Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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We report the isolation and molecular characterization of the mouse grb2 gene. The product of this gene, the Grb2 protein, is highly related to the Caenorhabditis elegans sem-5 gene product and the human GRB2 protein and displays the same SH3-SH2-SH3 structural motifs. In situ hybridization studies revealed that the mouse grb2 gene is widely expressed throughout embryonic development (E9.5 to P0). However, grb2 transcripts are not uniformly distributed, and in certain tissues (e.g., thymus) they appear to be regulated during development. Recent genetic and biochemical evidence has implicated the Grb2 protein in the signaling pathways that link cell surface tyrosine kinase receptors with Ras. We have investigated the association of the Grb2 protein with epidermal growth factor (EGF) and nerve growth factor (NGF) receptors in PC12 pheochromocytoma cells. EGF treatment of PC12 cells results in the rapid association of Grb2 with the activated EGF receptors, an interaction mediated by the Grb2 SH2 domain. However, Grb2 does not bind to NGF-activated Trk receptors. Mitogenic signaling of NGF in NIH 3T3 cells ectopically expressing Trk receptors also takes place without detectable association between Grb2 and Trk. These results suggest that whereas EGF and NGF can activate the Ras signaling pathway in PC12 cells, only the EGF receptor is likely to do so through a direct interaction with Grb2. Finally, binding studies with glutathione S-transferase fusion proteins indicate that Grb2 binds two distinct subsets of proteins which are individually recognized by its SH2 and SH3 domains. These observations add further support to the concept that Grb2 is a modular adaptor protein.
Mol Cell Biol 1993 Sep
PMID:Molecular cloning of the mouse grb2 gene: differential interaction of the Grb2 adaptor protein with epidermal growth factor and nerve growth factor receptors. 768 50

To gain insight into mechanisms of cell type-specific transcription of class mu-glutathione S-transferase genes, the gene encoding the Yb3 subunit was cloned. Yb3 subunits are selectively expressed at high levels in rat brain and testis but not in liver or kidney. The Yb3 subunit gene spans over 6 kb and consists of 8 exons and 7 introns and a sequence consisting of tandem direct repeat consensus octamer DNA binding motifs separated by a 6 base pair (bp) spacer was identified in its 5'-flanking region. Gel shift assays with a 40 bp segment of DNA containing the two consensus octamer sequences, revealed the presence of specific binding proteins in nuclear extracts of rat brain, testis and C6 glioma cells. DNA binding activity was greatly reduced in liver, kidney and HTC cells. Reporter vectors carrying segments of the 5'-flanking region of the Yb3 subunit gene fused to a luciferase gene were introduced into C6 glioma cells which express high levels of Yb3 subunits, and into HTC cells which do not. The plasmids consisting of the Yb3 gene promoter up to, but not including, the octamer motifs did not support luciferase transcription in the C6 glioma cells, but larger fragments that included the octamer repeat sequences, effectively directed transcription in the C6 glioma cells. With mutated octameric sequences transcriptional activity was greatly reduced, and none of the same Yb3 constructs directed substantial luciferase transcription in the HTC cells. The results show that octamer motifs in the 5'-flanking region of the Yb3 subunit gene are functional and are the principal cis-acting elements that account for its discrete cell type-selective expression. This gene is one of the few known targets for octamer DNA binding transcription factors in brain.
Brain Res Mol Brain Res 1995 Jan
PMID:Brain and testis selective expression of the glutathione S-transferase Yb3 subunit is governed by tandem direct repeat octamer motifs in the 5'-flanking region of its gene. 770 76

Arg15 is a conserved active-site residue in class Alpha glutathione transferases. X-ray diffraction studies of human glutathione transferase A1-1 have shown that N epsilon of this amino acid residue is adjacent to the sulfur atom of a glutathione derivative bound to the active site, suggesting the presence of a hydrogen bond. The phenolic hydroxyl group of Tyr9 also forms a hydrogen bond to the sulfur atom of glutathione, and removal of this hydroxyl group causes partial inactivation of the enzyme. The present study demonstrates by use of site-directed mutagenesis the functional significance of Arg15 for catalysis. Mutation of Arg15 into Leu reduced the catalytic activity by 25-fold, whereas substitution by Lys caused only a threefold decrease, indicating the significance of a positively charged residue at position 15. Mutation of Arg15 into Ala or His caused a substantial reduction of the specific activity (200 or 400-fold, respectively), one order of magnitude more pronounced than the effect of the Tyr9-->Phe mutation. Double mutations involving residues 9 and 15 demonstrated that the effects of mutations at the two positions were additive except for the substitution of His for Arg15, which appeared to cause secondary structural effects. The pKa value of the phenolic hydroxyl of Tyr9 was determined by UV absorption difference spectroscopy and was found to be 8.1 in the wild-type enzyme. The corresponding pKa values of mutants R15K, R15H and R15L were 8.5, 8.7 and 8.8, respectively, demonstrating the contribution of the guanidinium group of Arg15 to the electrostatic field in the active site. Addition of glutathione caused an increased pKa value of Tyr9; this effect was not obtained with S-methylglutathione. These results show that Tyr9 is protonated when glutathione is bound to the enzyme at physiological pH values. The involvement of an Arg residue in the binding and activation of glutathione is a feature that distinguishes class Alpha glutathione transferases from members in other glutathione transferase classes.
J Mol Biol 1995 Apr 07
PMID:Functional significance of arginine 15 in the active site of human class alpha glutathione transferase A1-1. 772 30

