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)

At fertilization, sea urchin eggs undergo a series of activation events, including a Ca2+ action potential, Ca2+ release from the endoplasmic reticulum, an increase in intracellular pH, sperm pronuclear formation, MAP kinase dephosphorylation, and DNA synthesis. To examine which of these events might be initiated by activation of phospholipase Cgamma (PLCgamma), which produces the second messengers inositol trisphosphate (IP3) and diacylglycerol, we used recombinant SH2 domains of PLCgamma as specific inhibitors. Sea urchin eggs were co-injected with a GST fusion protein composed of the two tandem SH2 domains of bovine PLCgamma and (1) Ca2+ green dextran to monitor intracellular free Ca2+, (2) BCECF dextran to monitor intracellular pH, (3) Oregon Green dUTP to monitor DNA synthesis, or (4) fluorescein 70-kDa dextran to monitor nuclear envelope formation. Microinjection of the tandem SH2 domains of PLCgamma produced a concentration-dependent inhibition of Ca2+ release and also inhibited cortical granule exocytosis, cytoplasmic alkalinization, MAP kinase dephosphorylation, DNA synthesis, and cleavage after fertilization. However, the Ca2+ action potential, sperm entry, and sperm pronuclear formation were not prevented by injection of the PLCgammaSH2 domain protein. Microinjection of a control protein, the tandem SH2 domains of the phosphatase SHP2, had no effect on Ca2+ release, cortical granule exocytosis, DNA synthesis, or cleavage. Specificity of the inhibitory action of the PLCgammaSH2 domains was further indicated by the finding that microinjection of PLCgammaSH2 domains that had been point mutated at a critical arginine did not inhibit Ca release at fertilization. Additionally, Ca2+ release in response to microinjection of IP3, cholera toxin, cADP ribose, or cGMP was not inhibited by the PLCgammaSH2 fusion protein. These results indicate that PLCgamma plays a key role in several fertilization events in sea urchin eggs, including Ca2+ release and DNA synthesis, but that the action potential, sperm entry, and male pronuclear formation can occur in the absence of PLCgamma activation or Ca2+ increase.
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PMID:Identification of PLCgamma-dependent and -independent events during fertilization of sea urchin eggs. 998 35

During a large-scale in vitro translation analysis of a human full-length cDNA bank, we found many clones producing in vitro translation products showing ladder bands on a fluorogram with the equidistance of about 9 kDa at the position larger than the molecular mass expected from the open reading frame. We have analyzed a clone showing a typical pattern of the ladder bands. This clone encoded a 188-amino acid polypeptide containing a putative transmembrane domain. A green fluorescent protein-tagged polypeptide expressed in COS7 cells was localized in the endoplasmic reticulum and the Golgi apparatus. The ladder bands were observed in a rabbit reticulocyte lysate system, but not in a wheat germ extract system. Addition of the glutathione S-transferase-fused ubiquitin into the lysate caused upward shifts of the ladder bands. Addition of microsomal membranes prevented the formation of the ladder bands. Time course experiments demonstrated that the in vitro translation products increased in the presence of microsomal membranes, but were gradually degraded in their absence. These results suggest that the ladder formation resulted from the ubiquitination of misfolded polypeptide that failed to translocate to its proper position, and that an exclusion mechanism of misfolded membrane protein works in the rabbit reticulocyte lysate system.
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PMID:Multi-ubiquitination of a nascent membrane protein produced in a rabbit reticulocyte lysate. 1039 20

Proteins of the Hsp70 family of ATPases interact with a conserved domain of their J-protein partners, the J-domain, to function in numerous cellular processes. We have studied the interaction of BiP, an Hsp70 family member in the lumen of the endoplasmic reticulum, with the J-domain of Sec63p, a component of the Sec complex involved in post-translational protein translocation across the endoplasmic reticulum membrane. In a real-time solid phase binding assay, BiP binds to the immobilized Sec complex or to a fusion protein of the J-domain and glutathione S-transferase in a reaction that requires ATP hydrolysis. In the final complex, BiP is bound in the ADP form with its peptide binding pocket occupied. An intact peptide binding pocket is required for this interaction. Our experiments suggest that the activation of BiP by the J-domain involves a transient contact between these components, and that in the absence of physiological substrates, J-activated BiP binds even to the J-proteins themselves.
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PMID:Interaction of BiP with the J-domain of the Sec63p component of the endoplasmic reticulum protein translocation complex. 1040 Jun 22

