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2003 Fiscal Year Final Research Report Summary

PROTEIN-ACTIVATING AND PROTEIN-CONJUGATING ENZYMES INVOLVED IN THE FORMATION OF NOVEL MEMBRANE SYSTEMS

Research Project

Project/Area Number 14380308
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Functional biochemistry
Research InstitutionJUNTENDO UNIVERSITY

Principal Investigator

UENO Takashi  JUNTENDO UNIVERSITY, DEPARTMENT OF BIOCHEMISTRY, ASSOCIATE PROFESSOR, 医学部, 助教授 (10053373)

Co-Investigator(Kenkyū-buntansha) MUNO Daisaku  JUNTENDO UNIVERSITY, DEPARTMENT OF BIOCHEMISTRY, RESEARCH ASSISTANT, 医学部, 助手 (00146771)
TANIDA Isei  JUNTENDO UNIVERSITY, DEPARTOEMTNOF BIOCHEMISTRY, ASSISTANT PROFESSOR, 医学部, 講師 (30296868)
Project Period (FY) 2002 – 2003
KeywordsAUTOPHAGY / LYSOSOME / MELANOSOME / PHAGOSOME / PROTEIN-ACTIVATING ENZYME / PROTEIN-CONJUGATING ENZYME / MEMBRANE FORMATION / ORGANELLE
Research Abstract

Autophagy is a universal mechanism, by which bulky cell proteins are degraded via lysosomal/vacuolar system. It has been demonstrated that more than 15 autophagy-related genes (ATG genes) are essential to this process and that numbers of the ATG gene products including Atg7, Atg3, Atg 10, Atgl2, Atg5, and Atg8 operate in two ubiquitin-like conjugation pathways. In this paper, we report that in some cultured melanoma cell lines such as B16-F0, B16-F1, M3, and G581, Atg7 and Atg12-Atg5 conjugate are abundantly expressed at extremely high levels. In order to clarify functional relevance of the elevated levels of these ATG gene products to possible enhancement of autophagy in melanomas, we further investigated the two Atg8 homologues, LC3 and GABARAP whose phospholipid-conjugated forms (LC3-II and GABARAP-II) are recruited to autophagosomal membranes and subsequently degraded after fusion of autophagosome with lysosome during starvation-induced autophagy. In B16-F1 melanoma, both LC3-II and GABARAP-II accumulated markedly when lysosomal proteolysis was inhibited with E64d and pepstatin. However, the accumulation occurred under not only nutrient-deprived condition but also nutrient-rich condition. Thus, LC3-II-or GABARAP-II-loaded membranes are continuously turned over via lysosomes in ordinary culture medium containing abundant amino acids and fetal calf serum. In cell fractionation analysis using discontinuous OptiPrep gradients, distribution of the accumulated LC3-II and GABARAP-II was found to well coincide with those of lysosomal makers (cathepsin L and LGP120) and melanosomal markers (tyrosinase and TRP-1). These biochemical data were further confirmed by immunofluorescence analyses. Taken together, these data indicate that LC3-II and GABARAP-II on melanosomal membranes facilitate melanosomes to fuse with lysosomes to be degraded in cultured B16-F1 melanoma.

  • Research Products

    (15 results)

All Other

All Publications (15 results)

  • [Publications] Tanida, I.: "Mammalian Apg12p, but not the Apg12p・Apg5p conjugate, facilitates LC3 processing."Biochem.Biophys.Res.Commun.. 296(5). 1164-1170 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Tanida, I.: "Murine Apg12p has a substrate preference for murine Apg7p over three Apg8p homologues・・・・"Biochem.Biophys.Res.Commun.. 292(1). 256-262 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Tanida, I.: "Human Apg3p/Aut1p homologue is an authentic E2 enzyme for multiple substrate, GATE-16, GABARAP・・・・"Journal of Biological Chemistry. 277(16). 13739-13744 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Tanida, I.: "GATE-16 and GABARAP are authentic modifiers mediated by Apg3 and Apg7."Biochem.Biophys.Res.Commun.. 300(3). 1164-1170 (2003)

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      「研究成果報告書概要(和文)」より
  • [Publications] Asanuma, K.: "MAP-LC3, a promising autophagosomal marker, is processed during the differentiation・・・・"FASEB Journal. 17(9). 1165-1167 (2003)

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      「研究成果報告書概要(和文)」より
  • [Publications] Nemoto. T.: "The Mouse APG10 Homologue, an E2-like Enzyme for Apg12p Conjugation, Facilitates MAP-LC3・・・・"Journal of Biological Chemistry. 278(41). 39517-39526 (2003)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Ueno, T.: "Autophagy(分担執筆)"Landes Bioscience, Eureka Com.. 23 (2004)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Tanida I, Tanida-Miyake E, Komatsu M, Ueno T, Kominami E.: "Human Apg3p/Autlp homologue is an authentic E2 enzyme for multiple substrates, GATE-16, GABARAP, and MAP-LC3, and facilitates the conjugation ofhApg12p to hApg5"J Biol Chem. 277(16). 13739-13744 (2002)

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      「研究成果報告書概要(欧文)」より
  • [Publications] Tanida I, Tanida-Miyake B, Nishitani T, Komatsu M, Yamazaki H, Ueno T, Kominami B.: "Murine Apg l2p has a substrate preference for murine Apg7p over three Apg8p homologs."Biochem Biophys Res Commun. 292. 256-262 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Tanida I, Nishitani T, Nemoto T, Ueno T, Kominami E.: "Mammalian Apg V2p, but not the Apgl2Apg5p conjugate, facilitates LC3 processing."Biochem Biophys Res Commun. 300. 1164-1170 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Tanida I, Komatsu M, Ueno T, Kominami E.: "GATE-16 and GABARAP are authentic modifiers mediated by Apg7 and Apg3."Biochem Biophys Res Commun. 300. 637-644 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Asanuma K, Tanida I, Shirato I, Ueno T, Takahara H, Nishitani T, Kominami E, Tomino Y.: "MAP-LC3, a promising autophagosomal marker, is processed during the differentiation and recovery of podocytes from PAN nephrosis."FASEB J. 17. 1165-1167 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Nemoto T, Tanida I, Tanida-Miyake E, Minematsu-Ikeguchi N, Yokota M, Ohsumi M, Ueno T, Kominami E.: "The mouse APG1O homologue, an E2-like enzyme for Apg12p conjugation, facilitates MAP-LC3 modification."J Biol Chem.. 278. 39517-39526 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Yamazaki-Sato H, Tanida I, Ueno T, Kominami E.: "The carboxylterminal 17 amino acids within Apg7 are essential for Apg8 lipidation, but not for Apg12 conjugation."FEBS Lett.. 551(1-3). 71-77 (2003)

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  • [Publications] Tanida I, Ueno T, Kominami E.: "LC3 conjugation system in mammalian autophagy"International Journal of Biochemistry. (in press).

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      「研究成果報告書概要(欧文)」より

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Published: 2005-04-19  

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