| Co-Investigator(Kenkyū-buntansha) |
TANIDA Isei Juntendo Univ., Dept.of Biochem., Assistant professor, 医学部, 講師 (30296868)
EZAKI Junji Juntendo Univ., Dept.of Biochem., Assistant professor, 医学部, 講師 (60232948)
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| Budget Amount *help |
¥14,900,000 (Direct Cost: ¥14,900,000)
Fiscal Year 2005: ¥6,000,000 (Direct Cost: ¥6,000,000)
Fiscal Year 2004: ¥8,900,000 (Direct Cost: ¥8,900,000)
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| Research Abstract |
The formation of autophagosomes, an essential step in autophagy, is mediated by two ubiquitylation-like modifications, Atg12-Atg5 conjugation and Atg8-lipidation. Lipidation of Atg8 is necessary for Atg8 to be recruited onto autophagosomal membarene, as an integral membrane component. In mammals, there are three Atg8 homologues, LC3, GABARAP, and GATE-16. Of these three, LC3 has been best characterized as a tissue-wide Atg8 homologue, however little is known about the lipidation of GABARAP ********* -melanoma cells, LC3 and GABARAP were abundantly expressed and accumulated when lysosomal proteolysis was inhibited with E64d and pepstatin, suggesting that the two homologues are involved in melanoma autophagy. Discontinuous OptiPrep gradient centrifugation revealed that, in the presence of E64d and pepstatin, the distribution of lipidated form of LC3 (LC3-II) and GABARAP (GABARAP-II) overlaps with that of lysosomal (LGP120) and melanosomal (tyrosinase and TRP-1) markers. The results were further supported by immunocytochemical and immuno electronmicrosopic anlyses showing that LC3-decorated melanosomes accumulate along the cell periphery, particularly on the tips of dendrites. The autophagic turnover of melanosomes occurred under both nutrient-rich and nutrient-deprived conditions. These findings indicate that GABARAP in addition to LC3 has an intrinsic role in melanosome autophagy.
Cultured B16 melanoma cells excrete LC3-positive vesicles in the medium. We purified these excreted vesicles with differential centrifugation and found that the vesicles possess lysosomal (LGP12O, cathepsinL, etc.) and melanosomal (TRP-1 andtyrosinase) markers, in addition to GABARAP and LC3. Electron micrographs of the vesicular fraction revealed that the vesicles are relatively small and homogeneous in size. From these data, we conclude that LC3- and GABARAP-positive vesicles fulfill the characteristics of exosome and secretory lysosomes.
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