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)

To assess the possibility that two conserved amino acids (glutamine 90 and asparagine 137) in O6-methylguanine-DNA methyltransferase (MGMT) are involved in protein-substrate contact and/or discrimination between favored and non-favored substrates, families of proteins mutant at these two sites were expressed in alkyltransferase-deficient bacteria and analyzed for stability, ability to repair O6-methylguanine (MG)-containing DNA, and ability to differentially repair a preferred (MG-containing DNA) versus a non-preferred (free base MG) substrate. All seven proteins mutant at glutamine 90 (except a proline mutant) were stable in bacteria and repaired MG-containing DNA (> 50% of wild-type levels). A representative glutamine 90 mutant protein was not, however, significantly different from the wild-type protein in the preferential repair of MG-containing DNA versus MG free base. Of eight proteins mutant at asparagine 137, only glutamine and serine mutants repaired MG-containing DNA to any degree (8.5% and 0.8% of wild-type respectively) and only the glutamine mutant protein was detectable in bacterial sonicates by Western blot analysis. Alanine and leucine mutant alkyltransferases, inactive and unstable as non-fusion proteins, could, however, be stably expressed in bacteria as glutathione S-transferase fusion proteins, although the proteins were still inactive in repair. These results suggest that while glutamine 90 has no direct role in MG-DNA methyltransferase-mediated repair or free base/lesioned DNA substrate specificity, asparagine 137 is important in both the stability and activity of the protein and may contribute to the formation or function of the active site of the protein.
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PMID:The role of two conserved amino acids, glutamine 90 and asparagine 137, in O6-methylguanine-DNA methyltransferase stability, activity and substrate specificity. 792 83

The type III connecting segment (IIICS) within fibronectin is the major binding site for the integrin alpha 4 beta 1. Most integrin ligands have an essential acidic residue within their integrin binding site, in IIICS this residue is hypothesized to be the aspartic acid at position 21. Alanine scanning mutagenesis was used to determine the amino acid residues within the intact IIICS domain required for interaction with alpha 4 beta 1. IIICS was cloned and expressed as a fusion protein with glutathione S-transferase. This recombinant form of IIICS supports the adhesion of CHO cells that express human alpha 4 beta 1 in a cation dependent manner. Alanine scanning mutagenesis of the EILDVP sequence in recombinant IIICS demonstrated that only two of these residues are critical for adhesion of alpha 4 beta 1 expressing cells. Mutations of leucine at position 20 and aspartic acid at position 21 to alanine significantly reduced cell adhesion. Conservative mutations of aspartic acid at position 21 to asparagine or glutamic acid also reduced the ability of the recombinant protein to support cell adhesion, although not to the same extent as the corresponding alanine replacement. Most importantly, we show that although the mutation of asp 21 impairs cell adhesion, an examination of cell adhesion as a function of time demonstrated that asp 21 is not necessary for cell adhesion through alpha 4 beta 1. In comparison to wild type IIICS, the asp 21 to ala mutant supported minimal adhesion at early time points (10-30 min.), but was equivalent to wild type IIICS in supporting adhesion over one hour.
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PMID:The type III connecting segment of fibronectin contains an aspartic acid residue that regulates the rate of binding to integrin alpha 4 beta 1. 880 92

We have recently demonstrated that the nonvisual arrestins, beta-arrestin and arrestin3, interact with high affinity and stoichiometrically with clathrin, and we postulated that this interaction mediates 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 study, we localized the clathrin binding domain of arrestin3 using a variety of approaches. Truncation mutagenesis demonstrated that the COOH-terminal half of arrestin3 is required for clathrin interaction. Assessment of the clathrin binding properties of various glutathione S-transferase-arrestin3 fusion proteins indicated that the predominant clathrin binding domain is contained within residues 367-385. Alanine scanning mutagenesis further localized this domain to residues 371-379, and site-directed mutagenesis demonstrated the critical importance of both hydrophobic (Leu-373, Ile-374, and Phe-376) and acidic (Glu-375 and Glu-377) residues in the arrestin3/clathrin interaction. These results are complementary to the observation that hydrophobic and basic residues in clathrin are critical for its interaction with nonvisual arrestins (Goodman, O. B. , Jr., Krupnick, J. G., Gurevich, V. V., Benovic, J. L., and Keen, J. H. (1997) J. Biol. Chem. 272, 15017-15022). Lastly, an arrestin3 mutant in which Leu-373, Ile-374, and Phe-376 were mutated to Ala was significantly defective in its ability to promote beta2-adrenergic receptor internalization in COS-1 cells when compared with wild-type arrestin3. Taken together, these results implicate a discrete region of arrestin3 in high affinity binding to clathrin, an interaction critical for agonist-promoted internalization of the beta2-adrenergic receptor.
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PMID:Arrestin/clathrin interaction. Localization of the clathrin binding domain of nonvisual arrestins to the carboxy terminus. 916 76

