Cloning of molecules that activate caspase9 regardless of cytochrome c.

我开发了一种新的方法来克隆激活半胱天冬酶9的分子，而不管细胞色素c。酵母细胞（S.宫颈癌）缺乏许多凋亡相关分子，如半胱天冬酶、Bax和Bcl-2。我已经表达了无活性的半胱天冬酶9和一个反式激活因子，该因子通过细胞表面的半胱天冬酶9特异性底物序列与已知受体连接。如果将半胱天冬酶9激活剂引入细胞，则反式激活因子区域从受体释放，并最终激活一些报告基因（它们也被提前引入同一细胞）。用这种方法，我克隆了AATF（凋亡拮抗转录因子）和RIP 60（复制起始区蛋白）的C-末端片段，以及全长半胱天冬酶2、3和4。C-末端AATF片段和N-末端RIP 60片段激活半胱天冬酶9，而不管细胞色素c。AATF在1999年被克隆为Dlk（ZIK激酶）-和Par 4-依赖性细胞死亡中的抑制剂（Page等人）。我已经揭示了AATF的C端片段激活caspase 9，但不是全长AATF。AATF可能在引导细胞促凋亡（通过半胱天冬酶9活化）或抗凋亡（通过Dlk和Par 4）方面发挥关键作用。我现在阐明开关机制。据报道，RIP 60是DNA复制组装和激活的必要因子。RIP 60的N-末端片段激活caspase 9，但不激活全长RIP 60。我发现RIP 60可以通过未知的机制诱导细胞死亡。应阐明其机制。

I have developed a new method to clone molecules that activate caspase9 regardless of cytochrome c. Yeast cells (S. cerviciae) are lacking many apoptosis-related molecules such as caspases, bax, and bcl-2. I have expressed inactive caspase9 and a transactivator that is connected to a known receptor via caspase9-specific substrate sequence on the cell surface. If a caspase9 activator is introduced into the cell, the transactivator region is released from the receptor, and eventually activates some reporters (They are also introduced into the same cell in advance). The cells containing caspase9 activator are detected in a selection medium for the reporter assays.Using this method, I have cloned a C-terminal fragment of AATF (Apoptosis antagonizing Transcription Factor) and RIP60 (Replication Initiation Region Protein), in addition to full-length caspases 2,3, and 4. The C-terminal AATF fragment and N-terminal RIP60 fragment activated caspase 9 regardless of cytochrome c.AATF was cloned as an inhibitor in the Dlk (ZIK kinase)-and Par4-dependent cell death in 1999 (Page, et al.). I have revealed that the C-termial fragment of AATF activates caspase9, but not the full-length AATF. AATF may play a key role to direct the cell to pro-apoptotic (via caspase9 activation) or to anti-apoptotic (via Dlk and Par4). I am now elucidating the switch mechanism.RIP60 was reported as a necessary factor in the assembly and activation of DNA replication. The N-terminal fragment of RIP60 activated caspase9, but not the full-length RIP60. I found that RIP60 can induce cell death by unknown mechanism. The mechanism should be elucidated.