Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.1.21 (thymidine kinase)
7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In vitro culture of H9 human lymphoid cells in the presence of 5.0 microM dideoxycytidine (ddC), for about 40-45 days, selected cells (H9-ddC cells), which were resistant to the drug and cross-resistant to AZT (zidovudine) and 5-fluoro-2'-deoxyuridine (FdUR). The major mechanism of cross-resistance to AZT and FdUR in these cells was low cellular activity of thymidine kinase (TK). To explore molecular mechanisms of the reduced TK activity in H9-ddC cells, the mRNA expression of TK1 and TK2 and western blot analysis of TK1 protein were performed. RT-PCR analysis revealed that in H9-ddC cells the expression of both TK1 and TK2 mRNA was reduced to 27.1% and 79.4%, respectively. The reduced TK1 gene expression was confirmed by an absence of a detectable TK1 protein band in western blot of H9-ddC cells. These results demonstrate that long-term treatment of H9 cells in the presence of ddC down-regulated TK1 and TK2 gene expression and reduced the expression and activity of TK in the resistant cells.
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PMID:2', 3'-Dideoxycytidine represses thymidine kinases 1 and 2 expression in T-lymphoid cells. 1465 72

Human cytosolic thymidine kinase (TK1) is tightly regulated in the cell cycle by multiple mechanisms. Our laboratory has previously shown that in mitotic-arrested cells human TK1 is phosphorylated at serine-13, accompanied by a decrease in catalytic efficiency. In this study we investigated whether serine-13 phosphorylation regulated TK1 activity and found that substitution of serine-13 with aspartic acid (S13D), which mimics phosphorylation, not only diminished the ATP-activating effect on the enzyme, but also decreased its thymidine substrate affinity. Our experimental results further showed that the S13D mutation perturbed ATP-induced tetramerization of TK1. Given that the dimeric form of TK1 is less active than the tetrameric, we propose that mitotic phosphorylation of serine-13 is of physiological importance, in that it may counteract ATP-dependent activation of TK1 by affecting its quaternary structure, thus attenuating its enzymatic function at the G2/M phase.
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PMID:Perturbation of ATP-induced tetramerization of human cytosolic thymidine kinase by substitution of serine-13 with aspartic acid at the mitotic phosphorylation site. 1469 31

Several thymidine analogues substituted with closo- and nido-carborane at the N-3 position were synthesized. The nido-carboranyl thymidine analogues were designed to be effective substrates for human thymidine kinase 1 in combination with an increased water solubility sufficient for clinical application in boron neutron capture therapy. This was done because N-3 substituted closo-carboranyl thymidine analogues previously synthesized in our laboratories were good TK1 substrates but were poorly water-soluble. Newly synthesized zwitterionic amino nido- and the corresponding neutral closo-m-carboranyl thymidine analogues exhibited excellent TK1 phosphorylation rates up to 75% relative to thymidine, indicating that these compounds were good substrates for thymidine kinase 1. Thin layer chromatographic studies were indicative of increased hydrophilicity of the synthesized nido-carboranyl thymidine analogues compared with their closo-carboranyl counterparts and previously reported closo-carboranyl thymidine analogues.
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PMID:The synthesis and biochemical evaluation of thymidine analogues substituted with nido carborane at the N-3 position. 1530 3

Cytosolic thymidine kinase 1, TK1, is a well known cell-cycle-regulated enzyme of importance in nucleotide metabolism as well as an activator of antiviral and anticancer drugs such as 3'-azido-3'-deoxythymidine (AZT). We have now determined the structures of the TK1 family, the human and Ureaplasma urealyticum enzymes, in complex with the feedback inhibitor dTTP. The TK1s have a tetrameric structure in which each subunit contains an alpha/beta-domain that is similar to ATPase domains of members of the RecA structural family and a domain containing a structural zinc. The zinc ion connects beta-structures at the root of a beta-ribbon that forms a stem that widens to a lasso-type loop. The thymidine of dTTP is hydrogen-bonded to main-chain atoms predominantly coming from the lasso loop. This binding is in contrast to other deoxyribonucleoside kinases where specific interactions occur with side chains. The TK1 structure differs fundamentally from the structures of the other deoxyribonucleoside kinases, indicating a different evolutionary origin.
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PMID:Structures of thymidine kinase 1 of human and mycoplasmic origin. 1561 77

