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)

Homeodomain-containing proteins are believed to function as sequence-specific DNA binding proteins, regulating gene expression. Specificity of sequence recognition is conferred by the homeodomain acting either alone or in conjunction with other conserved DNA binding domains as is the case for Pou domain and Paired domain proteins. The recent isolation of cDNAs encoding mammalian homologues of the Drosophila Cut homeodomain protein has revealed that the 72-amino acid Cut Repeats are conserved in evolution. We have investigated the biochemical activity of human Cut Repeats by expressing fusion proteins containing glutathione S-transferase linked to various combinations of Cut Repeats and Cut homeodomain. We show by gel retardation and DNase footprinting assays that Cut Repeats can function as DNA binding domains, either independently or in cooperation with the homeodomain. The binding affinity (KD) to a specific recognition site was estimated to be 8 x 10(-9) M for Cut Repeat 3 and 4 x 10(-10) M for Cut Repeat 1. When both Cut Repeat 3 and the Cut homeodomain were present in the fusion protein, the binding affinity was increased to 4 x 10(-11) M. These results define a novel class of proteins that contain in addition to the homeodomain a second conserved protein domain, the Cut Repeats, that also function as a DNA binding domain.
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PMID:Conserved cut repeats in the human cut homeodomain protein function as DNA binding domains. 790 99

Complementary DNA sequences encoding different portions of two sunflower homeodomain proteins were cloned in-frame in the expression vectors pRSET and pGEX-3X. When introduced into competent Escherichia coli cells and induced, the resulting plasmids directed the expression of large amounts (5-10% of total cellular protein) of the encoded polypeptides. As a rule, fusions in pRSET rendered insoluble proteins, while fusions in pGEX were soluble and could be purified in a single step by selective absorption onto glutathione-agarose beads, followed by elution with free glutathione. The purified proteins showed both glutathione S-transferase and DNA-binding activity, indicating that they retain their native conformation. The expression-purification protocol that was employed allowed the isolation of up to 0.7 mg of protein per gram of transformed cells. One of the fusion proteins, RH11 (which is a fusion of the homeodomain protein HAHR1 in pRSET), though insoluble, was able to bind DNA when spotted onto a nitrocellulose filter. This protein could also be simply purified in large amounts by electroelution from sodium dodecyl sulfate-polyacrylamide gels and used to elicit antibodies which recognized both the transgenic fusion and the native protein from sunflower nuclei. Our results clearly show that vector choice is a critical parameter for obtaining large amounts of a desired protein for particular purposes.
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PMID:Expression of sunflower homeodomain containing proteins in Escherichia coli: purification and functional studies. 963 21

SIX5 (previously known as myotonic dystrophy associated homeodomain protein - DMAHP ) is a member of the SIX [ sine oculis homeobox (Drosophila ) homologue ] gene family which encodes proteins containing a SIX domain adjacent to a homeo-domain. To investigate the DNA binding specificities of these two domains in SIX5, they were expressed as GST fusion proteins, both separately and together. Affinity purified recombinant proteins and cell lysates from bacteria expressing the recombinant proteins were used in gel retardation assays with double stranded oligonucleotides representing putative DNA binding sites. The putative sites included two in the promoter region of DMPK (dystrophia myotonica protein kinase ) and the previously characterised murine Six4 DNA binding site in the Na(+)/K(+) ATPase alpha 1 subunit gene ( ATP1A1 ) regulatory element (ARE). None of the recombinant proteins showed any affinity for the two putative sites in DMPK. However, the two recombinant proteins containing the homeodomain both formed at least one specific complex with the ARE. The recombinant protein containing both domains formed a second specific complex with the ARE, assumed to be a dimer complex. Finally, a whole genome PCR-based screen was used to identify genomic DNA sequences to which SIX5 binds, as an initial stage in the identification of genes regulated by SIX5.
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PMID:Functional analysis of the homeodomain protein SIX5. 1075 85

