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)

Multiple physiological actions of the hormonal form of vitamin D3, 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3), are mediated by a genomic pathway which is initiated by the highly specific recognition and binding by its cognate receptor (vitamin D receptor, VDR) in the target cells. Thus, knowledge of the three-dimensional geometries of the ligand, i.e., 1,25(OH)2D3, and the 1,25(OH)2D3-binding domain of VDR is crucial for a better understanding of diverse physiological roles of this hormone. Recently our laboratory has developed 1 alpha,25-dihydroxyvitamin D3-3 beta-bromoacetate (1,25(OH)2 D3-3-BE) as an affinity labeling reagent for covalently modifying the hormone binding domain of native VDRs from calf thymus and rat osteosarcoma cells and baculovirus-expressed recombinant human VDR (hVDR). In the present report, we report affinity labeling of the hormone binding domain of hVDR, expressed in Escherichia coli as a glutathione S-transferase fusion partner, site-specific cleavage of the affinity-labeled VDR with 3-bromo-3-methyl-2-(2-nitrophenylmercapto)- 3H-indole, and identification of the C-terminal subdomain of human VDR containing the putative hormone binding site.
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PMID:Identification of the subdomain in the nuclear receptor for the hormonal form of vitamin D3, 1 alpha,25-dihydroxyvitamin D3, vitamin D receptor, that is covalently modified by an affinity labeling reagent. 939 Jan 78

Crystallographic structures of the ligand-binding domains for the retinoid X (RXR) and estrogen receptors have identified conserved surface residues that participate in dimer formation. Homologous regions have been identified in the human vitamin D receptor (hVDR). Mutating Lys-386 to Ala (K386A) in hVDR significantly reduced binding to glutathione S-transferase-RXRalpha in solution, whereas binding of an I384R/Q385R VDR mutant was almost undetectable. The K386A mutant formed heterodimers with RXRalpha on DR-3 (a direct repeat of AGGTCA spaced by three nucleotides), whereas the I384R/Q385R mutant completely eliminated heterodimer formation. Wild type hVDR effected a 3-fold induction of DR-3-dependent thymidine kinase-luciferase activity in cultured neonatal rat atrial myocytes, an effect that was increased to 8-9-fold by cotransfected hRXRalpha. Induction by K386A, in the presence or absence of RXRalpha, was only slightly lower than that seen with wild type VDR. On the other hand, I384R/Q385R alone displayed no stimulatory activity and less than 2-fold induction in the presence of hRXRalpha. Qualitatively similar findings were observed with the negative regulation of the human atrial natriuretic peptide gene promoter by these mutants. Collectively, these studies identify specific amino acids in hVDR that play a critical role in heterodimer formation and subsequent modulation of gene transcription.
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PMID:Vitamin D-dependent suppression of human atrial natriuretic peptide gene promoter activity requires heterodimer assembly. 1019 14

Steroid hormone receptors (SHR) form complexes with heat shock proteins (hsps). The 1alpha,25-dihydroxyvitamin D(3) receptor (VDR) has not been previously shown to interact with hsps. During expression and purification of VDR-glutathione S-transferase (VDR-GST) fusion proteins encompassing full-length, DNA, and ligand-binding domains of the VDR (FL-VDR, DBD-VDR, and LBD-VDR), we observed binding of bacterial hsps with VDR-GST constructs. All VDR constructs bound DnaK in amounts greater than GST alone and bound smaller amounts of DnaJ or GrpE. GroEL bound only to FL-VDR. GroES did not bind to VDR. When VDR-GST constructs were incubated with a reticulocyte lysate system that has been used previously to examine SHR-hsp interactions, eukaryotic hsc70 was detected bound to FL-VDR and DBD-VDR. Binding of hsp90 to VDR was not detected. However, geldanamycin, an hsp90 inhibitor, reduced 1alpha,25-dihydroxyvitamin D(3)-mediated gene activation in osteoblasts. Our data show that the bacterial and eukaryotic hsps associate with the VDR and might be involved in VDR function.
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PMID:Association of prokaryotic and eukaryotic chaperone proteins with the human 1alpha,25-dihydroxyvitamin D(3) receptor. 1040 88

