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)

A novel rat gene, Mipu1, encodes a 608 amino acid protein with an amino-terminal KRAB domain and 14 carboxyl-terminal C2H2 zinc finger motifs. Mipu1 is localized to the nucleus through its KRAB domain or the linker adjacent to its zinc finger region. Using the GST-Mipu1 bound to glutathione-Sepharose beads, a consensus putative DNA binding site (5'-TGTCTTATCGAA-3') was extracted from a random oligonucleotide library. EMSA and target detection assay showed that the probe containing the putative site can bind to purified GST-Mipu1 fusion protein. The oligonucleotide containing the putative site was inserted into the pGL3-promotor vector to produce a reporter construct. The expression of reporter gene was repressed by overexpression of Mipu1 in a dose-dependent manner. Mutation analysis of the consensus sequence indicated that the repression mediated by Mipu1 is sequence-dependent. These results suggest that Mipu1 is a nuclear protein, which functions as a transcriptional repressor.
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PMID:Functional analysis of a novel KRAB/C2H2 zinc finger protein Mipu1. 1739 2

The Nczf gene, which is identified as a target gene of Ncx, encodes a novel Kruppel-associated box (KRAB) zinc finger protein, which functions as a sequence-specific transcriptional repressor. We generated a fusion protein of the zinc finger domain of Nczf and glutathione S-transferase to identify Nczf-binding consensus DNA sequences with random oligonucleotides of 15 and 35 bases. The consensus binding sequence of core nucleotides contains (A/T/C)CTTT(A/G)TTNT. In a gel mobility shift assay, the probe containing these sequences bound to the fusion protein. In silico analysis, these consensus sequences were found on regulatory regions of the endothelin receptor B and the microphthalmia-associated transcription factor genes, which are involved in neural crest development. These results suggest that Nczf functions as a sequence-specific transcription repressor to regulate neural crest cell development.
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PMID:Identification of the consensus DNA sequence for Nczf binding. 1757 Jul 63

G9a belongs to the subfamily of histone H3 lysine 9 (H3-K9)-specific methyltransferases. On amino acid sequence alignment of human and Drosophila G9a, we found that the N-terminal region from amino acids 532-605 to be evolutionarily conserved and named this the G9a homology domain (GHD). Using the GHD of human G9a (hG9a) as a bait, we isolated cDNA encoding a zinc finger protein 200 (ZNF200), which contains five C(2)H(2)-type zinc finger domains in tandem arrays. Interaction between G9a and ZNF200 could be demonstrated by in vitro binding assays and immunoprecipitation experiments using cultured human HEK293 cell extracts. GST pull-down assays using deletion derivatives of ZNF200 revealed that the interaction is through a region encompassing three of the five zinc finger domains. Furthermore, ZNF200 appear to co-localize with G9a in the nucleoplasm of HEK293 cells as discrete speckles. These results demonstrate that ZNF200 is a novel binding partner of G9a.
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PMID:Identification of ZNF200 as a novel binding partner of histone H3 methyltransferase G9a. 1758 99

This paper explores the use of surface-patterned nanohydrogels as a substrate for high-density and high-sensitivity protein arrays. Nanohydrogels were created by locally crosslinking dry amineterminated PEG 5000 thin films using a focused electron beam. Unirradiated polymer was subsequently washed away leaving behind gels approximately 200 nm in diameter with a dry height of about 50 nm which swell in water by a factor of about five. Two different protein assays involving the nucleic acid binding protein zinc finger 9 (ZNF9) were developed which covalently bind reagents to the amine groups within the PEG nanohydrogels. One directly binds ZNF9 while the other binds alpha-GST antibody to mediate attachment of GST-tagged ZNF9. In both cases 100 microm diameter spots containing 7500 discrete nanohydrogels were patterned into a format consistent with equivalent microarrays created by spotting reagents onto four different commercially available substrates. The arrays were interrogated using a fluorescently labeled oligonucleotide known to bind ZNF9. GST, beta-Gal, and BSA were used as negative controls. Using a standard microarray scanner the nanohydrogel arrays were shown to have a consistently higher combination of absolute signal, signal-to-background ratio, and signal-to-noise ratio than any of the four microarrays. We speculate that this behavior is due to a higher density of bound protein as well as a more accessible protein conformation. Fluorescence optical microscopy can resolve individual nanohydrogels opening the possibility that assays can be scaled from arrays of 100 microm diameter spots to arrays of single nanohydrogel spots. Such an advance can increase the spot density by a factor of approximately 10(4) and has significant implications for the highly efficient use of biological reagents in high throughput proteomic analysis.
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PMID:Hydrogel-based protein nanoarrays. 1768 76