Sequencing of polymerase chain reaction-amplified cDNAs from cultured cells of three patients with mevalonate kinase deficiency revealed a G --> A transversion at nucleotide 1000 of the coding region, converting alanine to threonine at position 334 (A334T). To characterize this defect, we expressed wild-type and mutant cDNAs in Escherichia coli as the glutathione S-transferase fusion proteins, with purification by affinity chromatography. SDS-polyacrylamide gel electrophoresis analysis for wild-type and mutant fusion proteins indicated an expected molecular mass of 42-43 kDa. Kinetic characterization of the wild-type fusion protein yielded Km values of 150 +/- 23 and 440 +/- 190 microM (mean +/- S.E.) for substrates (RS)-mevalonate and ATP, respectively. Expressed wild-type mevalonate kinase (MKase) had a maximum velocity of 13.6 +/- 1.4 units/mg of protein (n = 22, +/-S.E.), whereas the A334T mutation yielded an enzyme with average Vmax of 0.26 +/- 0.02 unit/mg of protein (n = 6, +/-S.E.), representing a decrease to 1.4% of control Vmax. Restriction digestion with HhaI, in conjunction with direct sequencing of cDNAs, revealed that two patients were homozygous and one heterozygous for the A334T allele, establishing autosomal recessive inheritance within families. Although the A334T enzyme had a normal Km for ATP of 680 +/- 226 microM (n = 3, +/-S.E.), the Michaelis constant for (RS)-mevalonate was increased >30-fold to 4623 +/- 1167 microM (n = 4, +/-S.E.) under standard assay conditions. Comparable kinetic results were obtained using extracts of lymphoblasts, which were homozygous for the A334T allele. Alanine 334 is invariant in MKase from bacteria to man and located in a glycine-rich region postulated to have homology with ATP-binding sequences. Our results indicate that the bacterial expression system for human MKase will provide a useful model system in which to analyze inherited mutations and identify the first active site residue in MKase associated with stabilization of mevalonate binding.
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PMID:Identification of an active site alanine in mevalonate kinase through characterization of a novel mutation in mevalonate kinase deficiency. 933 62

The Gcn4p activation domain contains seven clusters of hydrophobic residues that make additive contributions to transcriptional activation in vivo. We observed efficient binding of a glutathione S-transferase (GST)-Gcn4p fusion protein to components of three different coactivator complexes in Saccharomyces cerevisiae cell extracts, including subunits of transcription factor IID (TFIID) (yeast TAFII20 [yTAFII20], yTAFII60, and yTAFII90), the holoenzyme mediator (Srb2p, Srb4p, and Srb7p), and the Adap-Gcn5p complex (Ada2p and Ada3p). The binding to these coactivator subunits was completely dependent on the hydrophobic clusters in the Gcn4p activation domain. Alanine substitutions in single clusters led to moderate reductions in binding, double-cluster substitutions generally led to greater reductions in binding than the corresponding single-cluster mutations, and mutations in four or more clusters reduced binding to all of the coactivator proteins to background levels. The additive effects of these mutations on binding of coactivator proteins correlated with their cumulative effects on transcriptional activation by Gcn4p in vivo, particularly with Ada3p, suggesting that recruitment of these coactivator complexes to the promoter is a cardinal function of the Gcn4p activation domain. As judged by immunoprecipitation analysis, components of the mediator were not associated with constituents of TFIID and Adap-Gcn5p in the extracts, implying that GST-Gcn4p interacted with the mediator independently of these other coactivators. Unexpectedly, a proportion of Ada2p coimmunoprecipitated with yTAFII90, and the yTAFII20, -60, and -90 proteins were coimmunoprecipitated with Ada3p, revealing a stable interaction between components of TFIID and the Adap-Gcn5p complex. Because GST-Gcn4p did not bind specifically to highly purified TFIID, Gcn4p may interact with TFIID via the Adap-Gcn5p complex or some other adapter proteins. The ability of Gcn4p to interact with several distinct coactivator complexes that are physically and genetically linked to TATA box-binding protein can provide an explanation for the observation that yTAFII proteins are dispensable for activation by Gcn4p in vivo.
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PMID:The Gcn4p activation domain interacts specifically in vitro with RNA polymerase II holoenzyme, TFIID, and the Adap-Gcn5p coactivator complex. 948 88