We have identified mouse and human FKBP60, a new member of the FKBP gene family. FKBP60 shares strongest homology with FKBP65 and SMAP. FKBP60 contains a hydrophobic signal peptide at the N-terminus, 4 peptidyl-prolyl cis/trans isomerase (PPIase) domains and an endoplasmic reticulum retention motif (HDEL) at the C-terminus. Immunodetection of HA-tagged FKBP60 in NIH-3T3 cells suggests that FKBP60 is segregated to the endoplasmic reticulum. Northern blot analysis shows that FKBP60 is predominantly expressed in heart, skeletal muscle, lung, liver and kidney. With N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate, recombinant GST-FKBP60 is shown to accelerate effectively the isomerization of the peptidyl-prolyl bond. This isomerization activity is inhibited by FK506. mFKBP60 binds Ca2+ in vitro, presumably by its C-terminal EF-hand Ca2+ binding motif, and is phosphorylated in vivo. hFKBP60 has been mapped to 7p12 and/or 7p14 by fluorescence in situ hybridization (FISH).
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PMID:Biochemical analysis of mouse FKBP60, a novel member of the FKPB family. 1052 4

In order to examine whether fenbendazole has tumor-promoting activity, a total of 70 male Fischer 344 rats were initiated with a single intraperitoneal injection of 100 mg/kg of diethylnitrosamine (DEN) or were given the saline vehicle alone; beginning 1 wk later, rats were given a diet containing 3,600; 1,800; 600; 200; 70; or 0 ppm of fenbendazole for 8 wk. Subgroups of 5 rats each from the DEN+ 1,800; DEN+0; 1,800; and 0 ppm groups were euthanatized after 1 wk of fenbendazole treatment, and the remaining animals were euthanatized at 8 wk. After 1 wk, relative liver weights (ratios to body weights) were significantly increased in the DEN+ 1,800 and 1,800 ppm groups, and based on light microscopy, periportal hepatocellular hypertrophy was evident in these groups. After 8 wk, relative liver weights were significantly increased in the groups given > or =600 ppm with or without DEN initiation. Periportal hepatocellular hypertrophy, characterized by a marked increase in smooth endoplasmic reticulum, was observed in the groups given > or =600 ppm with or without DEN initiation. Induction of cytochrome P-450 (CYP) 1A2, 2B1, or 4A1 was noted in the fenbendazole-treated groups with or without DEN initiation; that associated with CYP 1A2 was most marked. Positive immunostaining for anti-CYP 1A1/2 or CYP 2B1/2 was observed diffusely in the livers of animals in the DEN+1,800 and DEN+3,600 ppm groups. The numbers and areas of connexin 32 (Cx32)-positive spots per square centimeter in centrilobular hepatocytes were significantly decreased in an almost dose-dependent manner with fenbendazole treatment after DEN initiation. In situ hybridization for Cx32 mRNA revealed a remarkable decrease in its expression in the centrilobular hepatocytes in the DEN+70 ppm group. The numbers of glutathione S-transferase placental-form positive single cells (plus mini foci) were significantly increased in the DEN+ 1,800 and DEN+3,600 ppm groups. Since those agents that induce CYP 2B1/2 isozymes and reduce Cx32 in centrilobular hepatocytes have been suggested to be liver tumor promoters, the present results indicate that fenbendazole may be a liver tumor promoter.
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PMID:Liver tumor promoting effects of fenbendazole in rats. 1052 35

