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)

The maltose transporter FGK2 complex of Escherichia coli was purified with the aid of a glutathione S-transferase molecular tag. In contrast to the membrane-associated form of the complex, which requires liganded maltose binding protein (MBP) for ATPase activity, the purified detergent-soluble complex exhibited a very high level of ATPase activity. This uncoupled activity was not due to dissociation of the MalK ATPase subunit from the integral membrane protein MalF and MalG subunits. The detergent-soluble ATPase activity of the complex could be further stimulated by wild-type MBP but not by a signaling-defective mutant MBP. Wild-type MBP increased the V(max) of the ATPase 2.7-fold but had no effect on the K(m) of the enzyme for ATP. When the detergent-soluble complex was reconstituted in proteoliposomes, it returned to being dependent on MBP for activation of ATPase, consistent with the idea that the structural changes induced in the complex by detergent that result in activation of the ATPase are reversible. The uncoupled ATPase activity resembled the membrane-bound activity of the complex also with respect to sensitivity to NaN(3), as well as a mercurial, p-chloromercuribenzosulfonic acid. Verapamil, a compound that activates the ATPase activity of the multiple drug resistance P-glycoprotein, activated the maltose transporter ATPase as well. The activation of this bacterial transporter by verapamil suggests that a structural feature that is conserved among both eukaryotic and prokaryotic ATP binding cassette transporters is responsible for this activation.
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PMID:The detergent-soluble maltose transporter is activated by maltose binding protein and verapamil. 1064 25

beta-Dystroglycan is a ubiquitously expressed integral membrane protein that undergoes tyrosine phosphorylation in an adhesion-dependent manner. However, it remains unknown whether tyrosine-phosphorylated beta-dystroglycan interacts with SH2 domain containing proteins. Here, we show that the tyrosine phosphorylation of beta-dystroglycan is constitutively elevated in v-Src transformed cells. We next reconstituted this phosphorylation event in vivo by transiently coexpressing wild-type c-Src with a fusion protein containing full-length beta-dystroglycan. Our results demonstrate that Src-induced tyrosine phosphorylation of beta-dystroglycan is strictly dependent on the presence of a PPxY motif at its extreme C-terminus. In the nonphosphorylated state, this PPxY motif is normally recognized as a ligand by the WW domain; phosphorylation at this site blocks the binding of certain WW domain containing proteins. Using a GST fusion protein carrying the cytoplasmic tail of beta-dystroglycan, we identified five SH2 domain containing proteins that interact with beta-dystroglycan in a phosphorylation-dependent manner, including c-Src, Fyn, Csk, NCK, and SHC. We localized this binding activity to the PPxY motif by employing a panel of beta-dystroglycan-derived phosphopeptides. In addition, tyrosine phosphorylation of beta-dystroglycan in vivo resulted in the coimmunoprecipitation of the same SH2 domain containing proteins, and this binding event required the beta-dystroglycan C-terminal PPxY motif. We discuss the possibility that tyrosine phosphorylation of the PPxY motif within beta-dystroglycan may act as a regulatory switch to inhibit the binding of certain WW domain containing proteins, while recruiting SH2 domain containing proteins.
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PMID:Tyrosine phosphorylation of beta-dystroglycan at its WW domain binding motif, PPxY, recruits SH2 domain containing proteins. 1172 72

Lamina-associated polypeptide 2beta (LAP2beta) of vertebrates is an integral membrane protein of the inner nuclear membrane that is generated by alternative splicing from the LAP2 gene. In the majority of Xenopus somatic cells including cultured kidney epithelial cells (A6 cells) there is only one major LAP2 isoform expressed that has the highest similarities with the mammalian LAP2beta whereas isoforms corresponding in size to the mammalian LAP2gamma and alpha are not detectable. We selected A6 cells and A6 cells stably expressing GFP fusion proteins of Xenopus LAP2beta (XLAP2Pbeta) as a model system to study interactions between LAP2beta and lamins. In vitro binding experiments with GST-XLAP2beta fusion proteins and immunoprecipitations with antibodies to GFP revealed that XLAP2beta is part of a complex that contains A- and B-type lamins. For the targeting to the nuclear envelope and the in vivo formation of this complex, GFP fusion proteins were sufficient comprising only the carboxyterminal 135 amino acids of XLAP2beta or the comparable region of zebrafish LAP2beta. A highly conserved 36 amino acids long sequence is located in this region of LAP2beta that is part of the lamina-binding domain previously identified in rat LAP2beta. GFP-LAP2beta fusion proteins of Xenopus, zebrafish, and rat that contained this sequence do compete with endogenous LAP2 in transfected cells for the same binding sites in the lamina. Our data indicate that the lamina-binding site of LAP2beta has been highly conserved during vertebrate evolution and suggests that this region of LAP2beta mediates the interactions between polymers of A- and B-type lamins.
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PMID:Lamina-associated polypeptide 2beta (LAP2beta) is contained in a protein complex together with A- and B-type lamins. 1269 Dec 63

