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プロテオミクスによるキナーゼ基質同定法およびチロシンホスファターゼ大規模解析法の開発に関する研究

新苗, 智也 京都大学 DOI:10.14989/doctor.k23832

2022.03.23

概要

タンパク質の可逆的リン酸化修飾は、プロテインキナーゼ(以下、キナーゼ)およびプロテインホスファターゼ(以下、ホスファターゼ)を介して行われる。リン酸化修飾は、酵素活性やタンパク質局在などの調節を通じて、様々な細胞機能において重要な役割を果たしており、ヒトにおいては約500種のキナーゼ、約200種のホスファターゼによって厳密に制御されている。その破綻はがん、免疫疾患および代謝疾患など種々の疾患の一因となるため、キナーゼ・ホスファターゼは有力な創薬標的・薬効マーカー候補となりうる。一部のキナーゼに対する阻害薬が上市されているものの、細胞内リン酸化ネットワークにはいまだに不明な点も多く、キナーゼの生理的基質の大規模同定法や、ホスファターゼの発現量や活性に対する大規模解析法といったネットワーク全体のプロファイリングに必要な基盤技術は確立されていない。

 ショットガンプロテオミクスは、タンパク質混合物をプロテアーゼによりペプチド断片化後に液体クロマトグラフィー/タンデム質量分析(LC/MS/MS)に導入し、ペプチドイオンのアミノ酸配列情報を取得する。ショットガンプロテオミクスはタンパク質やタンパク質翻訳後修飾の大規模同定が可能であり、様々な技術と組み合わせて用いることで、スループットや網羅性を向上させる。

 以上の背景を踏まえて、本研究では、プロテオミクスを用いたキナーゼ基質同定法(第1章)およびチロシンホスファターゼ大規模解析法(第2章)の開発を行った。

第1章 キナーゼ生理的基質同定法の開発
 LC/MS/MSを用いたリン酸化プロテオミクスの発展により、一度の分析で数万種のリン酸化部位が定量できるようになった。しかし、20万種程度知られているヒトプロテオーム中のリン酸化部位のうち、責任キナーゼが分かっているものは少ない(~5%)。本章では、細胞内リン酸化プロファイリング、モチーフ配列評価および近接依存ビオチン標識法を用いてキナーゼの細胞内リン酸化基質同定を試みた。さらに、リン酸化基質周辺におけるアミノ酸置換がキナーゼと基質の関係に及ぼす影響についても評価した。その結果、キナーゼの新規基質の同定に成功し、がん細胞において、基質周辺のアミノ酸置換が責任キナーゼによるリン酸化を変動させる可能性を示した。

第2章 非加水分解性リン酸化チロシンミミック体を用いたチロシンホスファターゼ濃縮に関する研究
 リン酸化シグナルの破綻は様々な疾患の原因となるため、キナーゼおよびホスファターゼは創薬標的や薬効マーカーとして重要である。しかし、一般的なグローバルプロテオミクスでは安定にキナーゼ・ホスファターゼを同定することは困難である。キナーゼについては、汎キナーゼ阻害剤固定化樹脂を用いたキナーゼ濃縮プラットフォームにより、200種以上のキナーゼの濃縮が可能となっている。一方、ホスファターゼについてはいまだ汎用的な濃縮法が存在しない。本章ではホスファターゼ全体を解析対象とするホスファトミクスのための基盤技術開発を目的とし、非加水分解性リン酸化チロシンミミックアミノ酸であるF2Pmpを含むペプチドプローブを用いたホスファターゼ濃縮法の開発を検討した。F2Pmpペプチドを用いて、組換え体チロシンホスファターゼまたは細胞抽出物に対してプルダウン実験を行い、F2Pmpペプチド配列依存性やホスファターゼ活性との相関について評価した。その結果、F2Pmpペプチドはチロシンホスファターゼ活性に応じて結合し、そのチロシンホスファターゼに対する選択性はペプチド配列により大きく異なることが明らかとなった。

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