Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.5.1.18 (glutathione S-transferase)
 22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Sec1 is a hydrophilic protein that plays an essential role in exocytosis from the yeast Saccharomyces cerevisiae. Two high copy suppressors of mutations in the Sec1 gene, SSO1 and SSO2, were recently identified that encode proteins of the syntaxin family. Syntaxin (a T-SNARE), together with SNAP-25 and synaptobrevin/VAMP (a T- and a V-SNARE, respectively), is thought to form the core of the docking-fusion complex in synaptic vesicle exocytosis. Proteins that exhibit similarity to Sec1 were identified in the nervous system of Drosophila melanogaster (Rop) and Caenorhabditis elegans (UNC18). Based on the amino acid sequence alignment of Sec1, Rop, and UNC18, we have used a PCR-based approach to isolate a rat brain cDNA encoding a Sec1 homologue. The cDNA hybridizes to a 3.5-kb brain-specific mRNA by Northern blot analysis and encodes a protein of 593 amino acids (rbSec1). Antibodies raised against a central portion of rbSec1 recognize a 67.5-kDa protein in total homogenates of rat brain but not of nonneuronal tissues. When incubated with a Triton X-100 brain extract, rbSec1-glutathione S-transferase (GST) fusion protein, but not GST protein alone, specifically interacts with syntaxin but not with SNAP-25 or synaptobrevin/VAMP. We conclude that the function of proteins of the Sec1 family in membrane fusion involves an interaction with a T-SNARE.
 ...
PMID:A rat brain Sec1 homologue related to Rop and UNC18 interacts with syntaxin. 813 39

In adipocytes, insulin stimulates the translocation of the glucose transporter, GLUT4, from an intracellular storage compartment to the cell surface. Substantial evidence exists to suggest that in the basal state GLUT4 resides in discrete storage vesicles. A direct interaction of GLUT4 storage vesicles with the plasma membrane has been implicated because the v-SNARE, vesicle-associated membrane protein-2 (VAMP2), appears to be a specific component of these vesicles. In the present study we sought to identify the cognate target SNAREs for VAMP2 in mouse 3T3-L1 adipocytes. Membrane fractions were isolated from adipocytes and probed by far Western blotting with the cytosolic portion of VAMP2 fused to glutathione S-transferase. Two plasma membrane-enriched proteins, p25 and p35, were specifically labeled with this probe. By using a combination of immunoblotting, detergent extraction, and anion exchange chromatography, we identified p35 as Syntaxin-4 and p25 as the recently identified murine SNAP-25 homologue, Syndet (mSNAP-23). By using surface plasmon resonance we show that VAMP2, Syntaxin-4, and Syndet form a ternary SDS-resistant SNARE complex. Microinjection of anti-Syndet antibodies into 3T3-L1 adipocytes, or incubation of permeabilized adipocytes with a synthetic peptide comprising the C-terminal 24 amino acids of Syndet, inhibited insulin-stimulated GLUT4 translocation to the cell surface by approximately 40%. GLUT1 trafficking remained unaffected by the presence of the peptide. Our data suggest that Syntaxin-4 and Syndet are important cell-surface target SNAREs within adipocytes that regulate docking and fusion of GLUT-4-containing vesicles with the plasma membrane in response to insulin.
 ...
PMID:Syndet, an adipocyte target SNARE involved in the insulin-induced translocation of GLUT4 to the cell surface. 966 52

Syntaxin 1, synaptobrevins or vesicle-associated membrane proteins, and the synaptosome-associated protein of 25 kDa (SNAP-25) are key molecules involved in the docking and fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, cell biological, and biochemical characterization of a 32-kDa protein homologous to both SNAP-25 (20% amino acid sequence identity) and the recently identified SNAP-23 (19% amino acid sequence identity). Northern blot analysis shows that the mRNA for this protein is widely expressed. Polyclonal antibodies against this protein detect a 32-kDa protein present in both cytosol and membrane fractions. The membrane-bound form of this protein is revealed to be primarily localized to the Golgi apparatus by indirect immunofluorescence microscopy, a finding that is further established by electron microscopy immunogold labeling showing that this protein is present in tubular-vesicular structures of the Golgi apparatus. Biochemical characterizations establish that this protein behaves like a SNAP receptor and is thus named Golgi SNARE of 32 kDa (GS32). GS32 in the Golgi extract is preferentially retained by the immobilized GST-syntaxin 6 fusion protein. The coimmunoprecipitation of syntaxin 6 but not syntaxin 5 or GS28 from the Golgi extract by antibodies against GS32 further sustains the preferential interaction of GS32 with Golgi syntaxin 6.
 ...
PMID:GS32, a novel Golgi SNARE of 32 kDa, interacts preferentially with syntaxin 6. 988 Mar 31

