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)

Glutathione S-transferase has been purified from bovine erythrocytes by affinity chromatography. The enzyme has an isoelectric point of 7.2, behaves as a 48-kDa protein composed of two identical subunits, and has an N-terminal sequence of PPYTIVYFPVQGR?EAMRMLL. This sequence, the amino acid composition, and the kinetic parameters suggest that the enzyme belongs to the pi-class of transferases. Hemins, porphyrins, and fatty acids form complexes with the enzyme and serve as effective inhibitors. Treatment of the transferase with N-ethylmaleimide, 3-amino-1,2,4-triazole, diethyl pyrocarbonate, or 2,3-butanedione inhibits transferase activity without altering tetrapyrrole binding. The role of the complexation and inhibition of glutathione S-transferase in erythroid metabolism has yet to be elucidated.
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PMID:Bovine erythrocyte glutathione S-transferase: purification, inhibition, and complex formation. 150 63

Ankyrin mediates the attachment of spectrin to transmembrane integral proteins in both erythroid and nonerythroid cells by binding to the beta-subunit of spectrin. Previous studies using enzymatic digestion, 2-nitro-5-thiocyanobenzoic acid cleavage, and rotary shadowing techniques have placed the spectrin-ankyrin binding site in the COOH-terminal third of beta-spectrin, but the precise site is not known. We have used a glutathione S-transferase prokaryotic expression system to prepare recombinant erythroid and nonerythroid beta-spectrin from cDNA encoding approximately the carboxy-terminal half of these proteins. Recombinant spectrin competed on an equimolar basis with 125I-labeled native spectrin for binding to erythrocyte membrane vesicles (IOVs), and also bound ankyrin in vitro as measured by sedimentation velocity experiments. Although full length beta-spectrin could inhibit all spectrin binding to IOVs, recombinant beta-spectrin encompassing the complete ankyrin binding domain but lacking the amino-terminal half of the molecule failed to inhibit about 25% of the binding capacity of the IOVs, suggesting that the ankyrin-independent spectrin membrane binding site must lie in the amino-terminal half of beta-spectrin. A nested set of shortened recombinants was generated by nuclease digestion of beta-spectrin cDNAs from ankyrin binding constructs. These defined the ankyrin binding domain as encompassing the 15th repeat unit in both erythroid and nonerythroid beta-spectrin, amino acid residues 1,768-1,898 in erythroid beta-spectrin. The ankyrin binding repeat unit is atypical in that it lacks the conserved tryptophan at position 45 (1,811) within the repeat and contains a nonhomologous 43 residue segment in the terminal third of the repeat. It also appears that the first 30 residues of this repeat, which are highly conserved between the erythroid and nonerythroid beta-spectrins, are critical for ankyrin binding activity. We hypothesize that ankyrin binds directly to the nonhomologous segment in the 15th repeat unit of both erythroid and nonerythroid beta-spectrin, but that this sequence must be presented in the context of a properly folded spectrin "repeat unit" structure. Future studies will identify which residues within the repeat unit are essential for activity, and which residues determine the specificity of various spectrins for different forms of ankyrin.
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PMID:Ankyrin binds to the 15th repetitive unit of erythroid and nonerythroid beta-spectrin. 183 9

Human erythrocyte glutathione S-transferase activity is inhibited, probably competitively, by hemin with a Ki of 10(-7) M. It is postulated that glutathione S-transferase functions physiologically as a hemin-binding and/or transport protein in developing erythroid cells.
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PMID:Binding of heme by glutathione S-transferase: a possible role of the erythrocyte enzyme. 712 73

Erythropoietin (Epo) regulates the proliferation and differentiation of erythroid precursors. The phosphorylation of proteins at tyrosine residues is critical in the growth signaling induced by Epo. This mechanism is regulated by the activities of both protein-tyrosine kinases and protein tyrosine phosphatases. The discovery of phosphotyrosine phosphatases that contain SH2 domains suggests roles for these molecules in growth factor signaling pathways. We found that Syp, a phosphotyrosine phosphatase, widely expressed in all tissues in mammals became phosphorylated on tyrosine after stimulation with Epo in M07ER cells engineered to express high levels of human EpoR. Syp was complexed with Grb2 in Epo-stimulated M07ER cells. Direct binding between Syp and Grb2 was also observed in vitro. Furthermore, Syp appeared to bind directly to tyrosine-phosphorylated EpoR in M07ER cells. Both NH2-terminal and COOH-terminal SH2 domains of Syp, made as glutathione S-transferase fusion proteins, were able to bind to the tyrosine-phosphorylated EpoR in vitro. These results suggest that Syp may be an important signaling component downstream of the EpoR and may regulate the proliferation and differentiation of hematopoietic cells.
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PMID:Involvement of SH2-containing phosphotyrosine phosphatase Syp in erythropoietin receptor signal transduction pathways. 753 99

