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)

We studied the effects of a zinc-inducible metallothionein-ras fusion gene (MTrasT24) in cultured rat liver epithelial (RLE) cells on expression of two genes induced during liver carcinogenesis in vivo: gamma-glutamyltransferase [(5-glutamyl)-peptide:amino acid 5-glutamyltransferase, EC 2.3.2.2] and glutathione S-transferase-P (RX:glutathione R-transferase, EC 2.5.1.18). Expression of MTrasT24 increased steady-state RNA levels of gamma-glutamyltransferase and glutathione transferase-P 6- to 100-fold and 1.6- to 6-fold, respectively; in contrast, levels of alpha-tubulin RNA fell slightly or were unchanged. RNA gel blots verified that gamma-glutamyltransferase and glutathione transferase-P RNAs were of the appropriate size, and results from immunocytochemistry on transfected cells demonstrated that RLE cells carrying MTrasT24 synthesized immunoreactive, appropriately localized gamma-glutamyltransferase and glutathione transferase-P. Zinc induction studies indicated that gamma-glutamyltransferase and glutathione transferase-P RNA levels were directly dependent on MTrasT24 RNA levels. These data suggest that expression of gamma-glutamyltransferase and glutathione transferase-P expression are part of a reorientation of cellular gene expression during carcinogenesis and that activated ras expression, like chemical carcinogens, can bring about this change.
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PMID:MTrasT24, a metallothionein-ras fusion gene, modulates expression in cultured rat liver cells of two genes associated with in vivo liver cancer. 289 74

During activation, T lymphocytes become motile cells, switching from a spherical to a polarized shape. Chemokines and other chemotactic cytokines induce lymphocyte polarization with the formation of a uropod in the rear pole, where the adhesion receptors intercellular adhesion molecule-1 (ICAM-1), ICAM-3, and CD44 redistribute. We have investigated membrane-cytoskeleton interactions that play a key role in the redistribution of adhesion receptors to the uropod. Immunofluorescence analysis showed that the ERM proteins radixin and moesin localized to the uropod of human T lymphoblasts treated with the chemokine RANTES (regulated on activation, normal T cell expressed, and secreted), a polarization-inducing agent; radixin colocalized with arrays of myosin II at the neck of the uropods, whereas moesin decorated the most distal part of the uropod and colocalized with ICAM-1, ICAM-3, and CD44 molecules. Two other cytoskeletal proteins, beta-actin and alpha-tubulin, clustered at the cell leading edge and uropod, respectively, of polarized lymphocytes. Biochemical analysis showed that moesin coimmunoprecipitates with ICAM-3 in T lymphoblasts stimulated with either RANTES or the polarization- inducing anti-ICAM-3 HP2/19 mAb, as well as in the constitutively polarized T cell line HSB-2. In addition, moesin is associated with CD44, but not with ICAM-1, in polarized T lymphocytes. A correlation between the degree of moesin-ICAM-3 interaction and cell polarization was found as determined by immunofluorescence and immunoprecipitation analysis done in parallel. The moesin-ICAM-3 interaction was specifically mediated by the cytoplasmic domain of ICAM-3 as revealed by precipitation of moesin with a GST fusion protein containing the ICAM-3 cytoplasmic tail from metabolically labeled Jurkat T cell lysates. The interaction of moesin with ICAM-3 was greatly diminished when RANTES-stimulated T lymphoblasts were pretreated with the myosin-disrupting drug butanedione monoxime, which prevents lymphocyte polarization. Altogether, these data indicate that moesin interacts with ICAM-3 and CD44 adhesion molecules in uropods of polarized T cells; these data also suggest that these interactions participate in the formation of links between membrane receptors and the cytoskeleton, thereby regulating morphological changes during cell locomotion.
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PMID:Moesin interacts with the cytoplasmic region of intercellular adhesion molecule-3 and is redistributed to the uropod of T lymphocytes during cell polarization. 929 94

The I kappaB alpha protein is a key molecular target involved in the control of NF-kappaB/Rel transcription factors during viral infection or inflammatory reactions. This NF-kappaB-inhibitory factor is regulated by posttranslational phosphorylation and ubiquitination of its amino-terminal signal response domain that targets I kappaB alpha for rapid proteolysis by the 26S proteasome. In an attempt to identify regulators of the I kappaB alpha inhibitory activity, we undertook a yeast two-hybrid genetic screen, using the amino-terminal end of I kappaB alpha as bait, and identified 12 independent interacting clones. Sequence analysis identified some of these cDNA clones as Dlc-1, a sequence encoding a small, 9-kDa human homolog of the outer-arm dynein light-chain protein. In the two-hybrid assay, Dlc-1 also interacted with full-length I kappaB alpha protein but not with N-terminal-deletion-containing versions of I kappaB alpha. I kappaB alpha interacted in vitro with a glutathione S-transferase-Dlc-1 fusion protein, and RelA(p65) did not displace this association, demonstrating that p65 and Dlc-1 contact different protein motifs of I kappaB alpha. Importantly, in HeLa and 293 cells, endogenous and transfected I kappaB alpha coimmunoprecipitated with Myc-tagged or endogenous Dlc-1. Indirect immunofluorescence analyzed by confocal microscopy indicated that Dlc-1 and I kappaB alpha colocalized with both nuclear and cytoplasmic distribution. Furthermore, Dlc-1 and I kappaB alpha were found to associate with the microtubule organizing center, a perinuclear region from which microtubules radiate. Likewise, I kappaB alpha colocalized with alpha-tubulin filaments. Taken together, these results highlight an intriguing interaction between the I kappaB alpha protein and the human homolog of a member of the dynein family of motor proteins and provide a potential link between cytoskeleton dynamics and gene regulation.
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PMID:I kappaB alpha physically interacts with a cytoskeleton-associated protein through its signal response domain. 937 68

