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1999 Fiscal Year Final Research Report Summary

Molecular mechanism of cellular processing of tumor necrosis factor (TNF)

Research Project

Project/Area Number 10660092
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field 応用微生物学・応用生物化学
Research InstitutionYamaguchi University

Principal Investigator

UTSUMI Toshihiko  Faculty of Agriculture, Yamaguchi University, Associate Professor, 農学部, 助教授 (20168727)

Co-Investigator(Kenkyū-buntansha) 内海 俊彦  山口大学, 農学部, 助教授 (20168727)
Project Period (FY) 1998 – 1999
Keywordstumor necrosis factor (TNF) / processing / membrane protein / secretion
Research Abstract

To determine the minimum requirement in the 76-residue leader sequence of pro-tumor necrosis factor (TNF) for membrane translocation across the endoplasmic reticulum (ER) and for the maturation of pro-TNF, we have constructed pro-TNF mutants in which a part of the transmembrane domain of pro-TNF was directly linked to the N-terminus of the mature domain, and evaluated their translocational behavior across the ER-membrane and their secretion from the transfected cells.
The in vitro translation/translocation assay using a canine pancreatic microsomal membrane system with a mutant, Δ-75--47,-32--1, revealed that the N-terminal half of the transmembrane domain of pro-TNF consisting of 14 residues functioned as a cleavable signal sequence ; it generated a cleaved form of TNF having a molecular mass similar to that of mature TNF. Analysis of the cleavage site by site-directed mutagenesis indicated that the site was inside of the leader sequence of this mutant. When the mutant Δ-75--47, -32--1 was expressed in COS-1 cells, efficient secretion of a biologically active soluble TNF was observed. Further deletion of the hydrophobic domain from this mutant inhibited the translocation, indicating that some extent of hydrophobicity is indispensable for the membrane translocation of the mature domain of TNF. Thus, the N-terminal half of the transmembrane domain of pro-TNF could function as a cleavable signal sequence when linked to the mature domain of TNF, and secretion of biologically active secretory form of TNF could be achieved with this 14-residue hydrophobic segment.
In intact pro-TNF, however, this 14-residue sequence could not function as a cleavable signal sequence during intracellular processing, indicating that the remainder of the 76-residue leader sequence of pro-TNF inhibits the signal peptide cleavage and enables the leader sequence to function as a type II signal-anchor sequence that generates a transmembrane form of TNF.

  • Research Products

    (14 results)

All Other

All Publications (14 results)

  • [Publications] Ishisaka,R.,Utsumi,T.et al.: "Actiyation of caspase-3-like protease by digitonin-treated lysosomes"FEBS Letters. 435. 233-236 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Ishisaka,R.,Utsumi,T.et al.: "A part of the transmembrane domain of pro-TNF can function as a cleavable signal sequence that generates a biologically active secretory forrn of TNT"J.Biochem.. 126. 413-420 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 石坂瑠美、内海俊彦ら: "リソソームとアポトーシス"生体の科学. 50. 117-126 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Ishisaka,R.,Utsumi,T.et al.: "Participation of a cathepsin L-type protease in the auctivation of caspase-3"Cell Struct.Funct.. 24. 465-470 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 内海俊彦: "TNF-αの分泌型へのプロセシングの分子機構"バイオサイエンスとインダストリー. 57. 39-40 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Arita,K.,Utsumi,T. et al.: "Mechanism of dibucaine-induced apoptosis in promyelocytic leukemia cells(HL-60)"Bicchemical Pharmacol.. (in press).

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] T.Utsumi et al.: "Neutrophils and priming.isolation of neutrophils/assay of O_2 generation by cytochrome-reduction.in Exprimental Protocols for reactive oxygen and nitrogen species"Oxford University Press(in press).

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] R. Ishisaka, T. Utsumi, M. Yabuki, T. Kanno, T. Furuno, M. Inoue, K. Utsumi: "Activation of caspase-3-like protease by digitonin-treated lysosomes."FEBS Letters. 435. 233-236 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] R. Ishisaka, N. Sato, K. Tanaka, T. Takeshige, H. Iwata, J. Klostergaard, T. Utsumi: "A part of the transmembrane domain of pro-TNF can function as a cleavable signal sequence that generates a biologically active secretory form of TNF."J. Biochem.. 126. 413-420 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] R. Ishisaka, T. Utsumi, M. Yabuki, N. Katunuma, K. Utsumi: "Apoptosis, Involvement of lysosomal enzymes."Seitai no Kagaku. 50. 117-126 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] R. Ishisaka, T. Utsumi, T. Kanno, K. Arita, N. Katunuma, J. Akiyama, K. Utsumi: "Participation of a cathepsin L-type protease in the activation of caspase-3"Cell Struct. Funct.. 24. 465-470 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] T. Utsumi: "Molecular mechanism for the cellular processing of TNF-α"Bioscience and Industry. 57. 39-40 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] K. Arai, T. Utsumi, A. Kato, T. Kanno, H. Kobuchi, B. Inoue, J. Akiyama, K. Utsumi: "Mechanism of dibucaine-induced apoptosis in promyelocytic leukemia cells (HL-60)"Biochem. Pharmacol.. in press.

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] T. Utsumi, K. Utsumi: "Neutrophils and priming. Isolation of neutrophils/assay of OィイD22ィエD2- generation by cytochrome-c reduction in Experimental protocols for reactive oxygen and nitrogen species (ed. By N. Taniguchi and J. Gutteridge)"Oxford University Press.

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

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Published: 2001-10-23  

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