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激活 caspase9 的分子，无论细胞色素 c 是什么。酵母细胞 (S. cerviciae) 缺乏许多与凋亡相关的分子，如 caspase、bax 和 bcl-2。我表达了无活性的 caspase9 和反式激活因子，该反式激活因子通过细胞表面上的 caspase9 特异性底物序列连接到已知受体。如果将caspase9激活剂引入到细胞中，反式激活区就会从受体中释放出来，并最终激活一些报告基因（它们也被提前引入到同一个细胞中）。在报告分析的选择培养基中检测含有 caspase9 激活剂的细胞。使用这种方法，除了全长 caspase 2,3 和 4 之外，我还克隆了 AATF（凋亡拮抗转录因子）和 RIP60（复制起始区蛋白）的 C 末端片段。无论怎样，C 末端 AATF 片段和 N 末端 RIP60 片段都会激活 caspase 9 细胞色素 c.AATF 于 1999 年被克隆为 Dlk（ZIK 激酶）和 Par4 依赖性细胞死亡的抑制剂（Page 等人）。我发现 AATF 的 C 端片段会激活 caspase9，但不会激活全长 AATF。 AATF 可能在引导细胞促凋亡（通过 caspase9 激活）或抗凋亡（通过 Dlk 和​​ Par4）方面发挥关键作用。我现在正在阐明开关机制。据报道，RIP60 是 DNA 复制组装和激活的必要因素。 RIP60 的 N 端片段激活 caspase9，但不激活全长 RIP60。我发现RIP60可以通过未知的机制诱导细胞死亡。应阐明其机制。

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)。

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)。

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)。

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)。