The thymidine mimics isocarbostyril nucleosides and difluorophenyl nucleosides were tested as deoxynucleoside kinase substrates using recombinant human cytosolic thymidine kinase (TK1) and deoxycytidine kinase (dCK), and mitochondrial thymidine kinase (TK2) and deoxyguanosine kinase (dGK). The isocarbostyril nucleoside compound 1-(2-deoxy-beta-D-ribofuranosyl)-isocarbostyril (EN1) was a poor substrate with all the enzymes. The phosphorylation rates of EN1 with TK1 and TK2 were <1% relative to Thd, where as the phosphorylation rates for EN1 were 1.4% and 1.1% with dCK and dGK relative to dCyd and dGuo, respectively. The analogue 1-(2-deoxy-beta-D-ribofuranosyl)-7-iodoisocarbostyril (EN2) showed poor relative-phosphorylation efficiencies (kcat/Km) with both TK1 and dGK, but not with TK2. The kcat/Km value for EN2 with TK2 was 12.6% relative to that for Thd. Of the difluorophenyl nucleosides, 5-(1'-(2'-deoxy-beta-D-ribofuranosyl))-2,4-difluorotoluene (JW1) and 1-(1'-(2'-deoxy-beta-D-ribofuranosyl))-2,4-difluoro-5-iodobenzene (JW2) were substrates for TK1 with phosphorylation efficiencies of about 5% relative to that for Thd. Both analogues were considerably more efficient substrates for TK2, with kcat/Km values of 45% relative to that for Thd. 2,5-Difluoro-4-[1-(2-deoxy-beta-L-ribofuranosyl)]-aniline (JW5), a L-nucleoside mimic, was phosphorylated up to 15% as efficiently as deoxycytidine by dCK. These data provide a possible explanation for the previously reported lack of cytotoxicity of the isocarbostyril- and difluorophenyl nucleosides, but potential mitochondrial effects of EN2, JW1 and JW2 should be further investigated.
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PMID:Phosphorylation of isocarbostyril- and difluorophenyl-nucleoside thymidine mimics by the human deoxynucleoside kinases. 1562 45

Probing of a cDNA expression library from multicellular development of Dictyostelium discoideum using a recombinant radiolabelled calmodulin probe (35S-VU1-CaM) led to the isolation of a cDNA encoding a putative CaM-binding protein (CaMBP). The cDNA contained an open reading frame of 951 bp encoding a 227aa polypeptide (25.5 kDa). Sequence comparisons led to highly significant matches with cytosolic thymidine kinases (TK1; EC 2.7.1.21) from a diverse number of species including humans (7e-56; 59% Identities; 75% Positives) indicating that the encoded protein is D. discoideum TK1 (DdTK1; ThyB). DdTK1 has not been previously characterized in this organism. In keeping with its sequence similarity with DdTK1, antibodies against humanTK1 recognize DdTK1, which is expressed during growth but decreases in amount after starvation. A CaM-binding domain (CaMBD; 20GKTTELIRRIKRFNFANKKC30) was identified and wild type DdTK1 plus two constructs (DdTK deltaC36, DdTK deltaC75) possessing the domain were shown to bind CaM in vitro but only in the presence of calcium while a construct (DdTK deltaN72) lacking the region failed to bind to CaM. Thus, DdTK1 is a Ca2+-dependent CaMBP. Sequence alignments against TK1 from vertebrates to viruses show that CaM-binding region is highly conserved. The identified CaMBD overlaps the ATP-binding (P-loop) domain suggesting CaM might affect the activity of this kinase. Recombinant DdTK is enzymatically active and showed stimulation by CaM (113+/-0.5%) an in vitro enhancement that was prevented by co-addition of the CaM antagonists W7 (91.2+/-0.8%) and W13 (96.6+/-0.6%). The discovery that TK1 from D. discoideum, and possibly other species including humans and a large number of human viruses, is a Ca2+-dependent CaMBP opens up new avenues for research on this medically relevant protein.
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PMID:Isolation and characterization of Dictyostelium thymidine kinase 1 as a calmodulin-binding protein. 1588 42

