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Selective degradation of p53 mutant by an engineered ScFv-linked ubiquitin ligase

Research Project

Project/Area Number 13671261
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field General surgery
Research InstitutionSt. Marianna University School of Medicine

Principal Investigator

FUKUDA Mamoru  St. Marianna University School of Medicine, Department of Surgery, Professor, 医学部, 助教授 (50081724)

Co-Investigator(Kenkyū-buntansha) OHTA Tomohiko  St. Marianna University School of Medicine, Department of Surgery, Assistant Professor, 医学部, 講師 (60233136)
Project Period (FY) 2001 – 2002
Project Status Completed (Fiscal Year 2002)
Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2001: ¥1,800,000 (Direct Cost: ¥1,800,000)
Keywordsgene therapy / protein degradation / scFv / ubiquitin ligase / p53 / RING finger / scFv
Research Abstract

We have attempted to create an engineered ubiquitin ligase with ScFv as a substrate recognition site that target and degrade only p53 mutant but not the wild type. The ScFv was cloned from cDNA library of a hybridoma cells that express Pab240, an antibody recognizes mutant p53 such as R273P, R175H, or V143A. We first tested a double RING finger ubiquitin ligase whether it actually targets the intended specific substrates. The engineered ligase contains the RING finger domains of both BRCA1 and BARD1 linked to a substrate recognition site PCNA, which is known to interact with cyclin dependent kinase inhibitor p57. The double RING finger ubiquitin ligase formed a homo-oligomer complex and exhibited significant ligase activity. Co-transfection of the ligase reduced the expression of transfected p57 to the back ground level in proteasome dependent manner, and restored the colony formation ability of U2OS cells that is otherwise inhibited by overexpressed p57. The results indicate the ability of the engineered double RING ubiquitin ligase to target the intended substrate. Next we tested the double RING ubiquitin ligase linked to the ScFv cloned from Pab240. However so far it has not been successful most likely because of the affinity problem between the ScFv and p53 mutants. To overcome the problem a new ScFv has been made by DNA synthesize method.

Report

(3 results)
  • 2002 Annual Research Report   Final Research Report Summary
  • 2001 Annual Research Report
  • Research Products

    (21 results)

All Other

All Publications (21 results)

  • [Publications] Rintaro Hashizume: "The RING heterodimer BRCA1-BARD1 is a ubiquitin ligase inactivated by a breast cancer-derived mutation"Journal of Biological Chemistry. 276(18). 14537-14540 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Ichiro Maeda: "In vitro Ubiquitination of Cyclin D1 by ROC1-CUL1 and ROC1-CUL3"FEBS letters. 494(2). 181-185 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] 太田智彦: "BRCA1のもう一つの機能 -ユビキチンリガーゼ活性-"細胞工学. 20(6). 854-856 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Daisuke Oyake: "Targeted substrate degradation by an engineered double RING ubiquitin ligase"Biochem. Biophys. Res. Commun.. 295(2). 370-375 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] 太田智彦: "BRCA1遺伝子と予防的両側乳房切除術"乳癌の臨床. 17(3). 210-216 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Peter S.Brzovic: "Binding and Recognition in the Assembly of an Active BRCA1-BARD1 Ubiquitin Ligase Complex"Proc. Natl. Acad. Sci. USA. (In press). (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Hashizume, R., Fukuda, M., Maeda, I., Nishikawa, H., Oyake, D., Yabuki, Y., Ogata, H., and Ohta, T.: "The RING heterodimer BRCA1-BARD1 is a ubiquitin ligase inactivated by a breast cancer-derived mutation"J. Biol. Chem.. 276. 14537-14540 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Maeda, I., Ohta, T., Koizumi, H., and Fukuda, M.: "In vitro ubiquitination of cyclin D1 by ROC1-CUL1 and ROC1-CUL3"FEBS Lett.. 494. 181-185 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Ohta, T., Hashizume, R., Fukuda: "The novel function of BRCA1 -ubiquitin ligase activity-"Cell Technology (Japanese). 20. 854-856 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Oyake D, Nishikawa H, Koizuka I, Fukuda M, and Ohta T: "Targeted substrate degradation by an engineered double RING ubiquitin ligase"Biochem. Biophys. Res. Commun.. 295. 370-375 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Ohta, T., Hashizume, R., Fukuda: "BRCA1 and prophylactic bilateral mastectomy"Jpn. J. Breast Cancer(Japanese). 17. 210-216 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Brzovic P, Keeffe J, Nishikawa H, Miyamoto K, Fox D, Fukuda M, Ohta T, and Klevit R: "Binding and Recognition in the Assembly of an Active BRCA1-BARD1 Ubiquitin Ligase Complex"Proc. Natl. Acad. Sci. USA. In Press. (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Rintaro Hashizume: "The RING heterodimer BRCA1-BARD1 is a ubiquitin ligase inactivated by a breast cancer-derived mutation"Journal of Biological Chemistry. 276(18). 14537-14540 (2001)

    • Related Report
      2002 Annual Research Report
  • [Publications] Ichiro Maeda: "In vitro Ubiquitination of Cyclin D1 by ROC1-CUL1 and ROCI-CUL3"FEBS letters. 494(3). 181-185 (2001)

    • Related Report
      2002 Annual Research Report
  • [Publications] 太田智彦: "BRCA1のもう一つの機能 -ユビキチンリガーゼ活性-"細胞工学. 20(6). 854-856 (2001)

    • Related Report
      2002 Annual Research Report
  • [Publications] Daisuke Oyake: "Targeted substrate degradation by an engineered double RING ubiquitin ligase"Biochem. Biophys. Res. Commun.. 295(2). 370-375 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] 太田智彦: "BRCA1遺伝子と予防的両側乳房切除術"乳癌の臨床. 17(3). 210-216 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Peter S.Brzovic: "Binding and Recognition in the Assembly of an Active BRCA1-BARD1 Ubiquitin Ligase Complex"Proc. Natl. Acad. Sci. USA. (In Press). (2003)

    • Related Report
      2002 Annual Research Report
  • [Publications] Rintaro Hashizume: "The RING heterodimer BRCA1-BARD1 is a ubiquitin ligase inactivated by a breast cancer-derived mutation"Journal of Biological Chemistry. 276(18). 14537-14540 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Ichiro Maeda: "In vitro Ubiquitination of Cyclin D1 by ROC1-CUL1 and ROC1-CUL3"FEBS letters. 494(3). 181-185 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] 太田智彦: "BRCA1のもう一つの機能,-ユビキチンリガーゼ活性-"細胞工学. 20(6). 854-856 (2001)

    • Related Report
      2001 Annual Research Report

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Published: 2001-04-01   Modified: 2016-04-21  

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