The recombinant proteins of Lol p VA and Lol p VB expressed in E. coli reacted with IgE antibodies from sera of allergic patients and mAbs FMC A7 and PpV1. Cross-absorption analyses using these recombinant proteins showed that Lol p VA and Lol p VB possess both similar and unique IgE binding determinants. Gene fragmentation was utilized to localize the antigenic and allergenic determinants of Lol p VB. When full-length cDNA of Lol p VB was digested into three fragments and expressed as the fusions from the glutathione transferase of pGEX vectors, fragments Met1-Val196 and Asp197-Val339 bound IgE while fragment Met1-Pro96 did not. The data suggest that there are at least two IgE binding determinants in Lol p VB. In addition, only fragment Met1-Val196 reacted with mAb PpV1. The localization of these determinants was further resolved using random fragment expression libraries. The mAb PpV1 determinant was near the N-terminal region of Lol p VB molecule. The IgE binding determinants were distributed in the central region: region I (amino acids 111-195) and II (199-254). These IgE binding determinants are conserved in Lol p VA.
Mol Immunol 1995 Mar
PMID:Mapping of the antigenic and allergenic epitopes of Lol p VB using gene fragmentation. 772 75

GATA-1, the founding member of a distinctive family of transcription factors, is expressed predominantly in erythroid cells and participates in the expression of numerous erythroid cell-expressed genes. GATA-binding sites are found in the promoters and enhancers of globin and nonglobin erythroid genes as well as in the alpha- and beta-globin locus control regions. To elucidate how GATA-1 may function in a variety of regulatory contexts, we have examined its protein-protein interactions. Here we show that GATA-1 self-associates in solution and in whole-cell extracts and that the zinc finger region of the molecule is sufficient to mediate this interaction. This physical interaction can influence transcription, as GATA-1 self-association is able to recruit a transcriptionally active but DNA-binding-defective derivative of GATA-1 to promoter-bound GATA-1 and result in superactivation. Through in vitro studies with bacterially expressed glutathione S-transferase fusion proteins, we have localized the minimal domain required for GATA-1 self-association to 40 amino acid residues within the C-terminal zinc finger region. Finally, we have detected physical interaction of GATA-1 with other GATA family members (GATA-2 and GATA-3) also mediated through the zinc finger domain. These findings have broad implications for the involvement of GATA factors in transcriptional control. In particular, the interaction of GATA-1 with itself and with other transcription factors may facilitate its function at diverse promoters in erythroid cells and also serve to bring together, or stabilize, loops between distant regulatory elements, such as the globin locus control regions and downstream globin promoters. We suggest that the zinc finger region of GATA-1, and related proteins, is multifunctional and mediates not only DNA binding but also important protein-protein interactions.
Mol Cell Biol 1995 May
PMID:Self-association of the erythroid transcription factor GATA-1 mediated by its zinc finger domains. 773 29

Two glutathione S-transferase (GST) clones from a larval midgut cDNA library of the tobacco hornworm, Manduca sexta were sequenced. The nucleotide sequence of the first clone, M. sexta GST1, encoded a protein of 217 amino acids with a predicted molecular weight of 24,644 and isoelectric point of 4.8. The M. sexta GST1 was 45.9-48.6% identical to GSTs from Musca domestica and several Drosophila species. The M. sexta GST2 cDNA encoded a protein of 203 amino acids with a predicted molecular weight of 23,596 and isoelectric point of 5.5. The M. sexta GST2 shared 44.8-50.0% sequence identity to a second cluster of insect GSTs from M. domestica, D. melanogaster and Anopheles gambiae. GST1 and GST2 were only 24.1% identical in amino acid sequence. The divergence of these two classes of insect GSTs occurred before the radiation of Diptera and Lepidoptera. Northern analysis of the expression of these GSTs showed increased GST1 mRNA levels in midguts of larvae fed diets containing 2-undecanone, or phenobarbital. Midgut and fat body cytosolic GST activities were induced when larvae were fed diets containing 2-tridecanone, 2-undecanone, or phenobarbital. Partial purification of midgut GSTs by size-exclusion and glutathione affinity chromatography resulted in a series of isoelectric focusing bands, with the major one corresponding to the predicted isoelectric point of the M. sexta GST1. In summary, two midgut GSTs have been identified on the basis of cDNA sequence and one of these, GST1, was inducible by dietary chemicals.
Insect Biochem Mol Biol 1995 Apr
PMID:Glutathione S-transferases from larval Manduca sexta midgut: sequence of two cDNAs and enzyme induction. 774 33

