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)

Soluble N -ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), including synaptosome-associated proteins of 25 kDa (SNAP25), syntaxins, and vesicle-associated membrane proteins (VAMP), are essential for regulated exocytosis of synaptic vesicles in neurotransmission. We identified a cDNA coding for a novel protein of 266 amino acids that we have named SIP30 (S NAP25 interacting protein of 30 kDa). SIP30 is expressed abundantly in brain and slightly in testis and kidney. In brain, SIP30 is highly expressed in the inferior and superior colliculi, which contain important relay nuclei of the auditory and visual systems. GST-pull-down and immunoprecipitation assays showed direct binding of SIP30 to SNAP25. Although SIP30 does not directly interact with syntaxin based on pull-down assays, syntaxin does co-immunoprecipitate with SIP30 suggesting that syntaxin is indirectly associated with SIP30, perhaps through SNAP25.
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PMID:Identification of a novel SNAP25 interacting protein (SIP30). 1206 81

Mastoparan, a hormone receptor-mimetic peptide isolated from wasp venom, stimulates insulin release from pancreatic beta-cells in a Ca(2+)-independent but GTP-dependent manner. In this report, the role of the Rho family GTP-binding protein Cdc42, in the mastoparan stimulus-secretion pathway, was examined. Overexpression of wild-type Cdc42 in beta HC-9 cells, an insulin-secreting mouse-derived cell line, resulted in a 2-fold increase in mastoparan-stimulated insulin release over vector-transfected beta HC-9 cells. This effect was not seen with secretagogues such as glucose that stimulate secretion via Ca(2+)-dependent pathways. GDP/GTP exchange assay data and studies with pertussis (PTX) toxin suggest that mastoparan may work directly to activate Cdc42 and not via PTX-sensitive heterotrimeric GTP-binding proteins. Using bacterial glutathione S-transferase-Cdc42 fusion proteins and co-immunoprecipitation and transient transfection studies, Cdc42 was shown to be an upstream regulator of the exocytotic protein, syntaxin. These results suggest that the GTP-dependent signal underlying the mastoparan effect acts at a "distal site" in stimulus-secretion coupling on one of the SNARE proteins essential for exocytosis. In vitro binding assays, using purified Cdc42 and syntaxin proteins, show that Cdc42 mediates the GTP signal through an indirect association with syntaxin. The H3 domain at the C-terminus of syntaxin, which participates in the formation of the ternary SNARE complex with the core proteins, SNAP-25 and synaptobrevin, is also required for the association with Cdc42. Thus, these studies indicate that Cdc42 could be a putative GTP-binding protein thought to be involved in the mastoparan-stimulated GTP-dependent pathway of insulin release.
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PMID:A link between Cdc42 and syntaxin is involved in mastoparan-stimulated insulin release. 1213 88

Intercalated and inner medullary collecting duct (IMCD) cells of the kidney mediate the transport of H+ by a plasma membrane H+-ATPase. The rate of H+ transport in these cells is regulated by exocytic insertion of H+-ATPase-laden vesicles into the apical membrane. We have shown that the exocytic insertion of proton pumps (H+-ATPase) into the apical membrane of rat IMCD cells, in culture, involves SNARE proteins (syntaxin (synt), SNAP-23, and VAMP). The membrane fusion complex observed in IMCD cells with the induction of proton pump exocytosis not only included these SNAREs but also the H+-ATPase. Based on these observations, we suggested that the targeting of these vesicles to the apical membrane is mediated by an interaction between the H+-ATPase and a specific t-SNARE. To evaluate this hypothesis, we utilized a "pull-down" assay in which we identified, by Western analysis, the proteins in a rat kidney medullary homogenate that complexed with glutathione S-transferase (GST) fusion syntaxin isoforms attached to Sepharose 4B-glutathione beads. The syntaxin isoforms employed were 1A, 1B, 2, 4, 5, and also 1A that was truncated to exclude the H3 SNARE binding domain (synt-1ADeltaH3). All full-length syntaxin isoforms formed complexes with SNAP-23 and VAMP. Neither GST nor synt-1ADeltaH3 formed complexes with these SNAREs. H+-ATPase (subunits E, a, and c) bound to syntaxin-1A and to a lesser extent to synt-1B but not to synt-1ADeltaH3 or synt-2, -4, and -5. In cultured IMCD cells transfected to express syntaxin truncated for the membrane binding domain (synt-DeltaC), expression of synt-1ADeltaC, but not synt-4DeltaC, inhibited H+-ATPase exocytosis. In conclusion, because all full-length syntaxins examined bound VAMP-2 and SNAP-23, but only non-H3-truncated syntaxin-1 bound H+-ATPase, and synt-1ADeltaC expression by intact IMCD cells inhibited H+-ATPase exocytosis, it is likely that the H+-ATPase binds directly to the H3 domain of syntaxin-1 and not through VAMP-2 or SNAP-23. Interaction between the syntaxin-1A and H+-ATPase is important in the targeted exocytosis of the proton pump to the apical membrane of intercalated cells.
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PMID:Syntaxin isoform specificity in the regulation of renal H+-ATPase exocytosis. 1265 53

