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A facile screening strategy to construct auto-fluorescent protein-based biosensors

Tajima, Shunsuke 京都大学 DOI:10.14989/doctor.k23998

2022.03.23

概要

生体内のクリーンで高効率なエネルギー利用・変換機構である代謝反応は、酵素が関与する並列した多段階の反応により成り立っている。これらの反応はシグナル伝達により時空間的に制御されているため、生体内の複雑な代謝システムを理解し活用するためには、生体内のシグナル伝達機構の理解が重要である。蛍光バイオセンサーを用いたシグナル伝達物質の生細胞内での可視化は、時空間分解能の高い手法であり、シグナル伝達機構の解明に広く用いられてきたが、バイオセンサーの開発には、大規模なライブラリーや多段階のスクリーニングが不可避であり、多大な時間と労力を要する。本論文は、蛍光タンパク質を用いてタンパク質の機能発現に伴う構造変化を可視化する手法と、迅速で簡便なスクリーニングによってバイオセンサーを作製する方法を論じたもので、6 章からなっている。

第 1 章は序論であり、シグナル伝達物質の生細胞内での可視化に用いられる蛍光バイオセンサーが時空間分解能の高い手法であり、シグナル伝達機構の解明に広く用いられてきた背景を論述するとともに、バイオセンサーの構造的な特徴と、応用例、さらに、目的とする機能を発揮するバイオセンサーを構築するうえでの問題点を論じている。そのうえで、蛍光タンパク質を用いてタンパク質の機能発現に伴う構造変化を可視化する手法と、迅速で簡便なスクリーニングによってバイオセンサーを作製する方法を開発するという本論文の目的を述べている。

第 2 章では、チャネルタンパク質 TRPC5 が NO に応答した際に誘起される構造変化を、蛍光タンパク質により可視化した。TRPC5 は、Cys553 が NO によりニトロソ化された後、近傍のCys558 と分子内ジスルフィド結合を形成して、チャネルが開口すると推定されている。 Cys553 と Cys558 を含みチャネル開口部を形成する TRPC5 部分構造を、EGFP の蛍光団近傍に導入した EGFP-TRPC5 を作製した。チオール基濃度定量と蛍光強度の測定から、NO に応答したジスルフィド結合形成による蛍光強度比の増加が観測された。即ち、Cys553 と Cys558 によるジスルフィド結合の形成により TRPC5 部分構造の構造が変化し、その構造変化が蛍光タンパク質の蛍光団まで伝播することによって、NO による TRPC5 部分構造の構造変化が可視化できることを示した。

第 3 章では、蛍光タンパク質を用いたバイオセンサーの蛍光強度変化を増大させるための、簡便な二段階スクリーニング法を開発した。第 2 章で作製したEGFP-TRPC5 の NO に応答した蛍光強度変化は微小ではあったが、EGFP-TRPC5 を もとにして NO バイオセンサーが構築できる可能性が示唆された。TRPC5 部分構造を短くし、Cys553 と Cys558 のジスルフィド結合形成部位を EGFP に近づけることで、ジスルフィド結合形成に伴う構造変化がより効率的に EGFP に伝わり、蛍光変化が増大すると考えた。しかし、TRPC5 部分構造を N 末端と C 末端の両端あるいは片方の端から一アミノ酸ずつ短くしていくだけでも計 47 個もの候補が考えられる。従来のスクリーニング法では、数多くの変異体を実際に作製し、何段階もの機能検証を経て最適なバイオセンサーを得る。本手法では、一段階目に in silico でのシミュレーションを用いて、各変異体の構造変化の大きさをジスルフィド結合形成前後の RMSD の大きさから評価し、蛍光強度変化の増大が期待される 10 個の変異体を効率的に選出した。二段階目として、ジスルフィド結合を形成した変異体を大腸菌を用いて作製し、簡易精製の後にジスルフィド結合開裂による蛍光強度変化を評価した結果、EGFP-TRPC5 より 2〜4 倍の大きな蛍光強度変化比を示す 3 つの変異体を選出した。

第 4 章では、第 3 章で得た 3 つの変異体が、実際に NO を検出するセンサーとして機能するかを試験管内で評価した。3 つの変異体全てについて、ジスルフィド結合形成により蛍光強度比が増加し、NO を検出することが確認された。また、還元によるジスルフィド結合開裂により蛍光強度比は減少し、可逆的なセンサーとなる可能性が示された。これらの変異体は EGFP-TRPC5 と同様に過酸化水素に対しても応答したため、レドックスセンサーとして利用できる。また、TRPC5 の Cryo-EM 像をもとにすると、抽出した部分構造は細胞膜近傍の細胞外に露出しているため、TRPC5部分構造自身ではなく、細胞膜の疎水性が TRPC5 の NO の選択性に寄与している可能性が示唆された。

第 5 章では、第 4 章、第 3 章で作製、評価したレドックスセンサーが、生細胞内でも機能するかを検証した。レドックスセンサーは、HEK293 細胞内において計測可能な蛍光強度を示すことが確認された。レドックスセンサーを発現させた HEK293細胞に外部から過酸化水素を添加すると、各細胞で試験管内での結果と同じ程度に蛍光強度比が増大することが確認された。これらの結果から、レドックスセンサーが細胞内で利用できることを実証した。

第 6 章は総括である。本研究では、蛍光タンパク質型バイオセンサーの検出原理を応用して、TRPC5 の機能発現過程で予想された構造変化を可視化した。さらに、二段階スクリーニング法を開発して、細胞内でも利用可能な検出感度を有するバイオセンサーを作製した。この二段階スクリーニング法は、一般的な蛍光タンパク質型バイオセンサーの構築にも不可欠な標的認識部位の導入位置の最適化に適用できるため、効率的な蛍光タンパク質型バイオセンサー構築を促進するものである。本研究で開発した手法は、特に標的認識部位の構造変化情報が乏しい場合に有効であるため、今後細胞内でのエネルギー利用過程に関与する数多くのタンパク質の機能検証を加速すると期待できる。

参考文献

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