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 cDNA clone encoding a rac-like small GTP binding protein was isolated from a cDNA library of Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower buds and named Brac1. The Brac1 cDNA contains an open reading frame encoding 198 amino acid residues with an estimated molecular mass of 21,690 Da and this coding region has conserved residues and motifs unique to the Rho subfamily of proteins. The deduced amino acid sequence of the Brac1 protein is closely related to that of Arabidopsis thaliana Arac3 (91%), but it shares relatively little homology with other members of the Ras superfamily (about 30% identity). To further characterize Brac1, a pGBrac1 expression vector consisting of PCR-amplified Brac1 cDNA plus glutathione S-transferase (GST) and pBKS(+)II was used to purify the protein. Using a PEI-cellulose/TLC plate, GTPase activity of this protein was confirmed and competition binding studies, using the guanine nucleotides, ATP, UTP and CTP, revealed that the di- and triphosphate forms of guanine nucleotides strongly bind Brac1. Membrane-bound PLD activity was synergistically enhanced by Brac1 in the presence of protein kinase C, but not in the presence of ARF (ADP-ribosylation factor). Genomic analysis indicated that Brac1 belongs to a multigene family. Brac1 transcripts were expressed in all the organs of Brassica, but were especially prevalent in flower buds.
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PMID:A rac-like small G-protein from Brassica campestris activates a PKC-dependent phospholipase D. 1469 72

The cytotoxic necrotizing factors (CNF)1 and CNF2 from pathogenic Escherichia coli strains activate RhoA, Rac1, and Cdc42 by deamidation of Gln63 (RhoA) or Gln61 (Rac and Cdc42). Recently, a novel cytotoxic necrotizing factor termed CNFY was identified in Yersinia pseudotuberculosis strains (Lockman, H. A., Gillespie, R. A., Baker, B. D., and Shakhnovich, E. (2002) Infect. Immun. 70, 2708-2714). We amplified the cnfy gene from genomic DNA of Y. pseudotuberculosis, cloned and expressed the recombinant protein, and studied its activity. Recombinant GST-CNFY induced morphological changes in HeLa cells and caused an upward shift of RhoA in SDS-PAGE, as is known for GST-CNF1 and GST-CNF2. Mass spectrometric analysis of GST-CNFY-treated RhoA confirmed deamidation at Glu63. Treatment of RhoA, Rac1, and Cdc42 with GST-CNFY decreased their GTPase activities, indicating that all of these Rho proteins could serve as substrates for GST-CNFY in vitro. In contrast, RhoA, but not Rac or Cdc42, was the substrate of GST-CNFY in culture cells. GST-CNFY caused marked stress fiber formation in HeLa cells after 2 h. In contrast to GST-CNF1, formation of filopodia or lamellipodia was not induced with GST-CNFY. Accordingly, effector pull-down experiments with lysates of toxin-treated cells revealed strong activation of RhoA but no activation of Rac1 or Cdc42 after 6 h of GST-CNFY-treatment. Moreover, in rat hippocampal neurons, GST-CNFY results in the retraction of neurites, indicating RhoA activation. In contrast, no activation of Rac or Cdc42 was found. Altogether, our data suggest that CNFY from Y. pseudotuberculosis is a strong, selective activator of RhoA, which can be used as a powerful tool for constitutive RhoA activation without concomitant activation of Rac1 or Cdc42.
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PMID:The Yersinia pseudotuberculosis cytotoxic necrotizing factor (CNFY) selectively activates RhoA. 1476 41

Dynamin 2 (Dyn2) is a large GTPase involved in vesicle formation and actin reorganization. In this study, we report a novel role for Dyn2 as a component of the centrosome that is involved in centrosome cohesion. By light microscopy, Dyn2 localized aside centrin and colocalized with gamma-tubulin at the centrosome; by immunoelectron microscopy, however, Dyn2 was detected in the pericentriolar material as well as on centrioles. Exogenously expressed green fluorescent protein (GFP)-tagged Dyn2 also localized to the centrosome, whereas glutathione S-transferase (GST)-tagged Dyn2 pulled down a protein complex(es) containing actin, alpha-tubulin and gamma-tubulin from liver homogenate. Furthermore, gel overlay and immunoprecipitation indicated a direct interaction between gamma-tubulin and a 219-amino-acid middle domain of Dyn2. Reduction of Dyn2 protein levels with small-interfering RNA (siRNA) resulted in centrosome splitting, whereas microtubule nucleation from centrosomes was not affected, suggesting a role for Dyn2 in centrosome cohesion. Finally, fluorescence recovery after photobleaching (FRAP) analysis of a GFP-tagged Dyn2 middle domain indicated that Dyn2 is a dynamic exchangeable component of the centrosome. These findings suggest a novel function for Dyn2 as a participant in centrosome cohesion.
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PMID:Dynamin 2 binds gamma-tubulin and participates in centrosome cohesion. 1504 27