The Pax6 genes encode evolutionary conserved transcription factors that act high up in the regulatory hierarchy controlling development of central organs such as the eyes and the central nervous system. These proteins contain two DNA-binding domains. The N-terminal paired domain is separated from a paired-type homeodomain by a linker region, and a transactivation domain is located C-terminal to the homeodomain. Vertebrate Pax6 genes express a paired-less isoform of Pax6 (Pax6DeltaPD) from an internal start codon in the coding region between the paired domain and homeodomain. We now provide evidence for an interaction between the full-length isoform and Pax6DeltaPD, which enhances the transactivation activity of Pax6 from paired domain-binding sites. The paired-like homeodomain protein Rax behaved similarly to Pax6DeltaPD. Both Pax6DeltaPD and Rax bound to the homeodomain of Pax6 in vitro in the absence of specific DNA binding. Coimmunoprecipitation experiments following cotransfection confirmed the existence of complexes between Pax6 and Pax6DeltaPD, Pax6 and Rax, and Pax6DeltaPD and Rax in vivo. Interestingly, the C-terminal subdomain of the paired domain and the homeodomain can interact with each other. The paired domain can also interact with itself. Surprisingly, GST pull-down assays revealed that the homeodomains of such diverse proteins as Chx10, Six3, Lhx2, En-1, Prep1, Prox1, and HoxB1 could all bind to Pax6, and several of these enhanced Pax6-mediated transactivation upon coexpression. Since many homeodomain proteins are coexpressed with Pax6 in several tissues during development, our results indicate the existence of novel regulatory interactions that may be important for fine tuning of gene regulation.
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PMID:Superactivation of Pax6-mediated transactivation from paired domain-binding sites by dna-independent recruitment of different homeodomain proteins. 1106 20

The homeodomain protein, Cdx-2, as transcription factor has been implicated in the transcriptional regulation of genes expressed in small intestine and the process of tumorgenesis. In current work, a conserved mouse Cdx-2 domain (mCdx-2D) coded by its cDNA fragment, which was amplified and cloned into the expression vector pGEX-4T1, was expressed as a fusion protein with GST (GST-mCd x-2D) and purified by one step of affinity chromatography. A polyclonal antibody against Cdx-2 was raised by using the recombinant fusion protein GST-mCdx-2D as antigen and was fractionated from the rabbit anti-serum. Western blot and EMSA (electrophoretic mobility shift assay) demonstrate that the natural and denatured Cdx-2s from different species (mouse and human) can be detected by the prepared anti-Cdx-2 antibody. Most notably, we found that the Cdx-2 in human intestine cell line Caco-2 is expressed in a differentiation-dependent manner and can efficiently bind to the mouse and human acat2 (acyl-coenzyme A: cholesterol acyltransferase 2) promoter regions, suggesting that the transcriptional factor Cdx-2 may play a role in regulating the acat2 expression in the intestinal cells.
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PMID:Preparation of an anti-Cdx-2 antibody for analysis of different species Cdx-2 binding to acat2 promoter. 1251 21

NRL (neural retina leucine zipper) is a basic motif leucine zipper transcription factor of the Maf-subfamily. Multiple phosphorylated isoforms of NRL are detected specifically in rod photoreceptors. NRL regulates the expression of several rod-specific genes, including rhodopsin and cGMP phosphodiesterase beta-subunit, in synergy with other transcription factors (e.g. the homeodomain protein CRX). Missense mutations in the human NRL gene are associated with autosomal dominant retinitis pigmentosa, whereas the loss of its function leads to rodless retina in Nrl-knockout mice that exhibit enhanced S-cone function. To further elucidate the molecular mechanism(s) underlying NRL-mediated transcriptional regulation, we used yeast two-hybrid screening to isolate NRL-interacting proteins in the retina and report the identification of Flt3-interacting zinc-finger protein, Fiz1. Interaction of Fiz1 and NRL-leucine zipper was validated by GST pulldown assays and co-immunoprecipitation from bovine retinal nuclear extracts. Fiz1 suppressed NRL- but not CRX-mediated transactivation of rhodopsin promoter activity in transiently transfected CV1 cells. The mRNA and the protein for both Fiz1 and its only other known interacting protein Flt3, a receptor tyrosine kinase, are expressed in the retina. Our results indicate potential cross-talk among signaling pathways in the retina and suggest that the function of NRL is modulated by its interaction with specific repressor proteins.
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PMID:Interaction of retinal bZIP transcription factor NRL with Flt3-interacting zinc-finger protein Fiz1: possible role of Fiz1 as a transcriptional repressor. 1256 83

