Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.5.1.18 (glutathione S-transferase)
 22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The muscle-specific intermediate filament protein desmin is up-regulated during skeletal muscle differentiation. When myoblasts leave the cell cycle and fusion into multinucleated myotubes starts, genes associated with myogenesis become activated. The activation is believed to be mediated by the muscle-specific determination factors. We present evidence that both MyoD and myogenin are able to activate the transcription of the hamster desmin gene. A proximal promoter fragment of 89 base pairs is sufficient for this transactivation process. The single E-box in this region is essential for desmin promoter activity in mouse C2 skeletal muscle cells and upon co-transfection of a myogenin expression vector also in human primary fibroblasts. Mutation of this MyoD binding site abrogates desmin transcription, and transactivation of the promoter no longer occurs. By using gel electrophoretic mobility shift assays, we were able to demonstrate that nuclear proteins from C2 muscle cells and myogenin/E12 glutathione S-transferase fusion proteins are able to bind to the functional E-box consensus sequence. A second E-box, situated in a more upstream regulatory region, which also binds to purified Helix-Loop-Helix proteins in vitro is only moderately affected by site-directed in vitro mutagenesis.
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PMID:A proximal promoter element in the hamster desmin upstream regulatory region is responsible for activation by myogenic determination factors. 827 97

The observation that adenovirus E1A gene products can inhibit differentiation of skeletal myocytes suggested that E1A may interfere with the activity of myogenic basic helix-loop-helix (bHLH) transcription factors. We have examined the ability of E1A to mediate repression of the muscle-specific creatine kinase (MCK) gene. Both the E1A12S and E1A13S products repressed MCK transcription in a concentration-dependent fashion. In contrast, amino-terminal deletion mutants (d2-36 and d15-35) of E1A12S were defective for repression. E1A12S also repressed expression of a promoter containing a multimer of the MCK high-affinity E box (the consensus site for myogenic bHLH protein binding) that was dependent, in C3H10T1/2 cells, on coexpression of a myogenin bHLH-VP16 fusion protein. A series of coprecipitation experiments with glutathione S-transferase fusion and in vitro-translated proteins demonstrated that E1A12S, but not amino-terminal E1A deletion mutants, could bind to full-length myogenin and E12 and to deletion mutants of myogenin and E12 that spare the bHLH domains. Thus, the bHLH domains of myogenin and E12, and the high-affinity E box, are targets for E1A-mediated repression of the MCK enhancer, and domains of E1A required for repression of muscle-specific gene transcription also mediate binding to bHLH proteins. We conclude that E1A mediates repression of muscle-specific gene transcription through its amino-terminal domain and propose that this may involve a direct physical interaction between E1A and the bHLH region of myogenic determination proteins.
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PMID:E1A-mediated inhibition of myogenesis correlates with a direct physical interaction of E1A12S and basic helix-loop-helix proteins. 839 37

Terminal differentiation of muscle cells results in opposite effects on gene promoters: muscle-specific promoters, which are repressed during active proliferation of myoblasts, are turned on, whereas at least some proliferation-associated promoters, such as c-fos, which are active during cell division, are turned off. MyoD and myogenin, transcription factors from the basic-helix-loop-helix (bHLH) family, are involved in both processes, up-regulating muscle genes and down-regulating c-fos. On the other hand, the serum response factor (SRF) is involved in the activation of muscle-specific genes, such as c-fos, as well as in the up-regulation of a subset of genes that are responsive to mitogens. Upon terminal differentiation, the activity of these various transcription factors could be modulated by the formation of distinct protein-protein complexes. Here, we have investigated the hypothesis that the function of SRF and/or MyoD and myogenin could be modulated by a physical association between these transcription factors. We show that myogenin from differentiating myoblasts specifically binds to SRF. In vitro analysis, using the glutathione S-transferase pull-down assay, indicates that SRF-myogenin interactions occur only with myogenin-E12 heterodimers and not with isolated myogenin. A physical interaction between myogenin, E12, and SRF could also be demonstrated in vivo using a triple-hybrid approach in yeast. Glutathione S-transferase pull-down analysis of various mutants of the proteins demonstrated that the bHLH domain of myogenin and that of E12 were necessary and sufficient for the interaction to be observed. Specific binding to SRF was also seen with MyoD. In contrast, Id, a natural inhibitor of myogenic bHLH proteins, did not bind SRF in any of the situations tested. These data suggest that SRF, on one hand, and myogenic bHLH, on the other, could modulate each other's activity through the formation of a heterotrimeric complex.
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PMID:Physical interaction between the mitogen-responsive serum response factor and myogenic basic-helix-loop-helix proteins. 861 11