Ras proteins are guanine nucleotide-binding proteins that are highly conserved among eukaryotes. They are involved in signal transduction pathways and are tightly regulated by two sets of antagonistic proteins: GTPase-activating proteins (GAPs) inhibit Ras proteins, whereas guanine exchange factors activate them. In this work, we describe Tfs1p, the first physiological inhibitor of a Ras GAP, Ira2p, in Saccharomyces cerevisiae. TFS1 is a multicopy suppressor of the cdc25-1 mutation in yeast and corresponds to the so-called Ic CPY cytoplasmic inhibitor. Moreover, Tfs1p belongs to the phosphatidylethanolamine-binding protein (PEBP) family, one member of which is RKIP, a kinase and serine protease inhibitor and a metastasis inhibitor in prostate cancer. In this work, the results of (i) a two-hybrid screen of a yeast genomic library, (ii) glutathione S-transferase pulldown experiments, (iii) multicopy suppressor tests of cdc25-1 mutants, and (iv) stress resistance tests to evaluate the activation level of Ras demonstrate that Tfs1p interacts with and inhibits Ira2p. We further show that the conserved ligand-binding pocket of Tfs1-the hallmark of the PEBP family-is important for its inhibitory activity.
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PMID:Tfs1p, a member of the PEBP family, inhibits the Ira2p but not the Ira1p Ras GTPase-activating protein in Saccharomyces cerevisiae. 1507 75

Ras-related small GTPases serve as critical regulators for a wide range of cellular signaling pathways and are activated by the conversion of the GDP-bound state to the GTP-bound conformation. Until recently, measurement of the GTP-bound active form of Ras-related G proteins involved immunoprecipitation of 32P-labeled protein followed by separation of the labeled GTP/GDP bound to GTPase. A new method based on the large affinity difference of the GTP- and GDP-bound form of Ras proteins for specific binding domains of effector proteins in vitro has been developed. By using glutathione S-transferase (GST) fusion proteins containing these binding domains, the GTP-bound form of the GTPase can be precipitated from cell lysates. In principle, this method can be used for all members of the Ras superfamily. Here we describe a general procedure to monitor the GTP-bound form of Ras-related GTPases.
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PMID:Nonradioactive methods for detecting activation of Ras-related small G proteins. 1517 15

We constructed an overexpression system for human ribosomal phosphoprotein P0, together with P1 and P2, which is crucially important for translation. Genes for these proteins, fused with the glutathione S-transferase (GST)-tag at the N-terminus, were inserted into baculovirus and introduced to insect cells. The fusion proteins, but not the proteins without the tag, were efficiently expressed into cells as soluble forms. The fusion protein GST.P0 as well as GST.P1/GST.P2 was phosphorylated in cells as detected by incorporation of (32)P and reactivity with monoclonal anti-phosphoserine antibody. GST.P0 expressed in insect cells, but not the protein obtained in Escherichia coli, had the ability to form a complex with P1 and P2 proteins and to bind to 28S rRNA. Moreover, the GST.P0-P1-P2 complex participated in high eEF-2-dependent GTPase activity. Baculovirus expression systems appear to provide recombinant human P0 samples that can be used for studies on the structure and function.
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PMID:Baculovirus-mediated expression and isolation of human ribosomal phosphoprotein P0 carrying a GST-tag in a functional state. 1533 36

Rac1 GTPase is implicated as a signaling mediator in various cellular events. In this study, we show that Rac1 contributes to IFN-gamma-induced inflammatory responses in rat astrocytes. We revealed that IFN-gamma rapidly stimulated activation of Rac1 in C6 astroglioma cells by investigating GST-PAK-PBD-binding ability. We also found that Rac1 deficiency led to attenuation of IFN-gamma-responsive transcriptional responses. Compared with levels in control cells, IFN-gamma-induced IFN-gamma-activated sequence promoter activity was markedly reduced in both C6 astroglioma cells and primary astrocytes expressing RacN17, a well-characterized Rac1-negative mutant. The expression of several IFN-gamma-responsive genes, such as MCP-1 and ICAM-1, was also reduced in cells expressing RacN17. Consistent with these observations, IFN-gamma-induced phosphorylation of STAT1 and STAT3 was lower in C6 cells expressing RacN17 (referred to as C6-RacN17) than in control cells. However, there was no difference in expression level of IFN-gammaRalpha subunit and IFN-gamma-induced phosphorylation of JAK1 between C6 control and C6-RacN17 cells. Interestingly, Rac1 appeared to associate with IFN-gammaRalpha and augment the interaction of IFN-gammaR with either STAT1 or STAT3 in response to IFN-gamma. Taken together, we suggest that Rac1 may serve as an auxiliary mediator of IFN-gamma-signaling, at least at the level of STAT activation, thus contributing to maximal activation of IFN-gamma-responsive inflammatory signaling in rat astrocytes.
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PMID:Rac1 contributes to maximal activation of STAT1 and STAT3 in IFN-gamma-stimulated rat astrocytes. 1549 21

