PubMed Health⌕ Search

Biomedical subjects

Noriko Shimazaki

Publications and source records attributed to Noriko Shimazaki.

16 recordsLinked to original sources
PubMed2006

Beta-sitosterol-3-O-beta-D-glucopyranoside: a eukaryotic DNA polymerase lambda inhibitor.

Beta-sitosterol-3-O-beta-D-glucopyranoside (compound 1), a steroidal glycoside isolated from onion (Allium cepa L.) selectively inhibited the activity of mammalian DNA polymerase lambda (pol lambda) in vitro. The compound did not influence the activities of replicative DNA polymerases such as alpha, delta and epsilon, but also showed no effect even on the activity of pol beta which is thought to have a very similar three-dimensional structure to the pol beta-like region of pol lambda. Since parts of compound 1 such as beta-sitosterol (compound 2) and D-glucose (compound 3) did not influence the activities of any enzymes tested, the converted structure of compounds 2 and 3 might be important for pol lambda inhibition. The inhibitory effect of compound 1 on both intact pol lambda (i.e. residues 1-575) and a truncated pol lambda lacking the N-terminal BRCA1 C-terminus (BRCT) domain (133-575, del-1 pol lambda) was dose-dependent, and 50% inhibition was observed at a concentration of 9.1 and 5.4 microM, respectively. The compound 1-induced inhibition of del-1 pol lambda activity was non-competitive with respect to both the DNA template-primer and the dNTP substrate. On the basis of these results, the pol lambda inhibitory mechanism of compound 1 is discussed.

Animals↗
PubMed2006

Structural relationship of curcumin derivatives binding to the BRCT domain of human DNA polymerase lambda.

We previously reported that phenolic compounds, petasiphenol and curcumin (diferuloylmethane), were a selective inhibitor of DNA polymerase lambda (pol lambda) in vitro. The purpose of this study was to investigate the molecular structural relationship of curcumin and 13 chemically synthesized derivatives of curcumin. The inhibitory effect on pol lambda (full-length, i.e. intact pol lambda including the BRCA1 C- terminal [BRCT] domain) by some derivatives was stronger than that by curcumin, and monoacetylcurcumin (compound 13) was the strongest pol lambda inhibitor of all the compounds tested, achieving 50% inhibition at a concentration of 3.9 microm. The compound did not influence the activities of replicative pols such as alpha, delta, and epsilon. It had no effect on pol beta activity either, although the three-dimensional structure of pol beta is thought to be highly similar to that of pol lambda. Compound 13 did not inhibit the activity of the C-terminal catalytic domain of pol lambda including the pol beta-like core, in which the BRCT motif was deleted from its N-terminal region. MALDI-TOF MS analysis demonstrated that compound 13 bound selectively to the N-terminal domain of pol lambda, but did not bind to the C-terminal region. Based on these results, the pol lambda-inhibitory mechanism of compound 13 is discussed.

Amino Acid Motifs↗
PubMed2005

5-(Hydroxymethyl)-2-furfural: a selective inhibitor of DNA polymerase lambda and terminal deoxynucleotidyltransferase.

5-(Hydroxymethyl)-2-furfural (HMF), a pyrolysate of carbohydrate isolated from instant coffee (Coffea arabica L.), selectively inhibits the activities of mammalian DNA polymerase lambda (pol lambda) and terminal deoxynucleotidyltransferase (TdT) which are family X pols, in vitro. The compound influenced neither the activities of replicative DNA polymerases such as alpha, delta, and epsilon, nor even the activity of pol beta which is from the same family and thought to have a very similar three-dimensional structure to the pol beta-like region of pol lambda. Since parts of HMF such as furan, furfuryl alcohol, and 2-furaldehyde did not influence the activities of any enzymes tested, the substituted form of furan with a hyroxymethyl group and a formyl group might be important for the inhibition of pol lambda and TdT. The inhibitory effect of HMF on intact pol lambda (i.e., residues 1-575), a truncated pol lambda lacking the N-terminal BRCA1 C-terminus domain (133-575, del-1 pol lambda) and another truncated pol lambda lacking the N-terminal proline-rich region (245-575, del-2 pol lambda) was dose-dependent, and 50% inhibition was observed at a concentration of 26.1, 10.3, and 4.6 microM, respectively. The IC(50) value of HMF for TdT was the same as that for del-2 pol lambda (5.5 microM). The HMF-induced inhibition of both pol lambda and TdT activities was competitive with respect to both the DNA template-primer and the dNTP substrate. On the basis of these results, HMF was suggested to bind to the pol beta-like region of pol lambda and TdT.

