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)

In metazoans, CBL proteins are RING finger type ubiquitin-protein isopeptide (E3) ligases involved in the down-regulation of epidermal growth factor tyrosine kinase receptors (EGFR). Among the three CBL proteins described in humans, CBLC (CBL3) remains poorly studied. By screening in parallel a human and a Caenorhabditis elegans library using the two-hybrid procedure in yeast, we found a novel interaction between Hsa-CBLC and Hsa-AIP4 or its C. elegans counterpart Cel-WWP1. Hsa-AIP4 and Cel-WWP1 are also ubiquitin E3 ligases. They contain a HECT (homologous to E6-AP C terminus) catalytic domain and four WW domains known to bind proline-rich regions. We confirmed the interaction between Hsa-CBLC and Hsa-AIP4 by a combination of glutathione S-transferase pull-down, co-immunoprecipitation, and colocalization experiments. We show that these two E3 ligases are involved in EGFR signaling because both become phosphorylated on tyrosine following epidermal growth factor stimulation. In addition, we observed that CBLC increases the ubiquitination of EGFR, and that coexpressing the WW domains of AIP4 exerts a dominant negative effect on EGFR ubiquitination. Finally, coexpressing CBLC and AIP4 induces a down-regulation of EGFR signaling. In conclusion, our data demonstrate that two E3 ligases of different classes can interact and cooperate to down-regulate EGFR signaling.
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PMID:Interaction between two ubiquitin-protein isopeptide ligases of different classes, CBLC and AIP4/ITCH. 1222 85

Primary response transcription factor, Egr-1, is rapidly activated by a variety of extracellular stimuli. Activation of Egr-1 is shown to function as a master switch activated by ischemia to trigger expression of pivotal regulators of inflammation, coagulation and vascular hyperpermeability. Egr-1 is a short-lived protein, but the mechanism that regulates its stability has not yet been clarified. In this study, the yeast two-hybrid screening revealed that Egr-1 interacts significantly with PRC8 (proteasome component C8) and the specific interaction was confirmed by GST pull-down assay and coimmunoprecipitation. Interestingly, we found that the PRC8-mediated regulation of Egr-1 activity is associated with the proteasome pathway and PRC8 inhibits the transcriptional activity of Egr-1. In addition, Egr-1 protein was specifically multiubiquitinated by ubiquitin. These data strongly imply that Egr-1 protein is targeted for proteolysis by the ubiquitin-dependent proteasome pathway.
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PMID:Regulation of Egr-1 by association with the proteasome component C8. 1237 79

ADAM family plays important roles in neurogenesis. The cytoplasmic tail of ADAM19 (ADAM19-CT) contains 193 residues. The presence of two putative SH3 ligand-binding sites suggests potential interactions with cytosolic proteins, which could be possibly linked to the functions of ADAM19. To address these issues, a yeast two-hybrid screen was performed in human fetal brain cDNA library to isolate proteins that interact with the cytoplasmic tail of ADAM19. Four proteins were obtained, ArgBP1, beta-cop, ubiquitin and a novel protein. GST-Pulldown assay has confirmed the interaction between AdAM19 and ArgBP1. By constructing series of deletion mutants of ADAM19-CT and ArgBP1 respectively, the interaction regions have been identified. They are the SH3 binding sites in ADAM19-CT and the P4 region in ArgBP1. And the interaction is specific. ArgBP1 does not bind to ADAM22, ADAM29 or ADAM9 (mouse). ArgBP1 may be the key protein, which accounts for the physiological function of ADAM19.
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PMID:Screen and identification of proteins interacting with ADAM19 cytoplasmic tail. 1246 24

NF-kappaB1 p105 functions both as a precursor of NF-kappaB1 p50 and as a cytoplasmic inhibitor of NF-kappaB. Following the stimulation of cells with tumor necrosis factor alpha (TNF-alpha), the IkappaB kinase (IKK) complex rapidly phosphorylates NF-kappaB1 p105 on serine 927 in the PEST region. This phosphorylation is essential for TNF-alpha to trigger p105 degradation, which releases the associated Rel/NF-kappaB subunits to translocate into the nucleus and regulate target gene transcription. Serine 927 resides in a conserved motif (Asp-Ser(927)-Gly-Val-Glu-Thr-Ser(932)) homologous to the IKK target sequence in IkappaBalpha. In this study, TNF-alpha-induced p105 proteolysis was revealed to additionally require the phosphorylation of serine 932. Experiments with IKK1(-/-) and IKK2(-/-) double knockout embryonic fibroblasts demonstrate that the IKK complex is essential for TNF-alpha to stimulate phosphorylation on p105 serines 927 and 932. Furthermore, purified IKK1 and IKK2 can each phosphorylate a glutathione S-transferase-p105(758-967) fusion protein on both regulatory serines in vitro. IKK-mediated p105 phosphorylation generates a binding site for betaTrCP, the receptor subunit of an SCF-type ubiquitin E3 ligase, and depletion of betaTrCP by RNA interference blocks TNF-alpha-induced p105 ubiquitination and proteolysis. Phosphopeptide competition experiments indicate that betaTrCP binds p105 more effectively when both serines 927 and 932 are phosphorylated. Interestingly, however, betaTrCP affinity for the IKK-phosphorylated sequence on p105 is substantially lower than that on IkappaBalpha. Thus, it appears that reduced p105 recruitment of betaTrCP and subsequent ubiquitination may contribute to delayed p105 proteolysis after TNF-alpha stimulation relative to that for IkappaBalpha.
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PMID:betaTrCP-mediated proteolysis of NF-kappaB1 p105 requires phosphorylation of p105 serines 927 and 932. 1248 91