ZIC3, a GLI superfamily transcription factor, is involved in establishing normal embryonic left-right patterning. Multiple abnormalities in the central nervous system (CNS) and axial skeleton have also been observed in mice bearing a Zic3 null allele, mice with a Zic3 overexpression allele, and the majority of patients carrying ZIC3 mutations. Previous studies indicate that ZIC3 protein can bind to the GLI consensus binding site (GLIBS) and physically interact with GLI3, a transcription factor involved in multiple aspects of neural and skeletal development. We investigated in vitro interactions of ZIC3 with GLI3 and the effect of ZIC3 mutations identified in patients with either heterotaxy or isolated cardiovascular malformations. Electrophoresis mobility shift assay (EMSA) revealed that all five intact zinc finger (ZF) domains were necessary for binding of ZIC3 to GLIBS. Inclusion of GLIBS upstream of a basal TK promoter had no effect on the activation of the promoter by ZIC3 alone, but it enhanced the synergistic activation of ZIC3 and GLI3. Wild-type (WT) ZIC3 showed specific binding to GLI3 in GST-pull-down assays. Nonsense and frameshift ZIC3 mutants lacking one or more of the zinc finger domains did not physically interact with GST-GLI3; however, two missense mutants c.1213A>G (p.K405E, fifth ZF domain), and c.649C>G (p.P217A, conserved N-terminal domain) retained binding. Luciferase reporter assays indicated that both p.P217A and p.K405E mutants also retained coactivation with GLI3 of reporter gene expression activity, while all the GLI3-nonbinding ZIC3 mutants lacked this activity. Interestingly, no CNS or skeletal abnormalities were observed in patients bearing the p.P217A or p.K405E mutations.
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PMID:Characterization of the interactions of human ZIC3 mutants with GLI3. 1776 85

FOG-2 is a transcriptional co-regulator that is required for cardiac morphogenesis as mice deficient in this factor die during mid-gestation of cardiac malformations. FOG-2 interacts with GATA4 to attenuate GATA4-dependent gene expression. The first 12 amino acids of FOG-2 (the FOG Repression Motif) are necessary to mediate this repression. To determine the mechanism by which the FOG Repression Motif functions, we identified 7 polypeptides from rat cardiac nuclear extracts that co-purified with a GST-FOG-2 fusion protein. All proteins identified are members of the NuRD nucleosome remodeling complex. Using in vitro binding and co-immunoprecipitation assays, we demonstrate that Metastasis-Associated proteins (MTA)-1, 2 and 3 and Retinoblastoma binding proteins RbAp46 and RbAp48 interact with FOG-2, but not with a mutant form of FOG-2 that is unable to repress transcription. Furthermore, we define a novel domain located in the C-terminal portion of MTA-1 that mediates the FOG-2/MTA-1 interaction. We also demonstrate that knockdown of MTA protein expression dramatically impairs the ability of FOG-2 to repress GATA4 activity. Finally, we show that the zinc finger domain of MTA-1 is required for FOG-2-mediated transcriptional repression and that this domain interacts with RbAp46 and RbAp48 subunits of the NuRD complex. Together, these results demonstrate the importance of FOG-2/MTA/RbAp interactions for FOG-2-mediated transcriptional repression and further define the molecular interactions between the FOG Repression Motif and the NuRD complex.
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PMID:The zinc finger and C-terminal domains of MTA proteins are required for FOG-2-mediated transcriptional repression via the NuRD complex. 1806 19

The affinity isolation of pre-purified plasmid DNA (pDNA) from model buffer solutions using native and poly(ethylene glycol) (PEG) derivatized zinc finger-GST (Glutathione-S-Transferase) fusion protein was examined in PEG-dextran (DEX) aqueous two-phase systems (ATPSs). In the absence of pDNA, partitioning of unbound PEGylated fusion protein into the PEG-rich phase was confirmed with 97.5% of the PEGylated fusion protein being detected in the PEG phase of a PEG 600-DEX 40 ATPS. This represents a 1322-fold increase in the protein partition coefficient in comparison to the non-PEGylated protein (Kc = 0.013). In the presence of pDNA containing a specific oligonucleotide recognition sequence, the zinc finger moiety of the PEGylated fusion protein bound to the plasmid and steered the complex to the PEG-rich phase. An increase in the proportion of pDNA that partitioned to the PEG-rich phase was observed as the concentration of PEGylated fusion protein was increased. Partitioning of the bound complex occurred to such an extent that no DNA was detected by the picogreen assay in the dextran phase. It was also possible to partition pDNA using a non-PEGylated (native) zinc finger-GST fusion protein in a PEG 1000-DEX 500 ATPS. In this case the native ligand accumulated mainly in the PEG phase. These results indicate good prospects for the design of new plasmid DNA purification methods using fusion proteins as affinity ligands.
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PMID:Affinity partitioning of plasmid DNA with a zinc finger protein. 1876 Jul 86