Animals↗
PubMed2005

Inhibitory effect of tocotrienol on eukaryotic DNA polymerase lambda and angiogenesis.

Tocotrienols, vitamin E compounds that have an unsaturated side chain with three double bonds, selectively inhibited the activity of mammalian DNA polymerase lambda (pol lambda) in vitro. These compounds did not influence the activities of replicative pols such as alpha, delta, and epsilon, or even the activity of pol beta which is thought to have a very similar three-dimensional structure to the pol beta-like region of pol lambda. Since delta-tocotrienol had the strongest inhibitory effect among the four (alpha- to delta-) tocotrienols, the isomer's structure might be an important factor in the inhibition of pol lambda. The inhibitory effect of delta-tocotrienol on both intact pol lambda (residues 1-575) and a truncated pol lambda lacking the N-terminal BRCA1 C-terminus (BRCT) domain (residues 133-575, del-1 pol lambda) was dose-dependent, with 50% inhibition observed at a concentration of 18.4 and 90.1microM, respectively. However, del-2 pol lambda (residues 245-575) containing the C-terminal pol beta-like region was unaffected. Tocotrienols also inhibited the proliferation of and formation of tubes by bovine aortic endothelial cells, with delta-tocotrienol having the greatest effect. These results indicated that tocotrienols targeted both pol lambda and angiogenesis as anti-cancer agents. The relationship between the inhibition of pol lambda and anti-angiogenesis by delta-tocotrienol was discussed.

Animals↗
PubMed2005

Monoacetylcurcumin: a new inhibitor of eukaryotic DNA polymerase lambda and a new ligand for inhibitor-affinity chromatography.

We previously reported that a phenolic compound, curcumin (diferuloylmethane), was a selective inhibitor of DNA polymerase lambda (pol lambda) in vitro [Y. Mizushina, M. Hirota, C. Murakami, T. Ishidoh, S. Kamisuki, N. Shimazaki, M. Takemura, M. Perpelescu, M. Suzuki, H. Yoshida, F. Sugawara, O. Koiwai, K. Sakaguchi, Some anti-chronic inflammatory compounds are DNA polymerase lambda-specific inhibitors, Biochem. Pharmacol. 66 (2003) 1935-1944.]. We also found that monoacetylcurcumin ([1E,4Z,6E]-7-(4''-acetoxy-3''-methoxyphenyl)-5-hydroxy-1-(4'-hydroxy-3'-methoxyphenyl)hepta-1,4,6-trien-3-on), a chemically synthesized derivative of curcumin, was a stronger pol lambda inhibitor than curcumin, achieving 50% inhibition at a concentration of 3.9microM. Monoacetylcurcumin did not influence the activities of replicative pols such as alpha, delta, and epsilon, and showed no effect even on the activity of pol beta, the three-dimensional structure of which is thought to be highly similar to that of pol lambda. The compound-induced inhibition of pol lambda activity was non-competitive with respect to both the DNA template-primer and the dNTP substrate. Monoacetylcurcumin did not inhibit the activity of the C-terminal catalytic domain of pol lambda including the pol beta-like core, in which the BRCT motif was deleted. The compound did not influence the activities of prokaryotic pols or other mammalian DNA metabolizing enzymes such as calf primase of pol alpha, calf terminal deoxynucleotidyl transferase, human telomerase, human immunodeficiency virus type-1 reverse transcriptase, T7 RNA polymerase, T4 polynucleotide kinase, and bovine deoxyribonuclease I. Therefore, we concluded that monoacetylcurcumin is a selective inhibitor of pol lambda and could be used as a chromatographic ligand to purify pol lambda. We then made a monoacetylcurcumin-conjugated column with epoxy-activated Sepharose 6B. In the column, pol lambda of full length was selectively adsorbed and eluted.

Animals↗
PubMed2005

DNA polymerase lambda directly binds to proliferating cell nuclear antigen through its confined C-terminal region.

DNA polymerase lambda (Pol lambda) was recently identified as a new member of the family X of DNA polymerases. Here, we show that Pol lambda directly binds to proliferating cell nuclear antigen (PCNA), an auxiliary protein for DNA replication and repair enzymes, both in vitro and in vivo. A pull-down assay using deletion mutants of Pol lambda showed that the confined C-terminal region of Pol lambda directly binds to PCNA. Furthermore, a synthetic peptide of 20-mers derived from the C-terminal region of Pol lambda competes with full-length Pol lambda for binding to PCNA. The residues between amino acids 518 and 537 of Pol lambda are required for binding to PCNA, and are different from the consensus PCNA interacting motif (PIM). Pol lambda associates with PCNA in vivo by immunoprecipitation analysis and EGFP-tagged Pol lambda co-localizes with PCNA as spots within a nucleus using fluorescent microscopy. Through direct binding, PCNA suppressed the distributive nucleotidyltransferase activity of Pol lambda. Pol micro, which also belongs to the family X of DNA polymerases, binds to PCNA by a pivotal amino acid residue.

