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)

The CCAAT-binding factor NF-Y (CBF/CP1) is a heteromeric transcription factor involved in the regulation of a variety of eukaryotic genes. We identified NF-Y as the CCAAT activity binding to the promoter region of the gene coding for the 28-kDa glutathione S-transferase of the human parasite Schistosoma mansoni (Sm28GST). We isolated the NF-YA cDNA from S. mansoni (SmNF-YA): the complete 268 amino acid sequence harbors a region in its C-terminal part that shows homology with the subunit interaction and DNA-binding domains of the mammalian NF-YA; the N-terminal region has an amino acid composition reminiscent of the mammalian and echinoderm counterparts, rich in glutamine and hydrophobic residues, but shows no sequence similarity at the primary level. In vitro synthesized SMNF-YA is able to associate with mammalian NF-YB/C subunits in the absence of DNA and to bind to the Sm28GST CCAAT box. Surprisingly, a monoclonal antibody directed against the non-conserved Q-rich activation domain of mammalian NF-YA supershifts and immunoprecipitates SMNF-YA, strongly suggesting structure conservation in the activation domain between divergent species.
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PMID:Cloning of Schistosoma mansoni transcription factor NF-YA subunit: phylogenic conservation of the HAP-2 homology domain. 881 62

The murine grg (Groucho-related gene) products are believed to interact with transcription factors and repress transcription, thereby regulating cell proliferation and differentiation. Most proteins in the grg family contain all of the domains found in the Drosophila Groucho protein, including the S/P (Ser-Pro-rich) domain required for interaction with transcription factors and the WD40 domain, which is thought to interact with other proteins. However, at least two Grg proteins contain only the amino-terminal Q (glutamine-rich) domain. We examined whether the Q domain is used for dimerization between Grg proteins, using the yeast two-hybrid system and binding assays with glutathione S-transferase fusion proteins. We found that Grg proteins are able to dimerize through the Q domain and that dimerization requires a core of 50 amino acids. Surprisingly, the dimerization does not require the leucine zipper located within the Q domain.
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PMID:Products of the grg (Groucho-related gene) family can dimerize through the amino-terminal Q domain. 895 48

Five neurodegenerative diseases are caused by proteins with expanded polyglutamine domains. Toxicity of these proteins has been previously identified only in mammals, and no simple model systems are available. In this paper, we demonstrate in E. coli that long polyglutamine domains (59-81 residues) as GST-fusion proteins inhibit growth while smaller glutamine (10-35 residues) or polyalanine (61 residues) domains have no effect. Analogously in humans, polyglutamine repeats less than 35-40 glutamines produce a normal phenotype, while expansion greater than 40 glutamines is always associated with disease. Expression of polyglutamine proteins in E. coli may help identify the molecular mechanism of pathogenesis of CAG trinucleotide repeat diseases and be a useful screen to identify potential therapeutic compound.
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PMID:Toxicity of expanded polyglutamine-domain proteins in Escherichia coli. 898 Jan 37

The Sulfolobus solfataricus, strain MT4, beta-glycosidase (Ss beta-gly) is a thermophilic member of glycohydrolase family 1. To identify active-site residues, glutamic acids 206 and 387 have been changed to isosteric glutamine by site-directed mutagenesis. Mutant proteins have been purified to homogeneity using the Schistosoma japonicum glutathione S-transferase (GST) fusion system. The proteolytic cleavage of the chimeric protein with thrombin was only obtainable after the introduction of a molecular spacer between the GST and the Ss beta-gly domains. The Glu387-->Gln mutant showed no detectable activity, as expected for the residue acting as the nucleophile of the reaction. The Glu206-->Gln mutant showed 10- and 60-fold reduced activities on aryl-galacto and aryl-glucosides, respectively, when compared with the wild type. Moreover, a significant Km decrease with p/o-nitrophenyl-beta-D-glucoside was observed. The residual activity of the Glu206-->Gln mutant lost the typical pH dependence shown by the wild type. These data suggest that Glu206 acts as the general acid/base catalyst in the hydrolysis reaction.
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PMID:Identification of two glutamic acid residues essential for catalysis in the beta-glycosidase from the thermoacidophilic archaeon Sulfolobus solfataricus. 901 Sep 32

