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Kengo Sakaguchi

Publications and source records attributed to Kengo Sakaguchi.

At least 19 recordsLinked to original sources
PubMed2007

Sulfoglycolipids as candidate antiangiogenic radiosensitizers.

Angiogenesis is considered an essential process for the growth of solid tumors and, accordingly, angiogenesis has been a focus of attention for cancer therapy. Although various antiangiogenic agents have been developed, adverse effects and limitations associated with antitumor therapies have recently become apparent. To overcome these problems, combining such agents with chemotherapy or radiotherapy is now strongly recommended in clinical practice. Provided such combination treatment, from the onset of therapy, different strategies in developing antiangiogenic agents should be used to enhance any combinatory effects and reduce adverse effects. By applying the concept of radiosensitizers, a new class of antiangiogenic treatments should now be possible. We recently developed sulfoglycolipids that possess such properties. In this review, we discuss the properties of antiangiogenic radiosensitizers and their potential usefulness.

Angiogenesis Inhibitors↗
PubMed2007

Structure-activity relationship of a glycolipid, sulfoquinovosyl diacylglycerol, with the DNA binding activity of p53.

The in vitro relationship between the human p53 DNA binding domain (p53 DBD) and glycolipids was investigated. We isolated the glycolipid fraction from spinach (Spinacia oleracea L.) and found that the fraction inhibited the double-stranded DNA (dsDNA) binding activity of p53 DBD. Since the fraction contained mainly three glycolipids, monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG) and sulfoquinovosyl diacylglycerol (SQDG), and each glycolipid was purified using silica gel column chromatography. Purified SQDG inhibited the activity, however, purified MGDG and DGDG had no influence. In this study, we demonstrated the structure-function relationship between chemically synthetic SQDG and p53 DBD. The major action is probably dependent on the fatty acid effect, although SQDG was a much stronger inhibitor than the fatty acid alone present in SQDG. The inhibitory activity of SQDG was weakened by the R248A mutant of p53 DBD, suggesting that R248 in the dsDNA binding site of p53 must be important for the inhibitory activity of SQDG. SQDG binding to p53 DBD could be reversed with a non-ionic detergent, Nonidet P-40. This is the first study of a glycolipid, SQDG, acting as a dsDNA binding inhibitor of p53, and it could be considered that a SQDG-containing thylakoid membrane in plant chloroplasts might regulate the activity of p53 for cell division, cell cycle checkpoint and tumor suppression.

Computer Simulation↗
PubMed2006

Proliferating cell nuclear antigen (PCNA) interacts with a meiosis-specific RecA homologues, Lim15/Dmc1, but does not stimulate its strand transfer activity.

PCNA is a multi-functional protein that is involved in various nuclear events. Here we show that PCNA participates in events occurring during early meiotic prophase. Analysis of protein-protein interactions using surface plasmon resonance indicates that Coprinus cinereus PCNA (CoPCNA) specifically interacts with a meiotic specific RecA-like factor, C. cinereus Lim15/Dmc1 (CoLim15) in vitro. The binding efficiency increases with addition of Mg(2+) ions, while ATP inhibits the interaction. Co-immunoprecipitation experiments indicate that the CoLim15 protein interacts with the CoPCNA protein in vitro and in the cell extracts. Despite the interaction between these two factors, no enhancement of CoLim15-dependent strand transfer activity by CoPCNA was found in vitro. We propose that the interaction between Lim15/Dmc1 and PCNA mediates the recombination-associated DNA synthesis during meiosis.

Animals↗
PubMed2006

Anti-tumor effects of dehydroaltenusin, a specific inhibitor of mammalian DNA polymerase alpha.

In the screening of selective inhibitors of eukaryotic DNA polymerases (pols), dehydroaltenusin was found to be an inhibitor of pol alpha from a fungus (Alternaria tennuis). We succeeded in chemically synthesizing dehydroaltenusin, and the compound inhibited only mammalian pol alpha with IC50 value of 0.5 microM, and did not influence the activities of other replicative pols such as pols delta and epsilon, but also showed no effect on pol alpha activity from another vertebrate, fish, or from a plant species. Dehydroaltenusin also had no influence on the other pols and DNA metabolic enzymes tested. The compound also inhibited the proliferation of human cancer cells with LD50 values of 38.0-44.4 microM. In an in vivo anti-tumor assay on nude mice bearing solid tumors of HeLa cells, dehydroaltenusin was shown to be a promising suppressor of solid tumors. Histopathological examination revealed that increased tumor necrosis and decreased mitotic index were apparently detected by the compound in vivo. Therefore, dehydroaltenusin could be of interest as not only a mammalian pol alpha-specific inhibitor, but also as a candidate drug for anti-cancer treatment.