The EVI1 gene is activated by chromosomal translocations and inversions in approximately 5% of human acute myeloid leukemia (AML) and by retroviral insertion in approximately 20% of murine myeloid leukemias. EVI1 encodes a nuclear DNA-binding protein having 10 zinc finger motifs in two noncontiguous domains consisting of an amino-terminal domain of seven fingers and a carboxyl domain containing three fingers. To evaluate the sequence specificity of Evi-1 binding and potentially identify genomic targets, whole-genome PCR was utilized to isolate multiple Sau3A fragments which specifically bind to the amino-terminal zinc finger domain. The majority of these clones represented single copy sequences and virtually all contained variable numbers of repeats of the GATA motif, the target sequence for the erythroid-specific transcription factor GATA-1. GST/Evi-1 fusion proteins containing the amino-terminal domain of zinc fingers bound the GATA motif in these clones as well as to those present in the human gamma-globin promoter, similar to the binding of purified GATA-1 protein. By obtaining corresponding large genomic clones for eight of these fragments, transcription units were found associated with two. One corresponded to the glyceraldehyde-3-phosphate dehydrogenase gene and its expression was not affected by Evi-1. The second is a novel gene whose expression is repressed in murine myeloid cell lines that express Evi-1.
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PMID:The Evi-1 zinc finger myeloid transforming protein binds to genomic fragments containing (GATA)n sequences. 762 27

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.
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PMID:Self-association of the erythroid transcription factor GATA-1 mediated by its zinc finger domains. 773 29

Protein 4.1's interaction with the erythroid skeletal proteins spectrin and actin and its essential role in regulating membrane strength are both attributable to expression of an alternatively spliced 63-nucleotide exon. The corresponding 21-amino acid (21-aa) cassette is within the previously identified spectrin-actin binding domain (10 kDa molecular mass) of erythroid protein 4.1. This cassette is absent, however, in several isoforms that are generated by tissue- and development-specific RNA splicing. Four isoforms of the 10-kDa domain were constructed for comparative assessment of functions particularly relevant to red cells. In vitro translated isoforms containing the 21-aa cassette, denoted 10k21 and 10k19,21, were able to bind spectrin, stabilize spectrin-actin complexes, and associate with red cell membrane. Isoforms replacing or lacking the 21-aa cassette, 10k19 and 10k0, did not function in these assays. A bacterially expressed fusion protein with glutathione-S-transferase, designated GST-10k21, congealed spectrin-actin into a network in vitro as found with purified protein 4.1. Additionally, incorporation of GST-10k21 into mechanically weak, 4.1-deficient membranes increased mechanical strength of these membranes to normal. GST-10k19 did not function in these assays. These results show that the 21-aa sequence in protein 4.1 is critical to mechanical integrity of the red cell membrane. These results also allow the role of protein 4.1 in membrane mechanics to be interpreted primarily in terms of its spectrin-actin binding function. Alternatively expressed sequences within the 10-kDa domain of nonerythroid protein 4.1 are suggested to have different, yet to be defined functions.
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PMID:Mechanochemistry of the alternatively spliced spectrin-actin binding domain in membrane skeletal protein 4.1. 846 54