Agrobacterium tumefaciens uses a type IV secretion system (T4SS) to transfer T-DNA and virulence proteins to plants. The T4SS is composed of two major structural components: the T-pilus and a membrane-associated complex that is responsible for translocating substrates across both bacterial membranes. VirB2 protein is the major component of the T-pilus. We used the C-terminal-processed portion of VirB2 protein as a bait to screen an Arabidopsis thaliana cDNA library for proteins that interact with VirB2 in yeast. We identified three related plant proteins, VirB2-interacting protein (BTI) 1 (BTI1), BTI2, and BTI3 with unknown functions, and a membrane-associated GTPase, AtRAB8. The three BTI proteins also interacted with VirB2 in vitro. Preincubation of Agrobacterium with GST-BTI1 protein decreased the transformation efficiency of Arabidopsis suspension cells by Agrobacterium. Transgenic BTI and AtRAB8 antisense and RNA interference Arabidopsis plants are less susceptible to transformation by Agrobacterium than are wild-type plants. The level of BTI1 protein is transiently increased immediately after Agrobacterium infection. In addition, overexpression of BTI1 protein in transgenic Arabidopsis results in plants that are hypersusceptible to Agrobacterium-mediated transformation. Confocal microscopic data indicate that GFP-BTI proteins preferentially localize to the periphery of root cells in transgenic Arabidopsis plants, suggesting that BTI proteins may contact the Agrobacterium T-pilus. We propose that the three BTI proteins and AtRAB8 are involved in the initial interaction of Agrobacterium with plant cells.
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PMID:Plant proteins that interact with VirB2, the Agrobacterium tumefaciens pilin protein, mediate plant transformation. 1549 53

In pancreatic beta cells, insulin granule exocytosis is regulated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein (SNAP) receptor) proteins, and this is coupled to cortical F-actin reorganization via the Rho family GTPase Cdc42 by an unknown mechanism. We investigated interactions among the target SNARE protein Syntaxin 1A and the vesicle-associated membrane SNARE protein (VAMP2) with Cdc42 and compared these structural interactions with their functional importance to glucose-stimulated insulin secretion in MIN6 beta cells. Subcellular fractionation analyses revealed a parallel redistribution of Cdc42 and VAMP2 from the granule fraction to the plasma membrane in response to glucose that temporally corresponded with the glucose-induced activation of Cdc42. Moreover, within these fractions Cdc42 and VAMP2 were found to co-immunoprecipitate under basal and glucose-stimulated conditions, suggesting that they moved as a complex. Furthermore, VAMP2 bound both GST-Cdc42-GTPgammaS and GST-Cdc42-GDP, indicating that the Cdc42-VAMP2 complex could form under both cytosolic GDP-bound Cdc42 and plasma membrane GTP-bound Cdc42 conformational conditions. In vitro binding analyses showed that VAMP2 bound directly to Cdc42 and that a heterotrimeric complex with Syntaxin 1A could also be formed. Deletion analyses of VAMP2 revealed that only the N-terminal 28 residues were required for Cdc42 binding. Expression of this 28-residue VAMP2 peptide in MIN6 beta cells resulted in the specific impairment of glucose-stimulated insulin secretion, indicating a functional importance for the Cdc42-VAMP2 interaction. Taken together, these data suggest a mechanism whereby glucose activates Cdc42 to induce the targeting of intracellular Cdc42-VAMP2-insulin granule complexes to Syntaxin 1A at the plasma membrane.
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PMID:A direct interaction between Cdc42 and vesicle-associated membrane protein 2 regulates SNARE-dependent insulin exocytosis. 1553 56

G protein-regulated inducer of neurite outgrowth 1 (GRIN1) was initially identified as a binding protein for guanosine 5'-3-O-(thio)triphosphate-bound Galphaz. GRIN1 is specifically expressed in brain and interacts selectively with activated alpha subunits of the Gi subfamily. GRIN1 colocalizes with Galphao at the growth cone of neuronal cells and promotes neurite extension in Neuro2a cells when coexpressed with constitutively active mutant GalphaoQ205L. These results suggest that GRIN1 functions as a downstream target for Galphao. However, GRIN1 does not contain domains that are homologous to known signaling motifs. To understand the mechanisms of Galphao-GRIN1 pathway, we analyzed functional domains of GRIN1 that are involved in binding with Galphao or with its targeting to the plasma membrane. Using pull-down assays with glutathione S-transferase-fused GRIN1 deletion mutants, Galphao binding regions were localized to amino acid residues 716 to 746 and 797 to 827 of GRIN1. The Galphao binding region of GRIN1 did not demonstrate GTPase accelerating activity for Galphao. GRIN1 localized in the cell periphery in Neuro2a cells, and two cysteine residues at C-terminal region of GRIN1 (Cys818 and Cys819) were shown to be critical for its membrane targeting. Coexpression of GRIN1 with GalphaoQ205L or GRIN1Delta(717-827), which lacks Galphao binding region, promoted microspike formation in Swiss 3T3 cells or neurite extension in Neuro2a cells. The dominant-negative mutant of Cdc42 blocked these morphological changes. Coexpression of GRIN1 and GalphaoQ205L stimulated the formation of GTP-bound Cdc42 in Swiss 3T3 cells. These results suggest that the binding of activated Galphao to GRIN1 induces activation of Cdc42, which leads to morphological changes in neuronal cells.
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PMID:Functional characterization of Galphao signaling through G protein-regulated inducer of neurite outgrowth 1. 1558 44


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