Human vitamin D receptor (hVDR) fused to glutathione S-transferase was utilized to detect a VDR-interacting protein (VIP) of approximately 170 kDa. VIP(170) is expressed in osteoblast-like ROS 17/2.8 cells and, to a lesser extent, in COS-7 and HeLa cells. VIP(170) may be a coactivator because it interacts only with 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) ligand-bound hVDR and because a mutation (E420A) in the activation function-2 (AF-2) of hVDR abolishes both receptor-mediated transactivation and VIP(170) binding. Unlike L254G hVDR, a heterodimerization mutant with an intact AF-2, the E420A mutant is only partially attenuated in its association with the retinoid X receptor (RXR) DNA-binding partner. Finally, the ability of overexpressed hVDR to squelch glucocorticoid receptor-mediated transactivation is lost in both the L254G and E420A mutants. These results suggest that several protein-protein interactions, including VDR association with RXR and VIP(170), are required for stabilization of a multimeric complex that transduces the signal for 1,25(OH)(2)D(3)-elicited transactivation.
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PMID:Biochemical evidence for a 170-kilodalton, AF-2-dependent vitamin D receptor/retinoid X receptor coactivator that is highly expressed in osteoblasts. 1067 74

Assessment of the risk of human exposure to man-made chemicals that bind to hormone receptors has emerged as a major public health issue. Among hormone receptors, nuclear receptors tend to be targets of xenobiotics because their endogenous ligands are small, fat-soluble molecules. Nuclear receptors are ligand-inducible transcriptional factors and regulate the transcriptional activity of various target genes. At the start of the initiation step of transcription, nuclear receptors interact with coactivators (TIF2, SRC1, ACTR, CBP/p300, etc.) in an agonist-dependent manner. Using the interaction of the nuclear receptor with a coactivator, we have developed a novel rapid ligand in vitro screening method that is easy to use and has high sensitivity. This method, called by us the CoA-BAP system, is applicable to most nuclear receptors and is suitable for high-throughput screening because the entire experimental operation can be carried out on a microplate. We used human TIF2 as a coactivator including LXXLL motifs expressed in Escherichia coli as a fusion protein with BAP and nuclear receptor LBD expressed in E. coli as a fusion protein with GST. On a GSH-coupled microplate these proteins were incubated with chemicals and the protein-protein interactions were detected as alkaline phosphatase activity. To date we have examined seven nuclear receptors (ERalpha/beta, TRalpha, RARalpha/gamma, RXRalpha,and VDR) and confirmed that the method works well.
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PMID:Basis of a high-throughput method for nuclear receptor ligands. 1286 36

A fractal analysis is used to model the binding and dissociation kinetics between analytes in solution and estrogen receptors (ER) immobilized on a sensor chip of a surface plasmon resonance (SPR) biosensor. Both cases are analyzed: unliganded as well as liganded. The influence of different ligands is also analyzed. A better understanding of the kinetics provides physical insights into the interactions and suggests means by which appropriate interactions (to promote correct signaling) and inappropriate interactions such as with xenoestrogens (to minimize inappropriate signaling and signaling deleterious to health) may be better controlled. The fractal approach is applied to analyte-ER interaction data available in the literature. Numerical values obtained for the binding and the dissociation rate coefficients are linked to the degree of roughness or heterogeneity (fractal dimension, D(f)) present on the biosensor chip surface. In general, the binding and the dissociation rate coefficients are very sensitive to the degree of heterogeneity on the surface. For example, the binding rate coefficient, k, exhibits a 4.60 order of dependence on the fractal dimension, D(f), for the binding of unliganded and liganded VDR mixed with GST-RXR in solution to Spp-1 VDRE (1,25-dihydroxyvitamin D(3) receptor element) DNA immobilized on a sensor chip surface (Cheskis and Freedman, Biochemistry 35 (1996) 3300-3318). A single-fractal analysis is adequate in some cases. In others (that exhibit complexities in the binding or the dissociation curves) a dual-fractal analysis is required to obtain a better fit. A predictive relationship is also presented for the ratio K(A)(=k/k(d)) as a function of the ratio of the fractal dimensions (D(f)/D(fd)). This has biomedical and environmental implications in that the dissociation and binding rate coefficients may be used to alleviate deleterious effects or enhance beneficial effects by selective modulation of the surface. The K(A) exhibits a 112-order dependence on the ratio of the fractal dimensions for the ligand effects on VDR-RXR interaction with specific DNA.
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PMID:A fractal analysis of analyte-estrogen receptor binding and dissociation kinetics using biosensors: environmental effects. 1290 31

