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 poly(A)-binding protein (PABP), in a complex with the 3'poly(A) tail of eukaryotic mRNAs, plays important roles in the control of translation and message stability. All known examples of PABP mRNAs contain an extensive A-rich sequence in their 5' untranslated regions. Studies in mammalian cells undergoing growth stimulation or terminal differentiation indicate that PABP expression is regulated at the translational level. Here we examine the hypothesis that synthesis of the PABP is autogenously controlled. We show that the endogenous inactive PABP mRNA in rabbit reticulocytes can be specifically stimulated by addition of low concentrations of poly(A) and that this stimulation is also observed with in vitro transcribed human PABP mRNA. By deleting the A-rich region from the leader of human PABP mRNA and adding it upstream of the initiator AUG in a reporter mRNA we show that the adenylate tract is sufficient and necessary for mRNA repression and poly(A)-mediated activation in the reticulocyte cell-free system. UV cross-linking experiments demonstrate that the leader adenylate tract binds PABP. Furthermore, addition of recombinant GST-PABP to the cell-free system represses translation of mRNAs containing the A-rich sequence in their 5'UTR, but has no effect on control mRNA. We thus conclude that in vitro PABP binding to the A-rich sequence in the 5' UTR of PABP mRNA represses its own synthesis.
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PMID:Autoregulation of poly(A)-binding protein synthesis in vitro. 761 48

In the yeast Saccharomyces cerevisiae, pre-mRNA 3'-end processing requires six factors: cleavage factor IA (CF IA), cleavage factor IB (CF IB), cleavage factor II (CF II), polyadenylation factor I (PF I), poly(A) polymerase (Pap1p) and poly(A)-binding protein I (Pab1p). We report the characterization of Pfs2p, a WD-repeat protein previously identified in a multiprotein complex carrying PF I-Pap1p activity. The 3'-end-processing defects of pfs2 mutant strains and the results of immunodepletion and immunoinactivation experiments indicate an essential function for Pfs2p in cleavage and polyadenylation. With a one-step affinity purification method that exploits protein A-tagged Pfs2p, we showed that this protein is part of a CF II-PF I complex. Pull-down experiments with GST fusion proteins revealed direct interactions of Pfs2p with subunits of CF II-PF I and CF IA. These results show that Pfs2p plays an essential role in 3'-end formation by bridging different processing factors and thereby promoting the assembly of the processing complex.
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PMID:The WD-repeat protein pfs2p bridges two essential factors within the yeast pre-mRNA 3'-end-processing complex. 1061 42

Oculopharyngeal muscular dystrophy (OPMD) is caused by short expansions of the GCG trinucleotide repeat encoding the polyalanine tract of the poly(A)-binding protein 2 (PABP2). PABP2 binds to the growing poly(A) tail, stimulating its extension during the polyadenylation process, and limits the length of the newly synthesized poly(A) tail. Whereas PABP2 is expressed ubiquitously, the clinical and pathological features of OPMD patients are restricted to the skeletal muscle. To elucidate the possible role of PABP2 in skeletal muscle, we established the stable C2 cell lines expressing human PABP2. These stable cell lines showed morphologically enhanced myotube formation accompanied by an increased expression of myogenic factors, MyoD and myogenin. In nuclear run-on assay, the transcription rate of the MyoD gene was significantly increased by PABP2 transfection. We found the N-terminal region of PABP2 was responsible for the up-regulation of these myogenic factors. Furthermore, Ski-interacting protein (SKIP) was isolated as a binding protein for PABP2 using the yeast two-hybrid system. The interaction of PABP2 and SKIP was confirmed by glutathione S-transferase-pulldown assay and immunoprecipitation. Confocal laser scanning showed PABP2 was co-localized with SKIP in nuclear speckles. The reporter assays showed that PABP2 co-operated with SKIP to synergistically activate E-box-mediated transcription through MYOD: Moreover, both PABP2 and SKIP were directly associated with MyoD to form a single complex. These findings suggest that PABP2 and SKIP directly control the expression of muscle-specific genes at the transcription level.
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PMID:The product of an oculopharyngeal muscular dystrophy gene, poly(A)-binding protein 2, interacts with SKIP and stimulates muscle-specific gene expression. 1137 6