The proline-rich homeodomain protein (PRH), also known as Hex, is a transcriptional repressor expressed in a variety of cell types. The PRH protein contains a proline-rich N-terminal domain that can repress transcription when attached to a heterologous DNA binding domain, a central homeodomain that mediates sequence-specific DNA binding, and an acidic C-terminal domain of unknown function. Although individual domains of PRH have been expressed in bacterial cells as GST- and histidine-tagged fusion proteins, attempts to express and purify the full-length protein have met with little success. Here we describe the purification of a histidine-tagged full-length PRH fusion protein. The protein described here will allow us to determine the mechanisms whereby PRH represses transcription.
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PMID:Purification of the proline-rich homeodomain protein. 1265 Sep 96

The PITX2 homeodomain protein is mutated in patients with Axenfeld-Rieger syndrome and is involved in the development of multiple organ systems, including the heart. We have examined the interaction of PITX2 isoforms with myocyte-enhancing factor 2A (MEF2A), which is a known regulator of cardiac development. A direct interaction between PITX2a and MEF2A was demonstrated using yeast two-hybrid and GST pull-down assays. To study the functional significance of this interaction, we used the atrial natriuretic factor (ANF) promoter. Coexpression of MEF2A and PITX2a or Pitx2c resulted in a strong synergistic activation of the ANF promoter in LS8 oral epithelial cells but not in other cell lines (NIH/3T3, Chinese hamster ovary, or C2C12). The synergism was dependent on promoter context, because it required MEF2 binding sites and was not seen with two other PITX2 target promoters. DNA binding by MEF2A was required but not sufficient for synergism. Upstream activators of p38 MAP kinases, MKK3 and MKK6, increased PITX2a and Pitx2c activity to yield up to 90-fold activation of the ANF promoter in LS8 cells. Because Axenfeld-Rieger syndrome is autosomal dominant and affects development of the oral epithelium, we tested one of the known PITX2 mutants. The PITX2a-K88E mutant protein suppressed wild type PITX2a synergism with MEF2A. These results demonstrate a promoter- and cell-specific functional interaction between PITX2 and MEF2A and suggest the possibility of coordinate control by these factors in the oral epithelium.
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PMID:Cell-specific activation of the atrial natriuretic factor promoter by PITX2 and MEF2A. 1546 16

The induction of pancreatic endocrine differentiation from undifferentiated precursor cells is a multi-step process regulated by the expression of several transcription factors. At E9.5 expression of homeodomain protein Pdx-1 defines the early pancreatic epithelium. Later, expression of the basic helix-loop-helix factor, Neurogenin 3 (Ngn3) marks the initiation of the endocrine lineage from the pancreatic precursor cells. A full understanding of these processes is essential in order to control development of insulin secreting beta-cells from embryonic stem cells in vitro. Since the expression of ngn3 is a key step, the aim of this work was to develop monoclonal antibodies for immunohistochemical (IHC) detection of Ngn3. All mice (RBF-strain) immunized with recombinant GST-mNGN3 fusion protein responded with a high antibody titer. The sera were further screened by IHC on paraffin sections of fetal (E13) mouse pancreas, and two hybridomas subsequently derived from one selected mouse produced antibodies with prominent and specific Ngn3 staining properties. These new antibodies provide novel useful tools for co-labeling studies using the large panel of existing rabbit polyclonal antibodies available against important transcription factors to further characterize the development of pancreatic endocrine progenitor cells.
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PMID:Generation of monoclonal antibodies against mouse neurogenin 3: a new immunocytochemical tool to study the pancreatic endocrine progenitor cell. 1568 67

Prox1 is a divergent homeodomain protein important for the development of the lens, retina, liver, pancreas, and lymphatic vasculature. Prox1 expression is highly upregulated in transformed hepatocytes and has been used as a marker to distinguish lymphatic from blood vasculature. We produced recombinant human Prox1 (amino acids 547-737) fused to glutathione S-transferase (GST) and used it to create two hybridomas, 5G10 and 4G10. Both of these hybridomas produced monoclonal antibodies able to detect Prox1 by immunofluorescence in lenses from diverse terrestrial vertebrates, including humans, rats, chickens, and lizards, although 5G10 was generally more sensitive in this application. Further, 4G10 was able to robustly detect endogenous and recombinant Prox1 in both cell and tissue extracts by Western blotting, while 5G10 was notably less sensitive for this purpose. These monoclonal antibodies will be useful for diverse studies on the role of Prox1 in both normal development and disease processes in terrestrial vertebrates.
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PMID:Production of monoclonal antibodies against Prox1. 1647 79


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