Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Thymidylate synthase (TS) and dCMP hydroxymethylase (CH) are homologous enzymes which catalyze the alkylation of C5 of pyrimidine nucleotides. One of the first catalytic steps is isomerization of the alkyl donor, methylenetetrahydrofolate, from its N5,N10 bridged form to the N5 iminium ion upon enzyme binding. Glu58 in TS has been postulated [Matthews et al. (1990) J. Mol. Biol. 214, 937-948] to be involved in this isomerization and the deprotonation of C5 of the nucleotide. Substitution by Asp or Gln of Glu58 in Escherichia coli TS, or of the corresponding Glu60 in CH from phage T4, decreases the activity of either enzyme. Alkylation is slowed much more than deprotonation, indicating uncoupling of steps which are tightly coupled for the wild-type enzymes. The data support minor roles for Glu58/60 in nucleotide binding and in isomerization of methylenetetrahydrofolate, but no major roles in nucleotide deprotonation, product dissociation, or hydration catalyzed by CH. The primary role of Glu58/60 is to accelerate bond cleavage between N5 of tetrahydrofolate and the methylene being transferred. The influence of Glu58/60 on the rate of bond cleavage is proposed to arise from electrostatic destabilization due to the proximity of the glutamyl carboxylate, of the anionic species formed when C5 of the nucleotide is deprotonated. The proposal explains the uncoupling of deprotonation and alkylation with the Glu58/60 variants and the reduced kinetic isotope effect on hydride transfer for TS(Glu58Gln). The inability of 5-deazatetrahydrofolate to stimulate enzyme-catalyzed tritium exchange from [5-(3H)]nucleotides into solvent suggests that N5 of tetrahydrofolate is the base which deprotonates the nucleotide.
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PMID:Electrostatic guidance of catalysis by a conserved glutamic acid in Escherichia coli dTMP synthase and bacteriophage T4 dCMP hydroxymethylase. 759 33

Thymidylate synthase (TS) is a homodimeric enzyme that catalyzes the reductive methylation of dUMP by N5,N10-methylene-5,6,7,8-tetrahydrofolic acid, to form dTMP. Inhibition of TS by the dUMP analog 5-fluoro-dUMP (FdUMP) occurs through the formation of a covalent ternary complex containing the nucleotide analog, N5,N10-methylene-5,6,7,8-tetrahydrofolic acid, and the enzyme; this complex is termed the inhibitory ternary complex (ITC). In the present report, the kinetics of FdUMP binding into an ITC with purified preparations of human TS were examined. Rapid chemical-quench techniques, as well as steady state binding methods, showed that the enzyme contains two distinct FdUMP binding sites with different affinities for the nucleotide analog. Binding to the first, or high affinity, site was rapid and reached a maximum stoichiometry of 1.0 mol of FdUMP/mol of dimer; binding to the second, or low affinity, site was much slower and reached a stoichiometry of 1.7 mol of FdUMP/mol of dimer. Rate constants for FdUMP binding to and dissociation from the ITC (kon and koff, respectively) were determined, as were equilibrium dissociation constants (Kd). A naturally occurring mutant form of TS, which contains a tyrosine to histidine substitution at residue 33 and renders cells relatively resistant to fluoropyrimidines, exhibited a lower affinity for FdUMP specifically at the second binding site, with little or no change at the first. Hill coefficients were < 1.0, with the His-33 enzyme having a significantly lower coefficient than the wild-type enzyme. The results, in total, indicate that the two FdUMP binding sites on the TS dimer are nonequivalent. We suggest that such nonequivalence may be due to negative cooperativity, where nucleotide binding to the first subunit elicits a conformational change that results in reduced affinity for ligand at the second subunit. This negative cooperativity may be stronger for the His-33 mutant. Thus, the relative fluoropyrimidine resistance conferred by the His-33 substitution may be due to enhanced negative cooperative effects on FdUMP binding into the ITC, thereby reducing the effectiveness of the pyrimidine analog as an inhibitor of thymidylate biosynthesis.
Mol Pharmacol 1995 Jul
PMID:Biphasic binding of 5-fluoro-2'-deoxyuridylate to human thymidylate synthase. 762 77