Erythropoietin (Epo), the primary in vivo stimulator of erythroid proliferation and differentiation, acts, in part, by altering the tyrosine phosphorylation levels of various intracellular signaling molecules. These phosphorylation levels are tightly regulated by both tyrosine kinases and tyrosine phosphatases. We have recently shown that the SH2 containing tyrosine phosphatase, Syp, binds directly to both the tyrosine phosphorylated form of the Epo receptor (EpoR) and to Grb2 after Epo stimulation of M07e cells engineered to express high levels of human EpoRs (T. Tauchi, et al: J Biol Chem 270:5631, 1995). To determine which tyrosine within the EpoR is responsible for binding Syp, we examined DA-3 cell lines expressing full-length mutant EpoRs bearing tyrosine to phenylalanine substitutions for each of the eight tyrosines within the intracellular domain of the EpoR. We found that: (1) all Epo-stimulated mutant EpoRs, except for the Y425F EpoR, coimmunoprecipitated with Syp; (2) all Epo-stimulated mutant EpoRs, except for the Y425F EpoR, bound to a GST-fusion protein containing both SH2 domains of Syp; (3) Jak2 could phosphorylate GST-Syp in vitro after Epo stimulation of wild-type (wt) EpoR expressing DA-3 cells; (4) Epo-stimulated tyrosine phosphorylation of Syp in vivo was markedly reduced in Y425F EpoR expressing DA-3 calls; and (5) DA-3 cells expressing the Y425F EpoR grow less well in response to Epo than wt EpoR expressing cells. These results suggest that Syp binds via its SH2 domains to phosphorylated Y425 within the EpoR and is then phosphorylated on tyrosine residues by Jak2. Moreover, Y425 in the EpoR reduces the Epo requirement for Syp tyrosine phosphorylation and promotes proliferation.
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PMID:Tyrosine 425 within the activated erythropoietin receptor binds Syp, reduces the erythropoietin required for Syp tyrosine phosphorylation, and promotes mitogenesis. 863 15

The trans-activator protein (Tat) of HIV-1 plays an important role in viral pathogenesis. Since Tat has been shown to alter expression of a number of host cellular genes, we have investigated the role of Tat in modulating gene expression and differentiation in hematopoietic progenitor cells. Tat protein was introduced in K562 cells, a human hematopoietic progenitor cell line, by either scrape-loading onto HeLa (HL)-tat cells or direct electroporation of an affinity-purified glutathione S-transferase (GST)-Tat fusion protein. Under these conditions, butyric acid-induced hemoglobin production in K562 cells was suppressed by 65 and 52%, respectively. However, coculturing with wild-type HeLa cells or electroporation with the control GST protein did not decrease hemoglobin production. To confirm the presence of bioactive Tat protein within K562 cells, the cells were transiently transfected with a pHIV/LTR-CAT prior to the introduction of Tat. A 30- to 40-fold induction in CAT gene expression was observed in the transfected K562 cells, which were either cocultured with HL-tat or were electroporated with GST-Tat. Simultaneous transient transfection of K562 cells with a TAR expression plasmid, to compete for the availability of Tat protein, significantly downregulated the HIV LTR trans-activation by Tat. In addition, overexpression of the TAR RNAs in K562 cells was able to downregulate the suppressive effect of Tat on butyric acid-induced differentiation. RT-PCR analysis of the total RNAs isolated from these cells demonstrated that Tat protein suppressed the butyric acid-induced gamma-globin gene expression by an average of 54% without affecting the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs. These data indicate that the viral Tat protein plays a significant role in abrogating erythroid differentiation in K562 cells.
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PMID:Effect of HIV type 1 Tat protein on butyric acid-induced differentiation in a hematopoietic progenitor cell line. 891 78

The induction of phase II detoxifying enzymes is an important defense mechanism against intake of xenobiotics. While this group of enzymes is believed to be under the transcriptional control of antioxidant response elements (AREs), this contention is experimentally unconfirmed. Since the ARE resembles the binding sequence of erythroid transcription factor NF-E2, we investigated the possibility that the phase II enzyme genes might be regulated by transcription factors that also bind to the NF-E2 sequence. The expression profiles of a number of transcription factors suggest that an Nrf2/small Maf heterodimer is the most likely candidate to fulfill this role in vivo. To directly test these questions, we disrupted the murine nrf2 gene in vivo. While the expression of phase II enzymes (e.g., glutathione S-transferase and NAD(P)H: quinone oxidoreductase) was markedly induced by a phenolic antioxidant in vivo in both wild type and heterozygous mutant mice, the induction was largely eliminated in the liver and intestine of homozygous nrf2-mutant mice. Nrf2 was found to bind to the ARE with high affinity only as a heterodimer with a small Maf protein, suggesting that Nrf2/small Maf activates gene expression directly through the ARE. These results demonstrate that Nrf2 is essential for the transcriptional induction of phase II enzymes and the presence of a coordinate transcriptional regulatory mechanism for phase II enzyme genes. The nrf2-deficient mice may prove to be a very useful model for the in vivo analysis of chemical carcinogenesis and resistance to anti-cancer drugs.
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PMID:An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. 924 Apr 32


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