Zinc deficiency affects hepatic functions and due to the central role of the liver in metabolism, this may contribute to metabolic alterations in other tissues in zinc deficiency. In addition to clinical manifestations of zinc deficiency, we used cDNA- and oligonucleotide-arrays to compare the expression of > 2500 different genes in liver of rats force-fed a zinc-adequate or a zinc-deficient diet for 11 d. Radio- or fluorescence-labeled cDNAs from liver of control and zinc-deficient rats were hybridized to arrays. Approximately 1550 mRNAs were detected above background levels; by comparing expression profiles of the two groups, the mRNA levels of 66 genes were found to be altered by zinc deficiency. Steady-state expression levels of 35 genes were reduced, whereas the mRNA-levels of 31 genes were elevated. Array data were verified by Northern blot analysis for 24 selected genes and 19 were confirmed to be up- or down-regulated. Among those, predominantly gene products that participate in growth (i.e., insulin-like growth factor binding proteins), lipid metabolism (long-chain acyl-CoA synthetase), xenobiotic metabolism (cytochrome P(450) isoenzymes), the stress response (glutathione transferase), nitrogen metabolism (cytosolic aspartate aminotransferase), intracellular trafficking (syntaxin isoforms) and signal transduction (G-protein-coupled receptors) were identified. Additionally, regulation of mRNA levels of genes important for porphyrin synthesis and collagen metabolism was observed. In conclusion, we have identified in vivo a number of mammalian genes from different cellular pathways whose expression changes in response to zinc depletion. The characterization of the identified genes and their products will allow a more comprehensive analysis of the role of zinc in metabolism; moreover, the mRNAs identified could be useful in establishing biomarkers for the determination of zinc status in mammals.
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PMID:Changes in rat hepatic gene expression in response to zinc deficiency as assessed by DNA arrays. 1267 11

The techniques of co-immunoprecipitation and immunocytochemical co-labelling are classically used to identify protein-protein interactions. We have used an antibody to the rat small conductance calcium-activated potassium channel subtype 1 (rSK1) to immunoprecipitate proteins from rat brain. A 35 kDa protein was recognized by two monoclonal antibodies to syntaxin 1 and a polyclonal antibody to syntaxin 1A, but not by antibodies to syntaxins 2, 3 or 4. These data suggested that syntaxin 1A is specifically associated with rSK1 in rat brain. A GST construct of the carboxyl terminus of rSK1 was able to pull-down syntaxin 1A from rat brain. Immunocytochemistry showed somatic labelling for both rSK1 and syntaxin 1A in acutely dissociated hippocampal CA1 neurons, confirming that these proteins could interact in vivo. However, control immunoprecipitations showed that antibodies to eight potassium channels could also immunoprecipitate syntaxin, even though some of these channels would not be expected to reside in the same subcellular compartment. Mock immunoprecipitations and pull-down assays showed that syntaxin 1 could directly interact with sepharose and agarose resins. Hence immunoprecipitation and pull-down assays do not provide evidence that syntaxin is specifically associating with a protein, placing doubt on a number of reported interactions with syntaxin 1A.
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PMID:False interaction of syntaxin 1A with a Ca(2+)-activated K(+) channel revealed by co-immunoprecipitation and pull-down assays: implications for identification of protein-protein interactions. 1268 80

