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)

Expression of cloned genes in prokaryotes such as Escherichia coli is a widely used technique in both basic research and biotechnology. Despite the availability of several E. coli expression vector systems, adequate levels of expression may not be achieved. Expressing proteins as fusions to the highly conserved eukaryotic protein ubiquitin has been reported by several investigators to enhance protein yield in both bacterial and eukaryotic systems. We have modified this technique by the co-expression in E. coli of a ubiquitin-fusion protein and the Saccharomyces cerevisiae ubiquitin-specific protease Ubp2. This allows the co-translational cleavage of engineered ubiquitin-fusion proteins expressed in E. coli. This system was used to express a human Pi class glutathione S-transferase (GST) GSTP1 as well as two mutant GSTP1 derivatives, Trp39-->Cys and Gln52-->Glu. The yield of these enzymes was improved 40-fold by using the ubiquitin-fusion/co-translational cleavage technique, and no uncleaved product was detected. The Trp39-->Cys mutant was totally devoid of GST activity, while the activity of the Gln52-->Glu mutant was reduced to 6% of wild-type GSTP1-1. As both of the mutated residues map within the glutathione-binding site, the reduced GST activity is consistent with a marked reduction in glutathione binding ability.
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PMID:Protein expression using cotranslational fusion and cleavage of ubiquitin. Mutagenesis of the glutathione-binding site of human Pi class glutathione S-transferase. 792 35

Transformation by the human papillomavirus (HPV) early gene products, E6 and E7, involves their interaction with cellular proteins p53 and Rb. Using glutathione S-transferase (GST) fusion proteins, we found that HPV E6 bound human p53 and that the relative efficiency of binding varied such that the GST-HPV type 16 E6 (16E6) protein bound p53 with highest affinity, followed by GST-31E6, GST-18E6, and GST-11E6. The GST-E6 fusion proteins were sufficient for binding p53 purified from a baculovirus expression system as well as in vitro translation sources, while no association was observed with GST-18E7 or a GST-16E6 mutant bearing a five-amino-acid deletion in E6. When the site-specific DNA binding activity of p53 was examined in the presence of GST-E6 proteins, an inhibition of DNA binding was observed. The degree of inhibition correlated with the relative affinity of different E6 proteins for p53; thus, GST-16E6 was the most potent inhibitor of p53 DNA binding activity, and GST-11E6 was the least effective. Prevention of p53 DNA binding is likely to play a role in the abrogation of the transcriptional activity of p53 by HPV E6 and provides a further mechanism for E6 disruption of p53 growth suppressor function in addition to its role in directing specific degradation of p53 through the ubiquitin-mediated pathway. The variation in inhibition of DNA binding seen with the various E6 proteins may thus contribute to the differences in oncogenic potential seen among the HPV types.
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PMID:Inhibition of p53 DNA binding by human papillomavirus E6 proteins. 820 1

Cytokines regulate cell growth by inducing the expression of specific target genes. Using the differential display method, we have cloned a cytokine-inducible immediate early gene, DUB-1 (for deubiquitinating enzyme). DUB-1 is related to members of the UBP superfamily of deubiquitinating enzymes, which includes the oncoprotein Tre-2. A glutathione S-transferase-DUB-1 fusion protein cleaved ubiquitin from a ubiquitin-beta-galactosidase protein. When a conserved cysteine residue of DUB-1, required for ubiquitin-specific thiol protease activity, was mutated to serine (C60S), deubiquitinating activity was abolished. Continuous expression of DUB-1 from a steroid-inducible promoter induced growth arrest in the G1 phase of the cell cycle. Cells arrested by DUB-1 expression remained viable and resumed proliferation upon steroid withdrawal. Our results suggest that DUB-1 regulates cellular growth by modulating either the ubiquitin-dependent proteolysis or the ubiquitination state of an unknown growth regulatory factor(s).
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PMID:DUB-1, a deubiquitinating enzyme with growth-suppressing activity. 862 27

A cDNA encoding the human Theta-class glutathione transferase GSTT2-2 was expressed in Escherichia coli as a ubiquitin fusion protein. The co-translational removal of the ubiquitin by a cloned ubiquitin-specific protease, Ubp1, generates enzymically active GSTT2-2 without any additional N-terminal residues. The recombinant isoenzyme was purified to apparent homogeneity by DEAE anion-exchange, gel filtration, dye ligand chromatography and high resolution anion-exchange chromatography on Mono Q FPLC. The recombinant enzyme had significant activity with a range of substrates, including cumene hydroperoxide and 1-menapthyl sulphate. The activity of GSTT2-2 with a range of secondary lipid peroxidation products such as the trans,trans-alka-2,4-dienals and trans-alk-2-enals, as well as its glutathione peroxidase activity with organic hydroperoxides, suggest that it may play a significant role in protection against the products of lipid peroxidation.
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PMID:Purification and characterization of a recombinant human Theta-class glutathione transferase (GSTT2-2). 864 50

