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

---

Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cervical cancers evolve from lesions generated by genital human papillomaviruses (HPV). "Low-risk" genital HPVs cause benign proliferations whereas "high-risk" types have the potential to progress into cancer. High-risk HPV E6 oncoproteins interact with the ubiquitin ligase E6AP and target several cellular proteins, including p53 and proteins of the MAGI family, towards ubiquitin-mediated degradation. E6AP, like other E6 binding proteins such as E6BP, IRF-3 and paxillin, interacts with E6 via a consensus leucine-charged motif. Here we have investigated the kinetics of the interactions of a 15-mer peptide containing the LxxvarphiLsh motif of E6AP with E6. For this we have developed a Biacore assay based on antibody-capture on the sensor surface of GST- and/or MBP-E6AP peptide constructs followed by E6 protein injection. Our experiments show that E6 oncoproteins from four major high-risk (16, 18, 33 and 58) HPV types bind to E6AP with equilibrium dissociation constants in the low micromolar range. The kinetic dissociation parameters of these interactions are remarkably similar. On the other hand, low-risk HPV 11 E6 does not interact with E6AP even at relatively high concentrations. We also show that the two zinc-binding domains of E6 are required for E6AP recognition. Finally, we have analysed the binding properties of site-directed mutants of the E6AP-derived peptide. We demonstrate the importance for binding of conserved aliphatic side-chains and the moderate role of the global negative charge of the peptide. This work provides the first quantitative data on an HPV E6-mediated interaction, which support the current models of E6AP-mediated degradation.
...
PMID:Kinetic analysis of the interactions of human papillomavirus E6 oncoproteins with the ubiquitin ligase E6AP using surface plasmon resonance. 1589 Feb 4

LINCR was identified as a glucocorticoid-attenuated response gene induced in the lung during endotoxemia. The LINCR protein has structural similarities to Drosophila Neuralized, which regulates the developmentally important Notch signaling pathway. Endotoxemia-induced LINCR expression in vivo was localized by in situ hybridization to alveolar epithelial type II cells, and shown to be induced by LPS and inflammatory cytokines in the T7 alveolar epithelial type II cell line. RING domain-dependent ubiquitin E3 ligase activity of LINCR was demonstrated using full-length FLAG-LINCR or a deletion mutant lacking the RING domain expressed in 293T cells, and using a GST-LINCR RING fusion protein expressed in Escherichia coli. LINCR preferentially interacted with the ubiquitin-conjugating enzyme UbcH6 and preferentially generated polyubiquitin chains linked via non-canonical lysine residues. We conclude that LINCR is a novel inflammation-induced ubiquitin E3 ligase expressed in alveolar epithelial type II cells, and discuss its potential role in the lung response to inflammation.
...
PMID:A novel inflammation-induced ubiquitin E3 ligase in alveolar type II cells. 1593 21

Disruption of components in the transforming growth factor-beta (TGF-beta) signalling cascade is a common occurrence in human cancers. TGF-beta pathway activation is accomplished via serine/threonine kinase receptors and intracellular Smad transcription factors. A key regulatory step involves specific ubiquitination by Smurfs that mediate the proteasomal degradation of Smads and/or receptors. Here, we report a novel interaction between Smads and ubiquitin C-terminal hydrolase UCH37, a deubiquitinating enzyme that could potentially reverse Smurf-mediated ubiquitination. In GST pull down experiments, UCH37 bound weakly to Smad2 and Smad3, and bound very strongly to Smad7 in a region that is distinct from the -PY- motif in Smad7 that interacts with Smurf ubiquitin ligases. Endogenous Smad7 and UCH37 formed a stable complex in U4A/JAK1 cells, and FLAG-Smad7 co-immunoprecipitated with HA-UCH37 in transfected HEK-293 cells. In addition, we show that UCH37 can deubiquitinate and stabilize the type I TGF-beta receptor. Furthermore, overexpression of UCH37 upregulates TGF-beta-dependent transcription, and this effect is reversed in cells subject to RNAi-mediated knockdown of endogenous UCH37. These findings support a new role for deubiquitinating enzymes in the control of the TGF-beta signalling pathway, and provide a novel molecular target for the design of inhibitors with therapeutic potential in cancer.
...
PMID:The deubiquitinating enzyme UCH37 interacts with Smads and regulates TGF-beta signalling. 1602 25