Animals↗
PubMed2006

Site-directed mutational analysis of structural interactions of low molecule compounds binding to the N-terminal 8 kDa domain of DNA polymerase beta.

We previously reported the mode of inhibition of DNA polymerase beta (pol. beta) by long chain fatty acids and a bile acid, involving binding analyses to the N-terminal 8-kDa DNA binding domain. Here we describe a site-directed mutational analysis in which the key amino acids (L11, K35, H51, K60, L77, and T79), which are direct interaction sites in the domain, were substituted with K, A, A, A, K, and A, respectively. And their pol. beta interactions with a C24-long chain fatty acid, nervonic acid (NA), and a bile acid, lithocholic acid (LCA), were investigated by gel mobility shift assay and NMR spectroscopy. In the case of K35A, there was complete loss of DNA binding activity while K60A hardly has any activity. In contrast the other mutations had no appreciable effects. Thus, K35 and K60 are key amino acid sites for binding to template DNA. The DNA binding activities of L11K, H51A, and T79A as well as the wild type were inhibited by NA to the same extent. T79A demonstrated a disturbed interaction with LCA. 1H-15N HSQC NMR analysis indicated that despite their many similarities, the wild-type and the mutant proteins displayed some significant chemical shift differences. Not only were the substituted amino acid residues three-dimensionally shifted, but some amino acids which are positioned far distant from the key amino acids showed a shift. These results suggest that the interaction surface was significantly distorted with the result that LCA could not bind to the domain. These findings confirm our previous biochemical and 3D structural proposals concerning inhibition by NA and LCA.

Amino Acids↗
PubMed2006

Sumoylation of a meiosis-specific RecA homolog, Lim15/Dmc1, via interaction with the small ubiquitin-related modifier (SUMO)-conjugating enzyme Ubc9.

Sumoylation is a post-translational modification system that covalently attaches the small ubiquitin-related modifier (SUMO) to target proteins. Ubc9 is required as the E2-type enzyme for SUMO-1 conjugation to targets. Here, we show that Ubc9 interacts with the meiosis-specific RecA homolog, Lim15/Dmc1 in the basidiomycete Coprinus cinereus (CcLim15), and mediates sumoylation of CcLim15 during meiosis. In vitro protein-protein interaction assays revealed that CcUbc9 interacts with CcLim15 and binds to the C-terminus (amino acids 105-347) of CcLim15, which includes the ATPase domain. Immunocytochemistry demonstrates that CcUbc9 and CcLim15 colocalize in the nuclei from the leptotene stage to the early pachytene stage during meiotic prophase I. Coimmunoprecipitation experiments indicate that CcUbc9 interacts with CcLim15 in vivo during meiotic prophase I. Furthermore, we show that CcLim15 is a target protein of sumoylation both in vivo and in vitro, and identify the C-terminus (amino acids 105-347) of CcLim15 as the site of sumoylation in vitro. These results suggest that sumoylation is a candidate modulator of meiotic recombination via interaction between Ubc9 and Lim15/Dmc1.

Adenosine Triphosphatases↗
PubMed2006

Characterization of four RecQ homologues from rice (Oryza sativa L. cv. Nipponbare).

The RecQ family of DNA helicases is conserved throughout the biological kingdoms. In this report, we have characterized four RecQ homologues clearly expressed in rice. OsRecQ1, OsRecQ886, and OsRecQsim expressions were strongly detected in meristematic tissues. Transcription of the OsRecQ homologues was differentially induced by several types of DNA-damaging agents. The expression of four OsRecQ homologues was induced by MMS and bleomycin. OsRecQ1 and OsRecQ886 were induced by H(2)O(2), and MitomycinC strongly induced the expression of OsRecQ1. Transient expression of OsRecQ/GFP fusion proteins demonstrated that OsRecQ2 and OsRecQ886 are found in nuclei, whereas OsRecQ1 and OsRecQsim are found in plastids. Neither OsRecQ1 nor OsRecQsim are induced by light. These results indicate that four of the RecQ homologues have different and specific functions in DNA repair pathways, and that OsRecQ1 and OsRecQsim may not involve in plastid differentiation but different aspects of a plastid-specific DNA repair system.

Adenosine Triphosphatases↗
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

Drosophila DNA polymerase zeta interacts with recombination repair protein 1, the Drosophila homologue of human abasic endonuclease 1.