Leukotrienes (LTs) are biologically active compounds derived from arachidonic acid which have important pathophysiological roles in asthma and inflammation. The cysteinyl leukotriene LTC(4) and its metabolites LTD(4) and LTE(4) stimulate bronchoconstriction, airway mucous formation and generalized edema formation. LTC(4) is formed by addition of glutathione to LTA(4), catalyzed by the integral membrane protein, LTC(4) synthase (LTCS). We now report the use of bioluminescence resonance energy transfer (BRET) to demonstrate that LTCS forms homo-oligomers in living cells. Fusion proteins of LTCS and Renilla luciferase (Rluc) and a variant of green fluorescent protein (GFP), respectively, were prepared. High BRET signals were recorded in transiently transfected human embryonic kidney (HEK 293) cells co-expressing Rluc/LTCS and GFP/LTCS. Homo-oligomer formation in living cells was verified by co-transfection of a plasmid expressing non-chimeric LTCS. This resulted in dose-dependent attenuation of the BRET signal. Additional evidence for oligomer formation was obtained in cell-free assays using glutathione S-transferase (GST) pull-down assay. To map interaction domains for oligomerization, GFP/LTCS fusion proteins were prepared with truncated variants of LTCS. The results obtained identified a C-terminal domain (amino acids 114-150) sufficient for oligomerization of LTCS. Another, centrally located, interaction domain appeared to exist between amino acids 57-88. The functional significance of LTCS homo-oligomer formation is currently being investigated.
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PMID:Leukotriene C4 synthase homo-oligomers detected in living cells by bioluminescence resonance energy transfer. 1288 Aug 68

The beta2 integrin CD11b/CD18 is an integral membrane protein that is present in the plasma membrane and secondary granules of neutrophils and functions as a major adhesion molecule. Upon cellular activation, there is translocation of intracellular pools of CD11b/CD18 to the plasma membrane in concert with enhanced cellular adhesion. Although much is known about the function of CD11b/CD18, how this protein is transported within the cell is less well defined. Here we report that CD11b/CD18 specifically binds to BAP31, a member of a novel class of sorting proteins regulating cellular anterograde transport. Through experiments aimed at identifying CD11b/CD18-binding proteins, we produced a monoclonal antibody termed E1B2 that recognizes a 28-kDa membrane protein that co-precipitates with CD11b/CD18. Microsequence analysis of the E1B2 antigen revealed that it is BAP31. Co-association of CD11b/CD18 and BAP31 was confirmed in co-immunoprecipitation and protein binding assays. Additional experiments revealed that the binding of BAP31 to CD11b/CD18 was not dependent on divalent cations nor mediated by the I-domain of CD11b. Using glutathione S-transferase fusion chimeras, we determined that binding of CD11b/CD18 to BAP31 is mediated through interactions with the cytoplasmic tail of BAP31. Immunolocalization studies revealed colocalization of BAP31 and CD11b/CD18 within neutrophil secondary granules. Subcellular fractionation studies in polymorphonuclear leukocytes (PMN) revealed similar patterns of redistribution of BAP31 and CD11b/CD18 from fractions enriched in secondary granules to the plasma membrane following stimulation with formylmethionylleucylphenylalanine (fMLP). Given the known sorting properties of BAP31, these findings suggest that BAP31 may play a role in regulating intracellular trafficking of CD11b/CD18 in neutrophils.
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PMID:Association of BAP31 with CD11b/CD18. Potential role in intracellular trafficking of CD11b/CD18 in neutrophils. 1529 14

