Cloning of molecules that activate caspase9 regardless of cytochrome c.

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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.