The poly(A)-binding protein (PABP), bound to the 3' poly(A) tail of eukaryotic mRNAs, plays critical roles in mRNA translation and stability. PABP autoregulates its synthesis by binding to a conserved A-rich sequence present in the 5'-untranslated region of PABP mRNA and repressing its translation. PABP is composed of two parts: the highly conserved N terminus, containing 4 RNA recognition motifs (RRMs) responsible for poly(A) and eIF4G binding; and the more variable C terminus, which includes the recently described PABC domain, and promotes intermolecular interaction between PABP molecules as well as cooperative binding to poly(A). Here we show that, in vitro, GST-PABP represses the translation of reporter mRNAs containing 20 or more A residues in their 5'-untranslated regions and remains effective as a repressor when an A61 tract is placed at different distances from the cap, up to 126 nucleotides. Deletion of the PABP C terminus, but not the PABC domain alone, significantly reduces its ability to inhibit translation when bound to sequences distal to the cap, but not to proximal ones. Moreover, cooperative binding by multiple PABP molecules to poly(A) requires the C terminus, but not the PABC domain. Further analysis using pull-down assays shows that the interaction between PABP molecules, mediated by the C terminus, does not require the PABC domain and is enhanced by the presence of RRM 4. In vivo, fusion proteins containing parts of the PABP C terminus fused to the viral coat protein MS2 have an enhanced ability to prevent the expression of chloramphenicol acetyltransferase reporter mRNAs containing the MS2 binding site at distal distances from the cap. Altogether, our results identify a proline- and glutamine-rich linker located between the RRMs and the PABC domain as being strictly required for PABP/PABP interaction, cooperative binding to poly(A) and enhanced translational repression of reporter mRNAs in vitro and in vivo.
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PMID:Identification of a C-terminal poly(A)-binding protein (PABP)-PABP interaction domain: role in cooperative binding to poly (A) and efficient cap distal translational repression. 1295 55

The C-terminal domain of poly(A)-binding protein (PABC) is a peptide-binding domain found in poly(A)-binding proteins (PABPs) and a HECT (homologous to E6-AP C-terminus) family E3 ubiquitin ligase. In protein synthesis, the PABC domain of PABP functions to recruit several translation factors possessing the PABP-interacting motif 2 (PAM2) to the mRNA poly(A) tail. We have determined the solution structure of the human PABC domain in complex with two peptides from PABP-interacting protein-1 (Paip1) and Paip2. The structures show a novel mode of peptide recognition, in which the peptide binds as a pair of beta-turns with extensive hydrophobic, electrostatic and aromatic stacking interactions. Mutagenesis of PABC and peptide residues was used to identify key protein-peptide interactions and quantified by isothermal calorimetry, surface plasmon resonance and GST pull-down assays. The results provide insight into the specificity of PABC in mediating PABP-protein interactions.
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PMID:Structural basis of ligand recognition by PABC, a highly specific peptide-binding domain found in poly(A)-binding protein and a HECT ubiquitin ligase. 1468 57

PRMT3 (protein arginine methyltransferase 3) is one of four type I arginine methyltransferases that catalyse the formation of asymmetric dimethylarginine. PRMT3 is unique in that its N-terminus harbours a C2H2 zinc-finger domain that is proposed to confer substrate specificity. In addition, PRMT3 is the only type I enzyme that is restricted to the cytoplasm. Known in vitro substrates for PRMT3 include GST-GAR (a glutathione S-transferase fusion protein containing the glycine- and arginine-rich N-terminal region of fibrillarin), Sam68 (Src-associated substrate during mitosis 68 kDa) and PABP-N1 [poly(A)-binding protein-N1; PABP2]. Here we report the identification of an in vivo substrate for mammalian PRMT3. We found that FLAG-tagged PRMT3 can 'pull down' a protein with a molecular mass of 30 kDa from HeLa cell extracts. MS identified this PRMT3-interacting protein as rpS2 (ribosomal protein S2). In vitro studies showed that the zinc-finger domain of PRMT3 is necessary and sufficient for binding to rpS2. In addition, rpS2 is methylated by PRMT3 in vitro and is also methylated in cell lines. Deletion analysis of the rpS2 amino acid sequence identified a N-terminal Arg-Gly repeat as the methylation site. Furthermore, both PRMT3 and rpS2 co-sediment with free ribosomal subunits. These studies implicate PRMT3 in ribosomal function and in the regulation of protein synthesis.
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PMID:Ribosomal protein S2 is a substrate for mammalian PRMT3 (protein arginine methyltransferase 3). 1547 65

Tob is a member of an emerging family of anti-proliferative proteins that suppress cell growth when over-expressed. tob mRNA is highly expressed in anergic T cells and over-expression of Tob suppresses transcription of interleukin-2 (IL-2) through its interaction with Smads. Here, we identified two types of cDNA clones coding for poly(A)-binding protein (PABP) and inducible PABP (iPABP) by screening an expression cDNA library with the GST-Tob probe. Co-immunoprecipitation and GST-pull down experiments showed that Tob associated with the carboxyl-terminal region of iPABP. We then found that iPABP, like PABP, was involved in regulation of translation: iPABP enhanced translation of IL-2 mRNA in vitro. The enhanced translation of IL-2 mRNA required the 3'UTR and poly(A) sequences. Tob abrogated the enhancement of translation through its interaction with carboxyl-terminal region of iPABP in vitro. Consistently, over-expression of Tob in NIH3T3 cells, in which exogenous iPABP was stably expressed, resulted in suppression of IL-2 production from the simultaneously transfected IL-2 expression plasmid. Finally, Tob, whose expression was induced by anergic stimulation, was co-immunoprecipitated with iPABP in human T cells. These findings suggest that Tob is involved in the translational suppression of IL-2 mRNA in anergic T cells through its interaction with iPABP.
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PMID:Interaction of anti-proliferative protein Tob with poly(A)-binding protein and inducible poly(A)-binding protein: implication of Tob in translational control. 1567 26