IPRI-MD-66 (MD-66) cells respond to 20-hydroxyecdysone (20E, 4 x 10(-6) M) in the medium by producing cytoplasmic extensions, clumping and attaching themselves to the substrate. These morphological changes are at a maximum by 6 days post treatment. Degenerate oligonucleotides, designed on the basis of conserved amino acid sequences in the DNA and ligand binding regions of the members of the steroid hormone receptor superfamily, were used in RNA-PCR to isolate two cDNA fragments, Malacosoma disstria hormone receptor 2 (MdHR2) and Malacosoma disstria hormone receptor 3 (MdHR3) from the MD-66 cells. Comparison of deduced amino acid sequences of these cDNA fragments with the members of the steroid hormone receptor superfamily showed that MdHR2 is most closely related to E75 proteins of Manduca sexta, Galleria mellonella and Drosophila melanogaster. The MdHR3 is most closely related to Manduca hormone receptor 3 (MHR3), Galleria hormone receptor 3 (GHR3) and Drosophila hormone receptor 3 (DHR3) proteins. At a concentration of 4 x 10(-6) M, 20E induces the expression of MdHR2 and MdHR3 beginning at 3 h, reaching maximum levels in 12 h and declining in 24 h. MdHR2 binds to a 2.5 kb mRNA, whereas MdHR3 binds to a 4.5 kb mRNA. Based on sequence similarity, RNA size and ecdysone inducibility, we conclude that these cDNA fragments, cloned from MD-66 cells, are regions of E75- (MdHR2) and MHR3- (MdHR3) like genes.
Insect Biochem Mol Biol 1995 Jun
PMID:Analysis of ecdysteroid action in Malacosoma disstria cells: cloning selected regions of E75- and MHR3-like genes. 762 1

Several lines of evidence suggest that molting in parasitic nematodes is controlled through the action of steroid molting hormones, or ecdysones. In other organisms, the central mediator of steroid hormone action is the hormone receptor. These receptor molecules are members of a superfamily of proteins called the nuclear hormone receptor family. Using an oligonucleotide derived from the amino-acid sequence of the Drosophila melanogaster ecdysone receptor, genes encoding homologues of the nuclear hormone receptor family were identified in the genome of the human filarial parasite Onchocerca volvulus. The O. volvulus genome contains at least three genes that encode putative members of the nuclear hormone receptor superfamily. A complete cDNA for one of these genes, designated OvNHR-1, has been isolated and characterized. The OvNHR-1 cDNA was 2378 bp in length, and contained a single open reading frame of 1104 bp. The open reading frame encoded a peptide with all of the features characteristic of a member of the nuclear hormone receptor superfamily of proteins. OVNHR-1 appeared to be encoded by a single-copy gene. Expression of the mRNA corresponding to OvNHR-1 was developmentally regulated, with maximal expression occurring during early embryogenesis. The polypeptide encoded by the OvNHR-1 open reading frame is antigenic in a minority of individuals exposed to O. volvulus.
Mol Biochem Parasitol 1995 Mar
PMID:Characterization of genes encoding members of the nuclear hormone receptor superfamily from Onchocerca volvulus. 763 1

Hepatocyte nuclear factor 4 (HNF-4), a highly conserved member of the steroid hormone receptor superfamily critical for development and liver-specific gene expression, is very similar to another superfamily member, retinoid X receptor alpha (RXR alpha), in overall amino acid sequence and DNA binding specificity. Since RXR alpha is known to heterodimerize with many other nuclear receptors, the formation of heterodimers between HNF-4 and RXR alpha was examined. With the electrophoretic mobility shift assay, coimmunoprecipitation, and transient transfection assays, it is shown that, unlike other nuclear receptors, HNF-4 does not form heterodimers with RXR alpha either in the presence or in the absence of DNA. We also show that in vitro-translated HNF-4 does not form heterodimeric complexes on DNA with a number of other receptors, including RXR beta, RXR gamma, retinoic acid receptor alpha, or thyroid hormone receptor alpha. To investigate the hypothesis that the lack of heterodimerization between HNF-4 and RXR alpha is due to a strong homodimerization activity of HNF-4, glycerol gradient sedimentation and kinetic analysis were used to show that HNF-4 is in fact a stable homodimer in solution. Finally, immunohistochemistry is used to show that the HNF-4 protein is found exclusively in the nuclei in both HepG2 cells, which express endogenous HNF-4, and transfected COS cells, which overexpress HNF-4. These findings lead us to propose that HNF-4 defines a new subclass of nuclear receptors which reside primarily in the nucleus and which bind DNA and regulate transcription as homodimers.
Mol Cell Biol 1995 Sep
PMID:Exclusive homodimerization of the orphan receptor hepatocyte nuclear factor 4 defines a new subclass of nuclear receptors. 765 30

