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2006 Fiscal Year Final Research Report Summary

New function of disulfide bonds in cytosolic proteins

Research Project

Project/Area Number 16570092
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Structural biochemistry
Research InstitutionNiigata University

Principal Investigator

ODANI Shoji  Niigata University, Institute of Science and Technology, Professor, 自然科学系, 教授 (60018702)

Co-Investigator(Kenkyū-buntansha) TAKAHASHI Yoshiaki  Niigata University, Institution of Medical and Dental Sciences, Professor, 医歯学系, 教授 (60115045)
Project Period (FY) 2004 – 2006
Keywordsfatty acid-binding protein / proteolysis / cysteine / oxidative stress / disulfide / glutathione / nematode / Caenorhabditis elegans
Research Abstract

A number of cytosolic proteins contain cysteine residues of unknown function. To understand the biological function of these cysteine residues and their disulfides, fatty acid-binding proteins (FABP) were chosen as model proteins. Liyer fatty acid-binding proteins (L-FABP) of rat, chick, lizard, frog, and fish possess cysteine residues at different positions in the amino acid sequence, and 3D modeling indicated that in fish L-FABP a pair of cysteine residues are located within the distance of sulfur atoms of a disulfide bond. Rat, chick and frog (Rana catesbeiana) liver FABPs were purified form the respective tissues by gel-filtration on Sephacryl columns, ion-exchange chromatography on DEAE-Sephacell, and HPLC on reversed-phase columns (octylsilane). Recombinant proteins were prepared for lizard (Anolis pukhelhis) and zebra fish (Brachydanio rerio). Lizard FABP cDNA was a gift of Prof. Morales, and zebrafish first strand cDNA library was prepared form the liver total RNA. They were li … More gated into expression vectors and transfected to E. coli BL21(DE3). Expressed proteins were purified by gel-filtration and HPLC. Mixed disulfides with glutathione and these proteins were prepared by incubation with diamide. They are defatted and lipid-binding activity was compared with untreated proteins. Lipid binding activity was measured by displacement of fluorescent probes, dansylaminoundecanoic acid and anilinonaphthalene sulfonate. Direct binding of a natural fluorescent fatty acid, cis-parinaric acid, was also measured. All these FABPs strongly bound various lipids regardless of bound glutathione. These results indicate that the purified and expressed FABPs are native and modification with glutathione does not affect biological function of the proteins, i.e., mixed disulfide formation did not alter the structure of the protein. However, it was found that mixed disulfide forms were rapidly digested with various proteinases, including intracellular proteases, regardless of the position of cysteine residues throughout the linear amino acid sequence. This strongly suggests that these mixed disulfide formation with glutathione is a process of tagging leading to proteolytic degradation of oxidatively damaged proteins. Since redox potential of a cysteine residue is under the influence of its location in the protein environment, above-mentioned mixed disulfide formation might be a determinant of life span of a protein.
The nematode, Caenorhabditis elegans also has nine genes coding FABPs with or without cysteine residues. This animal is useful for examine the effects of oxidative stress as culturing the worms in medium containing hydrogen peroxide readily simulates oxidative stresses. We first prepared 9 recombinant proteins of nematode FABP 1-9, and experimental conditions for in vitro glutathionylation was established. A similar glutathionylation experiment as described above was attempted first on FABP-5, and an dramatic increase of proteolytic susceptibility was again observed. We believe cytosolic disulfide formation is a hither-to-unknown marking event for protein degradation. Less

  • Research Products

    (8 results)

All 2007 2005

All Journal Article (8 results)

  • [Journal Article] Phosphorylated synaphin/complexin found in the brain exhibits enhanced SNARE complex binding.2007

    • Author(s)
      Shata, A.
    • Journal Title

      Biochemical and Biophysical Research Communications 354・3

      Pages: 808-813

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Phosphorylated synaphin/complexin found in the brain exhibits enhanced SNARE complex binding.2007

    • Author(s)
      Shata, A., Saisu, H., Odani, S., Abe, T.
    • Journal Title

      Biochemical and Biophysical Research Communications 354-3

      Pages: 803- 813

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Evidence for megalin-mediated proximal tubular uptake of L-FABP, a carrier of potentially nephrotoxic molecules2005

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

      Laboratory Investigations 85・1

      Pages: 522-531

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Ribosomal proteins cross-linked to the initiater AUG codon of a mRNA2005

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

      Journal of Biochemistry 138・1

      Pages: 41-46

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] A novel cysteine protease inhibitor with lectin activity from the epidermis of the Japanese eel Anguila japonica2005

    • Author(s)
      Saitoh, H.
    • Journal Title

      Comparative Biochemistry and Physiology 141・1

      Pages: 103-109

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Evidence for megalin- mediated proximal tubular uptake of L-FABP, a carrier of potentially nephrotoxic molecules.2005

    • Author(s)
      Oyama, Y., Takeda T, Hama H, et al.
    • Journal Title

      Laboratory Investigations 85-1

      Pages: 522-531

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Ribosomal proteins cross-linked to the initiator AUG codon of a mRNA.2005

    • Author(s)
      Takahashi, Y., Hirayama, S., Odani, S.
    • Journal Title

      Journal of Biochemistry 138-1

      Pages: 41-46

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] A novel cysteine protease inhibitor with lectin activity from the epidermis of the Japanese eel Anguila japnica.2005

    • Author(s)
      Saitoh, H., Isemura S., Chiba, A., Oka S., Odani, S.
    • Journal Title

      Comparative Biochemistry and Physiology 141-1

      Pages: 103-109

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 2008-05-27  

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