Elongation factor 3 (EF-3) is an essential requirement of the fungi for translational elongation. EF-3 is an ATPase, and the hydrolytic activity is stimulated 2 orders of magnitude by yeast ribosomes. Limited trypsinolysis of EF-3 results in the cleavage of a single peptide bond between residues 774 (Arg) and 775 (Gln), generating polypeptides of approximate molecular mass 90 and 30 kDa. The 90-kDa fragment is relatively resistant to proteolysis and retains ribosome-independent ATPase activity. The 30-kDa fragment is further proteolyzed into smaller fragments and retains the specificity for binding to yeast ribosomes. Both the intact EF-3 and the 30-kDa fragment are protected from proteolysis by yeast ribosomes. EF-3 is NH2 terminally blocked, and so is the 90-kDa fragment. The COOH terminally derived 30-kDa fragment contains glutamine (residue 775) at the NH2-terminal end. A construct was designed representing the COOH-terminal domain of EF-3 (30-kDa fragment), subcloned, and expressed as a glutathione S-transferase fusion in yeast. The glutathione S-transferase-30-kDa peptide remains stringently associated with ribosomes. Isolated fusion peptide rebinds to yeast ribosomes with high affinity. Based on these results, we propose that at least one of the ribosome-binding sites of EF-3 resides at the COOH-terminal end of the protein.
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PMID:Functional subdomains of yeast elongation factor 3. Localization of ribosome-binding domain. 904 59

We have expressed, purified, and analyzed the iron-containing superoxide dismutase (FeSOD) of Escherichia coli with mutations directed at tyrosine position 34 to introduce phenylalanine (SODY34F), serine (SODY34S), or cysteine (SODY34C). FeSOD and mutant enzymes were purified from SOD-deficient cells using a GST-FeSOD fusion protein intermediate which was subsequently cleaved with thrombin and repurified. Specific activities were measured using the xanthine-xanthine oxidase method and gave 3148 u/mg for wild-type FeSOD. The SODY34S mutation virtually inactivates the enzyme (42 u/mg); mutation to cysteine greatly reduces activity (563 u/mg), but the SODY34F mutant retains nearly 40% of the activity of wild type (1205 u/mg). Fusion protein intermediates were also shown to be active and were demonstrated to protect SOD-deficient E. coli cells from the induced effects of oxidative stress, with growth rates directly proportional to the specific activities of the expressed mutant enzymes. SODY34F exhibited decreased thermal stability, reduced activity at high pH, and a pronounced increase in sensitivity to the inhibitor sodium azide compared with wild-type FeSOD. These results suggest that tyrosine at position 34 is multifunctional and plays a structural role (probably through hydrogen bonding to glutamine at position 69) in maintaining the integrity of the active site, a stabilizing role at high pH, and a steric role in obstructing access to the active site of both substrate and inhibitor molecules.
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PMID:The conserved residue tyrosine 34 is essential for maximal activity of iron-superoxide dismutase from Escherichia coli. 912 14

Previously we demonstrated that nonvisual arrestins exhibit a high affinity interaction with clathrin, consistent with an adaptor function in the internalization of G protein-coupled receptors (Goodman, O. B., Jr., Krupnick, J. G., Santini, F., Gurevich, V. V., Penn, R. B., Gagnon, A. W., Keen, J. H., and Benovic, J. L. (1996) Nature 383, 447-450). In this report we show that a short sequence of highly conserved residues within the globular clathrin terminal domain is responsible for arrestin binding. Limited proteolysis of clathrin cages results in the release of terminal domains and concomitant abrogation of arrestin binding. The nonvisual arrestins, beta-arrestin and arrestin3, but not visual arrestin, bind specifically to a glutathione S-transferase-clathrin terminal domain fusion protein. Deletion analysis and alanine scanning mutagenesis localize the binding site to residues 89-100 of the clathrin heavy chain and indicate that residues 1-100 can function as an independent arrestin binding domain. Site-directed mutagenesis identifies an invariant glutamine (Glu-89) and two highly conserved lysines (Lys-96 and Lys-98) as residues critical for arrestin binding, complementing hydrophobic and acidic residues in arrestin3 which have been implicated in clathrin binding (Krupnick, J. G., Goodman, O. B., Jr., Keen, J. H., and Benovic, J. L. (1997) J. Biol. Chem. 272, 15011-15016). Despite exhibiting high affinity clathrin binding, arrestins do not induce coat assembly. The terminal domain is oriented toward the plasma membrane in coated pits, and its binding of both arrestins and AP-2 suggests that this domain is the anchor responsible for adaptor-receptor recruitment to the coated pit.
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PMID:Arrestin/clathrin interaction. Localization of the arrestin binding locus to the clathrin terminal domain. 916 77

