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

参与新型膜系统形成的蛋白质激活酶和蛋白质结合酶

基本信息

批准号：
14380308
负责人：
UENO Takashi
金额：
$ 9.15万
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：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14380308/
关键词：
AUTOPHAGY LYSOSOME MELANOSOME PHAGOSOME PROTEIN-ACTIVATING ENZYME PROTEIN-CONJUGATING ENZYME MEMBRANE FORMATION ORGANELLE

项目摘要

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.

自噬是一种普遍的机制，通过溶酶体/空泡系统降解巨大的细胞蛋白。已有的研究表明，超过15个自噬相关基因(ATG基因)参与了这一过程，ATG基因产物包括ATG7、ATG3、ATG10、ATG2、ATG5和ATG8在两条类似泛素的连接途径中起作用。在本文中，我们报道了一些培养的黑色素瘤细胞系，如B16-F0，B16-F1，M3，和G581，ATG7和ATG12-ATG5结合物在极高的水平上大量表达。为了阐明这些ATG基因产物水平升高与黑色素瘤自噬可能增强的功能相关性，我们进一步研究了两个ATG8同源物，LC3和GABARAP，它们的磷脂结合形式(LC3-II和GABARAP-II)被招募到自噬体膜上，并在饥饿诱导的自噬过程中自噬小体与溶酶体融合后降解。在B16-F1黑色素瘤中，当E64d和胃抑素抑制溶酶体蛋白降解时，LC3-II和GABARAP-II均显著积聚。然而，这种积累不仅发生在营养缺乏的条件下，而且发生在营养丰富的条件下。因此，在含有丰富氨基酸和胎牛血清的普通培养液中，LC3-II或GABARAP-II负载的膜通过溶酶体连续翻转。在使用不连续OptiPrep梯度的细胞分级分析中，累积的Lc3-II和GABARAP-II的分布与溶酶体标志物(组织蛋白酶L和LGP120)和黑素体标志物(酪氨酸酶和Trp-1)的分布很好地一致。免疫荧光分析进一步证实了这些生化数据。综上所述，这些数据表明，在培养的B16-F1黑色素瘤中，黑素体膜上的LC3-II和GABARAP-II促进黑素小体与溶酶体融合并被降解。