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Induction of apoptosis in oral squamous cell carcinoma by activating caspase

激活caspase诱导口腔鳞状细胞癌凋亡

基本信息

批准号：
13557172
负责人：
TOTSUKA Yasunori
金额：
$ 8.9万
依托单位：
Hokkaido University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13557172/
关键词：
Apoptosis Bnip3 Transmembrane domain E4orf6

项目摘要

Bnip3 is a pro-apoptotic pretein, which contains BH3 and transmembrane domains. We constructed several deletion mutants of Bnip3 and investigated their biological functions. The transmembrane domain of Bnip3 was not required fo the association of Bnip3 and ced-3 ; however, transmembrane domain deleted mutant could not initiate the apoptotic cascade. Immunofluorescence study demonstrated that wild type Bnip3 and ced-3 co-localized on mitochondria, and transmembrane domain deleted mutanat could not show the co-localization. These results suggest that recruitment of ced-3/caspase onto the mitochondrial membrane is essential for Bnip3 intiated apoptosis.The adenovirus E4orf6 is a viral oncoprotein know to cooperate with the E1A gene product in transforming primary murine cells. It has been shown to inhibit the apoptotic activities of p53 and p73 through direct binding to these proteins. Here, we demonstrate that the adenovirus E4orf6 protein inhibits apoptosis mediated by BNIP3 and Bik, which are BH3-only proteins of the Bcl-2 family. This activity was not mediated by p53 and p73 because E4orf6 had the same effect on the apoptosis in Saos-2 cells that do not express p53-related genes. It was also ascertained that E4orf6 could change the mitochondrial localization of BNIP3 and Bik. A mutant lacking the nuclear export signal of E4orf6 failed to inhibit apoptosis and to translocate BNIP3 protein from the mitochondria. Moreover, it was also established that E4orf6 was able to interact with BNIP3 and Bik in vitro. In BNIP3 protein, the region required for the interaction included the transmembrane domain, which is required for the localization of BNIP3 to the mitochondria. These results suggest that E4orf6 is exported from the nucleus to the cytoplasm, enabling it to interact with BH3-only proteins, eventualy leading to the inhibition of apoptotic activity.

Bnip 3是一种促凋亡蛋白，含有BH 3和跨膜结构域。我们构建了几个Bnip 3的缺失突变体，并研究了它们的生物学功能。Bnip 3与ced-3的结合不需要Bnip 3的跨膜结构域，但跨膜结构域缺失的突变体不能启动凋亡级联反应。免疫荧光研究表明野生型Bnip 3和ced-3共定位于线粒体上，而跨膜结构域缺失突变体Bnip 3和ced-3不能共定位。这些结果表明，ced-3/caspase募集到线粒体膜上对于Bnip 3诱导的细胞凋亡至关重要。腺病毒E4 orf 6是一种病毒癌蛋白，已知与E1 A基因产物合作转化原代小鼠细胞。已经显示通过直接结合p53和p73蛋白来抑制这些蛋白的凋亡活性。在这里，我们证明了腺病毒E4 orf 6蛋白抑制由BNIP 3和Bik介导的细胞凋亡，BNIP 3和Bik是Bcl-2家族的仅BH 3蛋白。这种活性不是由p53和p73介导的，因为E4 orf 6对不表达p53相关基因的Saos-2细胞的凋亡具有相同的作用。E4 orf 6可以改变BNIP 3和Bik的线粒体定位。缺乏E4 orf 6的核输出信号的突变体不能抑制细胞凋亡和从线粒体易位BNIP 3蛋白。此外，还确定了E4 orf 6能够在体外与BNIP 3和Bik相互作用。在BNIP 3蛋白中，相互作用所需的区域包括跨膜结构域，这是BNIP 3定位于线粒体所必需的。这些结果表明，E4 orf 6从细胞核输出到细胞质，使其能够与仅BH 3蛋白相互作用，最终导致凋亡活性的抑制。