Here we report the cloning and expression, in Escherichia coli, of PCR-amplified DNA encoding the 63-kDa stress-inducible protein of Neisseria gonorrhoeae strains VP1 and PID2, Neisseria meningitidis 2996 and the commensal Neisseria flavescens. DNA sequence analysis revealed in all cases one open reading frame of 541-544 amino acids corresponding to a protein of approximately 57,000 Da. The various neisserial proteins were > 96% identical at the amino acid level and showed extensive homology with proteins belonging to the Hsp60 heat-shock-protein family. We constructed defined glutathione S-transferase fusion polypeptides of the gonococcal Hsp60 homologue to locate antigenic domains on the recombinant protein. Variation in the immunoreactivity of two monoclonal antibodies recognizing a conserved and a neisseria-unique antigenic Hsp60 determinant, respectively, could thus be deduced to result from single amino acid substitutions. Analysis of the antibody response in patients' sera demonstrated reactivity with the same fusion polypeptides in six out of nine sera, indicating that neisserial Hsp60 is expressed during the natural infection and that distinct domains on the protein are immunodominant in vivo.
Mol Microbiol 1995 Jan
PMID:Construction of recombinant neisserial Hsp60 proteins and mapping of antigenic domains. 774 49

The GST (glutathione S-transferase)-NDK (nucleoside diphosphate kinase) fusion protein was expressed in Escherichia coli. The GST-NDK protein was capable of transferring gamma-phosphate from ATP to nucleoside diphosphates such as GDP, CDP, TDP and UDP. Western blot analysis using anti-NDK antibody indicated that NDK in endosperm gradually decreased during 36 h of imbibition. On the contrary, NDK in embryo increased during the same period. NDK activities in both tissues were in accord with these observations. Whereas the NDK protein in roots of rice seedlings during 7 days of imbibition remained constant, in shoots it declined after 5 days of imbibition. Thus, NDK may play a significant role in the cellular event modulated by adenylate energy charge level.
Plant Mol Biol 1995 Mar
PMID:Expression of functional proteins of cDNA encoding rice nucleoside diphosphate kinase (NDK) in Escherichia coli and organ-related alteration of NDK activities during rice seed germination (Oryza sativa L.). 776 75

Previously, we characterized nucleotide sequences of two cDNAs encoding adenylate kinase from rice plants (Oryza sativa L.). Each cDNA (Adk-a or Adk-b) was cloned into the expression vector pET 11d-GST to produce GST-AK fusion proteins in Escherichia coli. Recombinant proteins were cleaved by thrombin, and GST-free adenylate kinase proteins were obtained. Enzyme activity profiles of different pH and inhibition effects to the enzyme by Ap5A (adenosine-5'-pentaphospho-5'-adenosine) indicates that both adenylate kinase proteins have similar biochemical characteristics. Among the nucleoside monophosphates (AMP, CMP, GMP and UMP) investigated, only AMP reacted with ATP. Furthermore, using the antiserum against the rice adenylate kinase proteins, the cellular location of adenylate kinase proteins was examined by immunomicroscopic analysis in combination with a subcellular fractionation method. The results indicated that adenylate kinase proteins were distributed largely in cytosol of rice cells.
Plant Mol Biol 1995 Mar
PMID:Biochemical properties of rice adenylate kinase and subcellular location in plant cells. 776 84

An auxin-regulated gene, parA, comprises a gene family consisting of a handful genes which respond to various signals. Although Droog et al. (Plant Mol. Biol, 1993, 21, 965-972) postulated that the parA-related genes belong to the family of a cytoplasmic enzyme, glutathione S-transferase (GST), we detected a low level of GST activity in the parA products, whose value was below 1/30 of that of parB products encoding tobacco (Nicotiana tabacum L.) GST. Immunofluorescence studies using an antibody against parA protein revealed that the subcellular location of parA protein is the nucleus in cultured tobacco mesophyll protoplasts, while conventional GSTs' including the parB product were primarily located in the cytoplasm. Confocal laser scanning microscopy of tobacco BY-2 cells showed that the parA product was confined to the nucleus, but was excluded from the nucleolus. In addition, exon/intron organization of the parA family was appreciably different from that of conventional GSTs including parB. Furthermore, the parA protein is much more similar to a 24-kDa protein of Escherichia coli that is reported to bind to RNA polymerase. These different characteristics of parA compared with to the conventional GSTs, indicate that parA protein would have distinct functions, such as involvement in transcription, rather than functioning as a conventional GST. Transgenic tobacco plants that carried the parA promoter fused to a beta-glucuronidase gene were used to show that the parA gene is tissue-specific and also under developmental control.
 ...
PMID:Expression of the auxin-regulated parA gene in transgenic tobacco and nuclear localization of its gene products. 776 32


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