Synaptotagmins represent a family of neuronal proteins thought to function in membrane traffic. The best characterized synaptotagmin, synaptotagmin I, is essential for fast Ca2+-dependent synaptic vesicle exocytosis, indicating a role in the Ca2+ triggering of membrane fusion. Synaptotagmins contain two C2 domains, the C2A and C2B domains, which bind Ca2+ and may mediate their functions by binding to specific targets. For synaptotagmin I, several putative targets have been identified, including the SNARE proteins syntaxin and SNAP-25. However, it is unclear which of the many binding proteins are physiologically relevant. Furthermore, more than 10 highly homologous synaptotagmins are expressed in brain, but it is unknown if they execute similar binding reactions. To address these questions, we have performed a systematic, unbiased study of proteins which bind to the C2A domains of synaptotagmins I-VII. Although the various C2A domains exhibit similar binding activities for phospholipids and syntaxin, we found that they differ greatly in their protein binding patterns. Surprisingly, none of the previously characterized binding proteins for synaptotagmin I are among the major interacting proteins identified. Instead, several proteins that were not known to interact with synaptotagmin I were bound tightly and stoichiometrically, most prominently the NSF homologue VCP, which is thought to be involved in membrane fusion, and an unknown protein of 40 kDa. Point mutations in the Ca2+ binding loops of the C2A domain revealed that the interactions of these proteins with synaptotagmin I were highly specific. Furthermore, a synaptotagmin I/VCP complex could be immunoprecipitated from brain homogenates in a Ca2+-dependent manner, and GST-VCP fusion proteins efficiently captured synaptotagmin I from brain. However, when we investigated the tissue distribution of VCP, we found that, different from synaptic proteins, VCP was not enriched in brain and exhibited no developmental increase paralleling synaptogenesis. Moreover, binding of VCP, which is an ATPase, to synaptotagmin I was inhibited by both ATP and ADP, indicating that the native, nucleotide-occupied state of VCP does not bind to synaptotagmin. Together our findings suggest that the C2A-domains of different synaptotagmins, despite their homology, exhibit a high degree of specificity in their protein interactions. This is direct evidence for diverse roles of the various synaptotagmins in brain, consistent with their differential subcellular localizations. Furthermore, our results indicate that traditional approaches, such as affinity chromatography and immunoprecipitations, are useful tools to evaluate the overall spectrum of binding activity for a protein but are not sufficient to estimate physiological relevance.
 ...
PMID:Specificity of Ca2+-dependent protein interactions mediated by the C2A domains of synaptotagmins. 1071 14

The Brn-3a POU domain transcription factor is able to regulate the transcription of promoters containing a Brn-3 response element via its POU domain. In addition, the POU domain of Brn-3a has been shown to functionally interact with the estrogen receptor and regulate transcription from estrogen responsive promoters. The steroid receptor coactivator, Src-1, enhances transcription with a variety of steroid receptors. Here we describe a functional interaction between Brn-3a and Src-1. In glutathione S-transferase pull-down assays Src-1 was shown to specifically interact with Brn-3 proteins. Moreover, Src-1 co-immunoprecipitated from intact cells with Brn-3a. The transactivation potential of the Brn-3a/Src-1 complex was tested on both the Brn-3 responsive SNAP-25 promoter and the estrogen responsive vitellogenin promoter, in each of two different cell lines, the neuronal ND7 cell line, and the kidney BHK21 cell line. Src-1 consistently and strongly potentiated the activation of Brn-3a on the SNAP promoter construct in both the ND7 and BHK21 cell lines. The vitellogenin promoter construct, however, was only weakly activated by the Brn-3/Src-1 complex in the ND7 cells and there was even less effect on this promoter in the BHK21 cells. These results suggest a functional role for Src-1 in enhancing Brn-3a mediated transactivation, seemingly independent of nuclear hormone receptors, thus broadening the transcriptional repertoire of both Brn-3a and Src-1.
 ...
PMID:Functional interaction between Brn-3a and Src-1 co-activates Brn-3a-mediated transactivation. 1205 37

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.
 ...
PMID:A link between Cdc42 and syntaxin is involved in mastoparan-stimulated insulin release. 1213 88

Voltage-gated K(+) (Kv) 2.1 is the dominant Kv channel that controls membrane repolarization in rat islet beta-cells and downstream insulin exocytosis. We recently showed that exocytotic SNARE protein SNAP-25 directly binds and modulates rat islet beta-cell Kv 2.1 channel protein at the cytoplasmic N terminus. We now show that SNARE protein syntaxin 1A (Syn-1A) binds and modulates rat islet beta-cell Kv2.1 at its cytoplasmic C terminus (Kv2.1C). In HEK293 cells overexpressing Kv2.1, we observed identical effects of channel inhibition by dialyzed GST-Syn-1A, which could be blocked by Kv2.1C domain proteins (C1: amino acids 412-633, C2: amino acids 634-853), but not the Kv2.1 cytoplasmic N terminus (amino acids 1-182). This was confirmed by direct binding of GST-Syn-1A to the Kv2.1C1 and C2 domains proteins. These findings are in contrast to our recent report showing that Syn-1A binds and modulates the cytoplasmic N terminus of neuronal Kv1.1 and not by its C terminus. Co-expression of Syn-1A in Kv2.1-expressing HEK293 cells inhibited Kv2.1 surfacing, which caused a reduction of Kv2.1 current density. In addition, Syn-1A caused a slowing of Kv2.1 current activation and reduction in the slope factor of steady-state inactivation, but had no affect on inactivation kinetics or voltage dependence of activation. Taken together, SNAP-25 and Syn-1A mediate secretion not only through its participation in the exocytotic SNARE complex, but also by regulating membrane potential and calcium entry through their interaction with Kv and Ca(2+) channels. In contrast to Ca(2+) channels, where these SNARE proteins act on a common synprint site, the SNARE proteins act not only on distinct sites within a Kv channel, but also on distinct sites between different Kv channel families.
 ...
PMID:Syntaxin 1A binds to the cytoplasmic C terminus of Kv2.1 to regulate channel gating and trafficking. 1262 Oct 36

