蛍光基質を用いた細胞内プロテオリシスの可視化解析

使用荧光底物对细胞内蛋白水解进行可视化分析

• 批准号: 11780454
• 负责人: 西井 亘
• 金额: $ 1.41万
• 依托单位: Tokyo University of Pharmacy and Life Science
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Encouragement of Young Scientists (A)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11780454/
• 关键词: 蛍光基質; 可視化解析; 細胞死; ラット海馬; プロテオリシス; 細胞内イメージング

本研究では,細胞内プロテオリシスをリアルタイムで可視化検出しうる新規蛍光基質の開発,およびその細胞内イメージングを試みた.その結果,以下の実績を得た.(1)カスパーゼ1を標的とした新規プロテオリシス可視化試薬の開発カスパーゼ1は細胞死における情報伝達において,重要な役割を担うとされる細胞内プロテアーゼであり,その生体内における活性を可視化解析することは非常に意義深い.ここでは,カスパーゼ1の活性を可視化する試薬として,ペプチド鎖のN,C両端をtetramethylrhodaminおよびacryrodanで2重蛍光標識し,両蛍光基間で,FRET(Fluorescent Resonance Energy Transfer)がおこるようにデザインした新規基質を作製した.本基質は,カスパーゼ1の活性を受けて蛍光を発するので,その特異的な検出が可能である.また,Kmが14μMと親和性も高い.さらに,マイクロインジェクション等の煩雑な操作によらなくても容易に細胞内に導入可能である.これらの点で本基質はカスパーゼ1活性可視化試薬として極めて高性能であるといえる.(2)グルタミン酸刺激下のラット海馬スライス培養におけるカスパーゼ1活性の可視化ラット海馬は,グルタミン酸刺激により細胞死が誘導されることが知られている.本研究では上記の新規基質を用いてこの過程におけるカスパーゼ1活性の増減,局在の経時変化を解析した.グルタミン酸刺激後,一定時間経過後のラット海馬スライス培養に基質を導入し,蛍光顕微鏡で観察したところ,カスパーゼ1活性は,時間をおって増大し,15時間後にピークを迎えその後減少した.また活性は海馬CA1,CA2領域に局在していた(現在論文投稿準備中).

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