic cells.
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PMID:Glutathione homeostasis in human hepatic cells: overexpression of gamma-glutamylcysteine synthetase gene in cell lines resistant to buthionine sulfoximine, an inhibitor of glutathione synthesis. 961 Mar 71

Phorone, a glutathione (GSH) depletor, induces the expression of mRNAs of heme oxygenase-1 (HO-1) and c-jun by mediating the activation of activated protein-1 (AP-1) in rat livers. We have shown that phorone activates c-Jun N-terminal kinase (JNK), thus leading to c-Jun phosphorylation, and transactivation of AP-1 and HO-1 gene expression in the rat liver in response to oxidative stress. The in-gel kinase assay showed that phorone activated JNK1 predominantly in the rat liver nuclear extract. The JNK activation by phorone was slightly observed at 1 hr after administration and gradually increased with time. Ser73-phosphorylation of c-Jun catalyzed by JNK was significantly altered by changing hepatic GSH levels based on the results observed by the combined injection of buthionine sulfoximine (BSO) or GSH isopropyl ester (GIP) with phorone. Namely, BSO, an inhibitor of GSH biosynthesis, enhanced phorone-mediated c-Jun phosphorylation as well as AP-1 binding activity. However, GSH isopropyl ester prevented GSH depletion and abolished both c-Jun phosphorylation and the activation of AP-1 binding evoked by phorone. GSH isopropyl ester also suppressed phorone-produced HO-1 and c-jun gene expressions to 25 and 30% of the induced level. Perfluorodecanoic acid (PFDA) reduced GSH S-transferase activity, prevented phorone-mediated GSH depletion and abolished either HO-1 or c-jun mRNA induction by phorone. These results indicated that oxidative stress under GSH depletion produced by phorone could activate preferentially JNK and lead to the transcriptional activation of AP-1 and consequently to HO-1 gene expression. This study suggests that JNK activation could be one of the major signaling pathways to transmit intracellular events to the nuclei during oxidative stress via GSH depletion by phorone in rat livers.
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PMID:The expression of heme oxygenase-1 gene responded to oxidative stress produced by phorone, a glutathione depletor, in the rat liver; the relevance to activation of c-jun n-terminal kinase. 980 9

In the present study, we studied the signal transduction mechanism that is involved in the expression of c-Jun protein evident after exposure of rat liver epithelial RL34 cells to the major end product of oxidized fatty acid metabolism, 4-hydroxy-2-nonenal (HNE). HNE treatment of the cells resulted in depletion of intracellular glutathione (GSH) and in the formation of protein-bound HNE in plasma membrane. In addition, HNE strongly induced intracellular peroxide production, suggesting that HNE exerted oxidative stress on the cells. Potent expression of c-Jun occurred within 30 min of HNE treatment, which was accompanied by a time-dependent increase in activator protein-1 (AP-1) DNA binding activity. We found that HNE caused an immediate increase in tyrosine phosphorylation in RL34 cells. In addition, HNE strongly induced phosphorylation of c-Jun N-terminal kinases (JNK) and p38 mitogen-activated protein kinases and also moderately induced phosphorylation of extracellular signal-regulated kinases. The phosphorylation of JNK was accompanied by a rapid and transient increase in JNK and p38 activities, whereas changes in the activity of extracellular signal-regulated kinase were scarcely observed. GSH depletion by L-buthionine-S, R-sulfoximine, a specific inhibitor of GSH biosynthesis, only slightly enhanced peroxide production and JNK activation, suggesting that HNE exerted these effects independent of GSH depletion. This and the findings that (i) HNE strongly induced intracellular peroxide production, (ii) HNE-induced JNK activation was inhibited by pretreatment of the cells with a thiol antioxidant, N-acetylcysteine, and (iii) H2O2 significantly activated JNK support the hypothesis that pro-oxidants play a crucial role in the HNE-induced activation of stress signaling pathways. In addition, we found that, among the inhibitors of tyrosine kinases, cyclooxygenase, and Ca2+ influx, only quercetin exerted a significant inhibitory effect on HNE-induced JNK activation. In light of the JNK-dependent induction of c-jun transcription and the AP-1-induced transcription of xenobiotic-metabolizing enzymes, these data may show a potential critical role for JNK in the induction of a cellular defense program against toxic products generated from lipid peroxidation.
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PMID:Activation of stress signaling pathways by the end product of lipid peroxidation. 4-hydroxy-2-nonenal is a potential inducer of intracellular peroxide production. 989 Sep 86

