Cloning of molecules that activate caspase9 regardless of cytochrome c.

克隆激活 caspase9 的分子，与细胞色素 c 无关。

• 批准号: 14599009
• 负责人: HAYASHI Hideki
• 金额: $ 2.3万
• 依托单位: Nagasaki University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14599009/
• 关键词: cytochrome c; caspase; apoptosis

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.

我开发了一种新方法来克隆分子，无论细胞色素c如何激活caspase9。酵母细胞（宫颈链球菌）缺乏许多与凋亡相关的分子，例如胱天蛋白酶，BAX和BCL-2。我已经表达了通过CASPASE9特异性底物序列连接到已知受体的非活性caspase9和反式激活器。如果将caspase9激活剂引入细胞中，则从受体释放反式激活区域，并最终激活某些记者（它们也会提前引入同一细胞）。在选择培养基中检测到含有caspase9活化剂的细胞以进行报道。使用此方法，我还克隆了AATF（凋亡拮抗转录因子）和RIP60的C末端片段和RIP60（复制起始区域蛋白质）（复制起始区域蛋白质），除了全长caspase 2,3和4。在1999年，将细胞色素c.AATF克隆为DLK（Zik激酶）和PAR4依赖性细胞死亡中的抑制剂（Page等人）。我透露，AATF的C术片段激活CASPASE9，但没有激活全长AATF。 AATF可能会发挥关键作用，将细胞引导到促凋亡（通过CASPASE9激活）或抗凋亡（通过DLK和PAR4）。我现在正在阐明开关机构。RIP60是DNA复制组装和激活的必要因素。 RIP60的N末端片段激活了CASPASE9，而不是全长RIP60。我发现RIP60可以通过未知机制诱导细胞死亡。该机制应阐明。

项目成果

Kaibara, M.: "Identification of human Kir2. 2 (KCNJ12) gene encoding functional inward rectifier potassium channel in both mammalian cells and Xenopus oocytes"FEBS Lett.. 531・2. 250-254 (2002)

Kaibara, M.：“哺乳动物细胞和非洲爪蟾卵母细胞中编码功能性内向整流钾通道的人类 Kir2.2 (KCNJ12) 基因的鉴定”FEBS Lett.. 531・2 (2002)。

Kaibara, M.: "Identification of human Kir2.2 (KCNJ12) gene encoding functional inward rectifier potassium channel in both mammalian cells"FEBS Lett.. 531・2. 250-254 (2002)

Kaibara, M.：“两种哺乳动物细胞中编码功能性内向整流钾通道的人 Kir2.2 (KCNJ12) 基因的鉴定”FEBS Lett.. 531・2 (2002)。

Matsuo, K.: "Involvement of cholinergic neurons in orexin-induced contraction of guinea pig ileum"Eur.J.Pharmacol.. 452・1. 105-109 (2002)

Matsuo, K.：“胆碱能神经元参与食欲素诱导的豚鼠回肠收缩”Eur.J.Pharmacol.. 452・1 (2002)。

Hayashi, H.: "CARD6 is a modulator of NF-κB activation by Nod1- and Cardiak-mediated pathways"J.Biol.Chem.. 278・34. 31941-31949 (2003)

Hayashi, H.：“CARD6 是 Nod1 和 Cardiak 介导途径的 NF-κB 激活调节剂”J.Biol.Chem.. 278・34 (2003)。

Matsuo, K.: "Involvement of cholinergic neurons in orexin-induced contraction of guinea pig ileum."Eur. J. Pharmacol.. 452・1. 105-109 (2002)

Matsuo, K.：“胆碱能神经元参与食欲素诱导的豚鼠回肠收缩”。Eur. J. Pharmacol.. 452・1 (2002)。