Binding Sites↗
PubMed2005

Structural analysis of epolactaene derivatives as DNA polymerase inhibitors and anti-inflammatory compounds.

Epolactaene (compound 1), a neuritogenic compound found in human neuroblastoma cells, was found to show anti-inflammatory activity in vivo in this study. DNA polymerases and DNA topoisomerase II (topo II) were some of the major molecular targets of compound 1. Since the agent seems to be a potential pharmaceutical medicine, we synthesized derivatives chemically and obtained seven compounds, 1 to 7 to screen clinically more efficient epolactaene derivatives. A comparison of its structural derivatives revealed that the long alkyl side chain seemed to have an important role in the inhibitory effect. Notably, C18-alkyl chain conjugated epolactaene (compound 5) was the strongest inhibitor of DNA polymerase alpha, beta, lambda (pol alpha, beta, lambda) and topo II, with IC50 values of 13, 135, 4.4 and 5 microM, respectively, and 500 microg of compound 5 caused a marked reduction in TPA (12-O-tetradecanoylphorbol-13-acetate)-induced inflammation (inhibitory effect, 65.0%). Compound 5 did not influence the activities of plant or prokaryotic DNA polymerases, or of other DNA metabolic enzymes such as telomerase, RNA polymerase and deoxyribonuclease I. Based on these results, the relationship among the three-dimensional structure of epolactaene derivatives and the inhibition of polymerases and topo II, and anti-inflammation is discussed.

Animals↗
PubMed2004

A biochemically defined system for mammalian nonhomologous DNA end joining.

Nonhomologous end joining (NHEJ) is a major pathway in multicellular eukaryotes for repairing double-strand DNA breaks (DSBs). Here, the NHEJ reactions have been reconstituted in vitro by using purified Ku, DNA-PK(cs), Artemis, and XRCC4:DNA ligase IV proteins to join incompatible ends to yield diverse junctions. Purified DNA polymerase (pol) X family members (pol mu, pol lambda, and TdT, but not pol beta) contribute to junctional additions in ways that are consistent with corresponding data from genetic knockout mice. The pol lambda and pol mu contributions require their BRCT domains and are both physically and functionally dependent on Ku. This indicates a specific biochemical function for Ku in NHEJ at incompatible DNA ends. The XRCC4:DNA ligase IV complex is able to ligate one strand that has only minimal base pairing with the antiparallel strand. This important aspect of the ligation leads to an iterative strand-processing model for the steps of NHEJ.

Animals↗
PubMed2003

Some anti-chronic inflammatory compounds are DNA polymerase lambda-specific inhibitors.

We previously reported that a phenolic compound, petasiphenol, was a selective inhibitor of DNA polymerase lambda (pol lambda) in vitro. We found here that another phenolic compound, curcumin (diferuloylmethane), which is known as an anti-chronic inflammatory agent and is structurally quite similar to petasiphenol, was also a potent pol lambda inhibitor. The IC(50) values of petasiphenol and curcumin were 7.8 and 7.0 microM, respectively. Curcumin, as well as petasiphenol, did not influence the activities of replicative DNA polymerases, such as alpha, gamma, delta, and epsilon, but also showed no effect even on the pol beta activity belonging to the X family. Curcumin could prevent the growth of human NUGC-3 cancer cells with LD(50) values of 13 microM, and halted them at the G2/M phase in the cell cycle, whereas petasiphenol suppressed the cell growth at 66 microM and arrested the cells at the G1 phase. These data showed that curcumin and petasiphenol were slightly different functionally. We also previously reported that novel anti-inflammatory terpeno benzoic acids and triterpenoids were inhibitors of mammalian DNA polymerases. They could also efficiently inhibit the pol lambda activity, although they influenced the other polymerase species to the same extent, suggesting that there may be a physiological relationship between pol lambda inhibition and anti-12-O-tetradecanoylphorbol-13-acetate-induced inflammation. Expectedly, petasiphenol also showed an anti-12-O-tetradecanoylphorbol-13-acetate-induced inflammatory effect in mice. This finding may provide clues to investigating the molecular mechanism of inflammation.

Animals↗
PubMed2003

A sulphoquinovosyl diacylglycerol is a DNA polymerase epsilon inhibitor.