Cyclin-dependent kinase 4 (CDK4) is a key molecule in the regulation of cell cycle progression at the G1-S phase restriction point. Its activity is specifically regulated by p16 (also known as p16/CDKN2A, p16(INK4a), and MTS1), a tumor suppressor frequently altered in human cancers. A specific mutation in CDK4 codon 24 (Arginine to Cysteine) prevents p16 binding and thus inhibition by p16. This mutated CDK4 acts as a dominant oncogene and has been found in both sporadic and familial melanoma. To study the effects of other mutations in CDK4, we generated a panel of 18 CDK4 mutants using Charged-to-Alanine scanning mutagenesis, and investigated the p16-binding capacity of these mutants to identify novel sites involved in p16 binding. The mutant CDK4 proteins were generated by direct coupled transcription-translation in vitro and tested for binding to p16 using a p16-GST fusion protein. Several mutants demonstrated loss of p16 binding. In addition to the previously identified codon 24 mutants, alterations in and around codon 22, 25, 97, and 281 all showed loss of p16 binding capacity. These results indicate that several noncontiguous amino acid sequences on CDK4 are required for binding to p16, which suggests the existence of multiple sites of interaction with p16. Since p16-binding deficient CDK4 has oncogenic potential, these mutations may be present in melanomas or other human neoplasms.
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PMID:Several noncontiguous domains of CDK4 are involved in binding to the P16 tumor suppressor protein. 971 35

Activation of transcription at bacteriophage T4 late promoters and coupling of late transcription to concurrent replication requires a peculiar transcriptional activator, the gp45 sliding clamp of the T4 DNA polymerase. In order to activate transcription, the topologically DNA-linked trimeric gp45 must interact with two T4-encoded RNA polymerase-binding proteins, the gp33 co-activator, and the gp55 late sigma factor. The carboxy termini of gp55 and gp33 share a similar sequence, which has been shown to be required for response of late transcription to activation by gp45. Alanine-scanning mutagenesis of the C terminus of gp55 shows that residues within the short hydrophobic sequence L(D/A)FLYE, are necessary for gp55 to bind to gp45, and to respond maximally to transcriptional activation by gp45. When fused to GST, the peptide SLDFLYE suffices for specific gp45 binding. Thus, it constitutes the main gp55 epitope for gp45 interaction.
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PMID:Activator-sigma interaction: A hydrophobic segment mediates the interaction of a sigma family promoter recognition protein with a sliding clamp transcription activator. 981 12

Platelet-derived growth factor beta receptor (PDGFbetaR) is a transmembrane receptor tyrosine kinase involved in a variety of cellular functions. We have generated a constitutively activated murine PDGFbetaR containing a valine to alanine substitution at residue 536, located in the cytoplasmic juxtamembrane domain. When this mutant receptor (PR-V536A) was expressed in Ba/F3 cells, it allowed the cells to survive and proliferate in the absence of IL-3 or PDGF, and tyrosine phosphorylation of PR-V536A was increased markedly compared with that of the wild-type PDGFbetaR in the absence of ligand and similar to that observed in ligand-activated PDGFbetaR. PR-V536A displayed increased tyrosine kinase activity in vitro toward an exogenous substrate, and the tyrosine kinase activity of the receptor was required for the constitutive activation of the mutant. This valine to alanine substitution also activated a PDGFbetaR mutant unable to bind PDGF. Alanine substitutions at positions homologous to V536 of the murine PDGFbetaR also activated other members of the PDGF receptor subfamily. The amino acid sequence of this region revealed a strong similarity to WW domains present in other signal transduction proteins. Furthermore, GST fusion proteins containing the juxtamembrane region of the PDGFR specifically associated with peptides containing the WW domain consensus recognition sequence PPXY. The results suggest that the cytoplasmic juxtamembrane domain plays a role in the regulation of receptor activity and function, perhaps by participating in protein-protein interactions.
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PMID:A single amino acid substitution in a WW-like domain of diverse members of the PDGF receptor subfamily of tyrosine kinases causes constitutive receptor activation. 984 97