The interaction between elongation factor 1alpha (EF-1alpha) and alpha/beta-tubulins has been analyzed in vivo and in vitro. An association of both alpha- and beta-tubulins with EF-1alpha in the lysate of Tetrahymena pyriformis was detected by co-immunoprecipitation analysis. In contrast, in vitro biomolecular interaction analysis with glutathione S-transferase (GST) fusion proteins revealed that GST-beta-tubulin, but not GST-alpha-tubulin, can bind to GST-EF-1alpha. Two beta-tubulin binding sites have been identified to reside in the domains I and III of EF-1alpha. In addition, beta-tubulin itself seems to have two distinct interaction sites for each of the domains. Since domain II of EF-1alpha did not interact with beta-tubulin, we have re-evaluated the phylogenetic status of ciliates using EF-1alpha sequences devoid of domain II. The phylogenetic tree thus obtained was significantly different from that inferred from the whole sequence of EF-1alpha, suggesting the presence of functional constraints on the molecular evolution of EF-1alpha.
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PMID:Biochemical analysis of the interaction between elongation factor 1alpha and alpha/beta-tubulins from a ciliate, Tetrahymena pyriformis. 1040 69

Paxillin is a focal adhesion-associated protein that functions as a multi-domain adapter protein, binding several structural and signaling molecules. alpha-Tubulin was identified as an interacting protein in a two-hybrid screen using the paxillin C-terminal LIM domain as a bait. In vitro binding assays with glutathione S-transferase-paxillin demonstrated an interaction of alpha-tubulin with the C terminus of paxillin. Another member of the tubulin family, gamma-tubulin, bound to both the N and the C terminus of paxillin. The interaction between paxillin and both alpha- and gamma-tubulin in vivo was confirmed by co-immunoprecipitation from human T lymphoblasts. Immunofluorescence studies revealed that, in adherent T cells, paxillin localized to sites of cell-matrix interaction as well as to a large perinuclear region. Confocal microscopy revealed that this region corresponds to the lymphocyte microtubule organizing center, where paxillin colocalizes with alpha- and gamma-tubulin. The localization of paxillin to this area was observed in cells in suspension as well as during adhesion to integrin ligands. These data constitute the first characterization of the interaction of paxillin with the microtubule cytoskeleton, and suggest that paxillin, in addition to its well established role at focal adhesions, could also be associated with the lymphocyte microtubule network.
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PMID:Paxillin localizes to the lymphocyte microtubule organizing center and associates with the microtubule cytoskeleton. 1084 40

Tubulin-tyrosine ligase (TTL, EC 6.3.2.25) from porcine brain, which catalyses the readdition of tyrosine to the C-terminus of detyrosinated alpha-tubulin, was cloned and expressed in Escherichia coli as a glutathione S-transferase-fusion protein. Upon cleavage of the immobilised fusion protein, an electrophoretically homogeneous enzyme was obtained. Recombinant TTL, which exhibited similar catalytic properties as the mammalian enzyme purified from brain tissue, was capable of using nitrotyrosine as an alternative substrate in vitro. Incorporation of tyrosine into tubulin was competitively inhibited by nitrotyrosine with an apparent K(i) of 0.24 mM. The TTL-catalysed incorporation of nitrotyrosine as sole substrate into alpha-tubulin was clearly detectable at concentrations of 10 microM by immunological methods using nitrotyrosine specific antibodies. However, in competition with tyrosine 20-fold higher concentrations of nitrotyrosine were necessary before its incorporation became evident. Analysis of the C-terminal peptides of in vitro modified alpha-tubulin by MALDI-MS confirmed the covalent incorporation of nitrotyrosine into tubulin by TTL. In contrast to the C-terminal tyrosine, pancreatic carboxypeptidase A was incapable of cleaving nitrotyrosine from the modified alpha-tubulin.
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PMID:Incorporation of nitrotyrosine into alpha-tubulin by recombinant mammalian tubulin-tyrosine ligase. 1100 83