Sulphoquinovosyl diacylglycerol (SQDG) was reported as a selective inhibitor of eukaryotic DNA polymerases alpha and beta [Hanashima, Mizushina, Ohta, Yamazaki, Sugawara and Sakaguchi (2000) Jpn. J. Cancer Res. 91, 1073-1083] and an immunosuppressive agent [Matsumoto, Sahara, Fujita, Shimozawa, Takenouchi, Torigoe, Hanashima, Yamazaki, Takahashi, Sugawara et al. (2002) Transplantation 74, 261-267]. The purpose of this paper is to elucidate the biochemical properties of the inhibition more precisely. As expected, SQDG could inhibit the activities of mammalian DNA polymerases such as alpha, delta, eta and kappa in vitro in the range of 2-5 micro M, and beta and lambda in vitro in the range of 20-45 micro M. However, SQDG could inhibit only mammalian DNA polymerases epsilon (pol epsilon) activity at less than 0.04 micro M. SQDG bound more tightly to mammalian pol epsilon than the other mammalian polymerases tested. Moreover, SQDG could inhibit the activities of all the polymerases from animals such as fish and insect, but not of the polymerases from plant and prokaryotes. SQDG should, therefore, be called a mammalian pol epsilon-specific inhibitor or animal polymerase-specific inhibitor. To our knowledge, this represents the first report about an inhibitor specific to mammalian pol epsilon.

DNA Polymerase II↗
PubMed2003

Terminal deoxynucleotidyltransferase forms a ternary complex with a novel chromatin remodeling protein with 82 kDa and core histone.

BACKGROUND: Terminal deoxynucleotidyltransferase (TdT) is a DNA polymerase that enhances the Ig and TcR gene diversity in the N region at the junctions of variable (V), diversity (D) and joining (J) segments in B- and T-cells. TdT synthesizes the N region in concert with many proteins including DNA-PKcs, Ku70 and Ku86. To elucidate the molecular mechanism of the N region synthesis, we first attempted to isolate the genes with products that directly interact with TdT. RESULTS: Using a yeast two-hybrid system, we isolated a cDNA clone encoding a novel nuclear protein that interacts with TdT. This protein was designated as TdT interacting factor 2 (TdIF2). The confined region of the C-terminal in TdIF2 is involved in specific interaction with the entire C-terminal in TdT. TdIF2 contains an acidic region comprised of 42 residues. TdIF2 was shown to bind specifically to a core histone by pull down assay using specific antibodies against TdIF2. When a TdT/TdIF2 complex was applied on to a DNA-cellulose column, only TdT bound to the column while TdIF2 passed through. TdIF2 reduces the TdT activity to 46% of its maximum value in vitro assay system using activated DNA as primer. CONCLUSIONS: TdIF2 binds directly to TdT and core histone. Furthermore, TdT, TdIF2 and core histone form a ternary complex. TdIF2 liberates H2A/H2B from a core histone in correlation with PCNA. The enzymatic consequence of the TdIF2/TdT complex is the reduction of TdT activity in vitro. TdIF2 would function as a chromatin remodeling protein at the N region synthesis.

Amino Acid Sequence↗
PubMed2003

Molecular action mode of Hippospongic acid A, an inhibitor of gastrulation of starfish embryos.

Hippospongic acid A (HA-A) is a novel natural triterpene metabolite that exhibits inhibitory activity against the gastrulation of starfish embryos isolated from a marine sponge, Hippospongia sp. We succeeded in chemically synthesizing the natural enantiomer and the racemate HA-A. In this study, we examined its action mode in vitro. HA-A was a rare compound that could selectively but uniformly inhibit the activities of all the vertebrate DNA polymerases tested such as alpha, beta, delta, epsilon, eta, kappa, and lambda, in the IC(50) range of 5.9-17.6 microM, and interestingly also those of human DNA topoisomerases I and II (IC(50) = 15-25 microM). HA-A exhibited no inhibitory effect on DNA polymerases from insects, plants and prokaryotes, or on many other DNA metabolic enzymes. HA-A was an inhibitor specific to DNA polymerases and DNA topoisomerases from vertebrates, but not selective as to a subclass species among the enzymes. Since DNA polymerase beta is the smallest, we used it to analyze the biochemical relationship with HA-A. Biochemical, BIAcore and computer modeling analyses demonstrated that HA-A bound selectively to the N-terminal 8 kDa DNA template-binding domain of DNA polymerase beta, and HA-A inhibited the ssDNA binding activity. HA-A could prevent the growth of NUGC-3 cancer cells at both the G1 and G2/M phases, and induce apoptosis in the cells. The LD(50) value was 9.5 microM, i.e. in the same range as for the enzyme inhibition. Therefore, we concluded that one molecular basis of the gastrulation of starfish embryos is a process that requires DNA polymerases and DNA topoisomerases, and subsequently the gastrulation was inhibited by HA-A. We also discussed the in vivo role of HA-A.