Megakaryoblastic leukemia 1 (MKL1) was originally identified as a gene translocated in megakaryoblastic leukemia. It has been shown that MKL1 functions as a RhoA-regulated transcriptional coactivator of serum response factor (SRF). In order to identify a protein that regulates the function of MKL1, we performed yeast two-hybrid screening and isolated cDNA that encodes UBC9, an E2 enzyme of small ubiquitin-related modifier-1 (SUMO-1), as an MKL1-binding protein. UBC9 was found to physically interact with MKL1 by GST pull-down assay, and MKL1 was covalently modified with SUMO-1 in 293T cells and in vitro reconstitution system. MKL1 sumoylation is enhanced by either serum stimulation or co-expression of constitutively active form of RhoA. Mutational analysis showed that lysine residues at 499, 576, and 624 are the major acceptor sites for SUMO-1. In addition, reporter gene analysis revealed that mutation of the three sumoylation sites strongly enhances the transcriptional activity of MKL1. The covalent attachment of SUMO-1 to MKL1 by gene fusion represses MKL1-dependent transcription in a complementary manner. Finally, mutation of the sumoylation sites of MKL1 also enhances SRF-dependent transcription without affecting MKL1-SRF interaction. The combined results demonstrated that MKL1 is sumoylated and this modification represses transcriptional activity of MKL1.
...
PMID:Transcriptional activity of megakaryoblastic leukemia 1 (MKL1) is repressed by SUMO modification. 1609 47

We report here that microarrays comprised of several thousand peptoids (oligo-N-substituted glycines) are useful tools for the identification of proteins via a "fingerprinting" approach. By using maltose-binding protein, glutathione S-transferase, and ubiquitin, a specific and highly reproducible pattern of binding was observed when fluorescently labeled protein was hybridized to the array. A similar pattern was obtained when binding of an unlabeled protein to the array was visualized by secondary hybridization of a labeled antibody against that protein, showing that native proteins can be identified without the requirement for prior chemical labeling. This work suggests that small-molecule microarrays might be used for more complex fingerprinting assays of potential diagnostic value.
...
PMID:Protein "fingerprinting" in complex mixtures with peptoid microarrays. 1612 37

Using a C-terminal domain (PCT) of the measles virus (MV) phosphoprotein (P protein) as bait in a yeast two-hybrid screen, a cDNA identical to the recently described human p53-induced-RING-H2 (hPIRH2) cDNA was isolated. A glutathione S-transferase-hPIRH2 fusion protein expressed in bacteria was able to pull down P protein when mixed with an extract from P-expressing HeLa cells in vitro, and myc-tagged hPIRH2 could be reciprocally co-immunoprecipitated with MV P protein from human cells. Additionally, immunoprecipitation experiments demonstrated that hPIRH2-myc, MV P, and nucleocapsid (N) proteins form a ternary complex. The hPIRH2 binding site was mapped to the C-terminal X domain region of the P protein by using a yeast two-hybrid assay. The PCT binding site was mapped on hPIRH2 by using a novel yeast two-hybrid tagged PCR approach and by co-immunoprecipitation of hPIRH2 cysteine mutants and mouse/human PIRH2 chimeras. The hPIRH2 C terminus could mediate the interaction with MV P which was favored by the RING-H2 motif. When coexpressed with an enhanced green fluorescent protein-tagged hPIRH2 protein, MV P alone or in a complex with MV N was able to redistribute hPIRH2 to outside the nucleus, within intracellular aggregates. Finally, MV P efficiently stabilized hPIRH2-myc expression and prevented its ubiquitination in vivo but had no effect on the stability or ubiquitination of an alternative ubiquitin E3 ligase, Mdm2. Thus, MV P protein is the first protein from a pathogen that is able to specifically interact with and stabilize the ubiquitin E3 ligase hPIRH2 by preventing its ubiquitination.
...
PMID:Inhibition of ubiquitination and stabilization of human ubiquitin E3 ligase PIRH2 by measles virus phosphoprotein. 1614 Jul 59

Spectrin is important for the shape and the physical properties of the red blood cell, such as deformability and resistance to mechanical stress. Previous findings from our laboratory indicated that human erythrocyte alpha-spectrin can facilitate formation of ubiquitin-spectrin adducts and conjugates. Computer analysis revealed domains that contained significant homologies to known consensus catalytic E2 and E3 sequences, and allowed us to develop a model for alpha-spectrin ubiquitin conjugating enzyme (E2) and ubiquitin protein ligase (E3) enzymatic activities. In order to identify the precise E2/E3 site(s), in the present study, a GST-fusion alpha-spectrin (2005-2415) recombinant protein was tested using a cell free in vitro ubiquitination assay. We found that cysteine 2071 and cysteine 2100 are critical for alpha-spectrin (2005-2415) E2/E3 activity. Furthermore, together with testing an additional 13 site-specific mutants, we also demonstrated that both Cys2071 and Cys2100 are capable of transferring ubiquitin from an E1 enzyme to target sites within alpha-spectrin (2005-2415).
...
PMID:Erythrocyte spectrin's chimeric E2/E3 ubiquitin conjugating/ligating activity. 1617 54