The PABC domain is a peptide-binding domain that is specifically found in poly(A)-binding protein (PABP) and a HECT ubiquitin-protein isopeptide ligase (E3) known as HYD (hyperplastic discs), EDD (E3 isolated by differential display), or Rat100. The PABC domain of PABP recruits various regulatory proteins and translation factors to poly(A) mRNAs through binding of a conserved 12-amino acid peptide motif, PAM2 (PABP-interacting motif 2). In contrast, little is known about the specificity or function of the domain from HYD. Here, we used isothermal calorimetry and surface plasmon resonance titrations to show that the PABC domain of HYD binds PAM2 peptides with micromolar affinity. NMR chemical shift perturbations were used to map the peptide-binding site in the PABC domain of HYD. The structural features of binding are very similar to those of the interactions with the domain of PABP, which explains the overlapping peptide specificity and binding affinity. We identified the anti-proliferative Tob proteins as potential binding partners of HYD. This was confirmed by glutathione S-transferase pulldown and immunoprecipitation experiments demonstrating the interaction with full-length Tob2. Altogether, our results point to a role of the PABC domain as a protein-protein interaction domain that brings together the processes of translation, ubiquitin-mediated protein degradation, and cell cycle control.
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PMID:Comparative peptide binding studies of the PABC domains from the ubiquitin-protein isopeptide ligase HYD and poly(A)-binding protein. Implications for HYD function. 1655 97

In mammalian cells, mRNA decay begins with deadenylation, which involves two consecutive phases mediated by the PAN2-PAN3 and the CCR4-CAF1 complexes, respectively. The regulation of the critical deadenylation step and its relationship with RNA-processing bodies (P-bodies), which are thought to be a site where poly(A)-shortened mRNAs get degraded, are poorly understood. Using the Tet-Off transcriptional pulsing approach to investigate mRNA decay in mouse NIH 3T3 fibroblasts, we found that TOB, an antiproliferative transcription factor, enhances mRNA deadenylation in vivo. Results from glutathione S-transferase pull-down and coimmunoprecipitation experiments indicate that TOB can simultaneously interact with the poly(A) nuclease complex CCR4-CAF1 and the cytoplasmic poly(A)-binding protein, PABPC1. Combining these findings with those from mutagenesis studies, we further identified the protein motifs on TOB and PABPC1 that are necessary for their interaction and found that interaction with PABPC1 is necessary for TOB's deadenylation-enhancing effect. Moreover, our immunofluorescence microscopy results revealed that TOB colocalizes with P-bodies, suggesting a role of TOB in linking deadenylation to the P-bodies. Our findings reveal a new mechanism by which the fate of mammalian mRNA is modulated at the deadenylation step by a protein that recruits poly(A) nuclease(s) to the 3' poly(A) tail-PABP complex.
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PMID:Human TOB, an antiproliferative transcription factor, is a poly(A)-binding protein-dependent positive regulator of cytoplasmic mRNA deadenylation. 1778 42

Human pathogenic viruses manipulate host cell translation machinery to ensure efficient expression of viral genes and to thwart host cell protein synthesis. Viral strategies include cleaving translation factors, manipulating translation factor abundance and recruitment into translation initiation complexes, or expressing viral translation factor analogs. Analyzing translation factors in herpes simplex virus type 1 (HSV-1)-infected HeLa cells, we found diminished association of the polyadenylate-binding protein (PABP) with the cap-binding complex. Although total PABP levels were unchanged, HSV-1 infection prompted accumulation of cytoplasmic PABPC1, but not its physiologic binding partner PABP-interacting protein 2 (Paip2), in the nucleus. Using glutathione S-transferase-PABP pull-down and proteomic analyses, we identified several viral proteins interacting with PABPC1 including tegument protein UL47 and infected-cell protein ICP27. Transient expression of ICP27 and UL47 in HeLa cells suggested that ICP27 and UL47 jointly displace Paip2 from PABP. ICP27 expression alone was sufficient to cause PABPC1 redistribution to the nucleus. ICP27 and UL47 did not alter translation efficiency of transfected reporter RNAs but modulated transcript abundance and expression of reporter cDNAs in transfected cells. This indicates that redistribution of PABPC1 may be involved in co- and posttranscriptional regulation of mRNA processing and/or nuclear export by HSV-1 gene regulatory proteins.
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PMID:Herpes simplex virus proteins ICP27 and UL47 associate with polyadenylate-binding protein and control its subcellular distribution. 1986 86


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