We have characterized the mouse ortholog of the human ubiquitin-specific protease USP15. Mouse Usp15 consists of 981 amino acids with a predicted molecular mass of 112 kDa, contains the highly conserved Cys and His boxes present in all members of the UBP family of deubiquitinating enzymes, and is 98% identical/99% similar to human USP15. Usp15 shares 59.5% identity/75.5% sequence similarity with the mouse Unp(Usp4) oncoprotein. Recombinant Usp15 demonstrated ubiquitin-specific protease activity against engineered linear fusions of ubiquitin to glutathione S-transferase. Usp15 can also cleave the ubiquitin-proline bond, as can USP15 and Usp4. Alignment of mouse and human Usp15 and Usp4 protein sequences suggested that Usp15/USP15 may be alternately spliced in a manner analogous to Usp4. Sequence analysis of RT-PCR products from several human and mouse cell lines and tissues revealed alternate splicing in all cells studied. Northern blot analysis of both mouse and human Usp15 revealed two differently sized mRNAs in all tissues examined, owing to alternate polyadenylation sites spaced by 1.5 kb. Chromosomal mapping by interspecific backcross analysis localized the Usp15 gene to the distal region of mouse Chromosome (Chr) 10. This region is syntenic with human Chr 12q24, the location of human USP15, and a different location to Unp(Usp4) (Chr 9). Identification of the mouse Usp15 gene (>69.5 kb) and human USP15 gene (145 kb) sequences in genome databases reveals that both are composed of 22 exons with identical splice sites, and both have an exon/intron structure identical to the mouse Usp4 gene, including the alternately spliced exon. Phylogenetic studies suggest that a sequence currently identified as a chicken Usp4 ortholog is in fact a USP15 ortholog, while bona-fide chicken, cow, and rat Usp4 orthologs can be identified in EST databases.
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PMID:Isolation and characterization of the mouse ubiquitin-specific protease Usp15. 1253 66

The mechanism by which hypoxia induces gene transcription involves the inhibition of hypoxia-inducible factor (HIF)-1alpha prolyl hydroxylase activity, which prevents von Hippel-Lindau (vHL)-dependent targeting of HIF-1alpha to the ubiquitin-proteasome pathway. HIF-1alpha is stabilized, translocates to the nucleus, interacts with hypoxia-responsive elements, and promotes the activation of target genes. This report shows that cyclosporin A (CsA) interferes with the hypoxic signaling cascade in C6 glioma cells. CsA inhibits hypoxia-dependent gene transcription in a reporter gene assay and prevents the hypoxic accumulation of HIF-1alpha. Addition of the 530-603 C-terminal oxygen-dependent degradation (ODD) domain of HIF-1alpha to the green fluorescent protein (GFP) destabilized the protein in an oxygen-dependent manner. CsA prevented the hypoxic stabilization of an ODD.GFP fusion protein. An assay for 2-oxoglutarate-dependent dioxygenases was developed using a light mitochondrial kidney fraction as a source of enzyme. It uses the capacity of specific peptides to stimulate the degradation of [(14)C]2-oxoglutarate. CsA stimulated the enzymatic activity in the presence of a peptide that mimicked the 557-576 sequence of HIF-1alpha. The enzyme promoted [(35)S]vHL binding to glutathione S-transferase (GST).ODD fusion protein. This association increased in the presence of CsA. CsA effects were not observed when the proline residue corresponding to Pro-564 in the HIF-1alpha sequence was replaced by a hydroxyproline or an alanine residue. Finally, CsA increased vHL-ODD interaction during hypoxia. We conclude that CsA destabilizes HIF-1alpha by promoting hydroxylation of Pro-564 in the ODD domain. Such a mechanism may prevent hypoxic adaptation during CsA-induced nephrotoxicity and contribute to the adverse effects of this drug.
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PMID:Cyclosporin A prevents the hypoxic adaptation by activating hypoxia-inducible factor-1alpha Pro-564 hydroxylation. 1258 29