The active form of vitamin D, 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), exerts its effects through regulation of target gene transcription. Configuration at C-20 of 1,25(OH)(2)D(3) is important in determining potency, as shown by the high potency of analogs with inverted configuration at C-20 (20-epi compounds). Gemini analogs of 1,25(OH)(2)D(3) contain two side chains, combining a C-20-normal with a C20-epi side chain. We studied the potency of analogs combining double (Gemini) side chains with a 23-triple bond and a C-26,27-hexafluoro substitution in either the 20-epi (analog 20R) or 20-normal (analog 20S) side chain. These novel Gemini analogs were 8-50-fold more potent than 1,25(OH)(2)D(3) in inducing U937, HL-60G, and THP-1 differentiation and 5-50-fold more potent in inducing transcription from the osteocalcin vitamin D response element or the 25-hydroxyvitamin D(3)-24-hydroxylase (24OHase) promoter. In vivo, following i.p. injection in vitamin D-deficient mice, the 20S analog induced significantly higher levels of calbindin-D(9K) mRNA in intestine, and 24OHase and calbindin-D(28K) in kidney than 1,25(OH)(2)D(3) or analog 20R. Increased potency did not correlate with ligand-receptor binding affinity. In GST-pull down assays using in vitro translated VDR, Gemini analogs showed equivalent (or even attenuated) potency to 1,25(OH)(2)D(3) in recruiting cofactors DRIP205 and GRIP-1 to VDR. However, Gemini analogs were up to 15-fold more potent than 1,25(OH)(2)D(3) in recruiting the same cofactors to VDR in GST-pull down assays using equal amounts of VDR from nuclear extracts of VDR transfected and hormone treated (24 hr) COS-7 cells. Deletion of C-19 in either 20S or 20R Gemini analogs resulted overall in slightly less potent analogs compared to Gemini itself. We conclude that enhanced potency of the novel Gemini analogs is at least partly due to increased metabolic stability of the analogs, resulting in more cofactor binding and elevated levels of transcription.
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PMID:Novel Gemini analogs of 1alpha,25-dihydroxyvitamin D(3) with enhanced transcriptional activity. 1501 48

Resistance to the action of vitamin D (D) occurs in response to tumor necrosis factor-alpha (TNF-alpha), an effect mediated by nuclear factor kappa B (NfkappaB). To determine the mechanism of NfkappaB inhibition of D-stimulated transcription, chromatin immunoprecipitation assays (CHIP) were done in osteoblastic ROS 17/2.8 cells that had been treated with TNF-alpha or transfected with the p65 subunit of NfkappaB. These treatments caused stable incorporation of p65 into the transcription complex bound to the vitamin D response element (VDRE) of the osteocalcin promoter. Deletion analysis of p65 functional domains revealed that the p65 N-terminus and a midmolecular region were both required for the inhibitory action of p65. Pull-down assays were done using an immobilized glutathione S-transferase (GST)-VDR fusion protein to study the effect of p65 on VDR binding to steroid coactivator-1 (SRC-1), a major D-dependent coactivator. p65 inhibited VDR-SRC-1 binding in a dose-dependent manner. Mutations of p65 that abrogated the inhibitory effect on D-stimulated transcription also failed to inhibit VDR-SRC-1 binding. The inhibitory effect of p65 on VDR transactivation was not due to recruitment of a histone deacetylase (HDAC), since inhibition was not relieved by the HDAC inhibitors sodium butyrate or trichostatin A. Overexpression of SRC-1 or the general coactivators, Creb binding protein or SRC-3, also failed to relieve p65 inhibition of transcription. In addition, Chip assays revealed that TNF-alpha treatment prevented D recruitment of SRC-1 to the transcription complex. These results show that TNF-alpha inhibition of vitamin D-action includes stable integration of p65 in the VDR transcription complex. Once anchored to proteins within the complex, p65 disrupts VDR binding to SRC-1, thus decreasing the efficiency of D-stimulated gene transcription.
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PMID:Integration of the NfkappaB p65 subunit into the vitamin D receptor transcriptional complex: identification of p65 domains that inhibit 1,25-dihydroxyvitamin D3-stimulated transcription. 1521 79