Activity of thymidine kinase 1 in serum (STK) is a useful marker for leukaemia and lymphoma, but not for solid tumors. We investigate thymidine kinase 1 concentration in serum (S-TK1) as a potential tumor marker. S-TK1 concentration and STK activity levels were determined in 9 human malignant diseases (breast, gastric, rectal, colorectal, lung, brain cancer, hepatoma, lymphoma, leukaemia) and in benign and non-cancerous diseases, representing 850 preoperative cases. Healthy volunteers (n=43) were used as positive controls. S-TK1 concentration was determined by ECL dot blot assay and STK activity levels by an RIA assay. S-TK1 concentrations and STK activity levels in preoperative malignant patients were significantly higher than in healthy individuals, in patients with benign tumors and in those with non-cancerous diseases. Significant correlations between concentration and activity level were only found in healthy individuals, in patients with benign tumors, and in some patients with malignancies, i.e. leukaemia, and breast and gastric cancers. About 90-95 percent of the malignant patients showed S-TK1 concentrations above those of the healthy controls. The corresponding value for STK activity was about 75 percent. When sera from malignant patients were diluted with sera from healthy individuals, S-TK1 concentrations and STK activity levels decreased more than expected. This indicates the presence of a compound (or compounds) in the serum of healthy individuals that destabilises S-TK1. We conclude that S-TK1 concentration is a more sensitive tumor marker in solid malignancies than is STK activity.
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PMID:Concentration of thymidine kinase 1 in serum (S-TK1) is a more sensitive proliferation marker in human solid tumors than its activity. 1614 66

Thymidine kinases have been found in most organisms, from viruses and bacteria to mammals. Ureaplasma urealyticum (parvum), which belongs to the class of cell-wall-lacking Mollicutes, has no de novo synthesis of DNA precursors and therefore has to rely on the salvage pathway. Thus, thymidine kinase (Uu-TK) is the key enzyme in dTTP synthesis. Recently the 3D structure of Uu-TK was determined in a feedback inhibitor complex, demonstrating that a lasso-like loop binds the thymidine moiety of the feedback inhibitor by hydrogen bonding to main-chain atoms. Here the structure with the substrate deoxythymidine is presented. The substrate binds similarly to the deoxythymidine part of the feedback inhibitor, and the lasso-like loop binds the base and deoxyribose moieties as in the complex determined previously. The catalytic base, Glu97, has a different position in the substrate complex from that in the complex with the feedback inhibitor, having moved in closer to the 5'-OH of the substrate to form a hydrogen bond. The phosphorylation of and inhibition by several nucleoside analogues were investigated and are discussed in the light of the substrate binding pocket, in comparison with human TK1. Kinetic differences between Uu-TK and human TK1 were observed that may be explained by structural differences. The tight interaction with the substrate allows minor substitutions at the 3 and 5 positions of the base, only fluorine substitutions at the 2'-Ara position, but larger substitutions at the 3' position of the deoxyribose.
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PMID:Structure of the substrate complex of thymidine kinase from Ureaplasma urealyticum and investigations of possible drug targets for the enzyme. 1633 73