Humanin (HN) is a newly identified neuroprotective peptide that specifically suppresses Alzheimer's disease (AD)-related neurotoxicity. HN peptide has been detected in the human AD brain as well as in mouse testis and colon by immunoblot and immunohistochemical analyses. By means of yeast two-hybrid screening, we identified TRIM11 as a novel HN-interacting protein. TRIM11, which is a member of protein family containing a tripartite motif (TRIM), is composed of a RING finger domain, which is a putative E3 ubiquitin ligase, a B-box domain, a coiled-coil domain and a B30.2 domain. Deletion of the B30.2 domain in TRIM11 abolished the interaction with HN, whereas the B30.2 domain alone did not interact with HN. For their interaction, at least the coiled-coil domain was indispensable together with the B30.2 domain. The intracellular level of glutathione S-transferase-fused or EGFP-fused HN peptides or plain HN was drastically reduced by the coexpression of TRIM11. Disruption of the RING finger domain by deleting the first consensus cysteine or proteasome inhibitor treatment significantly diminished the effect of TRIM11 on the intracellular level of HN. These results suggest that TRIM11 plays a role in the regulation of intracellular HN level through ubiquitin-mediated protein degradation pathways.
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PMID:A tripartite motif protein TRIM11 binds and destabilizes Humanin, a neuroprotective peptide against Alzheimer's disease-relevant insults. 1267 Mar 3

Endocytosis of the growth hormone receptor (GHR) is regulated by the ubiquitin-conjugating system. A cytosolic 10 amino acid motif, referred to as the ubiquitin-dependent endocytosis (UbE) motif, is involved in the ubiquitination as well as in the endocytosis of the receptor. Proteins that are implicated in one of these processes have not been identified so far. Using a glutathione S-transferase (GST)-pulldown assay with a GST fusion protein encompassing the UbE motif of the GHR, a 35 kDa protein was purified. The protein was identified by MS as small glutamine-rich tetratricopeptide repeat (TPR)-containing protein (SGT). We found that GHR interacts with SGT. In vivo, both the precursor and the mature form of the receptor interacted with SGT. Inactivation of the ubiquitin-conjugating system did not affect the GHR-SGT interaction. Binding studies showed that the first TPR motif of SGT interacts with the UbE motif of the GHR. Taken together, these data show that SGT is a GHR-interacting protein, which binds independent of the ubiquitin-conjugating system.
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PMID:Small glutamine-rich tetratricopeptide repeat-containing protein (SGT) interacts with the ubiquitin-dependent endocytosis (UbE) motif of the growth hormone receptor. 1273 88

Polyubiquitination is required for retrotranslocation of proteins from the endoplasmic reticulum back into the cytosol, where they are degraded by the proteasome. We have tested whether the release of a polypeptide chain into the cytosol is caused by a ratcheting mechanism in which the attachment of polyubiquitin prevents the chain from moving back into the endoplasmic reticulum. Using a permeabilized cell system in which major histocompatibility complex class I heavy chains are retrotranslocated under the influence of the human cytomegalovirus protein US11, we demonstrate that polyubiquitination alone is insufficient to provide the driving force for retrotranslocation. Substrate release into the cytosol requires an additional ATP-dependent step. Release requires a lysine 48 linkage of ubiquitin chains. It does not occur when polyubiquitination of the substrate is carried out with glutathione S-transferase (GST)-ubiquitin, and this correlates with poly-GST-ubiquitin not being recognized by a ubiquitin-binding domain in the Ufd1-Npl4 cofactor of the ATPase p97. These data suggest that polyubiquitin does not serve as a ratcheting molecule. Rather, it may serve as a recognition signal for the p97-Ufd1-Npl4 complex, a component implicated in the movement of substrate into the cytosol.
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PMID:Polyubiquitin serves as a recognition signal, rather than a ratcheting molecule, during retrotranslocation of proteins across the endoplasmic reticulum membrane. 1281 30

A fine regulation of the amiloride-sensitive Epithelial Sodium Channel (ENaC), made of alpha, beta and gamma subunits, is crucial for maintenance of Na+ balance and blood pressure. Both beta- and gamma-ENaC participate in negative regulation by interacting with Nedd4-2, an E3 ubiquitin-ligase. Disruption of this interaction results in increased ENaC activity (Liddle syndrome). By two-hybrid screenings, we identified new potential partners of alpha-ENaC: WWP1 (E3 ubiquitin-ligase protein), UBC9 and TSG101 (E2 ubiquitin/SUMO-conjugating enzymes) and confirmed these interactions in GST pull-down assays. All these partners are implicated in protein trafficking and could be involved in the regulation of ENaC activity.
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PMID:Identification of new partners of the epithelial sodium channel alpha subunit. 1455 80


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