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)

Nhp6p is an architectural Saccharomyces cerevisiae non-histone chromosomal protein that bends DNA and plays an important role in transcription and genome stability. We used the split-ubiquitin system to isolate proteins that interact with Nhp6p in vivo, and we confirmed 11 of these protein-protein interactions with glutathione S-transferase pull-down experiments in vitro. Most of the Nhp6p-interacting proteins are involved in transcription and DNA repair. We utilized the ZDS1, PUR5 and UME6 genes, which are repressed by Nhp6p and its interacting partners Rpb4p and Med3p, to study the chromosomal localization of these three proteins in wild-type and gene deletion strains. Nhp6p, Med3p and Rpb4p were found at the promoters of ZDS1, PUR5 and UME6, indicating that the repressing effects the three proteins had on the expression of these three genes had been direct ones. We also found that Med3p inhibited promoter clearance of RNA polymerase II, which contained the dissociable subunit Rpb4p, while Nhp6p recruited Rpb4p to the basal promoters of ZDS1, PUR5 and UME6. Our results further suggest that Rpb4p inhibits transcription initiation but stimulates transcription elongation and that Nhp6p and Med3p regulate gene expression by controlling the local subunit composition of RNA polymerase II.
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PMID:Nhp6p and Med3p regulate gene expression by controlling the local subunit composition of RNA polymerase II. 1844 20

Actin, the major component of the cytoplasmic skeleton, has been shown to exist in the nucleus. Nuclear actin functions in several steps of the transcription process, including chromatin remodelling and transcription initiation and elongation. However, as a part of PICs (pre-initiation complexes), the role of actin remains to be elucidated. In the present study, we identified RHA (RNA helicase A) as an actin-interacting protein in PICs. Using immunoprecipitation and immunofluorescence techniques, we have shown that RHA associates with beta-actin in the nucleus. A GST (glutathione transferase) pulldown assay using different deletion mutants revealed that the RGG (Arg-Gly-Gly) region of RHA was responsible for the interaction with beta-actin, and this dominant-negative mutant reduced the recruitment of Pol II (RNA polymerase II) into PICs. Moreover, overexpression or depletion of RHA could influence the interaction of Pol II with beta-actin and beta-actin-involved gene transcription regulation. These results suggest that RHA acts as a bridging factor linking nuclear beta-actin with Pol II.
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PMID:RNA helicase A acts as a bridging factor linking nuclear beta-actin with RNA polymerase II. 1930 9

CDKN1C is a cyclin-dependent kinase inhibitor and is a candidate tumor suppressor gene. We previously found that the CDKN1C protein represses E2F1-driven transcription in an apparent negative feedback loop. Herein, we explore the mechanism by which CDKN1C represses transcription. We find that adenoviral-mediated overexpression of CDKN1C leads to a dramatic reduction in phosphorylation of the RNA polymerase II (pol II) C-terminal domain (CTD). RNA interference studies demonstrate that this activity is not an artifact of CDKN1C overexpression, because endogenous CDKN1C mediates an inhibition of RNA pol II CTD phosphorylation in HeLa cells upon treatment with dexamethasone. Surprisingly, we find that CDKN1C-mediated repression of RNA pol II phosphorylation is E2F1-dependent, suggesting that E2F1 may direct CDKN1C to chromatin. Chromatin immunoprecipitation assays demonstrate that CDKN1C is associated with E2F1-regulated promoters in vivo and that this association can dramatically reduce the level of RNA pol II CTD phosphorylation at both Ser-2 and Ser-5 of the C-terminal domain repeat. In addition, we show that CDKN1C interacts with both CDK7 and CDK9 (putative RNA pol II CTD kinases) and that CDKN1C blocks their ability to phosphorylate a glutathione S-transferase-CTD fusion protein in vitro. E2F1 and CDKN1C are found to form stable complexes both in vivo and in vitro. Molecular studies demonstrate that the E2F1-CDKN1C interaction is mediated by two E2F domains. A central E2F1 domain interacts directly with CDKN1C, whereas a C-terminal E2F1 domain interacts with CDKN1C via interaction with Rb. The results presented in this report highlight a novel mechanism of tumor suppression by CDKN1C.
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PMID:CDKN1C negatively regulates RNA polymerase II C-terminal domain phosphorylation in an E2F1-dependent manner. 2010 82