Human cytosolic thymidine kinase (hTK1) is the key enzyme of the pyrimidine salvage pathway and phosphorylates thymidine to thymidine monophosphate, a precursor building block of the DNA. Wild-type hTK1 (hTK1W) as well as a truncated form of the enzyme (hTK1M) carrying deletions at the N- and C-terminal regions were cloned as His(6)-tagged fusion proteins. Expression, isolation, and purification protocols have been established, leading to high yields of soluble and active wild type (approximately 35 mg) and truncated hTK1 (approximately 23 mg) per liter of culture. The protein was purified to near homogeneity. The chaperone DnaK was identified to be the major contaminant that could be removed by applying an additional ATP-MgCl(2) incubation and washing step. hTK1W was a permanent tetramer in solution, whereas the truncated construct hTK1M appears to be a dimer in absence and presence of substrates. Both hTK1W and hTK1M exhibit pronounced thermal stability with transition temperatures (T(m)) of 71.7 and 73.4 degrees C, respectively, when measured without adding substrates. The presence of substrates stabilized both hTK1W (DeltaT(m) ranging from 5.6 to 12.5 degrees C) and hTK1M (DeltaT(m) ranging from 0.8 to 5.3 degrees C). Both enzymes show high activity over a broad range of pH, temperature, and ionic strength. Kinetic studies determined a K(M) of 0.51 microM and a k(cat) of 0.28 s(-1) for wild-type hTK1. The truncated hTK1M has a K(M) of 0.87 microM and k(cat) of 1.65 s(-1), thus exhibiting increased catalytic efficiency. The availability of recombinant human TK1 will facilitate further biochemical and crystallographic studies.
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PMID:High-level expression and purification of human thymidine kinase 1: quaternary structure, stability, and kinetics. 1647 25

Toxicity induced by nitric oxide (NO(*)) has been extensively investigated in many in vitro and in vivo experimental models. Recently, our laboratories found that both concentration and cumulative total dose are critical determinants of cell death caused by NO(*). Here, we report results of studies designed to define total dose thresholds and threshold effects for several NO(*)-induced toxicity and cellular responses and to determine impacts of p53 on them. We exposed human lymphoblastoid TK6 cells harboring wild-type p53 and isogenic p53-null NH32 cells to NO(*) delivered by a membrane delivery system. Cells were exposed at a steady state concentration of 0.6 microM for varying lengths of time to deliver increasing cumulative doses (expressed in units of microM min), and several end points of cytotoxicity and mutagenesis were quantified. Threshold doses for NO(*)-induced cytotoxicity were 150 microM min in TK6 cells and 300 microM min in NH32 cells, respectively. Threshold doses for NO(*)-induced apoptosis were identical to those for cytotoxicity, but mitochondrial depolarization thresholds were lower than those for cytotoxicity and apoptosis in both cell types. To gain insight into underlying mechanisms, cells of both types were exposed to sublethal (33% of cytotoxicity threshold), cytotoxicity threshold, or toxic (twice the cytotoxicity threshold) doses of NO(*). In TK6 cells (p53), the sublethal threshold dose induced DNA double-strand breaks, but nucleobase deamination products (xanthine, hypoxanthine, and uracil) in DNA were increased only modestly (<50%) by toxic doses. Increased mutant fraction at the thymidine kinase gene (TK1) locus was observed only at the toxic dose of NO(*). Treatment of NH32 cells with NO(*) at the threshold or toxic dose elevated mutagenesis of the TK1 gene, but did not cause detectable levels of DNA double-strand breaks. At similar levels of cell viability, the frequency of DNA recombinational repair was higher in p53-null NH32 cells than in wild-type TK6 cells. NO(*) treatment induced p53-independent cell cycle arrest predominately at the S phase. Akt signaling pathway and antioxidant proteins were involved in the modulation of toxic responses of NO(*). These findings indicate that exposure to doses of NO(*) at or above the cytotoxicity threshold dose induces DNA double-strand breaks, mutagenesis, and protective cellular responses to NO(*) damage. Furthermore, recombinational repair of DNA may contribute to resistance to NO(*) toxicity and potentially increase the risk of mutagenesis. The p53 plays a central role in these responses in human lymphoblastoid cells.
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PMID:Threshold effects of nitric oxide-induced toxicity and cellular responses in wild-type and p53-null human lymphoblastoid cells. 1654 44


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