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Query: UNIPROT:P06889 (Mol)
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Ribonucleotide reductase activity is required for generating deoxyribonucleotides for DNA replication. Schizosaccharomyces pombe cells lacking ribonucleotide reductase activity arrest during S phase of the cell cycle. In a screen for hydroxyurea-sensitive mutants in S. pombe, we have identified a gene, liz1(+), which when mutated reveals an additional, previously undescribed role for ribonucleotide reductase activity during mitosis. Inactivation of ribonucleotide reductase, by either hydroxyurea or a cdc22-M45 mutation, causes liz1(-) cells in G2 to undergo an aberrant mitosis, resulting in chromosome missegregation and late mitotic arrest. liz1(+) encodes a 514-amino acid protein with strong similarity to a family of transmembrane transporters, and localizes to the plasma membrane of the cell. These results reveal an unexpected G2/M function of ribonucleotide reductase and establish that defects in a transmembrane protein can affect cell cycle progression.
Mol Biol Cell 1999 Feb
PMID:Liz1p, a novel fission yeast membrane protein, is required for normal cell division when ribonucleotide reductase is inhibited. 995 Jun 74

In an effort to understand biochemical features that are important to the selective antitumor activity of 2-chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine [Cl-F( upward arrow)-dAdo], we evaluated the biochemical pharmacology of three structurally similar compounds that have quite different antitumor activities. Cl-F( upward arrow)-dAdo was 50-fold more potent as an inhibitor of CEM cell growth than were either 2-chloro-9-(2-deoxy-2-fluoro-beta-D-ribofuranosyl)adenine [Cl-F( downward arrow)-dAdo] or 2-chloro-9-(2-deoxy-2, 2-difluoro-beta-D-ribofuranosyl)adenine [Cl-diF( upward arrow downward arrow)-dAdo]. The compounds were similar as substrates of deoxycytidine kinase. Similar amounts of their respective triphosphates accumulated in CEM cells, and the rate of disappearance of these metabolites was also similar. Cl-F( upward arrow)-dAdo was 10- to 30-fold more potent in its ability to inhibit the incorporation of cytidine into deoxycytidine nucleotides than either Cl-F( downward arrow)-dAdo or Cl-diF( upward arrow downward arrow)-dAdo, respectively, which indicated that ribonucleotide reductase was differentially inhibited by these three compounds. Thus, the differences in the cytotoxicity of these agents toward CEM cells were not related to quantitative differences in the phosphorylation of these agents to active forms but can mostly be accounted for by differences in the inhibition of ribonucleotide reductase activity. Furthermore, the inhibition of RNA and protein synthesis by Cl-F( downward arrow)-dAdo and Cl-diF( upward arrow downward arrow)-dAdo at concentrations similar to those required for the inhibition of DNA synthesis can help explain the poor antitumor selectivity of these two agents because all cells require RNA and protein synthesis.
Mol Pharmacol 1999 Mar
PMID:Comparison of the mechanism of cytotoxicity of 2-chloro-9-(2-deoxy-2- fluoro-beta-D-arabinofuranosyl)adenine, 2-chloro-9-(2-deoxy-2-fluoro- beta-D-ribofuranosyl)adenine, and 2-chloro-9-(2-deoxy-2,2-difluoro- beta-D-ribofuranosyl)adenine in CEM cells. 1005 35

Pseudomonas stutzeri is a facultative anaerobic bacterium with the capability of denitrification. In searching for regulators that control the expression of this trait in response to oxygen withdrawal, we have found an unprecedented multiplicity of four genes encoding transcription factors of the FNR family. The fnrA gene encodes a genuine FNR-type regulator, which is expressed constitutively and controls the cytochrome cbb3-type terminal oxidase (the cco operon), cytochrome c peroxidase (the ccp gene) and the oxygen-independent coproporphyrinogen III oxidase (the hemN gene), in addition to its previously demonstrated role in arginine catabolism (the arc operon). The fnr homologues dnrD, dnrE and dnrS encode regulators of a new subgroup within the FNR family. Their main distinctive feature is the lack of cysteine residues for complexing the [4Fe-4S] centre of redox-active FNR-type regulators. However, they form a phylogenetic lineage separate from the FixK branch of FNR proteins, which also lack this cysteine signature. We have studied the expression of the dnr genes under aerobic, oxygen-limited and denitrifying conditions. DnrD is a key regulator of denitrification by selective activation of the genes for cytochrome cd1 nitrite reductase and NO reductase. The dnrD gene is part of the 30 kb region carrying denitrification genes of P. stutzeri. Transcription of dnrD was activated in O2-limited cells and particularly strongly in denitrifying cells, but was not under the control of FnrA. In response to denitrifying growth conditions, dnrD was transcribed as part of an operon together with genes downstream and upstream of dnrD. dnrS was found about 9 kb upstream of dnrD, next to the nrdD gene for anaerobic ribonucleotide reductase. The transcription of dnrS required FnrA in O2-limited cells. Mutation of dnrS affected nrdD and the expression of ferredoxin I as an element of the oxidative stress response. The dnrE gene is part of the nar region encoding functions for respiratory nitrate reduction. We found the highest amount of dnrE transcripts in aerobically nitrate-challenged cells. The gene was transcribed from two promoters, P1 and P2, of which promoter P1 was under the control of the nitrate response regulator NarL. The multiplicity of FNR factors in P. stutzeri underlines the versatility of the FNR scaffold to serve for transcriptional regulation directed at anaerobic or nitrate-activated metabolic processes.
Mol Microbiol 1999 Mar
PMID:Multiple transcription factors of the FNR family in denitrifying Pseudomonas stutzeri: characterization of four fnr-like genes, regulatory responses and cognate metabolic processes. 1020 42