Botulinum neurotoxin type A (BoNT/A) is the etiological agent responsible for botulism, a disease characterized by peripheral neuromuscular blockade. BoNT/A is produced by Clostridium botulinum as a single chain protein that is activated by proteolytic cleavage to form a 50 kDa light chain (LC, 448 amino acids) and a disulfide bond-linked 100 kDa heavy chain (HC, 847 amino acids). Whilst HC comprises the receptor binding and translocation domains, LC is a Zn2+-endopeptidase that cleaves at a single glutaminyl-arginine bond corresponding to residues 197 and 198 at the C-terminus of SNAP25. Cleavage of SNAP25 uncouples the neural exocytosis docking/fusion machinery. LC/A (LC 1-448) and several C-terminal deletion proteins of LC/A were engineered and expressed as His-tagged fusion proteins in Escherichia coli. LC 1-448 was purified, but precipitated upon storage. Approximately 40% of LC 1-448 was a covalent dimer due to the formation of inter-chain disulfide bond formation at Cys430. Conversion of Cys430 to Ser abolished dimer formation of LC 1-448, but did not improve solubility. Three C-terminal deletion peptides were engineered; LC 1-425 and LC 1-418 were expressed and could be purified as soluble and stable proteins, whilst LC 1-398 was soluble, but not stable to storage. Kinetic studies showed that LC 1-448 and LC 1-425 efficiently cleaved GST-SNAP25 and the fluorescent substrate SNAPtide, while LC 1-418 catalyzed the cleavage of both the SNAP25 and the fluorescent substrate SNAPtide with a similar Km, but at a 10-fold slower kcat. Thus, regions within the C-terminus of LC/A contribute to solubility, stability, and catalysis.
 ...
PMID:The C-terminus of botulinum neurotoxin type A light chain contributes to solubility, catalysis, and stability. 1529 97

GS32/SNAP-29 is a SNAP-25-like SNARE and has been shown to interact with syntaxin 6. Using immobilized recombinant GS32, we have recovered EHD1 as a major GS32-interacting protein from total HeLa cell extracts. This interaction is mediated by the EH domain of EHD1 and the N-terminal NPF-containing 17-residue region of GS32. Co-immunoprecipitation suggests that GS32 could also interact with EHD1 in intact cells. When immobilized GST-EHD1 was used to fish out interacting proteins from total brain extracts, syndapin II was identified as a major interacting partner. Similar to the GS32-EHD1 interaction, syndapin II also interacts with the EH domain of EHD1 via its NPF repeat region. Interaction of endogenous EHD1 and syndapin II was also established by co-immunoprecipitation. Furthermore, interaction of GS32 and syndapin II with EHD1 was shown to be mutually exclusive, suggesting that EHD1 may regulate/participate in the functional pathways of both GS32 and syndapin II in a mutual exclusive manner. Opposing roles of GS32 and syndapin II in regulating the surface level of transferrin receptor (TfR) were observed.
 ...
PMID:Mutually exclusive interactions of EHD1 with GS32 and syndapin II. 1537 Oct 16

A protein labeling approach is employed for the localization of a zinc-responsive fluorescent probe in the mitochondria and in the Golgi apparatus of living cells. ZP1, a zinc sensor of the Zinpyr family, was functionalized with a benzylguanine moiety and thus converted into a substrate (ZP1BG) for the human DNA repair enzyme alkylguaninetransferase (AGT or SNAP-Tag). The labeling reaction of purified glutathione S-transferase tagged AGT with ZP1BG and the zinc response of the resulting protein-bound sensor were confirmed in vitro. The new detection system, which combines a protein labeling methodology with a zinc fluorescent sensor, was tested in live HeLa cells expressing AGT in specific locations. The enzyme was genetically fused to site-directing proteins that anchor the probe onto targeted organelles. Localization of the zinc sensors in the Golgi apparatus and in the mitochondria was demonstrated by fluorescence microscopy. The protein-bound fluorescence detection system is zinc-responsive in living cells.
 ...
PMID:Organelle-specific zinc detection using zinpyr-labeled fusion proteins in live cells. 1897 93


1 2 Next >>