Abasic (AP) sites are a threat to cellular viability and genomic integrity, since they impede transcription and DNA replication. In mammalian cells, DNA polymerase (pol) beta plays an important role in the repair of AP sites. However, it is known that many organisms, including Drosophila melanogaster, do not have a pol beta homologue, and it is unclear how they repair AP sites. Here, we screened for DNA polymerases that interact with the Drosophila AP endonuclease 1 homologue, Rrp1 (recombination repair protein 1), and found that Drosophila pol zeta (Dmpol zeta), DmREV3 and DmREV7 bound to Rrp1 in a protein affinity column. Rrp1 directly interacted with DmREV7 in vitro and in vivo but not with DmREV3. These findings suggest that the DNA polymerase partner for Rrp1 is Dmpol zeta and that this interaction occurs through DmREV7. Interestingly, DmREV7 bound to the N-terminal region of Rrp1, which has no known protein homologue, suggesting that this binding is a species-specific event. Moreover, DmREV7 could stimulate the AP endonuclease activity of Rrp1, but not the 3'-exonuclease activity, and form a homomultimer. DmREV3 could not incorporate nucleotides at the 5'-incised tetrahydrofran sites but did show strand displacement activity for one-nucleotide-gapped DNA, which was not influenced by either DmREV7 or Rrp1. Methyl methanesulfonate and hydrogen peroxide treatments increased mRNA levels of DmREV3 and DmREV7. On the basis of the direct interaction between DmREV7 and Rrp1, we suggest that Dmpol zeta may be involved in the repair pathway of AP sites in DNA.

Animals↗
PubMed2006

Alpha-sulfoquinovosylmonoacylglycerol is a novel potent radiosensitizer targeting tumor angiogenesis.

Angiogenesis is a promising target for the treatment of cancer, and varying types of antiangiogenic agents have been developed. However, limitations and problems associated with antiangiogenic therapy have recently arisen. Although radiotherapy can be combined with antiangiogenic compounds to overcome these difficulties, almost all previously described angiogenesis inhibitors could still cause side effects at effective doses, and only additive effects are seen in current combination therapy. In this study, we identified a member of the sulfoquinovosylacylglycerols, alpha-sulfoquinovosylmonoacylglycerol (alpha-SQMG), originally derived from sea urchins, as a potent radiosensitizer. The agent synergistically inhibits angiogenesis at low doses when combined with ionizing radiation. Combined treatment with alpha-SQMG and radiation seems to promote the adoption of a senescence-like phenotype by vascular endothelial cells. Finally, the agent remarkably enhances the radioresponse of human tumors transplanted into nude mice, accompanied by a significant reduction in the vascularity of the tumors. Collectively, alpha-SQMG may be a novel potent radiosensitizer targeting angiogenesis.

Angiogenesis Inhibitors↗
PubMed2006

Characterization of T-DNA insertion mutants and RNAi silenced plants of Arabidopsis thaliana UV-damaged DNA binding protein 2 (AtUV-DDB2).

The human UV-damaged DNA binding protein (UV-DDB), a heterodimeric protein composed of 127 kDa (UV-DDB1) and 48 kDa (UV-DDB2) subunits, has been shown to be involved in DNA repair. To elucidate the in vivo function of plant UV-DDB2, we have analyzed T-DNA insertion mutants of the Arabidopsis thaliana UV-DDB2 subunit (atuv-ddb2 mutants) and AtUV-DDB2 RNAi silenced plants (atuv-ddb2 silenced plants). atuv-ddb2 mutants and atuv-ddb2 silenced plants were both viable, suggesting that AtUV-DDB2 is not essential for survival. Interestingly, both plant types showed a dwarf phenotype, implying impaired growth of the meristem. To the best of our knowledge, this is the first occasion that a dwarf phenotype has been found to be associated with a UV-DDB2 mutation in either plants or animals. The mutants also demonstrated increased sensitivity to UV irradiation, methyl methanesulfonate and hydrogen peroxide treatment, indicating that AtUV-DDB2 is also involved in DNA repair. Our results lead us to suggest that not only does AtUV-DDB2 function in DNA repair, it also has a direct or indirect influence on cell proliferation in the plant meristem.

Arabidopsis↗
PubMed2006

The inhibitory action of long-chain fatty acids on the DNA binding activity of p53.