We report a novel human gene whose product specifically associates with the negative regulatory domain of the Wilms' tumor gene product (WT1) in a yeast two-hybrid screen and with WT1 in immunoprecipitation and glutathione S-transferase (GST) capture assays. The gene encodes a 17-kDa protein that has 56% amino acid sequence identity with yeast ubiquitin-conjugating enzyme (yUBC) 9, a protein required for cell cycle progression in yeast, and significant identity with other subfamilies of ubiquitin-conjugating enzymes. The human gene fully complements yeast that have a temperature-sensitive yUBC9 gene mutation to fully restore normal growth, indicating that we have cloned a functionally conserved human (h) homolog of yUBC9. Transcripts of hUBC9 of 4.4 kilobases (kb), 2.8 kb, and 1.3 kb were found in all human tissues tested. A single copy of the hUBC9 gene was found and localized to human chromosome 16p13.3. We conclude that hUBC9 retains striking structural and functional conservation with yUBC9 and suggest a possible link of the ubiquitin/proteosome proteolytic pathway and the WT1 transcriptional repressor system.
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PMID:Molecular cloning of the cDNA and chromosome localization of the gene for human ubiquitin-conjugating enzyme 9. 879 54

Poly(ADP-ribose) polymerase (PARP) has been suggested to play a regulatory role in vivo, in DNA replication and/or DNA repair based mainly on its capacity to bind to DNA strand breaks. This interaction is modulated through auto poly(ADP-ribosylation). However, the biological function of PARP may also involve interactions with proteins such as topoisomerase I or DNA polymerase alpha, which may or may not be themselves ADP-ribosylated. Using the yeast two-hybrid method search for other proteins interacting with PARP, we have isolated a full-length cDNA clone coding for a protein of 158 amino acid residues. This amino acid sequence is 66 and 56% identical to yeast ubiquitin-conjugating enzymes Hus5 and Ubc9 of Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. Moreover, we have demonstrated that the expressed protein complements a S. cerevisiae yeast strain deficient for Ubc9. The protein encoded by the isolated cDNA is thus a new human counterpart of the ubiquitin-conjugating enzyme family and has been called hUbc9. The hubc9 gene locus has been assigned to the chromosomal location 16p13.2-p13.3. By means of two-hybrid analysis it was discovered that hUbc9 interacts with the automodification domain of PARP. This interaction was further confirmed using GST (glutathione-S-transferase) tagged fusion proteins: (i) in vivo, by transfecting cos7 cells with hUbc9 cloned in an eukaryotic expression vector, and (ii) in vitro, by mixing purified PARP with hUbc9 purified and expressed in bacteria. The possible significance and function of this interaction is discussed while taking into account the possible intracellular role of hUbc9.
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PMID:Poly(ADP-ribose) polymerase interacts with a novel human ubiquitin conjugating enzyme: hUbc9. 919 46

Individual members of the conserved family of ubiquitin conjugating enzymes (E2s) mediate the ubiquitination and turnover of specific substrates of the ubiquitin-dependent degradation pathway. E2 proteins have a highly conserved core domain of approximately 150 amino acids which contains the active-site Cys. Certain E2s have unique terminal extensions, which are thought to contribute to selective E2 function by interacting either with substrates or with trans-acting factors such as ubiquitin-protein ligases (E3s). We used the mammalian ubiquitin conjugating enzyme E2-25K in a biochemical test of this hypothesis. The properties of two truncated derivatives show that the 47-residue tail of E2-25K is necessary for three of the enzyme's characteristic properties: high activity in the synthesis of unanchored K48-linked polyubiquitin chains; resistance of the active-site Cys residue to alkylation; and an unusual discrimination against noncognate (nonmammalian) ubiquitin activating (E1) enzymes. However, the tail is not sufficient to generate these properties, as shown by the characteristics of a chimeric enzyme in which the tail of E2-25K was fused to the core domain of yeast UBC4. These and other results indicate that the specific biochemical function of the tail is strongly dependent upon unique features of the E2-25K core domain. Thus, divergent regions within the conserved core domains of E2 proteins may be highly significant for function. Expression of truncated E2-25K as a glutathione S-transferase (GST) fusion protein resulted in the apparent recovery of E2-25K-specific properties, including activity in chain synthesis. However, the catalytic mechanism utilized by the truncated fusion protein proved to be distinct from the mechanism utilized by the wild-type enzyme. The unexpected properties of the fusion protein were due to GST-induced dimerization. These results indicate the potential for self-association to modulate the polyubiquitin chain synthesis activities of E2 proteins, and indicate that caution should be applied in interpreting the activities of GST fusion proteins.
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PMID:Structure and function of ubiquitin conjugating enzyme E2-25K: the tail is a core-dependent activity element. 926 33