Na(+) entry across the apical membranes of many absorptive epithelia is determined by the number (N) and open probability (P(o)) of epithelial sodium channels (ENaC). Previous results showed that the H3 domain of syntaxin-1A (S1A) binds to ENaC to reduce N, supporting a role for S1A in the regulation of ENaC trafficking. The aim of this study was to determine whether S1A-induced reductions in ENaC current also result from interactions between cell surface ENaC and S1A that alter ENaC P(o). Injection of a glutathione S-transferase (GST)-H3 S1A fusion protein into ENaC-expressing Xenopus oocytes inhibited whole cell Na(+) current (I(Na)) by 33% within 5 min. This effect was dose-dependent, with a K(i) of 7 ng/microl (approximately 200 nm). In contrast, injection of GST alone or a H3 domain-deleted GST-S1A fusion protein had no effect on I(Na). In cell-attached patch clamp experiments, GST-H3 acutely decreased ENaC P(o) by 30%, whereas GST-S1A Delta H3 was without effect. Further analysis revealed that ENaC mean closed time was significantly prolonged by S1A. Interestingly, GST-H3 had no effect on channel activity of an ENaC pore mutant that constitutively gates open (P(o) approximately equal 1.0), supporting the idea that S1A alters the closed state of ENaC and indicating that the actions of S1A on ENaC trafficking and gating can be separated experimentally. This study indicates that, in addition to a primary effect on ENaC trafficking, S1A interacts with cell surface ENaC to rapidly decrease channel gating. This rapid effect of S1A may modulate Na(+) entry rate during rapid increases in ENaC N.
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PMID:Syntaxin 1A regulates ENaC channel activity. 1470 19

H(+) transport in the collecting duct is regulated by exocytic insertion of H(+)-ATPase-laden vesicles into the apical membrane. The soluble N-ethylmaleimide-sensitive fusion protein attachment protein (SNAP) receptor (SNARE) proteins are critical for exocytosis. Syntaxin 1A contains three main domains, SNARE N, H3, and carboxy-terminal transmembrane domain. Several syntaxin isoforms form SNARE fusion complexes through the H3 domain; only syntaxin 1A, through its H3 domain, also binds H(+)-ATPase. This raised the possibility that there are separate binding sites within the H3 domain of syntaxin 1A for H(+)-ATPase and for SNARE proteins. A series of truncations in the H3 domain of syntaxin 1A were made and expressed as glutathione S-transferase (GST) fusion proteins. We determined the amount of H(+)-ATPase and SNARE proteins in rat kidney homogenate that complexed with GST-syntaxin molecules. Full-length syntaxin isoforms and syntaxin-1ADeltaC [amino acids (aa) 1-264] formed complexes with H(+)-ATPase and SNAP23 and vesicle-associated membrane polypeptide (VAMP). A cassette within the H3 portion was found that bound H(+)-ATPase (aa 235-264) and another that bound SNAP23 and VAMP (aa 190-234) to an equivalent degree as full-length syntaxin. However, the aa 235-264 cassette alone without the SNARE N (aa 1-160) does not bind but requires ligation to the SNARE N to bind H(+)-ATPase. When this chimerical construct was transected into inner medullary collecting duct cells it inhibited intracellular pH recovery, an index of H(+)-ATPase mediated secretion. We conclude that within the H3 domain of syntaxin 1A is a unique cassette that participates in the binding of the H(+)-ATPase to the apical membrane and confers specificity of syntaxin 1A in the process of H(+)-ATPase exocytosis.
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PMID:Syntaxin 1A has a specific binding site in the H3 domain that is critical for targeting of H+-ATPase to apical membrane of renal epithelial cells. 1587 13

Synaptic core complex formation between syntaxin and synaptosome-associated protein of 25 kDa (SNAP25) on the plasma membrane and synaptobrevin on the vesicle membrane is responsible for membrane fusion and neurotransmitter release. A radiolabeled protein binding assay for synaptic core complex formation was developed. The components of this assay included recombinant glutathione S-transferase (GST)-syntaxin immobilized on glutathione agarose beads, SNAP25 and (125)I-labeled synaptobrevin. Reactions were performed in tubes containing filter inserts to facilitate removal of unbound protein. The radiolabeled protein bound was then quantified by gamma counter. A K(d) of 1.6 microM was determined for the GST-syntaxin/SNAP25/synaptobrevin complex, and a K(d) of 12 microM was determined for the GST-syntaxin/synaptobrevin complex. The assay was used to screen 14 herbal extracts for effectors of core complex formation. Herbs traditionally used to treat neurological conditions such as depression, anxiety, and stress were chosen. A Hypericum perforatum extract was found to have a nonspecific effect via protein complexation, whereas an Albizzia julibrissin extract was found to reduce the level of core complex formation. The assay was used to further investigate the effect of the A. julibrissin extract. The discovery of an inhibitor of core complex formation demonstrates the efficacy of the assay in screening natural products for substances that affect core complex formation.
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PMID:A radioassay for synaptic core complex assembly: screening of herbal extracts for effectors. 1682 72