Glucocorticoid hormones positively regulate human papilloma virus (HPV) type 16 gene expression, and we have previously shown that this regulation is through three glucocorticoid response elements (GREs). The GRE at nucleotide 7640 is a composite GRE (cGRE) containing an overlapping activator protein-1 (AP-1) motif for the c-jun homodimer and c-jun/c-fos heterodimer. This report examined the effects of c-jun and/or c-fos AP-1 protooncogenes and the glucocorticoid hormone dexamethasone on expression of the HPV 16 cGRE in AP-1-deficient P19 embryonal carcinoma cells. The activity of the full-length HPV 16 enhancer was progressively increased with increasing levels of c-jun. The hormone induced an additional response. For the c-jun/c-fos heterodimer, the response to hormone was progressively diminished. Site-specific mutations of the cGRE revealed that the regulation by AP-1 and hormone required both GRE and the AP-1 motif. An enhancer fragment containing the cGRE and excluding the two simple GREs gave similar results. Two disruption mutations of the AP-1 site confirmed the requirement of this site for hormone response. A cGRE oligonucleotide construct substantiated the effect of c-jun for response to hormone. For heterodimer, activity and hormone response were both also progressively increased. The results reveal a unique cross-talk between the distinct AP-1- and hormone-signaling pathways, suggesting the involvement of a complex interaction of c-jun and c-fos and glucocorticoid hormone receptor with the HPV 16 cGRE, resulting in novel control patterns for regulating viral expression.
Mol Endocrinol 1994 Dec
PMID:Differential regulation by c-jun and c-fos protooncogenes of hormone response from composite glucocorticoid response element in human papilloma virus type 16 regulatory region. 770 58

An insect steroid hormone, 20-hydroxyecdysone (20E), plays an important role in regulating egg maturation in mosquitoes. To better understand its role, we cloned the cDNA coding for the putative ecdysteroid receptor from the mosquito, Aedes aegypti (AaEcR). The 4158 bp AaEcR cDNA has an open reading frame of 675 amino acids with 10 potential glycosylation sites and a putative phosphorylation polyserine domain. The AaEcR has a DNA binding domain with two zinc fingers and a ligand binding domain characteristic of members of the steroid hormone receptor superfamily. These AaEcR domains share 97 and 87% identities with the respective domains of the Drosophila ecdysteroid receptor (DmEcR). However, the A/B region of the AaEcR shares 35% identity with that of DmEcR-B1 isoform. The F region, located at the carboxyl-terminal of the AaEcR, has only 9% identity with the corresponding region of DmEcR. Potential nuclear targeting and dimerization signals are also present in the AaEcR sequence. There are three AaEcR transcripts of 4.2 kb, 6 kb and 11 kb in adult mosquitoes. 4.2 kb mRNA is predominantly expressed in female mosquitoes during vitellogenesis. In both the fat body and ovaries of the female mosquito, the level of AaEcR mRNA is high at the previtellogenic period and after the onset of vitellogenesis (6 h post blood meal, PBM).
Insect Biochem Mol Biol 1995 Jan
PMID:Mosquito ecdysteroid receptor: analysis of the cDNA and expression during vitellogenesis. 771 47