A number of enzymes use an amino acid free radical cofactor. Tyrosyl and tryptophanyl radicals react with nitric oxide (NO) with an almost diffusion-limited rate. The catalytically competent tyrosyl radical in ribonucleotide reductase (RR) and prostaglandin H synthase (PGHS) recombines with NO in a radical-radical reaction. The unstable adduct formed can dissociate to regenerate the tyrosyl radical. However, upon prolonged incubation with NO, the diiron center of mouse RR leaks out, while the adduct is successively oxidized into an iminoxyl radical and a nitrotyrosine in PGHS. These data provide a plausible mechanism for the physiological inactivation of RR observed in various models, and may help in understanding the inhibition of PGHS reported in some cases. Reversible combination with NO is an intrinsic property of tyrosyl radicals, which also occurs with Y(D) and Y(Z) in photosystem II, where NO has been useful in the analysis of the oxygen-evolving complex.
Cell Mol Life Sci 1999 Jul
PMID:Nitric oxide: a radical molecule in quest of free radicals in proteins. 1048 63

The reduction equivalents necessary for the ribonucleotide reductase (RNR)-catalyzed production of deoxyribonucleotides are provided by glutaredoxin (Grx) or thioredoxin (Trx). The initial location for transfer of reducing equivalents to RNR is located at the C terminus of the B1 subunit and involves the reduction of a disulfide between Cys754 and Cys759. We have used a 25-mer peptide corresponding to residues 737-761 of RNR B1 (C754-->S) to synthesize a stable mixed disulfide with Escherichia coli Grx-1 (C14-->S) resembling the structure of an intermediate in the reaction. The high-resolution solution structure of the mixed disulfide has been obtained by NMR with an RMSD of 0.56 A for all the backbone atoms of the protein and the well-defined portion of the peptide. The binding interactions responsible for specificity have been identified demonstrating the importance of electrostatic interactions in this system and providing a rationale for the specificity of the Grx-RNR interaction. The disulfide is buried in this complex, implying a solely intra-molecular mechanism of reduction in contrast to the previously determined structure of the glutathione complex where the disulfide was exposed; mutagenesis studies have shown the relevance of intermolecular reduction processes. Substantial conformational changes in the helices of the protein are associated with peptide binding which have significant mechanistic implications for protein disulfide reduction by glutaredoxins.
J Mol Biol 1999 Sep 10
PMID:Binding specificity and mechanistic insight into glutaredoxin-catalyzed protein disulfide reduction. 1049 64

We raised a specific antiserum against the recombinant M2 subunit protein of ribonucleotide reductase of Leishmania mexicana amazonensis in rabbit. This antiserum was used to study the expression and cellular location of the M2 protein in wildtype as well as hydroxyurea-resistant variants (HuR) of the parasite. The protein increased with increasing dose of the drug used for selection of resistance. The increase in protein level was accompanied by an increase in the copy numbers of mRNA of the M2 gene in the variants. In contrast to mammalian cells, the M2 protein of Leishmania is located in the nucleus rather than in the cytoplasm. The number of cells expressing M2 protein is also different in mammalian cells versus Leishmania. In mammalian cells, expression of M2 protein is a strictly S-phase-correlated event and in exponentially growing cells only approximately 50% of the cells are in S-phase and only these cells synthesize M2 protein. In L. m. amazonensis, however, almost all exponentially growing cells are positive for M2 protein. This makes it unlikely that M2 protein expression in Leishmania is S-phase dependent. In view of these findings, a fresh look in the future into the regulatory mechanisms of synthesis and the site of action of RNR in L. m. amazonensis is warranted.
Mol Biochem Parasitol 1999 Aug 20
PMID:Expression and cellular localization of ribonucleotide reductase small subunit M2 protein in hydroxyurea-resistant Leishmania mexicana amazonensis. 1049 82

Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides; this step is rate-limiting in DNA precursor synthesis. A number of regulatory mechanisms ensure optimal deoxyribonucleotide pools, which are essential for cell viability. The best studied mechanisms are transcriptional regulation of the RNR genes during the cell cycle and in the response to DNA damage, and the allosteric regulation of ribonucleotide reductase by nucleoside triphosphates. Recently, another mode of RNR regulation has been hypothesized in yeast. A novel protein, Sml1, was shown to bind to the Rnr1 protein of the yeast ribonucleotide reductase; this interaction was proposed to inhibit ribonucleotide reductase activity when DNA synthesis is not required (Zhao, X., Muller, E.G.D., and Rothstein, R. (1998) Mol. Cell 2, 329-340). Here, we use highly purified recombinant proteins to directly demonstrate that the Sml1 protein is a strong inhibitor of yeast RNR. The Sml1p specifically binds to the yeast Rnr1p in a 1:1 ratio with a dissociation constant of 0.4 microM. Interestingly, Sml1p also specifically binds to the mouse ribonucleotide reductase R1 protein. However, the inhibition observed in an in vitro mouse ribonucleotide reductase assay is less pronounced than the inhibition in yeast and probably occurs via a different mechanism.
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PMID:Yeast Sml1, a protein inhibitor of ribonucleotide reductase. 1059 72

Thioredoxin is a small multifunctional protein which acts as a dithiol hydrogen donor for ribonucleotide reductase in DNA synthesis. Thioredoxin participates in the regulation of different metabolic processes, such as changes in the activity of different enzymes, receptors or transcription factors. The aim of the present study was to determine possible differences in the expression of thioredoxin between myometrium and fibroids in women during different periods of life. Thioredoxin mRNA concentrations were determined in myometrial and fibroid tissues obtained from women during the menstrual cycle, during treatment with an analogue of gonadotrophin releasing hormone (GnRH agonist), in the postmenopausal period (PMP) and during pregnancy. The concentration of thioredoxin mRNA was measured by a solution hybridization method. The localization of thioredoxin protein was examined by immunohistochemistry. There were significantly lower levels of thioredoxin expression in both fibroids and myometrium from GnRH agonist treated and PMP women in comparison with the pregnant women. No difference in thioredoxin expression was found between myometrium and fibroids from the same woman or between myometria from uteri with or without fibroids in the same patient group. Thioredoxin expression in uterine fibroids does not seem to be up-regulated, but changes in response to the endocrine conditions in a similar way to that observed in the myometrium.
Mol Hum Reprod 2000 Jan
PMID:Thioredoxin expression in human myometrium and fibroids. 1061 Dec 62

An osmotic-remedial, temperature-sensitive conditional mutant (un-24) was generated by Repeat Induced Point mutation (RIP) from a cross between a wild-type N. crassa strain and a strain carrying a approximately 250-kb duplication of the left arm of linkage group II (LGII). The mutation was mapped to the duplicated segment, within 2.6 map units of the heterokaryon incompatibility locus het-6. DNA transformation identified a 3.75-kb fragment that complemented the temperature-sensitive phenotype. A large ORF within this fragment was found to have a high degree of sequence identity to the large subunit of ribonucleotide reductase (RNR) from diverse organisms. Conserved amino acids at the active site and the allosteric activity sites are also evident. An unusual feature of the Neurospora sequence is a large insertion near the C-terminus relative to otherwise homologous sequences from other organisms. Three transition mutations, indicative of RIP, were identified in the N-terminal region of the temperature-sensitive mutant allele. One of these mutations results in a non-conservative amino acid substitution within the four-helix bundle that is important in the allosteric control of ribonucleotide reductase activity. This substitution appears to disrupt proper folding of the allosteric activity site during synthesis of the protein.
Mol Gen Genet 2000 Jan
PMID:An osmotic-remedial, temperature-sensitive mutation in the allosteric activity site of ribonucleotide reductase in Neurospora crassa. 1066 63

Thioltransferase (TTase), also known as glutaredoxin (Grx), is an enzyme that catalyzes the reduction of a variety of disulfide compounds, including protein disulfides, in the presence of reduced glutathione. TTase acts as a cofactor for various enzymes such as ribonucleotide reductase. We previously purified a TTase from Schizosaccharomyces pombe and its molecular size was determined. In the present study, a cDNA coding TTase was isolated from a cDNA library of Schizosaccharomyces pombe by colony hybridization, which was constructed in a plasmid vector pGAD GH, and its corresponding insert was confirmed by Southern hybridization. The nucleotide sequence of the 375 bp long cDNA clone reveals an open reading frame, which encodes a protein of 101 amino acids. The coding region of the original clone was transferred after the lac promoter of pUC13 vector for expression in E. coli, and simultaneously, a suitable Shine-Dalgarno (SD) sequence was added in front of the coding region by PCR. The two primers used for PCR also separately contained BamHI and HindIII restriction sites. The E. coli strain (A434) harboring the pUC13 derivative pKU10 showed a 17.3-fold increase in TTase activity compared to the strain with only the vector plasmid.
Mol Cells 1999 Dec 31
PMID:Cloning, nucleotide sequence and expression of thioltransferase (glutaredoxin) cDNA from Schizosaccharomyces pombe. 1067 36


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