Glutathione (GSH) is an important physiological antioxidant in lung epithelial cells and lung lining fluid. We studied the regulation of GSH synthesis in response to the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) and the anti-inflammatory agent dexamethasone in human alveolar epithelial cells (A549). TNF-alpha (10 ng/ml) exposure increased GSH levels, concomitant with a significant increase in gamma-glutamylcysteine synthetase (gamma-GCS) activity and the expression of gamma-GCS heavy subunit (gamma-GCS-HS) mRNA at 24 h. Treatment with TNF-alpha also increased chloramphenicol acetyltransferase (CAT) activity of a gamma-GCS-HS 5'-flanking region reporter construct, transfected into alveolar epithelial cells. Mutation of the putative proximal AP-1-binding site (-269 to -263 base pairs), abolished TNF-alpha-mediated activation of the promoter. Gel shift and supershift analysis showed that TNF-alpha increased AP-1 DNA binding which was predominantly formed by dimers of c-Jun. Dexamethasone (3 microM) produced a significant decrease in the levels of GSH, decreased gamma-GCS activity and gamma-GCS-HS mRNA expression at 24 h. The increase in GSH levels, gamma-GCS-HS mRNA, gamma-GCS-HS promoter activity, and AP-1 DNA binding produced by TNF-alpha were abrogated by co-treating the cells with dexamethasone. Thus these data demonstrate that TNF-alpha and dexamethasone modulate GSH levels and gamma-GCS-HS mRNA expression by their effects on AP-1 (c-Jun homodimer). These data have implications for the oxidant/antioxidant balance in inflammatory lung diseases.
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PMID:Molecular mechanism of the regulation of glutathione synthesis by tumor necrosis factor-alpha and dexamethasone in human alveolar epithelial cells. 998 57

We previously reported that antisense c-jun suppressed apoptosis induced by serum deprivation in F-MEL cells. To elucidate the molecular mechanisms responsible for this suppression of apoptosis we investigated the activities and protein expression of antioxidant materials in the cell under serum deprivation. In the parental F-MEL cells enzyme activities of catalase, glutathione S-transferase (GST), and glutathione peroxidase (GPx) increased to reach the maximum at 24-72 h after removal of serum and then decreased to initial levels or a little less. Superoxide dismutase (SOD) maintained the initial level for 72 h and increased 1.5- to 2-fold at 96 h. Glutathione (GSH) levels increased at 24 h and then dropped significantly to one-third the initial level. On the other hand, in c-junAS (+) cells, in which antisense c-jun was expressed and c-Jun protein expression was reduced to undetectable level. We found 1.9-, 2.7-, 4.8-, and 15. 8-fold increase in the activities of catalase, GST, SOD, and GPx, respectively, at 96 h. GSH maintained almost the same level as the initial. Enhancement of these enzyme activities in c-junAS (+) cells was induced under serum deprivation. Western blottings for catalase, GST, and SOD also showed enhanced increase in protein expression, supporting the increase in enzyme activities. Cellular peroxide level under serum deprivation was monitored by flow cytometry using DCFH-DA as a probe. We found that the peroxide level increased at 24 h and then decreased at 72 and 96 h in c-junAS (+) cells, and reduction of the peroxide level coincided with an increase in antioxidant enzyme activities. These results indicate that antioxidant materials such as catalase, GST, SOD, GPx, and GSH are induced by serum deprivation when c-jun expression is inhibited in F-MEL cells. The link between inhibition of c-jun expression and enhancement of cellular antioxidant defense is discussed.
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PMID:Inhibition of c-Jun expression induces antioxidant enzymes under serum deprivation. 1066 16

Glutathione S-transferases (GSTs, EC 2.5.1.18) belong to a large family of functionally different enzymes that catalyze the S-conjugation of glutathione with a wide variety of electrophilic compounds including carcinogens and anticancer drugs. Drug resistance may result from reduction in apoptosis of neoplastic cells when exposed to antineoplastic drugs. The c-Jun N-terminal Kinase (JNK) belongs to the family of stress kinases and has been shown to be required for the maximal induction of apoptosis by DNA-damaging agents. Recently, an inhibition of JNK activity by GST P1-1, which was reversed by polymerization induced by oxidative stress, has been reported in 3T3-4A mouse fibroblast cell lines. The finding that GST P1-1 might inhibit JNK activity and that it is frequently highly expressed in tumor tissues suggests its possible implication in "apoptosis resistance" during antineoplastic therapy. We investigated the modulation of GST P1-1 during apoptosis in a neoplastic T-cell line (Jurkat) induced by hydrogen peroxide and etoposide. Apoptosis was paralleled by the appearance of a dimeric form of GST P1-1 on western blotting, associated with an increase in the Km(GSH) and a reduction in GST P1-1 specific activity toward 1-chloro-2,4-dinitrobenzene, which reached statistical significance only in H(2)O(2)-treated cells. Our data seem to suggest that H(2)O(2) and etoposide may partly act through a process of partial inactivation of the GST P1-1, possibly involving the "G" site in the process of dimerization, and thus favoring programmed cell death.
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PMID:Modulation of GST P1-1 activity by polymerization during apoptosis. 1077 20

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