A cleaved form of MAGE-A4 binds to Miz-1 and induces apoptosis in human cells
A cleaved form of MAGE-A4 binds to Miz-1 and induces apoptosis in human cells
复制标题
DOI：
10.1074/jbc.m310437200
复制
发表时间：
2004-04-09
影响因子：
4.8
通讯作者：
Fujita, J
中科院分区：
文献类型：
--
作者：
Sakurai, T;Itoh, K;Fujita, J
Gankyrin是最近发现的一种癌蛋白，由于它在大多数肝细胞癌中过表达，因此有望成为药物治疗的靶点。由于甘结蛋白与MAGE-A4相互作用，我们获得了几个MAGE-A4突变体，并评估了它们对细胞生长的影响。我们发现MAGE-A4的C端107个氨基酸(MAGE-A4DeltaN1)可诱导P53依赖和非P53依赖的细胞凋亡。MAGE-A4DeltaN1使P53蛋白水平升高，p21(Cip1)转录本和蛋白水平下降。在细胞凋亡过程中，Bclxl表达下调，线粒体完整性受到破坏。酵母双杂交鉴定MiZ-1为MAGE-A4DeltaN1结合蛋白。MAGE-A4DeltaN1通过与MiZ-1结合到p21Cip1启动子和下调p21(Cip1)的转录来招募。在293T细胞和U-2OS细胞中，全长MAGE-A4被加工成104个氨基酸的C末端片段，其活性与MAGE-A4DeltaN1相似。大范围的caspase抑制剂Z-VAD-FMK可以抑制MAGE-A4的加工，但不能通过对MAGE-A4中天冬氨酸的定点突变来抑制加工，这表明caspase(S)间接参与了加工。细胞暴露于阿霉素后，加工量增加。转导HIV Tat-MAGE-A4DeltaN1融合蛋白可在体外和体内抑制gankyrin过表达细胞的非锚定生长。这些结果表明，MAGE-A4的C末端片段至少部分地通过与MiZ-1结合而诱导细胞凋亡，该片段可能被开发为抗癌药物。此外，在人类细胞中发现全长MAGE-A4在基因毒性应激后产生一个具有促凋亡活性的C末端片段，这表明MAGE-A4具有新的功能。
Gankyrin, a recently discovered oncoprotein, is a promising target for drug therapy because it is overexpressed in most hepatocellular carcinomas. Since gankyrin interacts with MAGE-A4, we made several MAGE-A4 mutants and assessed their effects on cell growth. We found that the C-terminal 107 amino acids of MAGE-A4 (MAGE-A4DeltaN1) induced p53-dependent and p53-independent apoptosis. MAGE-A4DeltaN1 increased the p53 protein level, but decreased the p21(Cip1) transcript and protein levels. During apoptosis Bcl-xL was down-regulated and mitochondrial integrity was disrupted. A yeast two-hybrid screen identified Miz-1 as a MAGE-A4DeltaN1-binding protein. MAGE-A4DeltaN1 was recruited through association with Miz-1 to the p21Cip1 promoter and down-regulated transcription of p21(Cip1). In 293T cells and U-2 OS cells, full-length MAGE-A4 was processed to generate a C-terminal fragment of 104 amino acids with activities similar to MAGE-A4DeltaN1. Processing was inhibited with a broad range caspase inhibitor Z-VAD-FMK, but not by site-directed mutagenesis of aspartic acids in MAGE-A4, suggesting an indirect involvement of caspase(s) in the processing. The amount of the processed form was increased by exposure of cells to adriamycin. Transduction with a HIV Tat-MAGE-A4DeltaN1 fusion protein suppressed anchorage-independent growth of gankyrin-overexpressing cells in vitro and in vivo. These results demonstrate that the C-terminal fragment of MAGE-A4 induces apoptosis at least partly by binding to Miz-1, and that the fragment may be exploited as an anticancer agent. Furthermore, the finding that a C-terminal fragment with pro-apoptotic activity is generated from full-length MAGE-A4 after genotoxic stress in human cells suggests a novel function for MAGE-A4.