Ets transcription factors have Ets DNA binding domains, that have a winged helix-turn-helix structure. ETS-1, the founding member of the family, is regulated by the Ras and Ca2+ signaling pathways and is implicated in various physiological processes leading to cell growth, differentiation and apoptosis. We have identified ETS-1 interacting factors with a yeast two-hybrid screen. The majority of the positive clones turned out to encode the human homologue of the yeast ubiquitin-conjugating enzymes UBC9 and Hus5. In two different yeast assays, ETS-1 interacted with huUBC9. In an in vitro GST 'pull-down' assay, ETS-1 and several other Ets family members complexed with huUBC9. Interestingly, in mammalian cells, coexpression of huUBC9 resulted in a substantial increase in the transcriptional activity of ETS-1. Coexpressed huUBC9 did not affect the ETS-1 protein level, and moreover, a point mutation at Cys93, an amino acid known to be essential for ubiquitination, did not abolish the stimulation of the ETS-1 transcriptional activity. Our results indicate that the modulation of ETS-1 activity by huUBC9 results from processes other than ubiquitination and ETS-1 stabilization.
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PMID:Modulation of ETS-1 transcriptional activity by huUBC9, a ubiquitin-conjugating enzyme. 933 25

Sentrin is a ubiquitin-like molecule that has been shown to interact with the death domains of Fas and tumor necrosis factor receptor 1 (TNFR1), PML, Rad51, Rad52, and RanGAP1. We have reported previously that sentrin can be conjugated to other proteins in a manner analogous to protein ubiquitination (Kamitani, T., Nguyen, H. P., and Yeh, E. T. H. (1997) J. Biol. Chem. 272, 14001-14004). Furthermore, the conserved C-terminal Gly-Gly residues are required for sentrinization to occur. To identify enzymes which play a role in sentrinization, the yeast two-hybrid system was used to screen a human placenta cDNA library using sentrin as bait. A strong positive interacting clone was found to contain a cDNA insert encoding the ubiquitin-conjugating enzyme, Ubc9. The interaction between sentrin and Ubc9 required the ubiquitin domain and the C-terminal Gly-Gly residues of sentrin. This interaction appears to be specific because sentrin could only interact weakly with UbcH5B, but could not interact with HHR6B, UbcH6 nor E2-EPF. In vitro translated sentrin could be precipitated by a GST-Ubc9 fusion protein, but not by glutathione S-transferase. A beta-mercaptoethanol-sensitive Ubc9-sentrin conjugate could also be identified in the in vitro binding assay. Substitution of the conserved cysteine residue of Ubc9 by serine abolished the formation of the Ubc9-sentrin conjugate. Taken together, Ubc9 is a strong candidate to be the key conjugating enzyme in the sentrinization pathway.
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PMID:Preferential interaction of sentrin with a ubiquitin-conjugating enzyme, Ubc9. 935 68

Neuronal precursor cell-expressed developmentally down-regulated 4 (Nedd4) is a ubiquitin protein ligase (E3) containing a hect domain, 3 or 4 WW domains, and a putative C2 domain. We have recently demonstrated an association between the WW domains of Nedd4 and the proline-rich PY motifs (XPPXY) of the epithelial Na+ channel, as well as with PY motifs of several other proteins. The role of the putative C2 domain of Nedd4 has not been elucidated. Here we show that Nedd4, endogenously expressed in Madin-Darby canine kidney cells, was redistributed from the cytosolic to the particulate fraction in response to ionomycin plus Ca2+ treatment. A similar treatment of polarized Madin-Darby canine kidney cells led to an apical and lateral membrane localization of Nedd4, as determined by immunostaining and confocal microscopy. The C2 domain of Nedd4, expressed as a glutathione S-transferase (GST) fusion protein, was sufficient to bind cellular membranes in a Ca2+-dependent manner. Moreover, this GST-Nedd4-C2 domain was able to mediate Ca2+-dependent interactions with phosphatidylserine, phosphatidylinositol, and phosphatidylcholine liposomes in vitro. An epitope-tagged Nedd4 lacking its C2 domain and stably expressed in Madin-Darby canine kidney cells failed to mediate the Ca2+-induced plasma membrane localization seen in wild-type (epitope-tagged) Nedd4. These results indicate that the putative C2 domain of Nedd4 acts as a bona fide C2 domain which binds phospholipids and membranes in a Ca2+-dependent fashion and is involved in localizing the protein primarily to the apical region of polarized epithelial cells in response to Ca2+.
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PMID:The C2 domain of the ubiquitin protein ligase Nedd4 mediates Ca2+-dependent plasma membrane localization. 940 40


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