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Secretory lysosome : the roles of ATG gene products

分泌性溶酶体：ATG 基因产物的作用

基本信息

批准号：
16370063
负责人：
UENO Takashi
金额：
$ 9.54万
依托单位：
JUNTENDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

项目摘要

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.

自噬的形成是自噬过程中的一个重要步骤，它由两个泛素化修饰介导，即Atg12-ATG5结合和Atg8-脂化。Atg8的脂化是Atg8作为完整膜组分被募集到自噬体膜蛋白上所必需的。在哺乳动物中，有三个Atg8同源物，LC3，GABARAP和GATE-16。在这三种蛋白中，LC3被认为是组织范围内的ATG8同源物，但对GABARAP*-黑色素瘤细胞的脂化作用知之甚少，当溶酶体蛋白分解被E64d和胃抑素抑制时，LC3和GABARAP大量表达和积累，这表明这两种同源物参与了黑色素瘤的自噬。不连续的OptiPrep梯度离心法显示，在E64d和胃抑素存在的情况下，脂化形式的LC3(LC3-II)和GABARAP(GABARAP-II)的分布与溶酶体(LGP120)和黑素体(酪氨酸酶和色氨酸-1)标记的分布重叠。免疫细胞化学和免疫电子显微镜分析进一步支持了这一结果，表明LC3修饰的黑素小体沿着细胞外围聚集，特别是在树突的尖端。在营养丰富和营养匮乏的条件下，黑素小体的自噬周转都会发生。这些发现表明，除了Lc3，GABARAP在黑素体自噬中也有内在的作用。培养的B16黑色素瘤细胞在培养液中分泌Lc3阳性的囊泡。我们用差速离心法纯化了这些排出的小泡，发现这些小泡含有溶酶体(LGP120，cathepsinL等)。和黑素体(Trp-1和酪氨酸酶)标记物，以及GABARAP和LC3。囊泡组分的电子显微镜显示，囊泡相对较小，大小均匀。根据这些数据，我们认为Lc3和GABARAP阳性的囊泡具有外切体和分泌型溶酶体的特征。

项目成果

期刊论文数量（35）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Autophagy

DOI：
10.1007/978-3-030-21573-6_10048-1
发表时间：
2020
期刊：
Encyclopedia of Molecular Pharmacology
影响因子：
0
作者：
H. Simon;R. Friis
通讯作者：
H. Simon;R. Friis

Excess peroxisomes are degraded by autophagic machinery in mammals

DOI：
10.1074/jbc.m512283200
发表时间：
2006-02-17
期刊：
JOURNAL OF BIOLOGICAL CHEMISTRY
影响因子：
4.8
作者：
Iwata, J;Ezaki, J;Kominami, E
通讯作者：
Kominami, E

Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice.

DOI：
10.1083/jcb.200412022
发表时间：
2005-05-09
期刊：
The Journal of cell biology
影响因子：
0
作者：
Komatsu M;Waguri S;Ueno T;Iwata J;Murata S;Tanida I;Ezaki J;Mizushima N;Ohsumi Y;Uchiyama Y;Kominami E;Tanaka K;Chiba T
通讯作者：
Chiba T

^1H, ^<13>C and ^<15>N resonance assignments of human microtubule-associated protein light chain-3.

人微管相关蛋白轻链3的^ 1 H、^ 13 C和^ 15 N共振归属。