Induction of apoptosis in oral squamous cell carcinoma by activating caspase

Bnip3是一种促凋亡蛋白，包含BH3和跨膜结构域。我们构建了几个Bnip3缺失突变体，并研究了它们的生物学功能。Bnip3的跨膜结构域不需要Bnip3与ced-3的结合；而跨膜结构域缺失突变体不能引发细胞凋亡级联反应。免疫荧光研究表明，野生型Bnip3和ced-3在线粒体上共定位，跨膜结构域缺失突变体不能显示共定位。这些结果表明，ced-3/caspase在线粒体膜上的募集对于Bnip3引发的细胞凋亡至关重要。腺病毒E4orf6是一种已知与E1A基因产物合作转化原代小鼠细胞的病毒癌蛋白。它已被证明通过直接结合p53和p73蛋白来抑制凋亡活性。在这里，我们证明了腺病毒E4orf6蛋白抑制Bcl-2家族BH3-only蛋白BNIP3和Bik介导的细胞凋亡。这种活性不是由p53和p73介导的，因为E4orf6对不表达p53相关基因的Saos-2细胞的凋亡具有相同的作用。我们还确定E4orf6可以改变BNIP3和Bik的线粒体定位。缺乏E4orf6核输出信号的突变体不能抑制细胞凋亡，也不能将BNIP3蛋白从线粒体中转运。此外，我们还发现E4orf6能够在体外与BNIP3和Bik相互作用。在BNIP3蛋白中，相互作用所需的区域包括跨膜结构域，这是BNIP3定位到线粒体所必需的。这些结果表明，E4orf6从细胞核输出到细胞质，使其能够与BH3-only蛋白相互作用，最终导致细胞凋亡活性的抑制。

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.