Increasing evidence implicates cyclin-dependent kinase 5 (Cdk5) in neuronal synaptic function. We searched for Cdk5 substrates in synaptosomal fractions prepared from mouse brains. Mass spectrometric analysis after two-dimensional SDS-PAGE identified several synaptic proteins phosphorylated by Cdk5-p35; one protein identified was Sept5 (CDCrel-1). Although septins were isolated originally as cell division-related proteins in yeast, Sept5 is expressed predominantly in neurons and is implicated in exocytosis. We confirmed that Sept5 is phosphorylated by Cdk5-p35 in vitro and identified Ser17 of adult type Sept5 (Sept5_v1) as a major phosphorylation site. We found that Ser17 of Sept5_v1 is phosphorylated in mouse brains. Coimmunoprecipitation from synaptosomal fractions and glutathione S-transferase-syntaxin-1A pulldown assays of Sept5_v1 expressed in COS-7 cells showed that phosphorylation of Sept5_v1 by Cdk5-p35 decreases the binding to syntaxin-1. These results indicate that the interaction of Sept5 with syntaxin-1 is regulated by the phosphorylation of Sept5_v1 at Ser17 by Cdk5-p35.
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PMID:Phosphorylation of adult type Sept5 (CDCrel-1) by cyclin-dependent kinase 5 inhibits interaction with syntaxin-1. 1722 48

Islet beta-cell-specific ATP-sensitive K(+) (K(ATP)) channel openers thiadiazine dioxides induce islet rest to improve insulin secretion, but their molecular basis of action remains unclear. We reported that syntaxin-1A binds nucleotide binding folds of sulfonylurea receptor 1 (SUR1) in beta-cells to inhibit K(ATP) channels. As a strategy to elucidate the molecular mechanism of action of these K(ATP) channel openers, we explored the possibility that 6-chloro-3-(1-methylcyclobutyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (NNC55-0462) might influence syntaxin-1A-SUR1 interactions or vice versa. Whole-cell and inside-out patch-clamp electrophysiology was used to examine the effects of glutathione S-transferase (GST)-syntaxin-1A dialysis or green fluorescence protein/syntaxin-1A cotransfection on NNC55-0462 actions. In vitro pull-down binding studies were used to examine NNC55-0462 influence on syntaxin-1A-SUR1 interactions. Dialysis of GST-syntaxin-1A into the cell cytoplasm reduced both potency and efficacy of extracellularly perfused NNC55-0462 in a HEK cell line stably expressing Kir6.2/SUR1 (BA8 cells) and in rat islet beta-cells. Moreover, inside-out membrane patches excised from BA8 cells showed that both GST-syntaxin-1A and its H3 domain inhibited K(ATP) channels previously activated by NNC55-0462. This action on K(ATP) channels is isoform-specific to syntaxin-1A because syntaxin-2 was without effect. Furthermore, the parent compound diazoxide showed similar sensitivity to GST-syntaxin-1A inhibition. NNC55-0462, however, did not influence syntaxin-1A-SUR1 binding interaction. Our results demonstrated that syntaxin-1A interactions with SUR1 at its cytoplasmic domains can modulate the actions of the K(ATP) channel openers NNC55-0462 and diazoxide on K(ATP) channels. The reduced levels of islet syntaxin-1A in diabetes would thus be expected to exert a positive influence on the therapeutic effects of this class of K(ATP) channel openers.
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PMID:The actions of a novel potent islet beta-cell specific ATP-sensitive K+ channel opener can be modulated by syntaxin-1A acting on sulfonylurea receptor 1. 1749 34


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