Animals↗
PubMed2002

Petasiphenol: a DNA polymerase lambda inhibitor.

Petasiphenol, a bio-antimutagen isolated from a Japanese vegetable, Petasites japonicus, selectively inhibits the activities of mammalian DNA polymerase lambda (pol lambda) in vitro. The compound did not influence the activities of replicative DNA polymerases such as alpha, delta, and epsilon but also showed no effect even on the pol beta activity, the three-dimensional structure of which is thought to be highly similar to pol lambda. The inhibitory effect of petasiphenol on intact pol lambda including the BRCA1 C-terminus (BRCT) domain was dose-dependent, and 50% inhibition was observed at a concentration of 7.8 microM. The petasiphenol-induced inhibition of the pol lambda activity was noncompetitive with respect to both the DNA template-primer and the dNTP substrate. Petasiphenol did not only inhibit the activity of the truncated pol lambda including the pol beta-like core, in which the BRCT motif was deleted in its N-terminal region. BIAcore analysis demonstrated that petasiphenol bound selectively to the N-terminal domain of pol lambda but did not bind to the C-terminal region. On the basis of these results, the pol lambda inhibitory mechanism of petasiphenol is discussed.

Amino Acid Sequence↗
PubMed2002

Over-expression of human DNA polymerase lambda in E. coli and characterization of the recombinant enzyme.

BACKGROUND: DNA polymerase lambda (Pol lambda) was recently identified as a new member of the family X of DNA polymerases in eukaryotic cells. Pol lambda contains a nuclear localization signal (NLS), a BRCA1-C terminal (BRCT) domain, a proline-rich region, helix-hairpin-helix (HhH) and pol X motifs. Since the amino acid sequence for Pol lambda shares a high degree of homology to Pol beta, Pol lambda is considered to have a similar enzymatic nature to Pol beta. RESULTS: Recombinant human Pol lambda was shown to possess template-directed DNA polymerase activity in its monomeric form. Pol lambda required either Mn2+ or Mg2+ as a metal co-factor to catalyse this activity, and optimal activity was detected at pH 8.5-9.0. Pol lambda was insensitive to aphidicolin, but was sensitive to dideoxynucleoside triphosphates or N-ethylmaleimide. By constructing the truncated Pol lambda, the proline rich region was shown to act in a suppression of its polymerization activity. A chimeric enzyme comprised of the Pol lambda N-terminal region and Pol beta also showed a reduced Pol beta activity. Proliferating cell nuclear antigen (PCNA) directly interacts with Pol lambda through its Pol beta like region in vitro. CONCLUSIONS: Pol lambda possesses similar enzymatic nature to Pol beta; requirements of cations and optimal conditions for pH and NaCl concentration, aside from sensitivity to N-ethylmaleimide and template preference. The proline rich region of Pol lambda functions as a suppressor domain for its polymerization activity (SDPA). Pol lambda interacts directly with PCNA through its Pol beta like region. The functional consequence of this interaction is the negative regulation of Pol lambda activity.

Binding Sites↗
PubMed2001

A plant phytotoxin, solanapyrone A, is an inhibitor of DNA polymerase beta and lambda.

Solanapyrone A, a phytotoxin and enzyme inhibitor isolated from a fungus (SUT 01B1-2) selectively inhibits the activities of mammalian DNA polymerase beta and lambda (pol beta and lambda) in vitro. The IC50 values of the compound were 30 microm for pol beta and 37 microm for pol lambda. Because pol beta and lambda are in a family and their three-dimensional structures are thought to be highly similar to each other, we used pol beta to analyze the biochemical relationship with solanapyrone A. On pol beta, solanapyrone A antagonistically competed with both the DNA template and the nucleotide substrate. BIAcore analysis demonstrated that solanapyrone A bound selectively to the N-terminal 8-kDa domain of pol beta. This domain is known to bind single-stranded DNA, provide 5'-phosphate recognition of gapped DNA, and cleave the sugar-phosphate bond 3' to an intact apurinic/apyrimidinic (AP) site (i.e. AP lyase activity) including 5'-deoxyribose phosphate lyase activity. Solanapyrone A inhibited the single-stranded DNA-binding activity but did not influence the activities of the 5'-phosphate recognition in gapped DNA structures and the AP lyase. Based on these results, the inhibitory mechanism of solanapyrone A is discussed.

DNA↗