The mechanism by which hypoxia induces gene transcription involves the inhibition of HIF-1alpha (hypoxia-inducible factor-1 alpha subunit) PHD (prolyl hydroxylase) activity, which prevents the VHL (von Hippel-Lindau)-dependent targeting of HIF-1alpha to the ubiquitin/proteasome pathway. HIF-1alpha thus accumulates and promotes gene transcription. In the present study, first we provide direct biochemical evidence for the presence of a conserved hypoxic signalling pathway in Drosophila melanogaster. An assay for 2-oxoglutarate-dependent dioxygenases was developed using Drosophila embryonic and larval homogenates as a source of enzyme. Drosophila PHD has a low substrate specificity and hydroxylates key proline residues in the ODD (oxygen-dependent degradation) domains of human HIF-1alpha and Similar, the Drosophila homologue of HIF-1alpha. The enzyme promotes human and Drosophila [(35)S]VHL binding to GST (glutathione S-transferase)-ODD-domain fusion protein. Hydroxylation is enhanced by proteasomal inhibitors and was ascertained using an anti-hydroxyproline antibody. Secondly, by using transgenic flies expressing a fusion protein that combined an ODD domain and the green fluorescent protein (ODD-GFP), we analysed the hypoxic cascade in different embryonic and larval tissues. Hypoxic accumulation of the reporter protein was observed in the whole tracheal tree, but not in the ectoderm. Hypoxic stabilization of ODD-GFP in the ectoderm was restored by inducing VHL expression in these cells. These results show that Drosophila tissues exhibit different sensitivities to hypoxia.
...
PMID:Analysis of the hypoxia-sensing pathway in Drosophila melanogaster. 1617 82

Cdc34 is an E2-conjugating enzyme required for catalyzing the polyubiquitination reaction mediated by the Skp1.CUL1.F-box (SCF) protein E3 ubiquitin (Ub) ligase. Here, we show that the activity of human Cdc34 in the Ub-Ub ligation reaction was enhanced dramatically by SCF's core Ub ligase module, composed of a heterodimeric complex formed by the ROC1 RING finger protein and the CUL1 C terminus that contains a Nedd8 moiety covalently conjugated at K720. Unexpectedly, we found that N-terminal fusion of a GST moiety to human Cdc34 generated dimeric GST-Cdc34 that was constitutively active in supporting the assembly of K48-linked polyUb chains independently of SCF. Furthermore, fusion of a FK506-binding protein (FKBP) to the N terminus of human Cdc34 yielded FKBP-Cdc34 that was induced to form a dimer upon treatment with the chemical inducer AP20187. The AP20187-induced dimeric form of FKBP-Cdc34 was substantially more active than the monomer in catalyzing Ub-Ub ligation. Thus, juxtaposition of human Cdc34 activates its catalytic capability, suggesting that the SCF-mediated polyubiquitination reaction may require the conversion of Cdc34 from an inactive monomer to a highly active dimeric form.
...
PMID:Proximity-induced activation of human Cdc34 through heterologous dimerization. 1621 Feb 46

A general method for assaying deubiquitinating enzymes (DUBs) has been developed. This new method employs an indirect enzyme assay for determining the activity of DUBs using a linear fusion of polyHis-glutathione-S-transferase-ubiquitin-ecotin (His-GST-Ub-ecotin) as a substrate. Because ecotin, a trypsin inhibitor protein from Escherichia coli, is heat stable, the activity of DUBs can be assayed indirectly by determining the ability of ecotin to inhibit trypsin after incubation of any DUB with His-GST-Ub-ecotin followed by heating at 100 degrees. In the substrate construction, His-GST fusion to Ub was used for facilitation of the substrate purification as well as for assisting the heat precipitation of His-GST-Ub and uncleaved His-GST-Ub-ecotin, as Ub itself is also heat stable. This method can also be used for assaying the proteases that process Ub-like proteins (Ubls) using the substrates, in which Ub is replaced by Ubls.
...
PMID:Strategies for assaying deubiquitinating enzymes. 1627 54


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---