Ubiquitin regulator-X (UBX) is a discrete protein domain that binds p97/valosin-containing protein (VCP), a molecular chaperone involved in diverse cell processes, including endoplasmic-reticulum-associated protein degradation (ERAD). Here we characterize a human UBX-containing protein, UBXD2, that is highly conserved in mammals, which we have renamed erasin. Biochemical fractionation, immunofluorescence and electron microscopy, and protease protection experiments suggest that erasin is an integral membrane protein of the endoplasmic reticulum and nuclear envelope with both its N- and C-termini facing the cytoplasm or nucleoplasm. Localization of GFP-tagged deletion derivatives of erasin in HeLa cells revealed that a single 21-amino-acid sequence located near the C-terminus is necessary and sufficient for localization of erasin to the endoplasmic reticulum. Immunoprecipitation and GST-pulldown experiments confirmed that erasin binds p97/VCP via its UBX domain. Additional immunoprecipitation assays indicated that erasin exists in a complex with other p97/VCP-associated factors involved in ERAD. Overexpression of erasin enhanced the degradation of the ERAD substrate CD3delta, whereas siRNA-mediated reduction of erasin expression almost completely blocked ERAD. Erasin protein levels were increased by endoplasmic reticulum stress. Immunohistochemical staining of brain tissue from patients with Alzheimer's disease and control subjects revealed that erasin accumulates preferentially in neurons undergoing neurofibrillary degeneration in Alzheimer's disease. These results suggest that erasin may be involved in ERAD and in Alzheimer's disease.
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PMID:Characterization of erasin (UBXD2): a new ER protein that promotes ER-associated protein degradation. 1696 47

Peripherin/rds (p/rds), an integral membrane protein from the transmembrane 4 (TMF4) superfamily, possesses a multi-functional C-terminal domain that plays crucial roles in rod outer segment (ROS) disk renewal and structure. Here, we report that the calcium binding protein calmodulin (CaM) binds to the C-terminal domain of p/rds. Fluorescence spectroscopy reveals Ca2+-dependent association of CaM with a polypeptide corresponding to the C-terminal domain of p/rds. The fluorescence anisotropy of the polypeptide upon CaM titration yields a dissociation constant (KD) of 320 +/- 150 nM. The results of the fluorescence experiments were confirmed by GST-pull down analyses in which a GST-p/rds C-terminal domain fusion protein was shown to pull down CaM in a calcium-dependent manner. Moreover, molecular modeling and sequence predictions suggest that the CaM binding domain resides in a p/rds functional hot spot, between residues E314 and G329. Predictions were confirmed by peptide competition studies and a GST-p/rds C-terminal domain construct in which the putative Ca2+/CaM binding site was scrambled. This GST-polypeptide did not associate with Ca2+/CaM. This putative calmodulin domain is highly conserved between human, mouse, rat, and bovine p/rds. Finally, the binding of Ca2+/CaM inhibited fusion between ROS disk and ROS plasma membranes as well as p/rds C-terminal-domain-induced fusion in model membrane studies. These results offer a new mechanism for the modulation of p/rds function.
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PMID:Calcium-dependent association of calmodulin with the C-terminal domain of the tetraspanin protein peripherin/rds. 1732 25

Chlamydophila pneumoniae is an obligate intracellular bacterium that causes bronchitis, pharyngitis, and pneumonia and may be involved in atherogenesis and Alzheimer's disease. Genome sequencing has identified three eukaryote-type serine/threonine protein kinases, Pkn1, Pkn5, and PknD, that may be important signaling molecules in Chlamydia. Full-length PknD was cloned and expressed as a histidine-tagged protein in Escherichia coli. Differential centrifugation followed by sodium carbonate treatment of E. coli membranes demonstrated that His-PknD is an integral membrane protein. Fusions of overlapping PknD fragments to alkaline phosphatase revealed that PknD contains a single transmembrane domain and that the kinase domain is in the cytoplasm. To facilitate solubility, the kinase domain was cloned and expressed as a glutathione S-transferase (GST) fusion protein in E. coli. Purified GST-PknD kinase domain autophosphorylated, and catalytic mutants (K33G, D156G, and K33G-D156G mutants) and activation loop mutants (T185A and T193A) were inactive. PknD phosphorylated recombinant Cpn0712, a type III secretion YscD homolog that has two forkhead-associated domains. Thin-layer chromatography revealed that the PknD kinase domain autophosphorylated on threonine and tyrosine and phosphorylated the FHA-2 domain of Cpn0712 on serine and tyrosine. To our knowledge, this is the first demonstration of a bacterial protein kinase with amino acid specificity for both serine/threonine and tyrosine residues and this is the first study to show phosphorylation of a predicted type III secretion structural protein.
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PMID:Chlamydophila pneumoniae PknD exhibits dual amino acid specificity and phosphorylates Cpn0712, a putative type III secretion YscD homolog. 1776 19