The binding capacity (Cmax) of the glucocorticoid hormone receptor (GR) was affected by vitamin A status in rat liver. In rats fed on a vitamin A-overloaded diet as well as in rats administered with retinoic acid (RA) there was an increased ratio Cmax of nuclear GR (expressed as fmol/mg liver): Cmax of cytosolic GR (expressed as fmol/mg liver) while in rats fed on a vitamin A-deficient diet this ratio was decreased. These results suggested that an increased amount of RA, resulting from either metabolization of an increased amount of dietary retinol or RA administration, enhanced the translocation of GR from the cytosolic compartment to the nuclear compartment. Moreover such an increased amount of RA could also induce the observed decreased Cmax of the total GR that we observed. These observations were similar to the well known effects of dexamethasone administration on the properties of GR. It is probable that RA, similarly to dexamethasone treatment, induces a dissociation of the tetrameric form of the cytosolic GR and thus enhances translocation of the monomeric form from cytosol to nucleus and also resulting in an increased proteolytic degradation of the receptor.
J Steroid Biochem Mol Biol 1995 Apr
PMID:Retinoids modulate the binding capacity of the glucocorticoid receptor and its translocation from cytosol to nucleus in liver cells. 773 99

A recombinant baculovirus containing the diuretic hormone receptor cDNA from Manduca sexta was constructed. When Spodoptera frugiperda (Sf9) cells were infected with the virus, a time-dependent expression of the receptor appeared. The expressed receptor displayed high affinity for diuretic hormone, which was similar to the affinity observed in Malpighian tubules and transfected COS-7 cells. The receptor expression level was 77 pmol/mg protein, as compared to 3.1 pmol/mg protein in Malpighian tubules and 1.3 pmol/mg protein in transfected COS-7 cells. Chemical crosslinking of 125I-labeled Mas-DH to the expressed receptor revealed a protein of 48-52 kDa. Furthermore, Mas-DH stimulated cAMP synthesis in recombinant baculovirus infected Sf9 cells. The N-terminal truncated analog [13-41] Mas-DH bound to the expressed receptor with high affinity but did not stimulate cAMP synthesis. This suggests that the N-terminal region of Mas-DH is required for receptor activation but not receptor binding. The recombinant baculovirus provides an alternate system to study receptor function and will allow large scale production of receptor for biophysical characterization.
Insect Biochem Mol Biol 1995 Apr
PMID:Functional expression of a diuretic hormone receptor in baculovirus-infected insect cells: evidence suggesting that the N-terminal region of diuretic hormone is associated with receptor activation. 774 37

Although steroid hormone receptor activation has been known to be dependent on ligand binding, we report here ligand-independent transcriptional activation of the vitamin D receptor and retinoid receptors. In these studies, CV1 cells were transiently transfected with a human vitamin D receptor (VDR) expression vector and a reporter plasmid that contains multiple copies of the rat osteocalcin vitamin D response element up-stream of the bacterial chloramphenicol acetyltransferase (CAT) gene [osteocalcin (OC)VDREtkCAT]. Treatment of cells with 10(-8) M 1,25-dihydroxyvitamin D3 resulted in a 25-fold induction of CAT activity. When cells were treated with 5-50 nM okadaic acid (OA), an inhibitor of protein phosphatase-1 and -2A, significant inductions of CAT activity (18- to 57-fold) were observed. As VDR and dopamine receptors are colocalized in certain brain regions, we also examined whether VDR-mediated transcription can be activated by dopamine. VDR was found to activate CAT gene expression in cells treated with 200-500 microM dopamine (3- to 11-fold induction) or the selective D1 agonist SKF38393 (20-fold induction). Cells were also transfected with retinoic acid receptor (RAR) or retinoid-X receptor (RXR) expression vectors and reporter plasmids that contain either a retinoic acid response element or an RXR-specific response element. OA alone induced chloramphenicol acetyltransferase (CAT) activity in cells transfected with RAR alpha, RAR beta, RXR alpha, RXR beta, or RXR gamma (3- to 18-fold induction). However, OA did not affect transcription by RAR gamma, suggesting specificity of activation by OA among the retinoid receptors. Although the retinoid receptors have been detected in brain, maximum stimulation of transcription was not greater than 1.6-fold in the presence of 100-500 microM dopamine or 100 microM SKF38393 treatment. These data suggest specificity for dopamine activation among steroid hormone receptors and that phosphorylation alone, in the absence of ligand, can activate VDR- and retinoid receptor-mediated transcription.
Mol Endocrinol 1995 Feb
PMID:Ligand occupancy is not required for vitamin D receptor and retinoid receptor-mediated transcriptional activation. 777 73


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