Retroprocessed pseudogenes, calmodulin II (psi1, psi2, and psi3 CALMII), psi alpha-tubulin, pi-glutathione S-transferase (psi pi-GST) from rat, lactic acid dehydrogenase (psi LDH) from mouse, and heat shock protein 60 chaperonin (psi HSP60) from Chinese hamster, were examined for their presence in these species by polymerase chain reaction (PCR). Pseudogenes of these murine rodents were detected by PCR only in those species in which the genes were originally identified, suggesting that the selected pseudogene of one species arose too recently to be detected in the genomes of the other rodent species. The calculated ages of the rodent pseudogenes ranged from 1.7 Myr (psi alpha-tubulin) to 7.5 Myr (psi3 CALMII) when employing a homologous functional gene of the taxon as a reference in the relative rate test with the mouse or rat as the outgroup. Given the high rate of divergence of the genes of rodents relative to other species, selection of an outgroup with similar mutation rates seems warranted. To justify further the conclusion that the selected pseudogenes were indeed retroprocessed after these three taxa diverged, the presence of the pseudogenes in the genome of different rat species was examined. The existence of psi3 CALMII and psi alpha-tubulin pseudogenes of Rattus norvegicus among species belonging to Rattus sensu stricto is evidence for the common ancestry of this group.
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PMID:Age and detection of retroprocessed pseudogenes in murine rodents. 1173 3

We have characterized the cDNA for a Rho GTPase activating protein (GAP) mapping to chromosome 13q12. The cDNA was characterized by determining the complete sequence of a 4.8 kb cDNA clone that represents the 5' untranslated region (UTR), the translated region, and the 3' UTR. The protein has a sterile alpha-motif (SAM), a distinct GAP domain, and a conserved START (StAR related lipid transfer) domain. The cDNA has 5 instability motifs (ATTTA) in the 3' UTR and one motif in the translated region between GAP and START domains. The RhoGAP transcript is truncated in some breast carcinoma cell lines and it has low expression in other breast cancer cell lines as compared to a normal breast cell line. We have previously observed the absence of RhoGAP transcript in a breast tumor specimen. A GST-fusion of the RhoGAP was tested for its specificity on RhoA, Cdc42, and Rac1. The protein was most active for RhoA. Transfection of RhoGAP into MCF7 cells significantly inhibited cell growth. The introduction of the RhoGAP construct into MDAMB231 cells that had previously been transfected with a p21 construct did not affect cell proliferation, indicating the involvement of p21 in Rho-mediated proliferation of cancer cells. NIH3T3 cells overexpressing RhoGAP showed considerable inhibition of stress fiber formation. Several cDNAs were identified as RhoGAP interactors by using the yeast two-hybrid assay system. These cDNAs correspond to SWI/SNF, alpha-tubulin, HMG CoA reductase, and TAX1 binding protein (TAX1BP1). The interaction with HMG CoA reductase may partially explain the growth inhibition of breast carcinoma cells by statin class of cholesterol lowering drugs. The biological significance of the interacting proteins is discussed in the context of their involvement in tumorigenesis. Our results indicate that loss of RhoGAP or its altered activity suppresses the growth of breast tumor cells. The presence of various motifs in RhoGAP and its interaction with several other proteins suggest that the protein may regulate Rho signaling in multiple ways and possibly function in a Rho-independent manner.
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PMID:Chromosome 13q12 encoded Rho GTPase activating protein suppresses growth of breast carcinoma cells, and yeast two-hybrid screen shows its interaction with several proteins. 1498 79

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

Mutations in the gene encoding polycystin-2 (PC2) result in autosomal dominant polycystic kidney disease and defects in left-right asymmetry during embryogenesis. PC2 is a TRP-type Ca(2+)-permeable non-selective cation channel, which is expressed in kidney and other organs. PC2 is present and functional in microtubule-containing primary cilia of renal epithelial cells. However, no information is yet available as to whether PC2 interacts with microtubules. Here, we assessed the role of microtubular dynamics in regulating PC2 channel function in primary cilia. Isolated ciliary membranes from LLC-PK1 epithelial cells were reconstituted in a lipid bilayer system. The acute addition of the microtubular disrupter colchicine (15 mum) rapidly abolished, whereas the addition of the microtubular stabilizer paclitaxel (taxol, 15 mum) increased ciliary PC2 channel activity. The further addition of alpha-tubulin plus GTP also stimulated PC2 channel activity in ciliary membranes. However, alpha-tubulin and GTP had no effect on in vitro translated PC2. Using the yeast two-hybrid assay, we found that PC2 interacts with the microtubule-dependent motor kinesin-2 subunit KIF3A, a protein involved in polycystic kidney disease. The interaction occurred through the carboxyl termini domain of both proteins, which was further confirmed by in vitro glutathione S-transferase pull-down and dot blot overlay assays. Co-immunoprecipitation experiments showed that PC2 and KIF3A are in the same complex in native HEK293, Madin-Darby canine kidney cells (MDCK), and LLC-PK1 cells. Immunofluorescent staining also showed substantial PC2 and KIF3A co-localization in primary cilia of renal epithelial cells. The data indicate that microtubular organization regulates PC2 function, which may explain, among others, the regulatory role of PC2 in the sensory function of primary cilia.
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PMID:Polycystin-2 cation channel function is under the control of microtubular structures in primary cilia of renal epithelial cells. 1695 Jul 92


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