To examine the relationship between the decrease in connexin 32 (Cx32) and induction of P450 isozymes in the early phase of clofibrate hepatocarcinogenesis, a total of 20 male F344 rats were initiated with a single intraperitoneal injection of 150 mg/kg of diethylnitrosamine (DEN) or given the saline vehicle alone and starting 2 weeks later given diet containing 0.18, 0.09, and 0% clofibrate for 6 weeks. All animals were subjected to two-thirds partial hepatectomy at week 3 and killed at week 8. Absolute and relative (ratios to body weight) liver weights were significantly increased in the DEN + clofibrate groups compared with the DEN-alone group. Diffuse hepatocellular hypertrophy with granular cytoplasmic eosinophilia characterized by a marked increase in peroxisomes and smooth endoplasmic reticulum, was observed in the clofibrate treated rats. Induction of cytochrome P450 (CYP) 4A1 and 2B1/2 was noted in the DEN + clofibrate groups, this being most marked in the CYP 2B1 case. Immunohistochemically, positive immunostaining for anti-CYP 4A1 and CYP 2B1 were observed diffusely and centrilobularly, respectively. The numbers and areas of Cx32-positive spots per hepatocyte in the centrilobular areas in the treated rats were significantly decreased in an essentially dose-dependent manner, but no changes were observed in periportal areas. The numbers and areas of foci positive for glutathione S-transferase placental form (GST-P) were decreased in a dose dependent manner in the clofibrate treated groups. These results suggest that the CYP 2B1/2 induction and Cx32 decrease in centrilobular hepatocytes, similarly to those thought to be involved in the hepatic promotion mechanism of phenobarbital, may also play important roles in clofibrate actions in the liver, in addition to its causation of oxidative DNA injury.
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PMID:The relationship between decrease in Cx32 and induction of P450 isozymes in the early phase of clofibrate hepatocarcinogenesis in the rat. 1055 Apr 79

The interaction of tumor necrosis factor-alpha (TNFalpha) with its receptor sets in motion downstream signaling events including the activation of members of the mitogen-activated protein kinase (MAPK) family. In this study, we show that p42(mapk/erk2) phosphorylates sequences present within the cytoplasmic domain of CD120a (p55). By using a GST-CD120a-(207-425) fusion protein as substrate, phosphorylation was induced following stimulation of mouse macrophages with TNFalpha, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, and zymosan particles and was blocked by immunodepletion of p42(mapk/erk2) and by specific inhibition of p42(mapk/erk2) activation with PD098059. Transfection of COS-7 cells with CD120a (p55), wild-type p42(mapk/erk2), and constitutively active MEK-1 followed by metabolic labeling with [(32)P]orthophosphate indicated that p42(mapk/erk2) phosphorylated the cytoplasmic domain of CD120a (p55) in intact cells. As a consequence of phosphorylation, CD120a (p55) expression at the plasma membrane and Golgi apparatus was lost and the receptor accumulated in intracellular tubular structures associated with the endoplasmic reticulum. Mutation of the four Ser and Thr ERK consensus phosphorylation sites to Ala residues inhibited the ability of the receptor to redistribute to intracellular tubules in a p42(mapk/erk2)-dependent fashion; whereas mutation of the phosphorylation sites to Asp and Glu residues mimicked the effect of receptor phosphorylation. These findings thus indicate that the phosphorylation of CD120a (p55) alters the subcellular localization of the receptor and may thereby result in changes in its signaling properties.
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PMID:Phosphorylation of tumor necrosis factor receptor CD120a (p55) by p42(mapk/erk2) induces changes in its subcellular localization. 1055 65