The in vitro relationship between human p53 DNA binding domain (p53 DBD) and FA was investigated. We found that saturated and monounsaturated long-chain FA inhibited the double-stranded DNA (dsDNA) binding activity of p53 DBD. The strongest inhibitors of saturated and unsaturated FA were docosanoic acid (22:0) and cis-12-heneicosenoic acid (21:1n-9), respectively. n-Octadecane, trans-unsaturated FA, and FAME had no influence on the binding activity of p53 DBD, showing that the FA structures such as one or no double bond of cis configuration, hydrocarbon chain of length C20 to C22, and free carboxyl groups are important for the inhibition. The inhibitory effect of the R248A mutant of p53 DBD by saturated FA was as strong as that for wild-type p53 DBD. On the other hand, the inhibition of dsDNA binding activity of the same mutant by the cis-configuration of monounsaturated FA was weaker than that for the wild type. These results suggest that R248 in p53 DBD is important for binding to monounsaturated FA. This is the first report that long-chain FA act as a dsDNA binding inhibitor of p53, and it could be considered that FA in the cell membrane might regulate the activity of p53 for cell division, cell-cycle checkpoint, and tumor suppression.

Computer Simulation↗
PubMed2006

Identification of antibiotic clarithromycin binding peptide displayed by T7 phage particles.

Peptide libraries displayed by T7 phage, which contain random cDNA fragments insets, were screened for their ability to bind to a biotinylated derivative of clarithromycin. Phage particles bound to an immobilized derivative of the antibiotic were isolated and the inserted cDNA was amplified and sequenced. A common selected peptide sequence, composed of 19 amino acids, was obtained and a synthetic peptide with this sequence was produced. Surface plasmon resonance experiments showed that the synthetic peptide immobilized on a sensor chip bound to clarithromycin and the dissociation constant was determined to be 2.1 x 10(-3) M. The dissociation constants of other macrolide antibiotics, erythromycin, roxithromycin, azithromycin and josamycin were also determined to be 5.4 x 10(-3) M, 6.2 x 10(-5) M, 1.1 M and 3.4 x 10(-2) M, respectively. These results indicated that T7 phage display method might be useful to determine relatively weak interactions between small molecule drugs and the selected peptides which could represent a possible binding site conserved in binding proteins.

Amino Acid Sequence↗
PubMed2006

Drosophila damaged DNA binding protein 1 contributes to genome stability in somatic cells.

The damaged DNA-binding protein (DDB) complex consists of a heterodimer of p127 (DDB1) and p48 (DDB2) subunits and is believed to have a role in nucleotide excision repair (NER). We used the GAL4-UAS targeted expression system to knock down DDB1 in wing imaginal discs of Drosophila. The knock-down was achieved in transgenic flies using over-expression of inverted repeat RNA of the D-DDB1 gene [UAS-D-DDB1(650)-dsRNA]. As a consequence of RNA interference (RNAi), the fly had a shrunken wing phenotype. The wing spot test showed induced genome instability in transgenic flies with RNAi knock-down of D-DDB1 in wing imaginal discs. When Drosophila larvae with RNAi knock-down of D-DDB1 in wing imaginal discs were treated with the chemical mutagen methyl methanesulfonate (MMS), the frequency of flies with a severely shrunken wing phenotype increased compared to non-treated transgenic flies. These results suggested that DDB1 plays a role in the response to DNA damaged with MMS and in genome stability in Drosophila somatic cells.

Animals↗
PubMed2006

A higher plant has three different types of RPA heterotrimeric complex.

Replication protein A (RPA) is a protein complex composed of three subunits known as RPA70, RPA32, and RPA14. Generally, only one version of each of the three RPA genes is present in animals and yeast (with the exception of the human RPA32 ortholog). In rice (Oryza sativa L.), however, two paralogs of RPA70 have been reported. We screened the rice genome for RPA subunit genes, and identified three OsRPA70 (OsRPA70a, OsRPA70b and OsRPA70c), three OsRPA32 (OsRPA32-1, OsRPA32-2 and OsRPA32-3), and one OsRPA14. Through two-hybrid assays and pull down analyses, we showed that OsRPA70a interacted preferentially with OsRPA32-2, OsRPA70b with OsRPA32-1, and OsRPA70c with OsRPA32-3. OsRPA14 interacted with all OsRPA32 paralogs. Thus, rice has three types of RPA complex: OsRPA70a-OsRPA32-2-OsRPA14 (type A), OsRPA70b-OsRPA32-1-OsRPA14 (type B), and OsRPA70c-OsRPA32-3-OsRPA14 (type C). Subcellular localization analysis suggested that the type-A RPA complex is required for chloroplast DNA metabolism, whereas types B and C function in nuclear DNA metabolism.

Cloning, Molecular↗
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↗