The Drosophila nucleosome remodeling factor NURF utilizes the energy of ATP hydrolysis to perturb the structure of nucleosomes and facilitate binding of transcription factors. The ATPase activity of purified NURF is stimulated significantly more by nucleosomes than by naked DNA or histones alone, suggesting that NURF is able to recognize specific features of the nucleosome. Here, we show that the interaction between NURF and nucleosomes is impaired by proteolytic removal of the N-terminal histone tails and by chemical cross-linking of nucleosomal histones. The ATPase activity of NURF is also competitively inhibited by each of the four Drosophila histone tails expressed as GST fusion proteins. A similar inhibition is observed for a histone H4 tail substituted with glutamine at four conserved, acetylatable lysines. These findings indicate a novel role for the flexible histone tails in chromatin remodeling by NURF, and this role may, in part, be independent of histone acetylation.
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PMID:Role of histone tails in nucleosome remodeling by Drosophila NURF. 930 16

Tissue transglutaminase (TGase II) catalyzes the posttranslational modification of proteins by transamidation of available glutamine residues and is also a guanosinetriphosphatase (GTPase) and adenosinetriphosphatase (ATPase). Based on its homology with factor XIIIA, an extracellular transglutaminase, the structure of TGase II is likely composed of an N-terminal beta-sandwich domain, an alpha/beta catalytic core, and two C-terminally located beta-barrels. Here we used a domain-deletion approach to identify the GTP and ATP hydrolytic domains of TGase II. Full-length TGase II and two domain-deletion mutants, one retaining the N-terminal beta-sandwich and core domains (betaSCore) and the other retaining only the core domain, were expressed as glutathione S-transferase (GST) fusion proteins and purified. GST-Full and GST-betaSCore exhibited calcium-dependent TGase activity, whereas GST-Core had no detectable TGase activity, indicating the beta-sandwich domain is required for TGase activity but the C-terminal beta-barrels are not. All three GST-TGase II fusion proteins were photoaffinity-labeled with [alpha-32P]-8-azidoGTP and were able to bind GTP-agarose. The GTPase activity of GST-betaSCore was equivalent to that of GST-Full, whereas the ATPase activity was approximately 40% higher than GST-Full. GST-Core had approximately 50% higher GTPase activity and approximately 75% higher ATPase activity than GST-Full. The GTPase and ATPase activities of each of the GST-TGase II fusion proteins were inhibited in a dose-dependent manner by both GTPgammaS and ATPgammaS. These results demonstrate that the GTP and ATP hydrolysis sites are localized within the core domain of TGase II and that neither the N-terminal beta-sandwich domain nor the C-terminal beta-barrels are required for either GTP or ATP hydrolysis. Taken together with previous work [Singh, U. S., Erickson, J. W., & Cerione, R. A. (1995) Biochemistry 34, 15863-15871; Lai, T.-S., Slaughter, T. F., Koropchak, C. M., Haroon, Z. A., & Greenberg, C. S. (1996) J. Biol. Chem. 271, 31191-31195] the results of this study indicate that the GTP and ATP hydrolysis sites are localized to a 5. 5 kDa (47 amino acid) region at the start of the core domain.
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PMID:The core domain of the tissue transglutaminase Gh hydrolyzes GTP and ATP. 930 55

A 1.7-kilobase pair segment from the conjugative transfer region of plasmid R388 DNA was cloned and sequenced. It contained trwD, a gene essential for plasmid R388 conjugation, for expression of the conjugative W-pilus and for sensitivity to phage PRD1. The deduced amino acid sequence of TrwD showed homology to the PulE/VirB11 superfamily of potential ATPases involved in various types of transport processes. A fusion of trwD with the glutathione S-transferase (GST) was constructed, and the resulting fusion protein was purified from overproducing bacteria. Factor Xa hydrolysis of GST-TrwD and further purification rendered TrwD protein with more than 95% purity. Antibodies raised against TrwD localized it both in the soluble fraction and in the outer membrane of Escherichia coli. TrwD is probably a peripheral outer membrane protein because it could be solubilized by increasing salt concentration to 0.5 M NaCl in the lysis buffer. Both purified GST-TrwD and TrwD could hydrolize ATP. ATPase activity increased 2-fold in the presence of detergent-phospholipid mixed micelles. To study the importance of the nucleotide-binding site, Walker box A (GXXGXGK(T/S)), present in TrwD, the conserved lysine residue was replaced by glutamine. The mutant protein, expressed and purified under the same conditions as the wild type, did not exhibit ATPase activity. TrwD(K203Q) was not able to complement the mutation in trwD of the R388 mutant plasmid, suggesting the essentiality of the ATPase activity of the protein in the conjugative process. Furthermore, the dominant character of this mutation suggested that GST-TrwD(K432Q) was still able to interact either with itself or with other component(s) of the conjugative machinery.
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PMID:TrwD, a protein encoded by the IncW plasmid R388, displays an ATP hydrolase activity essential for bacterial conjugation. 932 77


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