DnaJ proteins are located in various compartments of the eukaryotic cell. As previously shown, peroxisomes and glyoxysomes possess a membrane-anchored form of DnaJ protein located on the cytosolic face. Hints as to how the membrane-bound co-chaperone interacts with cytosolic soluble chaperones were obtained by examining the affinity between the DnaJ protein and various potential partners of the Hsp70 family. Two genes encoding cytosolic Hsp70 isoforms were isolated and characterized from cucumber cotyledons. In addition, cDNAs encoding Hsp70 forms attributed to the cytosol, plastids and the lumen of the endoplasmic reticulum were prepared. His-tagged DnaJ proteins and glutathione S-transferase-Hsp70 fusion proteins were constructed. Using these tools, it was demonstrated that the soluble His-tagged form of DnaJ protein exclusively binds the cytosolic isoform 1 of Hsp70. This interaction was further analyzed by characterizing the interaction between the glyoxysome-bound form of the DnaJ protein and various isoforms of Hsp70. Specific binding to the glyoxysomal surface was only observed in the case of cytosolic isoform 1 of Hsp70. This interaction was strictly dependent on the presence of ADP. Glyoxysomes did not bind other cytosolic or plastidic isoforms or the BiP-related form of Hsp70. Analyzing the enzymatic properties of cytosolic Hsp70s, we showed that the ATPase-modulating activity of DnaJ was highest when isoform 1 was assayed. Collectively, the data indicate that the partner of the DnaJ protein anchored at the glyoxysomal membrane is the cytosolic isoform 1 of Hsp70. In addition to the chaperones located at the surface of glyoxysomes, two isoforms of Hsp70 and one soluble form of DnaJ protein were detected in the glyoxysomal matrix.
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PMID:The membrane-bound DnaJ protein located at the cytosolic site of glyoxysomes specifically binds the cytosolic isoform 1 of Hsp70 but not other Hsp70 species. 1065 11

To analyze the role of coat protein type II (COPII) coat components and targeting and fusion factors in selective export from the endoplasmic reticulum (ER) and transport to the Golgi, we have developed three novel, stage-specific assays. Cargo selection can be measured using a "stage 1 cargo capture assay," in which ER microsomes are incubated in the presence of glutathione S-transferase (GST)-tagged Sar1 GTPase and purified Sec23/24 components to follow recruitment of biosynthetic cargo to prebudding complexes. This cargo recruitment assay can be followed by two sequential assays that measure separately the budding of COPII-coated vesicles from ER microsomes (stage 2) and, finally, delivery of cargo-containing vesicles to the Golgi (stage 3). We show how these assays provide a means to identify the snap receptor (SNARE) protein rBet1 as an essential component that is not required for vesicle formation, but is required for vesicle targeting and fusion during ER-to-Golgi transport. In general, these assays provide an approach to characterize the biochemical basis for the recruitment of a wide variety of biosynthetic cargo proteins to COPII vesicles and the role of different transport components in the early secretory pathway of mammalian cells.
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PMID:Stage-specific assays to study biosynthetic cargo selection and role of SNAREs in export from the endoplasmic reticulum and delivery to the Golgi. 1072 Apr 62

The components within cytotoxic lymphocyte granules are responsible for a significant fraction of T and NK cell-mediated death. Perforin is stored in these granules together with calreticulin. Calreticulin has long been recognized as a chaperone protein of the endoplasmic reticulum (ER) and is the only resident ER protein to be found in the cytotoxic granules. Here we implicate a role for calreticulin in killing and report that it controls osmotic lysis mediated by purified perforin. Calreticulin, at a concentration of 2.2 x 10-7 M, completely blocked perforin-mediated lysis. Inhibition was stable and held over 5 h. Recombinant calreticulin, at a concentration of 8. 8 x 10-7 M, also blocked lysis, indicating the inhibition was due to calreticulin and not a copurifying protein in the native calreticulin preparations. Using calreticulin domain fragments (expressed as GST fusion proteins), we found inhibitory activity in the high-capacity calcium-binding C-domain, which does not bind perforin. The N- or P-domains, which can bind perforin, were unable to block lysis. The inhibition of lysis was independent of granzyme inactivation or the ability of calreticulin to sequester calcium. Our data indicate that calreticulin regulation of perforin-mediated lysis probably occurs without direct interaction with perforin. We propose a novel model in which calreticulin stabilizes membranes to prevent polyperforin pore formation.
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PMID:Perforin lytic activity is controlled by calreticulin. 1075 10


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