Role of heat shock protein (HSP) for the recovery of heat-inactivated DNA-PK

热休克蛋白 (HSP) 在恢复热失活 DNA-PK 中的作用

• 批准号: 13670950
• 负责人: MAKOTO Ihara
• 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: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670950/
• 关键词: DNA-PK; Ku70; Ku80; Recovery of hea-inactivation; Heat shock Protein; HSC73; 温熱処理; Heat shock protein

DNA-PK was inactivated by heat treatment. Heat-inactivated DNA-PK was recovered when cells were incubated at 37 ーC after heat treatment. Heat-inactivation of DNA-PK is a result of heat-inactivation of Ku70 and/or Ku80 of DNA-PK complex. Recovery of DNA-PK activity is also seen in the presence of cycloheximide. This result indicates that heat shock protein may participate the recovery of heat inactivated DNA-PK activity.In order to identify the heat sensitive, protein, cells expressing high order of Ku70 or Ku80 were created. The content of Ku70 or Ku80 after heat treatment was analyzed by western blotting.. The amount of Ku70 proteins decreased depending on the period of heat treatment. On the other hand, the amount of Ku80 was not influenced by heat treatment. This indicates the Ku70 is the heat sensitive component of DNA-PK.Next, the recovery of heat inactivated DNA-PK was examined using cells over expressing HSC73. Recovery of heat-inactivated DNA-PK was early in cells expressing high level of HSC73 compared with the normal cells.Extract of heat-treated cells was immunoprecipitated with anti-Ku antibody. The content of HSC73, co-immunoprecipitated with Ku, was analyzed by western blotting using anti HSC73 antibody. The content of HSC73 increased depending on the incubation time after heat treatment and reached maximum at 3h after heat treatment. This is in good agreement with the recovery time of heat inactivated DNA-PK activity.We are planning to use RNAi for inhibition of HSC73 and examine its effect of recovery of heat-inactivated DNA-PK.

通过热处理使DNA-PK失活。当热处理后将细胞在37 ℃下孵育时，热灭活的DNA-PK恢复。DNA-PK的热失活是DNA-PK复合物的Ku 70和/或Ku 80热失活的结果。在放线菌酮存在下也观察到DNA-PK活性的恢复。这一结果表明热休克蛋白可能参与了热失活的DNA PK活性的恢复。为了鉴定热敏感蛋白，建立了高表达Ku 70或Ku 80的细胞。采用Western blotting法分析热处理后的Ku 70或Ku 80含量。Ku 70蛋白的量随热处理时间的延长而减少。另一方面，Ku 80的量不受热处理的影响。这表明Ku 70是DNA-PK的热敏组分。接下来，使用过表达HSC 73的细胞检查热灭活的DNA-PK的恢复。热灭活的DNA-PK在高表达HSC 73的细胞中比正常细胞恢复得早。用抗HSC 73抗体通过Western印迹分析与Ku共沉淀的HSC 73的含量。HSC 73的含量随热处理后孵育时间的延长而增加，在热处理后3 h达到最大值。这与热灭活DNA-PK活性的恢复时间非常一致。我们计划使用RNAi抑制HSC 73，并检查其对热灭活DNA-PK的恢复效果。