HIPP26 from Arabidopsis thaliana belongs to a novel class of plant proteins, characterized by a heavy metal associated domain and an additional isoprenylation motif. It is induced during cold, salt and drought stress. The nuclear localization of HIPP26, predicted by a NLS motif, could be confirmed in onion epidermal cells overexpressing GFP-HIPP26. Experiments with modified HIPP26 indicate that the isoprenylation plays a role in the spatial distribution in the nucleus. Using promoter-GUS constructs, a tissue specific expression pattern of HIPP26 could be shown, with high expression in the vascular tissue. By a yeast-two-hybrid approach a strong interaction of HIPP26 with the zinc finger homeodomain transcription factor ATHB29, which is known to play a role in dehydration stress response could be detected. This was confirmed by GST pull-down assays. When using a modified HIPP26 lacking the two central cysteines of the heavy metal associated domain, ATHB29 was not bound in the GST pull-down assay, indicating that this structure is necessary for the interaction. Further yeast-two-hybrid analyses testing interaction of different members of the HIPP family with related zinc finger transcription factors revealed a specific interaction of ATHB29 with several HIPP proteins. A functional relationship between HIPP26 and ATHB29 is also indicated by experiments with mutants of HIPP26 showing altered expression levels of such genes known to be regulated by ATHB29.
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PMID:Stress induced and nuclear localized HIPP26 from Arabidopsis thaliana interacts via its heavy metal associated domain with the drought stress related zinc finger transcription factor ATHB29. 1897 36

Members of the super-class of zinc finger proteins are key regulators in early embryogenesis. Utilizing in silico mining of EST Databases for pre-implantation Embryo-Specific Zinc Finger Protein Genes, we characterized a novel zygotic mouse gene-tripartite motif family-like 1 (TRIML1), which expresses in embryo before implantation. Knocking down of TRIML1 resulted in the fewer cell number of blastocysts and failture to give rise to neonates after embryo transfer. The binding partner of TRIML1, Ubiquitin-specific protease 5 (USP5), was identified by yeast two-hybrid screening assay. The interaction was confirmed by GST pull-down and coimmunoprecipitation analysis. The role of TRIML1 in ubiquitin pathway during the development stage of mouse blastocyst was further discussed.
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PMID:Characterization and potential function of a novel pre-implantation embryo-specific RING finger protein: TRIML1. 1915 9

FBI-1, a member of the POK (POZ and Kruppel) family of transcription factors, plays a role in differentiation, oncogenesis, and adipogenesis. eEF1A is a eukaryotic translation elongation factor involved in several cellular processes including embryogenesis, oncogenic transformation, cell proliferation, and cytoskeletal organization. CCS-3, a potential cervical cancer suppressor, is an isoform of eEF1A. We found that eEF1A forms a complex with FBI-1 by co-immunoprecipitation, SDS-PAGE, and MALDI-TOF Mass analysis of the immunoprecipitate. GST fusion protein pull-downs showed that FBI-1 directly interacts with eEF1A and CCS-3 via the zinc finger and POZ-domain of FBI-1. FBI-1 co-localizes with either eEF1A or CCS-3 at the nuclear periplasm. CCS-3 enhances transcriptional repression of the p21CIP1 gene (hereafter referred to as p21) by FBI-1. The POZ-domain of FBI-1 interacts with the co-repressors, SMRT and BCoR. We found that CCS-3 also interacts with the co-repressors independently. The molecular interaction between the co-repressors and CCS-3 at the POZ-domain of FBI-1 appears to enhance FBI-1 mediated transcriptional repression. Our data suggest that CCS-3 may be important in cell differentiation, tumorigenesis, and oncogenesis by interacting with the proto-oncogene FBI-1 and transcriptional co-repressors.
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PMID:Eukaryotic translation initiator protein 1A isoform, CCS-3, enhances the transcriptional repression of p21CIP1 by proto-oncogene FBI-1 (Pokemon/ZBTB7A). 1947 Nov 3


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