Elongator is a multiprotein complex composed of two subcomplexes, Elp1-3 and Elp4-6. Elongator is highly conserved between yeast and humans and plays an important role in RNA polymerase II-mediated transcriptional elongation and many other processes, including cytoskeleton organization, exocytosis, and tRNA modification. Here, we determined the crystal structure of the Elp4-6 subcomplex of yeast. The overall structure of Elp4-6 revealed that Elp6 acts as a bridge to assemble Elp4 and Elp5. Detailed structural and sequence analyses revealed that each subunit in the Elp4-6 subcomplex forms a RecA-ATPase-like fold, although it lacks the key sequence signature of ATPases. Site-directed mutagenesis and biochemical analyses indicated that the Elp4-6 subcomplex can assemble into a hexameric ring-shaped structure in vitro and in vivo. Furthermore, GST pulldown assays showed that the ring-shaped assembly of the Elp4-6 subcomplex is important for its specific histone H3 binding. Our results may shed light on the substrate recognition and assembly of the holo-Elongator complex.
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PMID:Crystal structure of elongator subcomplex Elp4-6. 2255 26

Ubiquitylation is a highly diverse and complex post-translational modification for the regulation of protein function and stability. Studies of ubiquitylation have, however, been hampered by its rapid reversal in cell extracts, for example through the action of de-ubiquitylating enzymes (DUBs). Here we describe a novel ubiquitin-binding protein reagent, MultiDsk, composed of an array of five UBA domains from the yeast ubiquitin-binding protein Dsk2, fused to GST. MultiDsk binds ubiquitylated substrates with unprecedented avidity, and can be used as both an affinity resin to study protein ubiquitylation, and to effectively protect ubiquitylated proteins from the action of DUBs and the proteasome in crude cell extracts. We use the resin to show that the Def1 protein becomes ubiquitylated in response to DNA damage, and to isolate ubiquitylated forms of RNA polymerase II.
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PMID:MultiDsk: a ubiquitin-specific affinity resin. 2305 98

The UL4 gene is conserved within the genome of defective interfering particles of equine herpesvirus type 1 (EHV-1) that mediate persistent infection. Here, we show that the UL4 protein inhibits EHV-1 reporter gene expression by decreasing the level of transcribed mRNA. The UL4 protein did not bind any gene class of EHV-1 promoters in electromobility or chromatin immunoprecipitation assays, but directly interacted with the TATA box-binding protein (TBP) and the carboxy-terminal domain of RNA polymerase II both in vitro (GST-pulldown assays) and in infected cells (coimmunoprecipitation analyses). Microarray analyses of the expression of the 78 EHV-1 genes revealed that viral late genes important for virion assembly displayed enhanced expression in cells infected with UL4-null virus as compared to wild-type or UL4-restored EHV-1. Quantitative PCR analyses showed that viral DNA replication was not retarded in cells infected with the UL4-null virus as compared to wild-type EHV-1.
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PMID:The EHV-1 UL4 protein that tempers viral gene expression interacts with cellular transcription factors. 2441 34

Mediator is an evolutionary conserved multi-protein complex present in all eukaryotes. It functions as a transcriptional co-regulator by conveying signals from activators and repressors to the RNA polymerase II transcription machinery. The Arabidopsis thaliana Med25 (aMed25) ACtivation Interaction Domain (ACID) interacts with the Dreb2a activator which is involved in plant stress response pathways, while Human Med25-ACID (hMed25) interacts with the herpes simplex virus VP16 activator. Despite low sequence similarity, hMed25-ACID also interacts with the plant-specific Dreb2a transcriptional activator protein. We have used GST pull-down-, surface plasmon resonance-, isothermal titration calorimetry and NMR chemical shift experiments to characterize interactions between Dreb2a and VP16, with the hMed25 and aMed25-ACIDs. We found that VP16 interacts with aMed25-ACID with similar affinity as with hMed25-ACID and that the binding surface on aMed25-ACID overlaps with the binding site for Dreb2a. We also show that the Dreb2a interaction region in hMed25-ACID overlaps with the earlier reported VP16 binding site. In addition, we show that hMed25-ACID/Dreb2a and aMed25-ACID/Dreb2a display similar binding affinities but different binding energetics. Our results therefore indicate that interaction between transcriptional regulators and their target proteins in Mediator are less dependent on the primary sequences in the interaction domains but that these domains fold into similar structures upon interaction.
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PMID:Interaction studies of the human and Arabidopsis thaliana Med25-ACID proteins with the herpes simplex virus VP16- and plant-specific Dreb2a transcription factors. 2487 5