A biological role for MEF2 (myocyte enhancer factor 2) activity during mammalian myogenesis has been inferred but not directly proven because of its role in the transcriptional activation of many muscle-specific genes. Therefore, our purpose was to determine whether MEF2 activity is absolutely required for mammalian myogenesis. Using a dominant-negative approach to address this question, we constructed a mutated MEF2A protein comprised of the amino-terminal DNA binding/dimerization domain of MEF2A without its trans-activation domain as a bacterial fusion protein (GST-131) or in a eukaryotic expression vector (pcDNA-131). GST-131 and the protein encoded by pcDNA-131 bind specifically to the MEF2 cis element and abrogate trans-activation of a MEF2-responsive luciferase reporter gene by wild type MEF2A, thus serving a role as trans-dominant inhibitors of MEF2 function. In congruence with their ability to interfere with wild type MEF2 function, microinjection of GST-131 or pcDNA-131 into L6E9 or C2C12 myoblasts inhibited myotube formation. Immunofluorescence analysis showed that the expression of myogenin, myosin heavy chain, and MEF2A were inhibited in the GST-131 or pcDNA-131-injected cells compared with GST or pcDNA-injected controls. We also document that this trans-dominant MEF2 inhibitor impairs the myogenic conversion of C3H10T1/2 fibroblasts by MyoD. Thus, these data provide evidence that the trans-activation function of the MEF2 proteins during mammalian myogenesis is required for muscle-specific gene expression and differentiation.
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PMID:A dominant-negative form of transcription factor MEF2 inhibits myogenesis. 940 17

Nuclear receptor-mediated activation of transcription involves coactivation by cofactors collectively denoted the steroid receptor coactivators (SRCs). The process also involves the subsequent recruitment of p300/CBP and PCAF to a complex that synergistically regulates transcription and remodels the chromatin. PCAF and p300 have also been demonstrated to function as critical coactivators for the muscle-specific basic helix-loop-helix (bHLH) protein MyoD during myogenic commitment. Skeletal muscle differentiation and the activation of muscle-specific gene expression is dependent on the concerted action of another bHLH factor, myogenin, and the MADS protein, MEF-2, which function in a cooperative manner. We examined the functional role of one SRC, GRIP-1, in muscle differentiation, an ideal paradigm for the analysis of the determinative events that govern the cell's decision to divide or differentiate. We observed that the mRNA encoding GRIP-1 is expressed in proliferating myoblasts and post-mitotic differentiated myotubes, and that protein levels increase during differentiation. Exogenous/ectopic expression studies with GRIP-1 sense and antisense vectors in myogenic C2C12 cells demonstrated that this SRC is necessary for (1) induction/activation of myogenin, MEF-2, and the crucial cell cycle regulator, p21, and (2) contractile protein expression and myotube formation. Furthermore, we demonstrate that the SRC GRIP-1 coactivates MEF-2C-mediated transcription. GRIP-1 also coactivates the synergistic transactivation of E box-dependent transcription by myogenin and MEF-2C. GST-pulldowns, mammalian two-hybrid analysis, and immunoprecipitation demonstrate that the mechanism involves direct interactions between MEF-2C and GRIP-1 and is associated with the ability of the SRC to interact with the MADS domain of MEF-2C. The HLH region of myogenin mediates the direct interaction of myogenin and GRIP-1. Interestingly, interaction with myogenic factors is mediated by two regions of GRIP-1, an amino-terminal bHLH-PAS region and the carboxy-terminal region between amino acids 1158 and 1423 (which encodes an activation domain, has HAT activity, and interacts with the coactivator-associated arginine methyltransferase). This work demonstrates that GRIP-1 potentiates skeletal muscle differentiation by acting as a critical coactivator for MEF-2C-mediated transactivation and is the first study to ascribe a function to the amino-terminal bHLH-PAS region of SRCs.
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PMID:The steroid receptor coactivator, GRIP-1, is necessary for MEF-2C-dependent gene expression and skeletal muscle differentiation. 1081 56

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

Mutations in the EYA1 gene are responsible for branchio-oto-renal (BOR) syndrome as well as for other ocular defects. Most of the mutations are located within or in the vicinity of the EYA domain, which is highly conserved in the EYA protein family. The EYA domain is required for protein-protein interactions, which are important to the biological function of EYA proteins. To determine how EYA1 mutations cause BOR syndrome and/or ocular defects, we tested the effects of Eya1 mutations on interactions with Six. Dach, and G proteins by mammalian two-hybrid and GST-pulldown assays. Defective interactions were noted between BOR-type mutations S486P and L504R of Eya1 and Dach1, G proteins, and some Six proteins. These mutations impaired the activation of transcription from a Six-responsive gene, myogenin, with Six5. S486P and L504R showed an altered digestion pattern with trypsin, and L504R also decreased the sensitivity to V8 protease digestion and produced a peptide fragment with a different M(r). Our results suggest that defective protein-protein interactions of the mutations in the EYA domain underlie BOR syndrome and that SIX, DACH, and/or G proteins are possibly involved in the pathogenic processes.
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PMID:Impaired interactions between mouse Eyal harboring mutations found in patients with branchio-oto-renal syndrome and Six, Dach, and G proteins. 1195 62

