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Molecular analysis of C.elegans AAA proteins related to genetic diseases and developmental disorders

Research Project

Project/Area Number 16570147
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Molecular biology
Research InstitutionKUMAMOTO UNIVERSITY

Principal Investigator

YAMANAKA Kunitoshi  KUMAMOTO UNIVERSITY, INSTITUE OF MOLECULAR EMBRYOLOGY AND GENETICS, ASSISTANT PROFESSOR, 発生医学研究センター, 助教授 (90212290)

Co-Investigator(Kenkyū-buntansha) OGURA Teru  KUMAMOTO UNIVERSITY, INSTITUE OF MOLECULAR EMBRYOLOGY AND GENETICS, PROFESSOR, 発生医学研究センター, 教授 (00158825)
Project Period (FY) 2004 – 2005
Project Status Completed (Fiscal Year 2005)
Budget Amount *help
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2005: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2004: ¥1,700,000 (Direct Cost: ¥1,700,000)
KeywordsAAA protein / ATPase / C.elegans / hereditary spastic paraplegia / molecular chaperone / neurodegenerative disease / polyglutamine disease / p97 / VCP / spastin / fidgetin
Research Abstract

Caenorhabditis elegans possesses two homologues of p97/VCP/Cdc48p (CDC-48.1 and CDC-48.2), while humans and mice possess only one. To know their cellular functions, their mutant worms were analyzed. The cdc-48.1 deletion mutant showed slightly slow growth rate. Interestingly, we found that the cdc-48.1 deletion mutation decreased the brood size to half and concomitantly increased the number of non-fertilized eggs. Sperm in the cdc-48.1 mutant were exhausted at the stage earlier than that observed in the wild-type. These results together with the mating experiments suggest that C.elegans p97 functions in the switching processes from spermatogenesis to oogenesis. We also found that p97 plays a crucial role in unfolded protein response and ER network formation.
We found that spastin is involved in micritubule dynamics in vivo. We also found that purified spastin was able to directly bind tubulin in vitro and the ATPase activity of spastin was stimulated by tubiulin.
To elucidate the mechanism of ATP hydrolysis, we prepared several mutants of the C.elegans fidgetin homologue FIGL-1 carrying a mutation in each of three motifs, the Walker A and B motifs and the second region of homology. None of the constructed mutants showed ATPase activity. All the mutants except for K362A were able to bind ATP. Mixtures of E416A and R471A, or N461A and R471A led to the formation of hetero-hexamers with partially restored ATPase activities, providing direct, firm evidence for the intersubunit catalysis model. In addition, based on the results obtained with mixtures of K362A with wild-type or R471A subunits, we proposed that a conformational change upon ATP binding is required for proper orientation of the arginine fingers, which is essential for efficient hydrolysis of ATP bound to the neighboring subunit.

Report

(3 results)
  • 2005 Annual Research Report   Final Research Report Summary
  • 2004 Annual Research Report
  • Research Products

    (17 results)

All 2006 2004 Other

All Journal Article (17 results)

  • [Journal Article] An AAA protease FtsH can initiate proteolysis from internal sites of a model substrate, apo-flavodoxin.2006

    • Author(s)
      Okuno, T.
    • Journal Title

      Genes Cells 11(3)

      Pages: 261-268

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Annual Research Report 2005 Final Research Report Summary
  • [Journal Article] Flavodoxin, a new fluorescent substrate for monitoring proteolytic activity of FtsH lacking a robust unfolding activity.2006

    • Author(s)
      Okuno, T.
    • Journal Title

      J. Struc. Biol. (in press)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Characterization of mutants of the Escherichia coli AAA protease, FtsH, carrying a mutation in the central pore region.2006

    • Author(s)
      Okuno, T.
    • Journal Title

      J. Struc. Biol. (in press)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Mutational analysis of the functional motifs in the ATPase domain of C. elegans fidgetin homologue FIGL-1 : Firm evidence for an intersubunit catalysis mechanism of ATP hydrolysis by AAA ATPases.2006

    • Author(s)
      Yakushiji, Y.
    • Journal Title

      J. Struc. Biol. (in press)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] An AAA protease FtsH can initiate proteolysis from internal sites of a model substrate, apo-flavodoxin.2006

    • Author(s)
      Okuno, T., Yamanaka, K., Ogura, T.
    • Journal Title

      Genes Cells 11

      Pages: 261-268

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Flavodoxin, a new fluorescent substrate for monitoring proteolytic activity of FtsH lacking a robust unfolding activity.2006

    • Author(s)
      Okuno, T.
    • Journal Title

      J. Struct. Biol. (in press)

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Characterization of mutants of the Escherichia coli AAA protease, FtsH, carrying a mutation in the central pore region.2006

    • Author(s)
      Okuno, T.
    • Journal Title

      J. Struct. Biol. (in press)

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Mutational analysis of the functional motifs in the ATPase domain of C. elegans fidgetin homologue FIGL-1: Firm evidence for an intersubunit catalysis mechanism of ATP hydrolysis by AAA ATPases.2006

    • Author(s)
      Yakushiji, Y.
    • Journal Title

      J. Struct. Biol. (in press)

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Identification of a cysteine residue important for the ATPase activity of C. elegans fidgetin homologue.2004

    • Author(s)
      Yakushiji, Y.
    • Journal Title

      FEBS Lett. 578

      Pages: 191-197

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Identification of a cysteine residue important for the ATPase activity of C.elegans fidgetin homologue.2004

    • Author(s)
      Yakushiji, Y., Yamanaka, K., Ogura, T.
    • Journal Title

      FEBS Lett. 578

      Pages: 191-197

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Identification of a cysteine residue important for the ATPase activity of C.elegans fidgetin homologue.2004

    • Author(s)
      Yakushiji, Y.
    • Journal Title

      FEBS Lett. 578

      Pages: 191-197

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Analysis of the two p97/VCP/Cdc48p proteins of Caenorhabditis elegans and their suppression of polyglutamine-induced protein aggregation.2004

    • Author(s)
      Yamanaka, K.
    • Journal Title

      J.Struct.Biol. 146

      Pages: 242-250

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Spectrometric analysis of degradation of a physiological substrate σ^<32> by Escherichia coli AAA protease FtsH.2004

    • Author(s)
      Okuno, T.
    • Journal Title

      J.Struct.Biol. 146

      Pages: 148-154

    • Related Report
      2004 Annual Research Report
  • [Journal Article] 線虫を用いたAAA蛋白質に起因する神経変性疾患モデル2004

    • Author(s)
      山中 邦俊
    • Journal Title

      蛋白質 核酸 酵素 49

      Pages: 1124-1126

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Flavodoxin, a new fluorescent substrate for monitoring proteolytic activity of FtsH lacking a robust unfolding activity.

    • Author(s)
      Okuno, T., Yamanaka, K., Ogura, T.
    • Journal Title

      J.Struc.Biol. (in press)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Characterization of mutants of the Escherichia coli AAA protease, FtsH, carrying a mutation in the central pore region

    • Author(s)
      Okuno, T., Yamanaka, K., Ogura, T.
    • Journal Title

      J.Struc.Biol. (in press)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Mutational analysis of the functional motifs in the ATPase domain of C.elegans fidgetin homologue FIGL-1 : Firm evidence for an intersubunit catalysis mechanism of ATP hydrolysis by AAA ATPases.

    • Author(s)
      Yakushiji, Y., Nishikori, S., Yamanaka, K., Ogura, T.
    • Journal Title

      J.Struc.Biol. (in press)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary

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Published: 2004-04-01   Modified: 2016-04-21  

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