Bone-specific transcription of the osteocalcin (OC) gene is regulated principally by the Runx2 transcription factor and is further stimulated in response to 1alpha,25-dihydroxyvitamin D3 via its specific receptor (VDR). The rat OC gene promoter contains three recognition sites for Runx2 (sites A, B, and C). Mutation of sites A and B, which flank the 1alpha,25-dihydroxyvitamin D3-responsive element (VDRE), abolishes 1alpha,25-dihydroxyvitamin D3-dependent enhancement of OC transcription, indicating a tight functional relationship between the VDR and Runx2 factors. In contrast to most of the members of the nuclear receptor family, VDR possesses a very short N-terminal A/B domain, which has led to the suggestion that its N-terminal region does not contribute to transcriptional enhancement. Here, we have combined transient-overexpression, coimmunoprecipitation, in situ colocalization, chromatin immunoprecipitation, and glutathione S-transferase pull-down analyses to demonstrate that in osteoblastic cells expressing OC, VDR interacts directly with Runx2 bound to site B, which is located immediately adjacent to the VDRE. This interaction contributes significantly to 1alpha,25-dihydroxyvitamin D3-dependent enhancement of the OC promoter and requires a region located C terminal to the runt homology DNA binding domain of Runx2 and the N-terminal region of VDR. Together, our results indicate that Runx2 plays a key role in the 1alpha,25-dihydroxyvitamin D3-dependent stimulation of the OC promoter in osteoblastic cells by further stabilizing the interaction of the VDR with the VDRE. These studies demonstrate a novel mechanism for combinatorial control of bone tissue-specific gene expression. This mechanism involves the intersection of two major pathways: Runx2, a "master" transcriptional regulator of osteoblast differentiation, and 1alpha,25-dihydroxyvitamin D3, a hormone that promotes expression of genes associated with these terminally differentiated bone cells.
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PMID:Bone-specific transcription factor Runx2 interacts with the 1alpha,25-dihydroxyvitamin D3 receptor to up-regulate rat osteocalcin gene expression in osteoblastic cells. 1545 60

Calbindin-D(28k) has been reported to be a facilitator of calcium diffusion and to protect against apoptotic cell death. Most recently, we found that the presence of calbindin-D(28k) results in reduced calcium influx through voltage-dependent L-type Ca(2+) channels and enhanced sensitivity of the channels to calcium dependent inactivation. Co-immunoprecipitation and GST pull down assays indicate that calbindin-D(28k) interacts with the C-terminus of the L-type calcium channel alpha(1c) subunit (Ca(v)1.2). This is the first report of the binding of calbindin to a calcium channel and provides new insight concerning mechanisms by which calbindin acts to modulate intracellular calcium. Besides calbindin, another major target of 1,25(OH)(2)D(3) is 24(OH)ase, which is involved in the catabolism of 1,25(OH)(2)D(3). We reported that C/EBPbeta is a major transcriptional activator of 24(OH)ase that cooperates with CBP/p300 in regulating VDR mediated 24(OH)ase transcription. Recently, we found, in addition to p160 coactivators, that SWI/SNF complexes (that facilitate transcription by remodeling chromatin using the energy of ATP hydrolysis) are also involved in VDR mediated 24(OH)ase transcription and functionally cooperate with C/EBPbeta in regulating 24(OH)ase. These findings define novel mechanisms that may be of fundamental importance in understanding how 1,25(OH)(2)D(3) mediates its multiple biological effects.
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PMID:New insights into the function and regulation of vitamin D target proteins. 1725 25


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