Chlamydophila pneumoniae is a gram-negative obligate intracellular bacterial pathogen that causes pneumonia and bronchitis and may contribute to atherosclerosis. The developmental cycle of C. pneumoniae includes a morphological transition from an infectious extracellular elementary body (EB) to a noninfectious intracellular reticulate body (RB) that divides by binary fission. The C. pneumoniae genome encodes a type III secretion (T3S) apparatus that may be used to infect eukaryotic cells and to evade the host immune response. In the present study, Cpn0712 (CdsD), Cpn0704 (CdsQ), and Cpn0826 (CdsL), three C. pneumoniae genes encoding yersiniae T3S YscD, YscQ, and YscL homologs, respectively, were cloned and expressed as histidine- and glutathione S-transferase (GST)-tagged proteins in Escherichia coli. Purified recombinant proteins were used to raise hyper-immune polyclonal antiserum and were used in GST pull-down and copurification assays to identify protein-protein interactions. CdsD was detected in both EB and RB lysates by Western blot analyses, and immunofluorescent staining demonstrated the presence of CdsD within inclusions. Triton X-114 solubilization and phase separation of chlamydial EB proteins indicated that CdsD partitions with cytoplasmic proteins, suggesting it is not an integral membrane protein. GST pull-down assays indicated that recombinant CdsD interacts with CdsQ and CdsL, and copurification assays with chlamydial lysates confirmed that native CdsD interacts with CdsQ and CdsL. To the best of our knowledge, this is the first report demonstrating interactions between YscD, YscQ, and YscL homologs of bacterial T3S systems. These novel protein interactions may play important roles in the assembly or function of the chlamydial T3S apparatus.
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PMID:Interactions between CdsD, CdsQ, and CdsL, three putative Chlamydophila pneumoniae type III secretion proteins. 1828

Microsomal glutathione transferase 2 (mGST2) is an integral membrane protein involved in detoxication of xenobiotics, and has also been suggested to catalyze the biosynthesis of pro-inflammatory mediator leukotriene C4 (LTC4) as homologous to LTC4 synthase (LTC4S) in mammals. In the present study, a novel mGST2 homology was identified from Apostichopus japonicus (designated as AjmGST2) by RACE approaches. The full-length cDNA of AjmGST2 was of 1917bp encoding a polypeptide of 161 amino acids residues. Multiple sequences alignment and phylogenetic analysis together supported that AjmGST2 belonged to a new member in invertebrate mGSTs family and close to mammalian LTC4S. Spatial expression analysis revealed that AjmGST2 was ubiquitously expressed in all examined tissues with the larger magnitude in intestine. AjmGST2 transcripts in coelomocytes were slightly induced post 6h challenge of pathogenic Vibrio splendidus and reached the peak expression at 48h. The increased expression profiles of AjmGST2 were also detected in lipopolysaccharide (LPS) exposed primary coelomocytes. Consistently, LTC4 contents were also induced by a 1.56-fold increase in the same condition. Functional assay further revealed that AjmGST2 might be functioned as LTC4S to promote LTC4 synthesis. AjmGST2 knock-down by specific siRNA significantly depressed LTC4 contents with 27.0% decrease at 24h. Meantime, ROS levels were elevated by 40.1% in vitro. All of these results indicated that AjmGST2 performed dual functions roles as LTC4S and ROS eliminator in sea cucumber immune response.
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PMID:Microsomal glutathione transferase 2 modulates LTC4 synthesis and ROS production in Apostichopus japonicus. 2889 48


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