The coupling of transcription and associated processes has been shown to be dependent on the RNA polymerase II (RNAPII) C-terminal repeat domain (CTD) and the phosphorylation of the heptad repeats of which it is composed (consensus sequence Y1S2P3T4S5P6S7). Two primary S2 position CTD kinases have been identified in higher eukaryotes: P-TEFb and CDK12/CyclinK. The more recently discovered CDK12 appears to act at the 3'-end of the transcription unit and has been identified as a tumor suppressor for ovarian cancer; however much is still unknown about the in vivo roles of CDK12/CyclinK. In an effort to further characterize these roles we have purified to near homogeneity and characterized, full-length, active, human CDK12/CyclinK, and identified hCDK12-associated proteins via mass spectrometry. We find that employing a "2A" peptide-linked multicistronic construct containing CDK12 and CyclinK results in the efficient production of active, recombinant enzyme in the baculovirus/Sf9 expression system. Using GST-CTD fusion protein substrates we find that CDK12/CyclinK prefers a substrate with unmodified repeats or one that mimics prephosphorylation at the S7 position of the CTD; also the enzyme is sensitive to the inhibitor flavopiridol at higher concentrations. Identification of CDK12-associating proteins reveals a strong enrichment for RNA-processing factors suggesting that CDK12 affects RNA processing events in two distinct ways: Indirectly through generating factor-binding phospho-epitopes on the CTD of elongating RNAPII and directly through binding to specific factors.
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PMID:Expression, purification, and identification of associated proteins of the full-length hCDK12/CyclinK complex. 2542 6

AF4/AFF1 and AF5/AFF4 are both backbones for the assembly of "super elongation complexes" (SECs) that exert 2 distinct functions after the recruitment of P-TEFb from the 7SK snRNP: (1) initiation and elongation of RNA polymerase II gene transcription, and (2) modification of transcribed gene regions by distinct histone methylation patterns. In this study we aimed to investigate one of the initial steps, namely how P-TEFb is transferred from 7SK snRNPs to the SECs. In particular, we were interested in the role of DDX6 that we have recently identified as part of the AF4 complex. DDX6 is an evolutionarily conserved member of the DEAD-box RNA helicase family that is known to control miRNA and mRNA biology (translation, storage and degradation). Overexpressed DDX6 is associated with different cancer types and with c-Myc protein overexpression. We could demonstrate that DDX6 binds to 7SK snRNA and causes the release and transfer of P-TEFb to the AF4/AF4N SEC. DDX6 also binds stably to AF4 and AF4N as demonstrated by GST pull-down and co-immunoprecipitation experiments. As a consequence, overexpression of either AF4/AF4N or DDX6 resulted in a strong increase of mRNA production (5-6 fold), while their simultaneous expression increased the cellular mRNA production by 11-fold. Conversely, the corresponding knockdown of DDX6 decreased mRNA production by 70%. In conclusion, AF4/AF4N and DDX6 represent key molecules for the elongation process of gene transcription and a model will be proposed for the hand-over process of P-TEFb to SECs.
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PMID:DDX6 transfers P-TEFb kinase to the AF4/AF4N (AFF1) super elongation complex. 2767 41

The Tudor domain-containing proteins are characterized by their specific interactions with methylated protein motifs, including methyl-arginines and methyl-lysines. The Tudor domain-containing protein 3 (TDRD3) is one of the major methyl-arginine effector molecules that recognizes methylated arginine residues on histones and the C-terminal domain of RNA polymerase II, and activates transcription. However, majority of the cellular TDRD3 localizes to the cytoplasm and its functions there are still elusive. Here, we have identified ubiquitin-specific protease 9 X-linked (USP9X) as a TDRD3-interacting protein by GST (glutathione S-transferase) pull-down and co-immunoprecipitation. Detailed characterization suggests that the interaction between TDRD3 and USP9X is mediated through the Tudor domain of TDRD3 and the arginine methylation of USP9X. This interaction plays a critical role in TDRD3 protein stability, as knockdown of USP9X expression leads to increased TDRD3 ubiquitination. We also found that USP9X co-localizes with TDRD3 in cytoplasmic stress granules and this localization is diminished in Tdrd3-null mouse embryonic fibroblast cells, suggesting that TDRD3 is essential for USP9X stress granule localization. Furthermore, we found that one of the USP9X de-ubiquitination targets, myeloid cell leukemia protein 1, is regulated by TDRD3, indicating that TDRD3 potentially regulates USP9X de-ubiquitinase activity. Finally, we show that knockdown of TDRD3 expression sensitizes breast cancer cells to chemotherapeutic drug-induced apoptosis, likely due to its regulation of USP9X. This study provides a novel candidate strategy for targeting apoptosis pathways in cancer therapy.
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PMID:Arginine methylation of USP9X promotes its interaction with TDRD3 and its anti-apoptotic activities in breast cancer cells. 2810 74


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