Alternative splicing of the fibronectin gene transcript gives rise to a group of adhesive glycoproteins showing restricted spatial and temporal expression during embryonic development, tumor growth, and tissue repair. Alternative splicing occurs in three segments termed EIIIB, EIIIA, and V. The EIIIA (or ED-A) segment of fibronectin is expressed prominently but transiently in healing wounds coincident with fibroblast expression of an activation marker, smooth muscle cell alpha-actin. A monoclonal antibody (IST-9) to the EIIIA segment blocks transforming growth factor-beta-mediated smooth muscle cell alpha-actin expression by fibroblasts in culture. A second monoclonal antibody (DH1) blocks chondrocyte condensation in chicken embryos. We find that IST-9 and DH1 react with human, rat, and chicken but not with mouse or frog EIIIA, suggesting that His44 may be important for antibody binding. A series of deletion mutants of rat EIIIA, constructed as glutathione S-transferase fusion proteins, do not react with either IST-9, DH1, or a third monoclonal antibody (3E2). Mutations of pairs of amino acids to alanine have little effect, except for either (Val34Thr35) or (Tyr36Ser37), which are located in a beta strand upstream from His44. For these double mutants, the binding to all three monoclonal antibodies is markedly reduced. By contrast, single mutants at Thr35, Tyr36, or Ser37 retain full activity, suggesting that the epitope for these antibodies is determined in part by conformation. Alanine-scanning mutagenesis of rat EIIIA demonstrates the importance of Ile43 and His44 for binding. Mutation of frog EIIIA (normally Val43Lys44) to rat (Ile43His44) is sufficient to restore fully IST-9 binding and much of the activity of DH1 and 3E2. Our findings demonstrate that the function-blocking antibodies, IST-9 and DH1, bind to the Ile43 and His44 residues in a conformationally dependent fashion, implicating the loop region encompassing both residues as critical for mediating EIIIA function.
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PMID:Identification of two amino acids within the EIIIA (ED-A) segment of fibronectin constituting the epitope for two function-blocking monoclonal antibodies. 1036 33

Phosphorylation of the 20-kDa regulatory light chain of myosin catalyzed by a Ca(2+)/calmodulin-dependent myosin light chain kinase is important in the initiation of smooth muscle contraction and other contractile processes in non-muscle cells. It has been previously shown that residues 1-142 of smooth muscle myosin light chain kinase are necessary for high-affinity binding to actin-containing filaments in cells (1). To further localize the region of the kinase required for binding, a series of N-terminal deletion mutants as well as several N-terminal glutathione S-transferase fusion proteins were constructed. Cosedimentation assays showed that a peptide containing residues 1-75 binds to purified smooth muscle myofilaments. Furthermore, the N-terminal peptide was sufficient for high-affinity binding to actin stress fibers in smooth muscle cells in vivo. Alanine scanning mutagenesis in the fusion protein identified residues Asp-30, Phe-31, Arg-32, and Leu-35 as important for binding in vitro. There are two additional DFRXXL motifs located at residues 2-7 and 58-63. The DFR residues in these three motifs were individually replaced by alanine residues in the full-length kinase. Each of these mutations significantly decreased myosin light chain kinase binding to myofilaments in vitro, and each abolished high-affinity binding to actin-containing filaments in smooth muscle cells in vivo. These results identify a unique structural motif comprised of three repeat consensus sequences in the N terminus of myosin light chain kinase necessary for high-affinity binding to actin-containing filaments.
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PMID:Identification of a novel actin binding motif in smooth muscle myosin light chain kinase. 1050 6


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