The btg1 (B-cell translocation gene 1) gene coding sequence was isolated from a translocation break point in a case of B-cell chronic lymphocytic leukaemia. We have already shown that BTG1, considered as an antiproliferative protein, strongly stimulates myoblast differentiation. However, the mechanisms involved in this influence remained unknown. In cultured myoblasts, we found that BTG1 stimulates the transcriptional activity of nuclear receptors (T3 and all-trans retinoic acid receptors but not RXRalpha and PPARgamma), c-Jun and myogenic factors (CMD1, Myf5, myogenin). Immunoprecipitation experiments performed in cells or using in vitro-synthesized proteins and GST pull-down assays established that BTG1 directly interacts with T3 and all-trans retinoic acid receptors and with avian MyoD (CMD1). These interactions are mediated by the transactivation domain of each transcription factor and the A box and C-terminal part of BTG1. NCoR presence induces the ligand dependency of the interaction with nuclear receptors. Lastly, deletion of BTG1 interacting domains abrogates its ability to stimulate nuclear receptors and CMD1 activity, and its myogenic influence. In conclusion, BTG1 is a novel important coactivator involved in the regulation of myoblast differentiation. It not only stimulates the activity of myogenic factors, but also of nuclear receptors already known as positive myogenic regulators.
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PMID:Coactivation of nuclear receptors and myogenic factors induces the major BTG1 influence on muscle differentiation. 1567 37

MyoD initiates muscle differentiation and promotes skeletal myogenesis by regulating temporal gene expression. MyoD-interacting proteins induce regulatory effects, and the identification of new MyoD-binding partners may provide mechanistic insights into the regulation of gene expression during myogenesis. FHL3 is one of three members of the FHL protein family that are expressed in skeletal muscle, but its function in myogenesis is unknown. Overexpression of human FHL3 in mouse C2C12 cells retarded myotube formation and decreased the expression of muscle-specific regulatory genes such as myogenin but not MyoD. By contrast, short interfering RNA (siRNA)-mediated FHL3 protein knockdown enhanced myoblast differentiation associated with increased myogenin, but not MyoD protein expression, early during differentiation. We demonstrate that FHL3 is a MyoD-associated protein by direct binding assays, colocalisation in the nucleus of myoblasts and GST pull-down studies. Moreover, we determined that FHL3 interacts with MyoD, functioning as its potent negative co-transcriptional regulator. Ectopic expression of FHL3 in myoblasts impaired MyoD-mediated transcriptional activity and muscle gene expression. By contrast, siRNA-mediated FHL3 knockdown enhanced MyoD transcriptional activity in a dose-dependent manner. These findings reveal that FHL3 association with MyoD may contribute to the regulation of MyoD-dependent transcription of muscle genes and thereby myogenesis.
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PMID:FHL3 binds MyoD and negatively regulates myotube formation. 1738 85

Previous studies have suggested that vitamin B6 is an ergogenic factor. However, the role of dietary vitamin B6 in skeletal muscle has not been widely researched. The aim of the present study was to investigate the effects of dietary vitamin B6 on the gene expression of 19 myokines, 14 nuclear factor erythroid 2-related factor 2 (Nrf2)-regulated factors, 8 myogenesis-related factors and 4 heat shock proteins (HSPs), which may serve important roles in skeletal muscles. Rats were fed a diet containing 1 (marginal vitamin B6 deficiency), 7 (recommended dietary level) or 35 mg/kg of pyridoxine (PN) HCl/ for 6 weeks. Gene expressions were subsequently analysed using reverse transcription-quantitative polymerase chain reaction. Food intake and growth were unaffected by this dietary treatment. The rats in the 7 and 35 mg/kg PN HCl groups exhibited a significant increase in the concentration of pyridoxal 5'-phosphate in the gastrocnemius muscle compared with the 1 mg/kg PN HCl diet (P<0.01). The expressions of myokines, such as IL-7, IL-8, secreted protein acidic and rich in cysteine, IL-6, growth differentiation factor 11, myonectin, leukaemia inhibitory factor, apelin and retinoic acid receptor responder (tazarotene induced) 1, the expression of Nrf2 and its regulated factors, such as heme oxygenase 1, superoxide dismutase 2, glutathione peroxidase 1 and glutathione S-transferase, and the expression of myogenin and HSP60 were significantly elevated in the 7 mg/kg PN HCl group compared with the 1 mg/kg PN HCl diet (P<0.05). No significant differences in levels of these genes were observed between the 35 and 1 mg/kg PN HCl, with the exception of GDF11 and myonectin, whose expressions were significantly increased in the 35 mg/kg PN HCl (P<0.05). Notably, the majority of gene expressions that were affected responded to dietary supplemental vitamin B6 in a similar manner. The results suggest that compared with the marginal vitamin B6 deficiency, the recommended dietary intake of vitamin B6 upregulates the gene expression of a number of factors that promote the growth and repair of skeletal muscle.
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PMID:Dietary vitamin B6 modulates the gene expression of myokines, Nrf2-related factors, myogenin and HSP60 in the skeletal muscle of rats. 2891 74


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