To show that HAX-1 degradation is part of the apoptotic process and any involvement Omi may have, we used the ucf-101 inhibitor. ucf-101 is a specific inhibitor of the proteolytic activity of Omi and has been described previously (13). When HK-2 cells were treated with cisplatin in the presence of ucf-101, the percentage of apoptotic cells decreased and the inhibitor significantly blocked HAX-1 degradation. This effect was more pronounced when a higher concentration of the inhibitor was used.
To confirm the specificity of the inhibitor in this system and exclude the possibility that another protease rather than Omi is involved in HAX-1 cleavage, we used cell lines derived from mnd2 mice (9). The parent cell line (mnd2-MSCV) derived from mouse embryo fibroblasts has no detectable Omi proteolytic activity (9). The same cell line has been transfected with wild type human Omi cDNA (mnd2-MSCV-Omi) and expresses high levels of active Omi protein (14). We found that in mnd2-MSCV cells, when induced to undergo apoptosis with various stimuli, the number of apoptotic cells was very low. Furthermore, no detectable cleavage of HAX-1 was observed. This is in contrast with the mnd2-MSCV-Omi cells where apoptosis was robust, and HAX-1 levels were inversely proportional to the degree of apoptosis. This experiment clearly shows that Omi is solely responsible for HAX-1 cleavage, which is essential for apoptosis under the conditions used in these experiments. HAX-1 subcellular localization depends on cell type (21, 30) and has been reported to be present in the mitochondria, cytoplasm, or plasma membrane (10, 21, 22, 30). We performed subcellular fractionation to investigate where HAX-1 cleavage by Omi takes place. We found that, in HEK293 cells, HAX-1 was predominantly present in the mitochondria, and this localization did not change in response to apoptotic stimuli. This suggests that Omi can initiate apoptosis in the mitochondria by cleaving HAX-1 protein. This is in accord with a recent study that shows Omi can induce apoptosis in human neutrophils treated with TNF- without being released from the mitochondria (7). Although several studies clearly define HAX-1 as an anti-apoptotic protein, the mechanism of its function is unknown. HAX-1 has sequence similarity to Bcl-2 family of proteins (10, 22).
參考答案:为了表明HAX-1 的降解是凋亡过程的一部分以及OMI可能的参与，我们用ucf-101抑制剂。ucf-101是此前报道过的(13)卵母细胞成熟抑制因子蛋白酶解活性的一种特异性抑制剂。当HK-2 细胞在ucf-101的存在下用顺铂处理时，凋亡细胞百分比下降，而且这种抑制剂明显阻止了HAX-1的降解。当抑制剂浓度加大时这种效应更加突出。
为了证实这种抑制剂在本系统中的特异性，并排除其他蛋白酶而不是OMI 涉及HAX-1酶切的可能性，我们使用mnd2 小鼠衍生的细胞系(9)。小鼠胚胎成纤母细胞系(mnd2-MSCV)没有可检测的OMI 蛋白酶活性(9)。相同的细胞系被转染到野生型人类OMI的cDNA (mnd2-MSCV-卵母细胞成熟抑制因子) 中，表达了大量的活性OMI蛋白(14)。我们发现mnd2-MSCV细胞用各种刺激剂诱导凋亡时，凋亡细胞的数量非常低。 进一步讲，没有观察到可检测的HAX-1酶切。这和细胞凋亡很旺盛的mnd2-MSCV-OMI细胞相反，并且HAX-1含量和细胞凋亡水平呈反比关系。该试验清楚地表明了卵母细胞成熟抑制因子单独决定HAX-1的酶切，在本实验使用的条件下对于细胞凋亡是必须的。HAX-1的亚细胞位置取决于细胞形状(21, 30)，并且已经有报道说可以存在于线粒体、细胞质或质膜 (10、21、22、30)中。 我们对细胞亚组分进行了分离操作来研究OMI酶切HAX-1 蛋白的位点。我们发现HEK293 细胞中的HAX-1蛋白主要存在于线粒体，并且这个位点不随刺激剂反应的改变而变化。 这暗示着卵母细胞成熟抑制因子靠酶切HAX-1 蛋白来启动线粒体中的细胞凋亡。这和最近的研究是一致的，该研究表明卵母细胞成熟抑制因子能诱导用TNF处理的人类神经多肽的凋亡而不从线粒体中释放出来(7)。尽管几个研究清楚地把HAX-1界定为一种抗凋亡蛋白，它发挥功能的机理仍然未知。HAX-1 和Bcl-2蛋白家族有序列相似性 (10, 22)。
